extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Medicine

text:
Plant-Derived Products for Treatment of Vascular Intima Hyperplasia Selectively Inhibit Vascular Smooth Muscle Cell Functions

Natural products are used widely for preventing intimal hyperplasia (IH), a common cardiovascular disease. Four different cells initiate and progress IH, namely, vascular smooth muscle, adventitial and endothelial cells, and circulation or bone marrow-derived cells. Vascular smooth muscle cells (VSMCs) play a critical role in initiation and development of intimal thickening and formation of neointimal hyperplasia. In this review, we describe the different originating cells involved in vascular IH and emphasize the effect of different natural products on inhibiting abnormal cellular functions, such as VSMC proliferation and migration. We further present a classification for the different natural products like phenols, flavonoids, terpenes, and alkaloids that suppress VSMC growth. Abnormal VSMC physiology involves disturbance in MAPKs, PI3K/AKT, JAK-STAT, FAK, and NF-κB signal pathways. Most of the natural isolate studies have revealed G1/S phase of cell cycle arrest, decreased ROS production, induced cell apoptosis, restrained migration, and downregulated collagen deposition. It is necessary to screen optimal drugs from natural sources that preferentially inhibit VSMC rather than vascular endothelial cell growth to prevent early IH, restenosis following graft implantation, and atherosclerotic diseases.

Introduction
Intimal hyperplasia (IH) is a fibroproliferative disorder observed in vascular pathogenesis particularly in vessel anastomotic stenosis, atherosclerosis, blockage of vessel grafts, angioplasty, and in-stent restenosis [1]. IH is characterized by enhanced cell migration, proliferation, and differentiation that cause narrowing of the tunica intima. Several cells are associated with initiation and progression of IH, namely, vascular smooth muscle cells (VSMCs) [2], vascular adventitial cells [3], vascular endothelial cells (VEC) [4], and circulating bone marrow-derived cells [5]. These cells have different origins but may contribute to IH formation. For example, endothelial cells may undergo endothelial-tomesenchymal transition (EndMT) acquiring a fibroproliferative mesenchymal phenotype whereas adventitia-derived stem cells may migrate to the intimal lesion site and differentiate into fibroblasts. VSMCs play a critical role in the initiation and development of intimal thickening and formation of neointimal hyperplasia [6,7].
Many herbal medicines sourced from plants or foods have been used to prevent cardiovascular disease over the millennia. For example, green tea contains various flavanols that have antioxidative [8,9], anti-inflammatory 2 Evidence-Based Complementary and Alternative Medicine [10], antimicrobial [11], and hypolipidemic [9] effects. This pharmacological profile helps prevent atherosclerotic plaque formation caused by inflammation and oxidative stress. Red wine, another commonly enjoyed beverage, has long been believed to be rich in polyphenols [12], which act as powerful antioxidants. These assist in preventing lower density lipoprotein oxidation in heart disease and attenuating development of atherosclerotic disease in the hamster model [13,14]. Resveratrol (3,5,4 -trihydroxy-trans-stilbene), a typical polyphenol extracted from red wine, has been proven to inhibit proliferation of VSMCs in vitro [15]. Many natural compounds have been reported to be active and to have potential utility as clinical medicines. Tanshinone is an isolate from Salvia miltiorrhiza that has been used against cardiovascular disease in China [16]. Therefore, many active compounds with cosmopolitan distribution are being used as herbal medicines or foods, giving hope for screening for potential therapeutic agents against IH ( Figure 1).
Recent clinical studies have shown that rapamycin A, an VSMC inhibitor, prevents development of IH-induced vascular endothelial dysfunction [17]. This nonspecific cytotoxicity leads to stenosis and eventually to failure of vascular reconstruction after injury. Therefore, the ideal drug to prevent restenosis or IH is one that inhibits VSMC proliferation selectively while having minimal inhibitory effect on VEC proliferation.

Diverse Cells Involved in Vascular IH
As stated earlier, four different cell types are involved in the initiation and progression of IH. These are VSMCs, vascular adventitial cells, VECs, and circulating bone marrowderived cells (Figure 2). VSMCs play a critical role in the initiation of intimal thickening and the formation neointimal hyperplasia. Physiologically VSMCs exist in two phenotypes, i.e., differentiated cells and proliferating cells, which are responsible for maintaining the homeostasis and function of vascular vessels [2,6]. Stimulation by certain growth and inflammatory factors, such as platelet-derived growth factor, tumor necrosis factor-(TNF-), and thrombin, results in dedifferentiation into mature VSMCs [18,19]. Mature and differentiated VSMCs exhibit loss of contractility and increased proliferation and expression of ECM protein and various cytokines. This phenomenon is responsible for intimal thickening leading to the neointimal hyperplasia formation that is observed in early-phase atherosclerosis.
Endothelial-to-mesenchymal transition is a phenomenon where endothelial cells acquire a fibroproliferative mesenchymal phenotype through differential stimulation [20]. These transitioned endothelial cells mimic fibroblasts and have increased ECM production and migration capabilities. In vascular diseases, these transitioned endothelial cells can quickly migrate and differentiate into smooth muscle-like cells serving as a potential contributor to IH [21,22]. EMT is reported to be modulated by shear stress in an ERK5dependent manner, to contribute to neointimal hyperplasia, and to induce atherogenic differentiation [23]. In addition to adventitia-derived stem cells, circulating smooth muscle progenitor cells have also been implicated in the pathogenesis of neointimal hyperplasia [24] and in the recruitment of endothelial precursor cells after vascular trauma. The presence of bone marrow-derived cells in solid neointima or allograft lesions suggests their crucial involvement in lesion formation following vascular injury [5,25]. Although various cells contribute to IH pathogenesis, smooth muscle cells are the main culprits in lesion formation. Therefore, therapeutic strategies that maintain VSMCs in a terminally differentiated state and inhibit their proliferation and migration can be useful in preventing neointimal hyperplasia.

Antiproliferation, Migration, and Cellular Functions of Abnormal VSMCs as a Target to Decrease Intimal Hyperplasia
VSMCs in the normal vascular tunica media express a range of smooth muscle cell markers including smooth muscle cell myosin heavy chain (MYH11), 22-kDa SMC lineagerestricted protein (SM22 /tagln), alpha smooth muscle actin (ACTA2), and smoothelin. VSMCs in vitro and in atherosclerosis undergo phenotypic switching with reduced expression of these markers, while increasing capacity for cell proliferation, migration, and secretion of various ECM proteins and cytokines. These phenotypic switches have long been considered of fundamental significance in IH progression.
Most studies investigating inhibition of VSMCs adopt drugs like rapamycin, sirolimus, or tacrolimus to induce VSMC apoptosis and cell cycle arrest at G1/S phase, suppress ROS production, inhibit VSMC migration, and downregulate collagen deposition. These approaches do not recover the mature VSMC immunophenotypes, but they do decrease neointimal formation and prevent stenosis following vascular injury. To investigate the anticellular function of drugs on VSMCs many models have been established in vitro and in vivo. For the in vitro experiments, inflammatory cytokines like TNF-or some growth factors such as platelet-derived growth factor (PDGF) are used for inducing abnormal proliferation and migration of VSMCs. For the in vivo experiments, IH is usually induced using the vascular endothelial denudation model or carotid artery ligation injury.
Dietary supplements and traditional herbal medicines are complementary medication approaches used in every society and are widely used for preventing IH in Asia and in other developed countries [26]. Many herbal drugs and foods have been verified as suppressing abnormal VSMC growth and inhibiting intima formation. The positive effects of the herbal medicines and plants depend on their active natural compounds including phenols, flavonoids, terpenes, and alkaloids. These natural products are involved in different signaling pathways that regulate abnormal VMSCs to attenuate IH.

Typical Signal Pathways Involved in Growth and Physiology of VSMCs in IH Disease
The six signaling pathways involved in most drug inhibitory VSMC studies ( Figure 3) are mitogen-activated protein    MAPKs are involved in cell proliferation, differentiation, mitosis, cell survival, and apoptosis [27]. Three major families of MAPKs are extracellular signal-regulated kinase (ERK) [28], p38 kinase, and c-Jun N terminal kinase (JNK). These contribute to the two important signaling pathways, Ras/ERK-MAPK and JNK/p38-MAPK, which are involved in regulating VSMCs [29]. In antiproliferation studies of VSMCs, PI3K/Akt signaling pathway includes many key factors such as GSK3 , p21, and p27, which all inhibit cyclins and CDKs thereby interfering with cell cycle processes. GSK3 is one of the critical downstream molecules of the Akt signaling pathway involved in cell proliferation, metabolism, growth, and survival. It is reported that cyclin D is regulated by GSK3 [30] and that activation of GSK3 leads to exportation into cytoplasm for proteolysis, thus downregulating cyclin D1 expression [31]. The JAK-STAT signaling pathway transmits information from extracellular chemical signals to the nucleus resulting in DNA transcription and expression of genes involved in immunity, proliferation, differentiation, and apoptosis [32]. The downstream proteins in this pathway include cyclin D, p21, Bcl-2, and c-Myc, which are all directly involved in growth, apoptosis, and cell cycle progression in VSMC studies [33]. FAK is involved in cellular adhesion and migration [34]. FAK is typically located at structures known as focal adhesions, which are multiprotein structures including actin, filamin, and vinculin which link the ECM to the cytoplasmic cytoskeleton [35][36][37]. In addition, FAK interacts with PI3K and p53 [38,39] and with the PI3K/Akt and MAPKs signaling pathways that are involved in cell cycle regulation. NF-B controls many genes involved with inflammation which are crucial to progression of diseases including arthritis, asthma, and atherosclerosis [40,41]. Inflammation also mediates abnormal movement and growth of VSMCs, while suppressing inflammation could attenuate neointimal hyperplasia significantly [42][43][44][45]. Therefore, different signaling pathways are involved in VSMC inhibition, which provides preferential protein targets for future drug screening.

