text:
Dorsomorphin Promotes Survival and Germline Competence of Zebrafish Spermatogonial Stem Cells in Culture

Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish using a zebrafish ovarian feeder cell line (OFC3) that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC) survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae. In contrast, dorsomorphin inhibited the growth and survival of zebrafish female germline stem cells (FGSCs) in culture. In the presence of dorsomorphin, the spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 for at least six weeks in culture. Transplantation experiments revealed that 6 week-old spermatogonial cell cultures maintained in the presence of dorsomorphin were able to successfully colonize the gonad in 18% of recipient larvae and produce functional gametes in the resulting adult chimeric fish. Germline transmission was not successful when the spermatogonia were cultured 6 weeks in the absence of dorsomorphin before transplantation. The results indicate that Bmp signaling is detrimental to SSCs but required for the survival of zebrafish FGSCs in culture. Manipulation of Bmp signaling could provide a strategy to optimize culture conditions of germline stem cells from other species.

Introduction
The zebrafish embryo is an ideal model for in vivo analysis of germ cell determination and migration during early development [1,2], however complementary in vitro studies of zebrafish germ cell growth and differentiation have been lacking due to the absence of suitable cell culture systems. Mouse spermatogonial stem cell (SSC) and female germline stem cell (FGSC) cultures have proven to be valuable in vitro models for studies of germ cell differentiation and been used for the production of transgenic and knockout mice [3,4,5,6]. Recently, our lab used a drug selection strategy to establish zebrafish FGSC cultures that were initiated from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish. The Tg(piwil1: neo);Tg(piwil1:DsRed) fish express Neo and DsRed under the control of the piwil1 promoter enabling the use of G418 selection to isolate the piwil1:neo expressing FGSCs. Using this method a homogeneous population of FGSCs were selected and maintained for more than 6 weeks in culture during which the cells continued to express multiple germ cell markers. Following transplantation into recipient larvae, the cultured FGSCs successfully colonized the gonad of the host and produced functional gametes in the adult chimeric fish [7].
Since piwil1 is also expressed in the germ cell lineage of male zebrafish [8,9,10], in this paper, we apply the same drug selection strategy to establish zebrafish spermatogonial cell cultures from the gonad of male Tg(piwil1:neo);Tg(piwil1:DsRed) fish. Previous attempts to establish long-term spermatogonial cell cultures from zebrafish were ineffective due to spontaneous differentiation of the spermatogonia to non-proliferating spermatids [11,12] and to the cultures becoming overgrown by testicular somatic cells [13]. To avoid these problems, primary cultures were initiated from the testes of Tg(piwil1:neo);Tg(pi-wil1:DsRed) fish and treated with G418 to select Neo-expressing spermatogonia and eliminate somatic cells. A key component of the culture system was the addition of dorsomorphin to block Bmp signaling which prevented the spontaneous differentiation of the zebrafish spermatogonia and prolonged their survival in culture. The action of dorsomorphin on spermatogonia in culture is consistent with results of previous studies showing that mutation of the Bmp type I receptor impairs germ cell differentiation in zebrafish testis [14] and the addition of exogenous BMP promotes mouse spermatogonial differentiation in culture [15,16]. In our study, inhibition of Bmp signaling also enhanced the capacity of cultured spermatogonia to colonize the gonad following transplantation into recipient larvae and produce functional gametes in the adult chimeric fish.

Animals and Ethics
Zebrafish were maintained and staged as previously described [17]. All of the experimental procedures and protocols described in this study were approved by the Purdue University Animal Care and Use Committee and adhered to the National Research Council's Guide for Care and Use of Laboratory Animals.

Immunocytochemistry and Histology
Gonads dissected from zebrafish were fixed with either 4% paraformaldehyde in phosphate-buffered saline (PBS) or Bouin's fixative at 4uC overnight, and then processed through successive treatments of ethanol (50%, 70%, 95%, and 100%) followed by two xylene baths and embedded in paraffin. The serial paraffin sections (5 mm) were prepared for immunocytochemical staining to visualize Neo and Vasa within the testicular tissues of Tg(piwil1:neo) [7] according to published protocols [18,19]. Tissue sections were treated with mouse anti-neomycin phosphotransferase II monoclonal IgG (1:500 dilution, Abcam) and rabbit anti-zebrafish Vasa antiserum [20] (1:4000 dilution) and then stained with Alexa Fluor 488 AffiniPure goat anti-mouse IgG and Cy3 AffiniPure goat antirabbit IgG (1:500 dilution, Jackson ImmunoResearch Lab, Inc.). Neo and Vasa expression was evaluated by examining the tissue sections by fluorescence microscopy using a Nikon Eclipse TE200 microscope (Nikon, Tokyo, Japan) equipped with a RT Slider digital camera (Spot Imaging Solution, Sterling Heights, MI, USA). The sections were stained with hematoxylin-eosin for histological examination by light microscopy.

Cell Transplantation and Analysis
Cultured spermatogonia were dissociated (0.25% EDTA in PBS), washed, re-suspended in 50 ml of L-15 medium and immediately transplanted into two-week-old recipient larvae that were treated as embryos with a morpholino (MO) nucleotides against dnd [23]