Flavonoids Regulate Cell Cycle and Functions Inhibiting VSMCs Proliferation and Migration.
Flavonoids are distributed throughout the plant kingdom and fulfill a diverse range of biological and pharmacological effects such as antiinflammatory [46], antioxidant [47], antibacterial [48], antitumor [49], and antidiarrheal activities [50]. For treatment of cardiovascular disease, flavonoid studies have focused on reducing hypertension, risk of atherosclerosis, oxidative stress, and related signaling pathways in blood vessel cells, as well as modifying vascular inflammatory mechanisms [51,52]. In this review, we described the chemical structure, category, source, and mechanism of action of some typical flavonoids that suppress VSMC function and inhibit IH (Table 1). Nobiletin is widely distributed in citrus fruits and has been reported to inhibit VSMC proliferation and migration in vitro [44]. In addition, carotid balloon injured rats given nobiletin 25 mg/kg/day by gavage had significantly decreased neointimal hyperplasia via regulation of the ROS derived NF-B pathway and decreased serum TNF-and IL-6 concentrations [44]. Cyanidin-3-O-glucoside, an anthocyanin flavonoid, inhibited TNF--induced NoxA1 (a type of NADPH oxidase) and downregulated expression of both TNF-and NoxA1 at transcriptional and translational levels [53]. (2S)-Naringenin, a typical flavonoid isolated from Typha angustata, inhibited PDGF-BB-induced proliferation Evidence-Based Complementary and Alternative Medicine 5  [54]. Hu and colleagues found that icariin reduced the amount of ox-LDL-induced proliferation of VSMCs through suppression of PCNA expression and inactivation of ERK1/2 [55]. Puerarin, isolated from Radix puerariae, exerted inhibitory effects on high glucose-induced VSMC proliferation via interfering with PKC 2/Rac1-dependent ROS pathways, thus resulting in attenuation of neointimal formation [56]. Alpinetin is a well-known flavonoid isolated from a variety of plants such as Alpinia katsumadai, Amomum subulatum, and Scutellaria rivularis. It may have some protective effects on VSMCs as it decreases LDH leakage and inhibits production of NO in TNF--induced VSMC [57]. Hesperetin, a flavonoid, inhibits PDGFa-BB-induced pASMC proliferation via the AKT/GSK3 signaling pathway through upregulating p27 expression while suppressing cyclin D1/E, CDK2/4 and p38 [58]. Pinocembrin reduces the increased ERK1/2 phosphorylation that occurs in response to angiotensin II in both rat aortic rings ex vivo and VSMCs in vitro [59]. Glyceollins, which are isoflavonoids, inhibit PDGF-BB-induced hVSMC proliferation and migration by downregulating CDK2, cyclin D1, pPDGFr-, phospholipase C 1, Akt, and ERK1/2 and interfering with ROS generation, while upregulating p27 kip1 and p53 expression levels [60].
Morelloflavone is a biflavonoid, which has been found to block injury-induced neointimal hyperplasia via inhibition of VSMC migration and downregulation of FAK, Src, ERK and RhoA expression [61]. Some studies have demonstrated that a natural flavonoid, kaempferol, may induce miR-21. This results in downregulation of ROCK4, 5, and 7, which are critical for cytoskeletal organization and cell motility, leading to decreased cell migration [62]. Finally, green tea is beneficial for health due to its antioxidant, anticarcinogenic, anti-inflammatory, and antiradiation effects [63][64][65]. A large number of flavonoids, especially flavan-3-ols ("catechins"), inhibit IH in a rat balloon injury model through upregulation of TIMP-2 expression to modulate MMP activity [66]. From the above review, flavonoids are an important candidate compound type for screening natural drugs capable of inhibiting VSMC growth.  [67][68][69]. Some typical polyphenols prevent IH by restraining VSMC function including proliferation, migration, and fibrosis ( Table 2). Salvianolic acid B is a typical polyphenol that is usually isolated from Salvia miltiorrhiza. It markedly reduces neointimal thickness by inducing neointimal cell apoptosis through upregulating p53 expression levels [70].

Polyphenols as an Antioxidants
In another study, salvianolic acid B protected hAECs and neointimal formation through inhibition of LDL oxidation by reducing ROS generation [71]. Magnesium lithospermate B, a derivative of salvianolic acid B, prevented diabetic atherosclerosis via the Nrf2-ARE-NQO1 transcriptional pathway [72]. Magnolol (a phenol) is a powerful antioxidant that inhibited balloon injury-induced rabbit IH by downregulating MCP-1 expression [73]. In another work, magnolol inhibited VSMC migration via the cytoskeletal remodeling pathway through inhibition of 1-integrin expression, phosphorylation of FAK and MLC20, and activation of RhoA and Cdc42 [74]. Lithospermic acid, a polyphenol, arrested cell cycle progression at the G1 phase via downregulating expression of cyclin D1 and inhibiting ROS generation and ERK1/2 phosphorylation [75]. Moreover, lithospermic acid attenuated LPS-induced VSMC migration by inhibiting MMP-9 expression in a dose-dependent manner (25-100 mol/L). Hispolon blocked balloon injury-induced neointimal hyperplasia via inhibition of VSMC proliferation. It also inhibited VSMC migration by lowering MMP-2/9 expression and increasing TIMP-1/2 expression through suppression of the FAK signaling pathway [76]. Lim and colleagues were of the view that obovatol blocked the cell cycle in G1 phase by downregulating expression of cyclins and CDKs, while selectively upregulating expression of p21 Cip1 , a well-known CDK inhibitor, both in vitro and in vivo [77]. Some studies have shown that curcumin (diarylheptanoid phenol) has potent antioxidant properties, which can be used for attenuating neointimal hyperplasia [78]. Curcumin has also been shown to inhibit PDGF-induced VSMC migration, proliferation, and collagen synthesis in a concentrationdependent manner [79], with a concentration range of 0.01 to 10 mol/L inhibiting VSMC proliferation and migration. Curcumin-coated stents inhibited neointimal formation in the rabbit iliac artery stent model. Moreover, curcumin inhibited LPS-induced MMP-2 activity in rat VSMCs through Ras/MEK1/2 and NF-B signaling [80].
Curcumin shows the ideal biological effects of inhibiting abnormal VSMC proliferation and migration without compromising VEC proliferation or delaying reendothelialization after blood vessel injury. Curcumin inhibited platelet adhesion to brain microvascular endothelial cells by decreasing expression of P-selectin, E-selectin, and GPIIb/GPIIIa in a concentration-dependent manner (30-240 mol/L). Curcumin antagonized the detrimental effect of rapamycin on aortic endothelial cells in vitro, through upregulation of eNOS [81]. Hence, curcumin very selectively inhibited abnormal VSMC functions, such as PDGF-induced proliferation or migration, without impairing VECs. As a result, curcumin has been regarded as an ideal drug for attenuating atherosclerosis and restenosis. In summary, polyphenols exhibit beneficial and wide ranging biological effects relevant to prevention of IH. Polyphenols are worthy candidate compounds to be screened as natural drugs for inhibiting VSMCs.

Terpenes Suppress Abnormal VSMC Function against
Neointimal Formation. Terpenes are proven cell cycle inhibitors for various cell types, especially tumor cells [82,83]. Like similar compounds with active sites for regulating VSMC mitosis and DNA synthesis, terpenes lead cell proliferation and function arrest via cell cycle blockade or apoptosis induction (Table 3). Betulinic acid, a typical terpene, has been reported to inhibit growth and proliferation of VSMCs via arresting G1/S cell cycle in a dosedependent manner [84]. A monoterpene, (S)-(-)-perillic acid, has been reported to decrease protein prenylation leading to DNA synthesis and inhibition of VSMCs [85]. A sesquiterpene lactone, parthenolide, arrested VSMC G0/G1 cell cycle via upregulating p21 and p27. It also increased I B expression and reduced Cox-2 expression in a timedependent manner [86]. A special terpene, plumericin, arrested VSMCs in the G1/G0 phase of the cell cycle along with causing abrogated cyclin D1 expression, hindered Ser 807/811 -pRb protein [87], and blockade of STAT3 signaling via S-glutathionylation. Paclitaxel, a diterpenoid, has been used as an anticancer drug for decades and has been shown to prevent neointimal formation in oral administration studies [88]. Moreover, paclitaxel arrested VSMC G1/S phase by upregulating p21 and p53 in vitro [89]. Epothilone D is a paclitaxel-like microtubule-stabilizing agent that was isolated originally from the myxobacterium Sorangium cellulosum. It inhibits neointimal hyperplasia through blockade of VSMC CDK2 and pRb [90]. -Elemene protected VECs from injury induced by H 2 O 2 in vitro via downregulating MDA while upregulating T-AOC, SOD, GSH-Px, and CAT [91]. Meanwhile, -elemene selectively inhibited VSMC proliferation/migration and inhibited neointimal formation in vivo following vascular injuries [91]. Recent studies have indicated that artemisinin effectively inhibited VSMC proliferation induced by TNF-through apoptotic induction of the caspase pathway and cell cycle arrest [92,93]. It also significantly inhibited neointimal formation in rat balloon injured carotid arteries. Therefore, terpenes are also notable candidate compounds for screening natural drugs capable of inhibiting VSMCs.

Alkaloids Exhibit Antiproliferation Biological Effect on
VSMCs. Alkaloids are a group of naturally occurring chemical compounds that mostly contain basic nitrogen atoms. Alkaloids have diverse biological effects including those against tumors, hypertension, and pain. For vascular IH, some studies indicate that alkaloids hinder cell cycle progress, decrease ROS production, and inhibit VSMC migration (Table 4). A classic alkaloid, piperine, selectively inhibits VSMC proliferation with an IC50 of 11.8 mol/L without influencing VEC growth [94]. Coptisine was isolated from Coptis chinensis and suppresses VSMC proliferation selectively at lower concentrations with a GI 50 of 3.3 mol/L (1.2 g/mL) [95]. Vinpocetine, a potential derivative of vincamine, inhibits high glucose-induced proliferation of VSMCs by preventing ROS generation and affecting MAPK, PI3K/Akt, and NF-QB signaling, Wang, Wen, Peng, Li, Zhuang, Lu, Liu, Li, Li, and Xu [96]. Vinpocetine arrested G1/S phase of the cell cycle by downregulating cyclin D1 and pERK1/2. Alongside these effects, vinpocetine also inhibited VSMC migration and ROS production [97]. A quinazolinone alkaloid, halofuginone, selectively inhibited cell proliferation, ECM deposition, and type I collagen synthesis in VSMCs versus VECs, which attenuated injury-induced IH [98]. Carbazole or murrayafoline A inhibited PDGF-BB induced abnormal proliferation of VSMCs by downregulating cyclin D1/E, CDK2/4, and PCNA and phosphorylation of Rb [99].
Evidence-Based Complementary and Alternative Medicine 9 Review of these recent studies on the effects of alkaloids provides hope for identification of useful drugs capable of inhibiting VSMC growth and preventing IH.

Other Promising Natural Compounds for Preventing
Intima Hyperplasia. As shown in Table 5, emodin is a typical anthraquinone compound beneficial for prevention of atherosclerosis due to its effects against inflammation, proliferation, and migration and its ability to induce apoptosis in VSMCs [100]. Moreover, emodin arrested growth and induced apoptosis and autophagy via enhanced ROS production and upregulation of p53 expression [101]. Emodin inhibited VSMC proliferation induced by angiotensin II through downregulation of PCNA and c-myc expression [102]. Moreover, emodin showed anti-inflammatory effects by inhibiting Hcy-induced CRP generation, a key inflammatory molecule in atherogenesis in VSMCs [103]. Emodin has also been shown to inhibit TNF--induced hASMC proliferation via caspase signaling and a mitochondrial-dependent apoptotic pathway by downregulating Bcl-2 and upregulating Bax expression [104]. Additionally, emodin reduced TNFinduced migration of VSMCs by suppressing NF-B activation and MMP2/9 expression levels [105]. Our recent study demonstrated that emodin efficiently and concentrationdependently (0.05 to 5 mol/L) inhibited hVSMC proliferation more than hVEC proliferation in vitro, with less influence on reendothelialization of VECs in rat carotid artery balloon injury [106].
Methyl-protodioscin is a steroidal saponin that has been reported to inhibit neointimal formation by restraining VSMC proliferation and migration through downregulation of ADAM15, FAK, ERK, PI3K, Akt, and MMP-2/9 expression levels [107]. Salvia miltiorrhiza has been used to prevent cardiovascular diseases in traditional Chinese medicine over the millennia. Tanshinone-IIA is a principal active component of Salvia miltiorrhiza that suppresses abnormal VSMC proliferation by cell cycle arrest at G0/G1 phase and inhibits phosphorylation of ERK1/2 and c-fos expression [108]. It has been reported that ajoene (1-50 nol/L) interfered with progression of the G1 phase in the cell cycle and restrained rat VSMC proliferation via inhibiting protein prenylation [109]. Gastrodin influenced the S phase entry of VSMCs and stabilized p27 KIP1 expression. It also inhibited VSMC proliferation and attenuated neointimal hyperplasia by suppressing phosphorylation of ERK1/2, p38 MAPK, Akt, and GSK3 [110]. Genipin has been reported to inhibit TNFinduced VSMC proliferation and migration in a dosedependent manner by upregulating HO-1 expression, preventing ERK/MAPK and Akt phosphorylation, and additionally blocking generation of ROS [111]. Ginsenoside Rg1 is one of the main active components of Panax ginseng and is said to arrest G1/S phase in VSMCs by interfering with GRKs, PKC, and N-ras while upregulating p21 expression [112]. Vascular IH is significantly decreased when carotid artery balloon injured rats are intraperitoneally injected with ginsenoside Rg1 for 14 days [113]. Moreover, ginsenoside Rg1 significantly inhibited TNF--induced hASMC proliferation dose-dependently through downregulating cyclin D1, inactivating ERK1/2 and PKB, and upregulating expression of p53, p21 WAF/CIP1 , and p27 KIP1 [114]. A coumarin called ostruthin is a major bioactive constituent of Peucedanum ostruthium and inhibited serum (10%)-induced VSMC proliferation in a dose-dependent manner [115].
Most foods contain various biologically active constituents that act to prevent and cure neointimal hyperplasia by inhibiting abnormal VSMC proliferation and migration. A well-known carotenoid, lycopene, is abundant in tomatoes and its products and has been reported to inhibit neointimal hyperplasia in a rabbit restenosis model. It does this by  regulation of blood lipid concentrations and suppression of oxidative stress [116]. Sulforaphane, an organosulfur compound, mostly found in cruciferous vegetables significantly inhibited PDGF-BB-induced VSMC proliferation by upregulating p21 and p53 expression, while CDK2, cyclin E, and PCNA expression was suppressed [117].

Selective Inhibition of VSMCs versus VECs Shows Significant
Although many natural products inhibit VSMC function, most anti-smooth muscle proliferation drugs such as rapamycin (in-stent coating) also inhibit VEC proliferation and delay reendothelialization. This nonspecific cytotoxicity leads to restenosis and final graft or stent implantation failure. When screening for selective natural drugs that inhibit smooth muscle cell proliferation and migration, it is necessary to combine computer-aided design, bioinformatics, and a high-throughput screening platform. In this review, we selected certain drugs including chemosynthetic (idarubicin) and some natural ( -elemene, coptisine, halofuginone, piperine, and curcumin) compounds that possess specificity for suppressing proliferation of VSMCs over VECs. The chemical structure of the natural compounds has no typical similarity and cannot be analyzed using structural-activity relationships of molecular-protein binding sites. However, an online tool "Swiss Target Prediction" was used to predict potential targets of these compounds [118]. Most of the predicted targets of these drugs were membrane receptors, enzymes, kinases, proteases, or transporter proteins ( Table 6). The analyses showed that microtubule-associated protein TAU (MAPT) is the most frequent protein target among them (Figure 4). This stabilizes microtubules and influences transportation of cellular secretory proteins. Moreover, MAPT has been reported to accelerate cancer cell growth [119], while its inactivation through gene knockdown suppressed cell proliferation [120].
Therefore, it is speculated that the diverse affinity of a natural drug to different functional protein targets may be one of the key factors for different selectivity profiles on VSMCs or VECs. Common targets like MAPT could be used as one of the important indicators in screening selective inhibitory drugs in future studies.

Conclusion
This review highlighted the originating four cells that may contribute to IH and then focused on VSMCs due to their involvement in intima formation as a consequence of abnormal proliferation, migration, and physiology. It further summarized typical signaling pathways such as MAPKs, PI3K/Akt, JAK-STAT, FAK, and NF-B and their involvement in the abnormal activities of VSMCs. Based on these the above cell origins and pathways, we organized and classified different natural isolates including phenols, flavonoids, terpenes, and alkaloids that have suppressing effects on VSMCs.
In addition, many natural drugs not only induce apoptosis and arrest cell cycle in VSMCs, but also impair VECs leading to vascular restenosis and failure of blood vessel remodeling. Thus, it is crucial to screen desirable drugs from natural sources that preferentially inhibit VSMCs versus VECs to prevent IH in the early stages, restenosis following graft implantation, and even atherosclerotic diseases.

Conflicts of Interest
The authors declare that they have no conflicts of interest.

Authors' Contributions
Kang Xu, Mohanad Kh Al-ani, and Xin Pan designed the project, performed the experiments, collected the data, and wrote the manuscript. Qingjia Chi analyzed the data and wrote and revised the manuscript. Nianguo Dong and Xuefeng Qiu designed the project, gave financial support, and wrote and revised the manuscript. All authors read and approved the final manuscript.


extfieldsofstudy: Biology Medicine

text:
Detecting the Population Structure and Scanning for Signatures of Selection in Horses (Equus caballus) From Whole-Genome Sequencing Data

Animal domestication gives rise to gradual changes at the genomic level through selection in populations. Selective sweeps have been traced in the genomes of many animal species, including humans, cattle, and dogs. However, little is known regarding positional candidate genes and genomic regions that exhibit signatures of selection in domestic horses. In addition, an understanding of the genetic processes underlying horse domestication, especially the origin of Chinese native populations, is still lacking. In our study, we generated whole genome sequences from 4 Chinese native horses and combined them with 48 publicly available full genome sequences, from which 15 341 213 high-quality unique single-nucleotide polymorphism variants were identified. Kazakh and Lichuan horses are 2 typical Asian native breeds that were formed in Kazakh or Northwest China and South China, respectively. We detected 1390 loss-of-function (LoF) variants in protein-coding genes, and gene ontology (GO) enrichment analysis revealed that some LoF-affected genes were overrepresented in GO terms related to the immune response. Bayesian clustering, distance analysis, and principal component analysis demonstrated that the population structure of these breeds largely reflected weak geographic patterns. Kazakh and Lichuan horses were assigned to the same lineage with other Asian native breeds, in agreement with previous studies on the genetic origin of Chinese domestic horses. We applied the composite likelihood ratio method to scan for genomic regions showing signals of recent selection in the horse genome. A total of 1052 genomic windows of 10 kB, corresponding to 933 distinct core regions, significantly exceeded neutral simulations. The GO enrichment analysis revealed that the genes under selective sweeps were overrepresented with GO terms, including “negative regulation of canonical Wnt signaling pathway,” “muscle contraction,” and “axon guidance.” Frequent exercise training in domestic horses may have resulted in changes in the expression of genes related to metabolism, muscle structure, and the nervous system.

Introduction
The genetic diversity of domesticated animals changes gradually through selective processes in populations. Archaeological and genetic evidence suggested that the initial domestication of horses (Equus caballus) began 5000 to 6000 years ago and possible multiple horse domestication events occurred across Eurasia. 1,2 For the past 400 years, the establishment of formal breed registries and continuous breed specialization has focused on the preservation and improvement of traits related to riding, draft, aesthetics, and performance. The intensive selection for these traits has led to high athletic quality and high skeletal muscle mass. 3 Chinese native horses were formed under different ecological conditions and exhibit high levels of morphological and genetic diversity. 4,5 Kazakh horses are believed to have been developed by crossing native horses with Mongolian, Middle Asian, and European breeds raised together in herds. 6 They are generally small, rugged horses with different coat colors. The Kazakh horse is used for riding and pack as well as for milk and meat. The Lichuan breed is geographically distributed in mountainous areas of Southern China and is broadly used in light draft and riding for human usage. In this study, we selected only Kazakh horses and Lichuan breeds, with distinct variations in numerous phenotypes, to test whether they represent potential Northern type and Southern type of Chinese horse, respectively. Previous studies demonstrated that Northern/Southern Chinese distinct groups can be used to discriminate the Han Chinese 7 as well the domestic pig genetically. 8 Recent advancements in high-throughput sequencing technology and the assembly of the first horse genome sequence have allowed for in-depth analysis of the genetic variations present in horses. 9 Phenotypic changes associated with mutations 2 Evolutionary Bioinformatics accompany domestication as a result of the joint impact of natural selection and human-controlled selective breeding. Due to a strong selection of beneficial alleles, a selective sweep leads to a single genomic background, which results in a large reduction in genetic variations in local region of the genome. 10 Selective sweeps have been found in domestic and wild horse populations using genome scans. [11][12][13] Selective sweeps have been found in 33 domestic horse breeds using genome scans of nearly 50 000 markers in Equine SNP50 BeadChip (Illumina, San Diego, CA, USA). 13 However, the limited markers cannot give single base resolution of the entire genome. In this study, we describe the whole genome sequencing of 4 horses from 2 Chinese native breed and 44 horses with publicly available genome data from completely different breeds to find and annotate distinct genetic disparities in horses using extremely precise SNP calling approaches. The aim of this study was to highlight candidate genes and to trace footprints of horse selection at the genome level. Genes were selected in proximity to genomic regions and positions displaying signatures of selection by the composite likelihood ratio (CLR) method using sequencing data. 14 Moreover, the functions of genes under selection were studied by gene ontology (GO) annotation analysis.

Experimental animals and genome sequencing
We sequenced 4 Chinese native horses including one individual from the Lichuan breed and 3 from the Kazakh breed. Genomic DNA was extracted from blood tissue using a standard phenol-chloroform protocol. This study was conducted according to the regulations approved by the ethical committee of Huazhong Agricultural University and also the standing committee of Hubei People's Congress, P. R. China.

SNP calling
Horse gene sequences were retrieved from the NCBI database. Next-generation sequencing reads from the 48 samples were mapped individually to the reference genome using bwa-mem 18 with default parameters. BAM files for all animals were sorted, and duplicates were filtered using Picard-version-1.108 (http:// broadinstitute.github.io/picard/). RealignerTargetCreator and IndelRealigner were applied for local realignment, and individual sample SNP calling was performed using HaplotypeCaller (with the following parameters: -pairHMM VECTOR_ LOGLESS_CACHING -emitRefConfidence GVCF -variant_ index_type LINEAR -variant_index_parameter 128000) in GATK version 3.50. 19 Multisample SNP calling was performed to merge the GVCFs using GenotypeGVCFs with default settings from GATK version 3.50. 19 The identified SNPs in the VCF were filtered for downstream analysis by requiring a minimum depth of 5 and a minimum root mean square mapping quality score of 20. We also removed SNPs with a minor allele frequency <0.05 and with >10% missing genotypes among the 48 samples in the population.
The 15 341 213 high-quality SNPs identified in the horse were categorized according to their genomic locations, including in exons, introns, untranslated regions (UTRs), and intergenic regions, and SNPs located in exons were further divided into synonymous and nonsynonymous SNPs. The high-quality SNPs recovered here were used in the GO, SNP summary, target gene, and selective sweep analyses. The putative SNPs were functionally annotated primarily based on the SnpEff software system analysis 20 with standard settings. For each SNP, the position (5′ UTR, 3′ UTR, exonic, intronic, intergenic, splice acceptor or donor site, upstream or downstream) and also the useful annotation (nonsynonymous, synonymous) were identified based on the gene annotation of the horse reference genome from the NCBI database (EquCab2.0, annotation date November 20, 2015). 9

Population genetics analysis
We also conducted an individual-scale principal component analysis (PCA) for the 48 horses. Here, genotype likelihoods were estimated assuming Hardy-Weinberg equilibrium using the ANGSD package (with parameters: angsd -nInd 48 -doMajorMinor 1 -doMaf 1 -doPost 1 -doGeno 32 -doSaf 1). 21 Genotype likelihoods were used to compare horses via PCA using the function "ngsCovar" in ngsTools, 22 which implements a probabilistic approach to estimate the genotype covariance matrix (with parameters -nind 48 -nsites 12363534 -block_size 20000 -call 0 -minmaf 0.05). The population structure among various domestic populations was inferred using admixture. 23 We removed the Przewalski horses because they are the only wild population. After excluding the 3 Przewalski horses, we choose 422 881 high-quality autosomal SNPs randomly to infer the genetic structure of the domestic horses, using the program admixture, which includes a cross-validation procedure that allows identifying the value of K for which the model has best predictive accuracy.

Detection of positive selection
The method for detection of positive selection for horse is mainly similar to the previous one that we have used for ducks. 24 To identify sweeps, the CLR test was performed using allele frequencies, and the site frequency spectrum (SFS) of the complete chromosome was considered as the background SFS to calculate the combined likelihood of a recent selective sweep in each window. 14 The empirical CLR distributions were obtained by 1000 simulations of a neutral sequence equal to each chromosome in length using ms software 25 under a previously selected demographic model named "PSMC #1," which represents the basic model with a constant population size from t2 to t0. 11 In this PSMC profile, a mutation rate of 7.242 × 10 −9 per site per generation and a generation time of 8 years were used. 16 The CLR value and the corresponding A value in each alignment positions where the SweeD score is calculated for the horse and simulated genotypes were obtained from a SweeD run with a grid size of 10 kB. The corresponding α value of a CLR value is a function of the selection coefficient, the recombination rate, and the effective population size. The results from the simulated genotypes were used to calculate a critical threshold of CLR values for observed data in each chromosome. We identified candidate sites for selective sweep on the basis of P values after correction of multiple testing using a false discovery rate (FDR) of 0.05 (using "BH" method in the "p.adjust" function of R). We then identified annotated sequences overlapping with the selected regions as determined by SweeD.

Functional enrichment
Functional enrichment was performed on the list of lossof-function (LoF) genes and the genes under selection in horses as detected by the genome-wide selective sweep scans, respectively. The GO terms were obtained using the Databank for Annotation, Visualization and Integrated Discovery (DAVID). 26 DAVID was used to evaluate enrichment in the GO terms using known annotations of horse genes with Equus caballus selected as background. For further GO term analysis, a P value of .05 and a FDR of 25% were set.

Genomic variants
Overall, 15 341 213 high-quality SNPs were detected using GATK, representing the distinctive variants of 48 samples. The SNPs were functionally classified using SnpEff annotations with the boundaries of all neighboring genes ( Table 2). Most SNPs were located in intergenic regions, and intronic SNPs were most common in genic regions. SnpEff also found  Zhang et al 5 To gain insights into the SNPs underlying recessive traits, we clustered 5 high-impact severity types of SNPs into LoF variants, including splice acceptor, splice donor, stop gain, stop loss, and start loss SNPs in protein-coding genes. We detected 1390 LoF SNPs in protein-coding genes, all of which were heterozygous in at least one of the sampled animals. There were 981 genes with more than 1 LoF SNP. The GO enrichment analysis revealed that genes associated with "G protein-coupled receptor (GPCR) communication pathway," "antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex (MHC) class II," "detection of chemical stimulus involved in sensory perception," "immune response," and "signal transduction" were overrepresented in LoF-affected genes ( Table  3). G protein-coupled receptors have been found to be linked with signaling pathways such as transmembrane receptor activity and neurotransmission. 28 Two similar GO enrichment results for LoF-containing genes pointing to GPCR receptor activity and sensory perception were also found in humans 29 and cattle. 30 Interestingly, genes associated with GO terms "antigen processing and presentation of peptide or polysaccharide substance via MHC class II," "immune response," "defense response," and "cellular response to interferon-gamma" were significantly (P < .05) overrepresented in the LoF-affected gene set (Table 3). These GO terms are involved in immune responses. DRB, DQA, and DQB are classical MHC class II molecules involved in the development of adaptive immune responses.

Population genetic structure
Principal component analysis was used to explore the individual relationships within and among breeds. The first 2 principal components explained 8.53% of the variation of the studied horses ( Figure 1). The position of Przewalski clearly deviates from the domestic horse in PC1. PC1 also allows visualizing the proximity among Duelmener, Fjord, Icelandic, Kazakh, Lichuan, and Mongolian (where we defined group A) and the proximity among Hanoverian, Morgan, Quarter, Sorraia, and Standardbred (where we defined group B). Admixture proportions were assessed without user-defined population information to infer the presence of distinct populations among the samples (Figure 2). At K = 3 or K = 4, Franches-Montagnes and Arabian forms one unique cluster; at K = 5, Jeju pony forms one unique cluster. For other breeds, comparatively strong population structure exists among breeds, and they can be assigned to 2 (or 3) alternate clusters from K = 3 to K = 5 including group A (Duelmener, Fjord, Icelandic, Kazakh, Lichuan, and Mongolian) and group B (Hanoverian, Morgan, Quarter, Sorraia, and Standardbred). For group A, geographically this was unexpected, where Nordic breeds (Norwegian Fjord, Icelandic, and Duelmener) clustered with Asian breeds including the Mongolian. Previous results of mitochondrial DNA have revealed links between the Mongolian horse and breeds in Iceland, Scandinavia, Central Europe, and the British Isles. The Mongol horses are believed to have been originally imported from Russia subsequently became the basis for the Norwegian Fjord horse. 31 At K = 6, Sorraia forms one unique cluster. The Sorraia horse has no long history as a domestic breed but is considered to be of a nearly ancestral type in the southern part of the Iberian Peninsula. 32 However, our result did not support Sorraia as an independent ancestral type based on result from K = 2 to K = 5, and the unique cluster in K = 6 may be explained by the small population size and recently inbreeding programs. Genetic admixture of Morgan reveals that these breeds are currently or traditionally continually crossed with other breeds from K = 2 to K = 8. The Morgan horse has been a largely closed breed for 200 years or more but there has been some unreported crossbreeding in recent times. 33 Bayesian clustering and PCA demonstrated the relationships among the horse breeds with weak geographic patterns. The tight grouping within most native breeds and looser grouping of individuals in admixed breeds have been reported previously in modern horses using data from a 54K SNP chip. 33,34 Cluster analysis reveals that Arabian or Franches-Montagnes forms one unique cluster with relatively low K value, which is consistent with former study using 50K SNP chip. 33,34 Interestingly, Standardbred forms a unique cluster with relatively high K value in this study, different from previous study. 33 To date, no footprints are available to describe how the earliest domestic horses spread into China in ancient times. Our study found that Kazakh and Lichuan were assigned to the same lineage as other native Asian breeds, in agreement with previous studies on the origin of Chinese domestic horses. 4,5,35,36 The strong genetic relationship between Asian native breeds and European native breeds have made it more difficult to understand the population history of the horse across Eurasia. Low levels of population differentiation observed between breeds might be explained by historical admixture. Unlike the domestic pig in China, 8 we suggest that in China, Northern/Southern distinct groups could not be used to genetically distinct native Chinese horse breeds. We consider that during domestication process of horse, gene flow continued among Chinesedomesticated horses.

Genome-wide selection signatures
The CLR test scanned genomic regions to find signals of positive selection in the horse genome. Composite likelihood ratio is calculated by comparing the regional SFS with the background SFS that specifies the probability of a signal at each window of 10 kB in length across the horse genome. A total of 1052 windows obtained a P value less than .01, indicating that the CLR of those windows surpassed the CLRs achieved from the distribution of 1000 neutral simulations. The SNP and gene density values for these regions are presented in Figure 3. Some of the 1052 windows were adjacent to one another, which led to the designation of 933 distinct core regions. The regions with the largest CLR in the horse genome are located on chromosome 8 (Figure 3), including 9 adjacent 10-KB windows. The top-scoring 90-Kb region was chr8: 1.916 to 2.006, and this region contains 227 SNPs, all of which are intron variants of the LOC100065759 pseudogene.

Genes under selection involved in biological processes
We explored the functions of genes associated with various biological processes. The P value of .05 was considered significant for GO annotation. We recovered 21 enriched GO groups (P < .05; Table 4). The top 3 GO terms with the lowest P values were "negative regulation of canonical Wnt signaling pathway" (GO:0090090, P = 2.93E−03), "muscle contraction" (GO:0006936, P = 1.22E−02) and "axon guidance" (GO:0007411, P = 1.31E−02). The GO term "muscle contraction" was defined as a change in muscle geometry induced by the force generated within muscle tissue. Force generation requires a chemo-mechanical energy conversion process that is mediated by actin/myosin complex activity, which produces force through adenosine triphosphate hydrolysis. Myomesin genes (MYOM1 and MYOM2) are expressed in muscle cells to stabilize the 3-dimensional conformation of the thick filament, and tropomodulin1 (TMOD1) protein binds and restrains the minus end of actin, fine-tuning the length of actin filaments in muscle and nonmuscle cells. 37 A previous study in horses also found that genomic regions with the most genetic differentiation in domesticated horses were enriched in genes involved in metabolism, muscle contraction, reproduction, signaling pathways, and behavior, 11 which supports our result. The GO term "axon guidance" is a neural development subdomain in relation to this process, through which neurons send out axons to achieve the correct targets. In this GO term, the axon guidance receptor gene (ROBO1) is a candidate gene for developmental dyslexia, and ROBO1 polymorphisms are associated with functioning in the language acquisition system. 38,39 Abnormal expression of Contactin-4 (CNTN4) has been implicated in some cases of autism. The cadherin gene (CDH4) has a predictive pivotal role during brain segmentation and neuronal outgrowth. Therefore, genes in this GO term may be related to the trait of racing in domestic horses, which is supported by other genes related to exercise-induced stress that were found in selective sweep tests in thoroughbreds. 15 Previous studies have shown that intensive exercise training in thoroughbreds leads to gene expression changes related to metabolism and muscle structure. 40,41 Author Contributions SZ and XD conceived of and designed the experiments. CZ, JC, YM, and CX analyzed the data. MG, AB, DG, PN, and XL contributed to sampling. HIA, CZ, YF, and HW contributed to the writing of the manuscript. All the authors reviewed and approved the final manuscript.


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Domain architecture of BAF250a reveals the ARID and ARM-repeat domains with implication in function and assembly of the BAF remodeling complex

BAF250a and BAF250b are subunits of the SWI/SNF chromatin-remodeling complex that recruit the complex to chromatin allowing transcriptional activation of several genes. Despite being the central subunits of the SWI/SNF complex, the structural and functional annotation of BAF250a/b remains poorly understood. BAF250a (nearly 2200 residues protein) harbors an N-terminal DNA binding ARID (~110 residues) and a C-terminal folded region (~250 residues) of unknown structure and function, recently annotated as BAF250_C. Using hydrophobic core analysis, fold prediction and comparative modeling, here we have defined a domain boundary and associate a β-catenin like ARM-repeat fold to the C-terminus of BAF250a that encompass BAF250_C. The N-terminal DNA-binding ARID is found in diverse domain combinations in proteins imparting unique functions. We used a comparative sequence analysis based approach to study the ARIDs from diverse domain contexts and identified conserved residue positions that are important to preserve its core structure. Supporting this, mutation of one such conserved residue valine, at position 1067, to glycine, resulted in destabilization, loss of structural integrity and DNA binding affinity of ARID. Additionally, we identified a set of conserved and surface-exposed residues unique to the ARID when it co-occurs with the ARM repeat containing BAF250_C in BAF250a. Several of these residues are found mutated in somatic cancers. We predict that these residues in BAF250a may play important roles in mediating protein-DNA and protein-protein interactions in the BAF complex.

Introduction
Chromatin remodeling complexes are specialized cellular machinery consisting of a number of proteins that work in concert, to remodel the nucleosome (histone-DNA complex) structure during various cellular processes such as transcription, replication, and repair leading to PLOS  regulation of gene expression [1]. Both the histone modification enzymes and the ATP-dependent complexes play key roles in mediating this reorganization. While the former modify histones, the latter utilize ATP hydrolysis to drive the local disruption or alter association of histones with DNA. Such complexes, as observed in humans, yeast and drosophila typically involve as many as twelve to fifteen proteins [2,3]. Each protein is known to exist in more than one isoform, thus expanding the scope for the formation of a wide range of complexes that can function distinctly from each other. Indeed, it is suggested that the controlled expression of specific sub-units in various developmental stages is possibly a means to regulate the composition of individual complexes and their interactions with intended targets [4,5]. BAF250a (also known as ARID1A_HUMAN or P270) is a characteristic member of the ATP-dependent SWI/SNF chromatin remodeling complex in humans that is thought to recruit the complex to the chromatin through either protein-DNA or protein-protein interactions [6][7][8]. It is known to exist in the complexes in a mutually exclusive state with its paralogue BAF250b (or ARID1B_HUMAN) resulting in functionally distinct complexes: BAF-A (with BAF250a) or BAF-B (with BAF250b) SWI/SNF remodeling complexes [7,[9][10][11].
Recently, several studies have revealed that BAF250a mutations are observed in a variety of cancers [8,12,13]. Depletion of BAF250b in BAF250a mutant cell lines resulted in the dissociation of the other subunits of the SWI/SNF complexes showing that BAF250a and BAF250b are a central component of these complexes [14]. Other protein subunits of the SWI/SNF complex include actin (ACTB), SMARCA2, SMARCA4/BRG1/BAF190A, ACTL6A/BAF53, ACTL6B/ BAF53B, SMARCE1/BAF57, SMARCC1/BAF155, SMARCC2/BAF170, SMARCB1/SNF5/ INI1, and one or more of SMARCD1/BAF60A, SMARCD2/BAF60B, or SMARCD3/BAF60C [2]. Although the major players of the complex are known, the nature of their interaction either with themselves or with their targets are not clearly established.
The structure and functional annotation of BAF250a/b (> 2200 residues long proteins) remains poorly understood. The folded regions and domain boundaries of these lengthy proteins have not been clearly defined. Likewise, their DNA-binding specificities and potential protein-protein interactions have not been studied systematically [15]. BAF250a/b contains two annotated folded domains: an N-terminal AT-rich interacting domain (ARID,~110 residues) and a C-terminal domain of unknown function (DUF3518,~256 residues) in PFAM, recently annotated as BAF250_C [15]. Presently, no other domain has been associated with the region interspersed between these two domains although functionally annotated sites such as GR-binding LXXL motifs, NES (nuclear export signal), NLS (nuclear localization signal), etc., have been identified in this regions.
As mentioned earlier, BAF250a is believed to recruit the SWI/SNF complexes to specific genes either through its interaction with DNA or binding to transcription factors, transcriptional co-activators and transcriptional co-repressors complexes [8,[16][17][18]. The N-terminal ARID in BAF250a is primarily responsible for it's interaction with DNA. The evidences so far suggest that BAF250a ARID interacts with DNA without any sequence preference [19][20][21], therefore questioning the recruitment of SWI/SNF complexes by BAF250a to the target genes via its interaction with specific DNA sequences. BAF250a has also been linked to nuclear hormone induced transcription and expression of cell-cycle regulators. Several nuclear hormonebinding LXXLL motifs and a GR (glucocorticoid receptor)-binding region have been mapped to the C-terminal region of the protein that contains the BAF250_C [8].
In this study, using sequence and hydrophobic core analysis, we predict the existence of folded regions along the sequence of BAF250a [22]. This includes the N-terminal ARID, the C-terminal region that encompasses the BAF250_C, and 3 other regions of at least 100 residues length. The occurrence of the ARID in multiple domain contexts, with a variety of partnering domains, motivated us to probe for sequence-specific signatures on the ARID that could drive or dictate its folding and functions, such as DNA and protein interactions in BAF250a/b. We performed a systematic study of the alignments of ARIDs in 11 major domain contexts to determine if such architecture-dependent sequence signatures exist. Significantly, in a domain architecture where ARID always co-occurs with BAF250_C (e.g. in BAF250a/b), we examined alignments of homologues of BAF250a/b that conserve the same domain organization and identified a set of conserved residues in ARID, which could be further classified into two distinct sets. One set constitutes conserved residues that are required for the maintenance of the ARID core, while the other set constitutes conserved but surface exposed residues that are unique to ARID when in association with BAF250_C. Likely, such residues that are uniformly conserved in all the homologues may have important functional roles in driving the interactions with other regions of BAF250a/b or other proteins in the SWI/SNF complex. Similarly, our analysis also reveals conserved and surface exposed residues in BAF250_C when it cooccurs with ARID (in BAF250a/b). Such selective conservation of residues unique to this combination of domains may have a bearing on the globularity of BAF250a and also mediate BAF250a association with other members of SWI/SNF complex via their interaction with the BAF250_C, which is predicted to adopt the ARM repeat fold typically involved in protein-protein interactions. Through motif searches, we also recognize potential interactions sites in BAF60B, BRG1, BAF53B implying that these may be the interacting partners of BAF250_C in the SWI/SNF complex. Further, we explored the distribution of somatic mutations reported in the BAF250a gene to predict the consequences of such mutations on the structures of the ARID and modelled structure of BAF250_C. Majority of these residues are surface exposed and therefore mutation of these residues can potentially lead to impairment of DNA and/or protein binding ability of BAF250a.

Dataset
Homologues of the proteins containing the ARID were obtained by performing iterative jackhmmer searches [23] against the UniProt database (v.2017_03) at the EBI server [24]. Seed alignments of the ARID containing proteins from PFAM 31.0 were employed as an input to jumpstart the searches. Searches were performed for up to five iterations at E-value inclusion and reporting thresholds less than 0.0001 to identify 4414 proteins that contain the ARID domain. Domain assignments, using pfamscan [25], were performed for the identified hits to prune for false positives and to identity ARID containing proteins that co-occur with the Cterminal BAF250_C.

Domain alignments of the ARID and BAF250_C
239 ARID sequences were found to co-occur with the BAF250_C in our dataset. To retain only full-length sequences, sequences that were truncated/fragments were excluded and the remaining sequences were aligned using command-line MUSCLE (v3.8.31) [26]. Care was taken to include homologues of known structure, where applicable, and a total of 210 sequences from diverse organisms were considered for the ARID dataset. Likewise, cognate sequences of 210 BAF250_C terminal domain were extracted and aligned using MUSCLE. Here again, fragments and truncated domains were excluded.
Once aligned, unrooted phylogenetic trees were derived for the two domains separately to compare the sequences, using the Neighbour-joining algorithm in Phylip with 1000 bootstraps [27].

Domain contexts and derived architectures
The ARID family in PFAM is represented by 3169 sequences in the full alignment and 4414 proteins in the jackhmmer searches performed here. In all, based on domain assignments, these sequences are observed in 164 architectures or domain combinations, suggesting that the ARID occurs in diverse domain contexts. Careful examination of the various domain contexts showed that the same domains reappear in different orders or numbers in various domain contexts. For example, 13 sequences are observed to possess the JmjN, ARID, PHD, JmjC, zf-C5HC2, and three copies of PLU-1 (3X) domain combination while 17 sequences contain JmjN, ARID, PHD, JmjC, zf-C5HC2 and two copies of PLU-1 (2X) domains. Although the nature of the domains is identical in the two sets of sequences, the number of copies of the PLU-1 domains varies with the latter set containing two copies of the PLU-1 domains.
To evaluate if any sequence signals distinguish the various domain combinations, we extracted a subset of the 164 contexts of the ARID with a unique representation of domain combinations (S1 Table). In this way, we eliminated redundancies in the sequences representing the same domain combinations that differed in the number of copies of the individual domains in the dataset.
The eleven such unique domain contexts that were considered for further analysis are listed in S1 Table. As shown here, the number of sequences in each domain architecture varied. For instance there are nearly 1395 sequences in the domain context that contains only the ARID, while there are 239 sequences in the context that contain both the ARID and BAF250_ C domains and only 6 sequences that contain the Myb_DNA-bind_2, ARID, Rap1_C domain combinations. To remove redundant sequences and restrict the analysis to a representative set of sequences in each architecture, we clustered the sequences within each architecture using blastclust from the NCBI-BLAST package [28], at 80% identity over 90% of the length of each sequence. Architectures with fewer sequences were not subjected to clustering to retain a minimal meaningful set for sequence comparisons. We also determined the average length of the ARID in each domain architecture and considered only those sequences that were +/-15 residues length-deviant from the average ARID length in each architecture. Although stringent, this excluded sequences that were fragmented or very long in each architecture.

Generation of structure-guided multiple sequence alignments of ARIDs in each architecture
The regions corresponding to the ARID were extracted from each architecture and multiple sequence alignments were performed using the default options of command-line MUSCLE (v3.8.31) [26]. Conservation of residues in the alignment was derived based on identity, similarity and biochemical properties. The fingerprints derived for each architecture were compared using an alignment of the ARIDs across all architectures as a reference (data not shown, available on request). Since the structure of the ARID was available in some of the domain architectures, these were included in the cognate alignment, where applicable. Property and conservation-based fingerprints for each architecture were derived and visualized using Mview [29] and Espript [30].

Preparation of ARID WT and V1067G mutant proteins and DNA for CD and NMR spectroscopy
ARID (residues 968-1143) of BAF250a was cloned in pET28a vector that imparts an N-terminal 6XHis-tag to protein. Site-directed mutagenesis was performed using Q5 mutagenesis kit to produce valine (V) to glycine (G) mutation at 1067 residue position in ARID. Wild type (WT) and mutant (V1067G) proteins were expressed in E. Coli BL21(DE3)-Rosetta cells using 1 mM IPTG for induction at 20˚C for 16 h. The first step of purification involved 6XHis-tag Ni 2+ -NTA affinity chromatography followed by gel-filtration chromatography under a buffer consisting of: 50 mM NaH 2 PO 4 , pH 6, 0.025 mM EDTA. This buffer was used in CD spectroscopy, NMR spectroscopy, and ITC experiments. Uniformly 15 N labeled proteins were expressed in M9 media using 15 NH 4 Cl as sole source of nitrogen.
Palindromic 12mer Dickerson-dodecamer DNA (5'-CGCGAATTCGCG-3') sequence (DD12 dsDNA), used in this study, was commercially synthesized (Eurofin Inc). DNA was dissolved in water and solution was heated for 4 min at 95˚C and annealed using snap cooling on ice (4˚C) to generate a dsDNA solution.

CD and NMR spectroscopy
CD spectra for WT and V1067G proteins were recorded on a JASCO715 spectropolarimeter at 25˚C between 190nm-260nm wavelength. Thermal melting of proteins using CD was performed by thermal scans in which the protein samples (10 μM) were heated from 10˚C to 90˚C (at from 1˚C/min rate controlled by a JASCO programmable Peltier element) and the CD signal was recorded at 222 nm. NMR spectra were recorded on a BRUKER 700 MHz NMR spectrometer equipped with HCN cryoprobe. NMR data were recorded using Topspin 3.1. Datasets were processed using Topspin3.1 and analyzed using the SPARKY software (UCSF, CA, USA).

Isothermal titration calorimetry (ITC)
Isothermal titration calorimetry (ITC) experiments were performed using a MicroCal iTC200 machine (GE) at 25˚C. Proteins (ARID WT and V1067G mutant) and DD12 dsDNA were prepared in the same buffer consisting of: 50 mM NaH 2 PO 4 , pH 6, 0.025 mM EDTA. For the titrations the sample cell was filled with 20 μM of the DNA and was titrated with 200 μM of protein. 20 injections of the titrant were done at an interval of 180 seconds. Separate protein titration in buffer was performed to determine the protein heat of dilution. The heat of dilution was subtracted from the integrated heat and the data was then fit for a one site-binding model. All the parameters were kept floating during fitting. Experiments were repeated 2-3 times for the reproducibility.

Molecular dynamic (MD) simulation of ARID in BAF250a and V1067G mutant protein
The NMR structure of ARID domain (PDB ID: 1RYU) was used as the starting structure for the WT ARID. In the same protein, residue V1067 was replaced with G in-silico using mutate tool of WHAT IF server [31] to obtain the V1067G mutant system. The same MD simulation protocol was applied to both WT and mutant using the latest GROMACS 4.6.5 simulation package [32][33][34]. GROMOS96 45a3 united-atom force field was used to model the intramolecular protein interactions and the intermolecular interactions between the protein and solvent molecules [35]. Initial energy minimization and equilibration steps, were followed by a simulation with pressure coupling at 1 atm. During this phase, the velocities were reassigned according to a Maxwell's distribution at 300K. Finally, the production phase of MD simulation was run keeping the temperature, pressure and number of molecules of the ensemble invariant. Production phase using 0.002ps time step was continued up to 50 ns. All the subsequent analyses were performed using different programs of the GROMACS package.

SEG-HCA of BAF250a
We used a novel bioinformatics tool SEG-HCA (segmentation based-hydrophobic core analysis) for the prediction and characterization of the BAF250a full-length sequence for folded regions/domains [22]. SEG-HCA delineates foldable domains, by recognizing regions that are enriched with high densities of hydrophobic cluster, without employing any evolutionary information. SEG-HCA analysis predicted 10 regions in BAF250a sequence longer than 25 residues to harbor hydrophobic clusters, of which four regions that are longer than or equal to 100 residues can potentially fold into globular domains.

Modeling of BAF250_C
PFAM predicts the BAF250_C terminal domain in BAF250a that extends between 1974-2231 residues. The novel predicted region 10 (residues 1939-2282) that includes the BAF250_C was subjected to fold recognition using PHYRE2 server [36]. The top templates identified by the approach were crystal structure of armadillo (ARM) repeats domain of human adenomatous polyposis coli (APC) (PDB Id: 3NMW, chain A) and crystal structure of the myosin chaperone UNC-45 from C. elegans in complex with a Hsp70 peptide (PDB ID: 4I2W, chain A) with which the BAF250_C shows 18-20% sequence identity.
The predicted folded region was also evaluated using the Robetta server [37]. A folded ARM repeats containing model was derived using the crystal structure of the armadillo repeat domain of APC (PDB ID: 3AU3, chain A) as reference, with a confidence score of 0.3726.

Interacting partners in the SWI/SNF chromatin remodeling complex
We looked for the β-catenin-interacting sequence motif of DxθθxФx2-7E (where θ and Ф are hydrophobic and aromatic residues, respectively) in all the annotated members in SWI/SNF chromatin remodeling complex as the probable interacting partners of BAF250_C. The search was performed through Patmatdb program of EMBOSS [38] on the different members of the SWI/SNF chromatin remodeling complex.

Hydrophobic core analysis of BAF250a reveals folded regions in its sequence
SEG-HCA (segmentation based-hydrophobic core analysis) is a novel method that delineates foldable domains using only the amino acid sequence of the corresponding protein without any evolutionary information [22]. Essentially SEG-HCA relies on the identification of regions in protein sequence with high densities in hydrophobic clusters (hydrophobic cluster analysis) [39,40]. The hydrophobic clusters mainly corresponding to regular secondary structures may finally result in folded, globular domains. We used SEG-HCA tool to predict potential folded regions in the entire length of BAF250a (S1A and S1B Fig). SEG-HCA analysis revealed several regions in BAF250a sequence that can potentially fold with a hydrophobic core. There are total 10 regions of > 25 aa predicted to have a cluster of hydrophobic residues resulting in a hydrophobic core (S1A and S1B Fig). Of these, region 2 (aa 781-908, length 128 residues), region 4 (aa 1008-1107, length 100 residues), region 8 (aa 1659-1759, length 100 residues), and region 10 (aa 1939-2282, length 344 residues) are at least 100 residues long and can potentially form independently folded domains (Fig 1A, S1A and S1B Fig). Indeed, region 4 overlaps with ARID (aa 1000-1119) and region 10 encompass PFAM annotated BAF250_C (aa 1974-2231) domains. The length of SEG-HCA predicted region 10 (residues 1939-2282) is longer than the PFAM annotated domain (residues 1974-2231) and was therefore used as a domain boundary for BAF250_C in this study. The region between these two domains has been shown to contain protein-protein interaction motifs/domains including HIC1 (hypermethylated in cancer 1) binding domain (aa 1355-1451) and GR (glucocorticoid receptor) binding domains (aa 1635-2285), which partially overlaps with BAF250_C (aa 1974-2231) [8]. Other regions at the N-terminus (aa 1-600) and regions interspersed between the ARID and BAF250_C, excluding the boundaries predicted by SEG-HCA, are rich in repetitive sequences and predicted to be disordered. However, it is possible that these intrinsically disordered regions adopt stable structure upon binding to the targets [41].
Three-dimensional structure of the N-terminal ARID of human BAF250a (SEG-HCA region 4) was solved previously using solution NMR methods ( Fig 1B) [19]. However, the presumed domain in the C-terminal, reported as the longest foldable region (SEG-HCA region 10) in BAF250a, is functionally and structurally uncharacterized. Therefore, we have predicted Structural, sequence, and evolutionary analysis of BAF250a the fold of region 10 in BAF250a using intensive PHYRE2 [36] and profile-based searches such as HHPred [42]. In both the approaches, the Armadillo (ARM) repeats were found to be associated with the fold with nearly 238 residues (93%) predicted to adopt this fold at greater than 90% accuracy. Further, we used the ab-initio approach of the Robetta webserver to model this region [37]. The server proposed a folded ARM repeats containing model using crystal structures of the armadillo repeat domain of adenomatous polyposis coli (APC) (PDB ID: 3AU3, chain A) as reference. APC is a tumor suppressor protein that regulates Wnt signaling and other cellular processes through interaction of its ARM repeats with various proteins [43]. The proposed model predicts 5 ARM repeats in the C-terminal folded domain of BAF250a (Fig 1C).

ARIDs in different domain context cluster distinctly
Different kinds of structural and functional constraints operate on the amino acid residues in proteins during evolution. Therefore, the pattern of evolutionary constraints on amino acid positions in a protein family may differ depending on its partnering or another interacting domain in the protein [44]. Indeed, it is well appreciated that the domain contexts of a protein dictate its function, as often the same domain in different contexts results in an altogether different biological function [45]. The availability of the ARID in diverse domain contexts (as seen here in ARCH 1-11) therefore presents an opportunity to compare its sequences in each architecture. In PFAM 31.0, the ARID is found in combination with other domains in 164 domain architectures [15]. Of these, we have selected 11 major ARID containing architectures (termed ARCH1-11) (Fig 2 and S1 Table, also see Methods) to determine if the ARID in each of the architectures has a differential set of sequence signatures that can discriminate between the various domain contexts in which it occurs. Representative sequences of the ARID from each of the 10 architectures (excluding ARCH1 where it is not associated with any other domain) were aligned. The phylogenetic tree derived from this alignment through neighborjoining and with 1000 bootstraps showed a distinct sub-clustering of the ARID sequences in an architecture-specific manner. As shown in Fig 2, this distinct sub-clustering across architectures suggests that specific sequence signals exemplify ARID in each of the diverse domain architectures that it occurs in.

ARID and BAF250_C have co-evolved in BAF250a
BAF250a/b homologues in the SWI/SNF complexes are exclusively associated with the BAF250_C in ARCH2. Unlike ARID, which is associated with a number of other domains in various organisms; the BAF250_C is uniquely associated with the ARID (except for one instance where in addition to ARID it co-occurs with the mannosyl transferase in ARID1a of the King Cobra). Of the 236 proteins showing the BAF250_C and ARID domain combination (ARCH2), the majority of proteins are annotated as either ARID1a (BAF250a) or ARID1b (BAF250b)-like sequences or as subunits of the SWI/SNF-like ATP-dependent chromatin remodeling complexes in various eukaryotes. Some proteins are annotated as osa proteins while others are as yet uncharacterized proteins. Although the predicted domain contexts of both BAF250a and BAF250b proteins are similar, the two proteins share only 59% sequence identity along the entire length of the proteins. Unique association between ARID and BAF250_C domains in ARCH2 prompted us to examine the evolution of these two domains in ARCH2. For this, 210 sequences of the core ARID and those of the BAF250_C were aligned separately using MUSCLE [22]. Phylogenetic trees were generated using neighbor in the PHY-LIP suite with 1000 bootstraps and visualized through iTOL [38]. The trees derived separately from the ARID and BAF250_C domains, mirror each other's topologies (S2 Fig). This suggests that these domains are subjected to similar evolutionary pressures to co-evolve [46]. S2 Fig also shows that the trees of the core ARID and BAF250_C domains make a clear distinction between BAF250a and BAF250b-like sequences. Such a partitioning of the two paralogues and their as yet uncharacterized homologues is useful, since the tree offers clues to their proximity to BAF250a-like and BAF250b-like proteins. This partitioning could be employed in the potential annotation of a number of proteins in the dataset that are uncharacterized or unannotated. Based on this distinct partitioning, 40 sequences that were ambiguously annotated in the UniProt database were grouped into BAF250a and BAF250b like sub-clusters (S2

ARIDs in diverse domain contexts harbor distinct sequence fingerprints
In order to determine the sequence fingerprint of the ARID in each of 11 major domain combinations with simultaneous preservation of the ARID fold, we performed sequence analysis of the ARID in each of the different domain architectures. For this, the ARID sequences within each architecture were first aligned. Representative structural templates of the ARID, where Structural, sequence, and evolutionary analysis of BAF250a available, were included and a structure-guided multiple sequence alignment was derived using Promals3D [47]. We employed an Mview based representation to capture differences in biochemical properties of the sequences in each architecture. Indeed, a distinct consensus sequence pattern is derived for the ARID in each of the 11 architectures, suggesting that the ARID within the context of different architectures is diverse in sequence (data not shown, available on request). Subsequently, the distinct consensus signature sequences of the ARID derived from each architecture were aligned. Shown in Fig 3A, is the consensus alignment of the ARID sequence fingerprints from each architecture. The consensus alignment shows that there are a number of residue positions, which are invariant in all ARIDs in all domain contexts (residues in olive-green in Fig 3A). This suggests that these residues are likely to play a critical role in maintaining the highly conserved structure of the ARID fold across all domain combinations.
In a previous study, six residues: V1058, I1061, V1067, L1076, Y1096, and K1093 were shown to form hydrophobic core of BAF250a ARID (Fig 3B) [10]. Indeed these residue positions are well conserved in ARIDs across all domain architectures (Fig 3A, marked in red  box). Other absolutely invariant residues: P1042, L1051, G1062, and W1073 are found almost identically spaced in ARID across all architectures [20]. Previously, the conserved residue Y1097 in H5, invariant residue P1042 in loop L1, and conserved residue W1073 in H4 were shown to form an aromatic structural scaffold in ARID structure [19]. A biochemical property-based conservation at each residue position of BAF250a ARID across all architectures is summarized in S2 Table. There are residue positions where ARIDs vary from each other in an architecture-specific manner. Likely, these positions preserve the overall ARID structural fold and yet bring about specificity in each distinct architecture (residues marked in blue in Fig 3A and denoted as variable in S2 Table).

Mutation of a residue (V1067G) of the conserved hydrophobic core leads to unfolding of BAF250a ARID
Mutation of a residue, valine at position 1067 (V1067) to a glycine, that is part of the hydrophobic core described earlier, was shown to cause growth defects and ultimately growth arrest in mice (Fig 3A and 3B) [10]. V1067G mutation results in a protein that had reduced DNA binding affinity [19]. We expressed and purified ARID WT and V1067G mutant from E.coli BL21 (DE3) expression cells (please see Materials and Method). Both the WT and mutant protein eluted at approximate monomer size from a size-exclusion column (Fig 4A). The purity level and the identity of the proteins were ascertained using SDS-PAGE analysis and massspectrometry (Fig 4B and S3 Fig). We observed precipitation of V1067G mutant protein over time compared to the WT ARID that remained soluble unto 2-3 weeks. Fig 4C shows the CD spectra of WT and V1076G mutant of ARID. The CD spectra showed that both the proteins were α-helical at 25˚C, however there is loss of secondary structures in V1067G compared to the WT ARID. Further, we monitored thermal unfolding of proteins using CD spectroscopy. CD signal at 222 nm was monitored as a function of temperature from 10˚C to 90˚C and T m of the transition was calculated. Both WT and mutant proteins showed two-state transition but with varying T m . While T m for the WT was 56˚C, the T m for the V1067G mutant was 40˚C showing that V1067G mutation leads to destabilization of the ARID (Fig 4D).
We also recorded 1D 1 H and 2D 15 N-1 H HSQC NMR spectra of WT and V1067 mutant of ARID. The 1D 1 H spectra clearly showed that as compared to the WT there has been gross loss of folded structure in mutant V1067G as evident from the broadened resonance peaks in the amide and methyl protons region of the spectra (Fig 4E). This was further exemplified by the inspection and comparison of 2D 15 N-1 H HSQC spectra of WT and V1067G mutant (Fig 4F). The cross peaks in 2D 15 N-1 H HSQC spectra result from every N-H correlation in protein including the backbone amide group. Therefore, the 2D 15 N-1 H HSQC in the amide region acts as finger print of a protein. Well-dispersed and uniform intensity cross-peaks in 2D 15 N-1 H HSQC spectra indicate that protein is well folded under the experimental condition. WT ARID gave a 2D 15 N-1 H HSQC spectra of a typically well folded protein, however the 2D 15 N-1 H HSQC spectra of V1067G mutant showed disappearance of well dispersed cross-peaks and appearance of overlapped and broadened resonances indicating that mutant protein is only partially folded as compared to the WT protein.

MD simulations show that V1067A mutation results in destabilization of ARID
In order to gain a molecular insight into the effect of V1067G mutation on the structure of WT ARID, we performed an all-atom MD simulation of both WT (PDB ID: 1RYU) and an in Structural, sequence, and evolutionary analysis of BAF250a silico generated V1067G mutant, using GROMOS96 45a3 united atom force field [35]. Comparison of Root Mean Square Deviation (RMSD) between WT and mutant over the entire simulation time exhibits less stability and higher degree of flexibility for V1067G mutant (Fig 5A). Root Mean Square Fluctuation (RMSF) over 50 ns simulation showed that the mobility of WT increases selectively in regions involved in protein-DNA interaction: residues near the N-terminus, loop L1, part of H4 and H5 helices compared to the mutant, which may contribute to DNA binding ability of WT ARID (Fig 5B). It is clearly evident that over the entire simulation time, WT protein maintained its overall structure whereas in mutant V1067G, the structure underwent distortions. Helices H5 and H6 developed a knick in mutant (Fig 5C). The structural alterations significantly affected the orientation of previously reported DNA-binding loop (L1) and helices (H4 and H5). Significant structural deviations were shown in helix H2, H3 and H4 through the 50 ns simulation time course that likely resulted in unfolding of the Structural, sequence, and evolutionary analysis of BAF250a hydrophobic core in V1067G, which may lead to reduction in its DNA-binding affinity. Helix H1 also underwent significant positional changes in mutant over the simulation affecting the overall structure of the mutant protein.

V1067G mutation leads to decrease in DNA binding affinity of BAF250a ARID
We used isothermal titration calorimetry (ITC) to probe the interaction of WT and V1067G mutant with DNA. We used Dickerson Dodecamer sequence (DD12 dsDNA) that had been shown to interact with the ARID in a previous NMR study [19]. At 25˚C, WT ARID and DD12 dsDNA complex formation proceeds with very small but a net positive enthalpy change (ΔH = 1.51±0.05 kcal/mol) with an observed stoichiometry of~1 (Fig 6A and Table 1). Table 1 shows all the thermodynamic parameters determined from the ITC results. ARID interacts with DD12 dsDNA with K d (equilibrium dissociation constant) of 71.94±37.7 nM. This is in agreement with the previous study that had reported an apparent K d of~50 nM for ARID and  DNA interaction. It is however worth to note that a larger construct of protein that encompassed the ARID was used in that study [10]. In case of V1067G mutant ARID and DD12 dsDNA interaction, the complex formation proceeds with a net negative change in enthalpy (ΔH = -0.69±0.41 kcal/mol) at 25˚C (Fig 6B  and Table 1). The K d of the interaction is 694±331 nM, which is about 10 fold higher than that for the WT ARID, clearly showing that the V1067G mutation in ARID reduces the interaction between ARID and DNA dramatically. Taken together as a proof of principle, the MD simulation and biophysical studies including CD and NMR spectroscopy, and ITC titrations showed that a change in a residue that is conserved in all the ARIDs would result in distortion of the molecular structure, and results in loss of DNA binding. A detailed thermodynamic and mutational study would be required to determine whether the reported interaction between ARID and DNA is specific or non-specific.

A set of conserved residues that are unique for ARCH2 lie on exposed helices of BAF250a ARID involved in DNA binding
To identify sequence fingerprints that distinguish ARIDs in ARCH2 from ARIDs in other architectures, we compared the consensus alignment derived for ARCH2 (Fig 7) with the consensus alignment of the ARID derived for all the other domain architectures (Fig 3A). As shown here, while a number of residue positions are conserved in all ARIDs and likely involved in formation of the core (residues in yellow in Fig 7), some positions are uniquely conserved in ARCH2 (residues in red in Fig 7).
BAF250a ARID has previously been shown to interact with DD12 dsDNA using NMR chemical shift perturbation (CSP) methods [19]. The perturbed residues are from the N-terminal region, loop L1, followed by helix H4 and H5 of the ARID and include residues: L1041, V1044, R1046, K1047, K1072, W1073, R1074, S1091, L1092, K1093, K1094, 1096Y and I1097 (red and yellow residues in Fig 8B and S2 Table). Interestingly, residues L1041, R1046, K1072, R1074, S1091, K1094 and I1097 were identified as uniquely conserved to the ARID in ARCH2 (red residues in Fig 8B and S2 Table). Therefore, we argue that the associated DNA binding functionality is likely responsible for the conservation of these surface exposed residues in BAF250a.
A number of selectively conserved and surface exposed residues found on helix H1 were not shown to be involved in DNA-binding and remain uncharacterized (Fig 8A). We speculate that these ARCH2-specific residues in ARID might be involved in potential inter-domain interactions in BAF250a or with other proteins in the BAF complex.

Sequence and structural analysis reveal potential role of ARCH2-specific BAF250_C (in BAF250a) in mediating protein-protein interactions
A structure-based multiple sequence alignment of well-known ARM repeats containing proteins such as Adenomatous polyposis coli, p120 catenin, β-catenin and the BAF250_C domain (residues 1939-2282 of BAF250a) shows the signature three-helical Armadillo (ARM) repeats containing unit of this fold (S4 Fig and Fig 1C) (details in Materials and Methods). ARM repeats typically consist of three α-helices that are stacked in parallel resulting in a super-helical structure and are often associated with protein-binding and scaffolding functions [49,50]. The protein binding function of ARM repeat proteins involve residues from multiple repeats rather than a single repeat that has allowed this domain to diverge extensively in sequence [49,51]. The alignment also shows that the hydrophobic interactions between residues of each repeat unit, characteristic of ARM domain structures, are well conserved in BAF250_C domain (S4 Fig). Further, we aligned the BAF250_C domains of BAF250a homologues (in ARCH2) in our dataset and found the sequences to be well conserved along the entire length (S5 Fig). High sequence conservation and solvent accessibility are two criteria that can be employed to identify surface-exposed patches of residues in a structure that are involved in protein-protein interactions. Ideally such sites should be uniformly conserved and surface exposed in all the sequences in the alignment. Therefore, we identified conserved residues in the alignment of BAF250_C homologues in our dataset (S5 Fig). Further, we computed the solvent-exposed residues in the modeled BAF250_C using Naccess (see S5 Fig legend for description) [48]. As shown here, a number of these conserved but surface exposed residues were found to lie on repeats 1 and 2 at the N-terminus, a region that was extended in BAF250_C based on our SEG-HCA analysis (Fig 8C). Therefore, we postulate that these residues are likely to function in mediating interactions between BAF250_C and other domains of the protein or with other proteins in the remodeling complex.
ARM repeat-containing proteins such as β-catenin that are known to interact with APC, cadherins and Tcf family members have been observed with asymmetric charge distribution [52]. The surface charge distribution of modelled BAF250_C is distinct from other ARMrepeat proteins such as β-catenin, APC, and plakophilin and shows the presence of a number of negatively charged residues, which include several surface exposed and conserved residues in ARCH2 (S6 Fig). Possibly these uniquely conserved and surface exposed residues in BAF250_C may facilitate the interactions of BAF250a with other proteins in the chromatin remodeling complex.

Mapping cancer mutations of BAF250a
Increasingly, evidences of the role of BAF250a in various cancers through a number of recurrent mutations of the gene have been demonstrated [1,8,13,[53][54][55]. Since structures of the ARID and the modelled BAF250_C showed a number of uniquely conserved residues, we mapped these mutations on the structures of these two domains. The COSMIC database was searched for the most frequent somatic mutations involving the BAF250a gene distributed in 1904 unique samples [56]. COSMIC database stores the expert-curated somatic cancer mutations in proteins. Of the mutations that map to the ARID and the BAF250_C, a majority of the mutations were found to occur on surface-exposed residues (Fig 9A and S3 Table) and therefore unlikely to alter the overall structure of the individual domains but possibly alter the function of the protein. Indeed, on ARID, mutations were observed in three DNA-binding residues (T1004, R1046 and R1074). Seven observed somatic mutations involving residues V1982, R1989, W2050, L2106, G2087, L2088 and L2089 lie on the BAF250_C domain. Further, three mutations at M1022, R1026 and K1106 on the ARID occur in the residues which are conserved in ARCH2 (where ARID and BAF250_C co-occur). This raises the possibility that these mutations can potentially affect the predicted domain-domain interactions in BAF250a/b. In addition, the two LXXLL motifs on the BAF250_C domain also harbor five of the observed missense mutations, suggesting that binding sites involving interactions with nuclear hormone receptors are likely to be affected by these mutations. A large number of the missense mutations on BAF250_C are spread over the entire domain (Fig 9A and S3 Table). Indeed, a recent structure-based analysis has revealed that cancer missense mutations target protein interaction interfaces [57]. Since the ARM domain has an extensive interaction interface, different parts of which may be involved in interactions with various partnering proteins in the chromatinremodeling complex, mutations in these different interfaces may disrupt these interactions as well.

Discussion
Using hydrophobic core analysis based SEG-HCA approach, here we report several regions in BAF250a that harbor functionally important motifs, which are likely to be folded with a hydrophobic core. The predicted regions included the DNA binding ARID and a β-catenin like ARM-repeat fold containing BAF250_C domains. Using rigorous sequence and phylogenetic analysis, we showed that ARM-repeat fold in association with ARID is only found in BAF250a homologues (in ARCH2) suggesting that these two domains co-evolved in these proteins. While instances of the ARM repeat domains interacting with proteins that harbor a DNAbinding domain are known, their co-occurrence in a single protein is reported here for the first time [58,59]. We believe that these two domains co-occur to facilitate the interactions of the ARID containing proteins with other members of the SWI/SNF complex. Alignment of all ARID sequences (across all domain architectures) resulted in identification of a set of conserved residue positions that are critical for the maintenance of ARID fold. In a previous study a hydrophobic cluster was identified that consisted of conserved residues V1058, I1061, V1067, L1076, Y1096, and K1093. Here, we provide an experimental validation that mutation of a conserved residue (V1067G) results in gross destabilization of ARID fold that impairs its DNA binding functions. Using ITC experiments we showed that V1067G mutation leads to a drastic decrease (~10 fold) in the DNA binding affinity of ARID.
Further, alignment of ARID and BAF250_C sequence in 'ARCH2' resulted in identification of a specific set of conserved residues in these domains. Expectedly, this set includes residues that are important for fold maintenance but in addition, we found sets of conserved residues that are solvent exposed. This suggests that the conservation of these residues might be due to their functional importance such as DNA binding (in case of ARID) or in mediating proteinprotein interactions [19].
Both BAF250a and BAF250b contain ARID and BAF250_C domain combination. The proteins differ in length with BAF250a_HUMAN being 2285 residues in length and BAF250b_-HUMAN of 2236 residues length, but share 59% sequence identity along the length of the proteins. Furthermore, the sequence identity is 83% and 80% for the ARID and BAF250_C domains respectively. In spite of sharing this level of sequence identity either in full length or in folded regions, these two paralogues are functionally distinct [11,13,14]. In our sequence and phylogenetic analysis, we found that BAF250a and BAF250b sequences, though similar in domain architecture, partition into two distinct clusters. Therefore, the variable residue positions in folded ARID and BAF250_C in BAF250a and BAF250b may be functionally relevant resulting in different functional output of these proteins.
In order to test the direct binding of BAF250_C with the putative binding partners, we made attempts to express and purify the recombinant BAF250_C from E.coli. However, it expressed in insufficient quantities (in soluble fraction of E.coli lysate) hampering our attempts to purify the protein (S1 Text and S7 Fig). When interaction databases such as BIOGRID and IntAct were searched for the interactors of BAF250a we find that BRG1 is listed as a top interactor, and BAF60A as potential interactors of BAF250a [68,69] The interaction of BRG1 with BAF250a has been demonstrated elsewhere as well [18,70]. In a recent molecular modelling study, the C-terminal region of BAF250a was predicted to interact with BAF60A [71], although more experiments are needed to verify this. BAF250b was also shown to repress Wnt/β-catenin signaling through its interaction with BRG1 in a separate study. The BRG1 interacting residues were mapped to the C-terminal BAF250_C region of the protein in this study [72]. It has been suggested that the C-terminal BAF250_C containing region has putative functional motifs such as nuclear export signals (NES) and LXXLL-motif that are likely involved in mediating protein-protein interactions in several proteins including nuclear hormone receptor such as glucocorticoid receptor (GR) [71,73]. BAF250b has also been shown to acts as an E3 ubiquitin ligase that targets histone H2B at lysine 120 [74]. Thus far the ubiquitin ligase activity has not been directly shown for BAF250a, however this activity in BAF250b was mapped to the ARMrepeat containing BAF250_C that is present in both BAF250a and BAF250b. All these observations suggest that the ARM repeat containing BAF250_C in BAF250a/b is involved in multiple protein-protein interactions, both within the SWI/SNF complex as well as with other proteins (Fig 9B). Indeed, several of the observed somatic mutations in various cancers map to the large surface-exposed regions of both the ARM and ARID domains, likely interrupting such interactions (Fig 9A).
In conclusion, our analysis predicts that the BAF250a with its N-terminal ARID that binds DNA and ARM repeat BAF250_C that mediates its interactions with other proteins can recruit the human BAF complex to its targets through protein-DNA and protein-protein interactions (Fig 9B) [75]. Future experiments targeting these domains will help validate the predicted interactions of ARID and ARM repeat containing BAF250_C and reveal the function of BAF250a in the BAF complex.  (Fig 1). Only the seed sequences for this domain in PFAM are shown here. Solvent exposed as well as conserved residues across all sequences are highlighted at 70% conservation threshold and were derived by consulting the alignment of 236 BAF250_C homologues (S2 Fig). The symbols used in the consensus are as follows: uppercase-identity; lowercase-consensus level > 0.5; !-I,V; $ -L, M; %-F,Y; #-NDQEBZ. Solvent accessibility is shown at the bottom of the alignment along a bar with blue for solvent exposed residues, cyan for partially exposed residues and white for solvent inaccessible residues. (TIF)

S6 Fig. Surface charge properties of ARM repeat domain in various proteins. (A)
BAF250_C modelled in this study, shows predominantly negatively charged patches that is also uniquely conserved in homologs of BAF250_C (Fig 9A). (B) β-catenin, is known to bind multiple partners along the positively charged groove running through the entire surface. (C) In Adenomatous polyposis coli protein (APC), the positive groove might be the recognition site for APC-binding partners. The acidic patch is used to interact with β-catenin.  Table. Biochemical property-based conservation at 70% level for each residue in all ARIDs across 11 different domain architectures vs. ARID in ARCH2 (BAF250a ARID, PDB ID: 1RYU). If particular residue is found to be conserved, single letter amino acid code is written in bracket next to the biochemical nature of the amino acids. � Marked indicate the position that is selectively conserved in ARID in ARCH2 as compared to all other architectures. DNA binding residues from previous study (reference # 19 in the main text) were highlighted in bold face. Therefore, functionality of the uniquely conserved residues ( � ) may be attributed as DNA binding where it is written in bold. (DOCX) S3 Table. List of missense mutations reported for the ARID1A in the COSMIC database. Only mutations that map to the ARID and BAF250_C terminal domains are listed here. (DOCX) S1 Text. Details of cloning, expression, and purification attempts on BAF250_C. (DOCX)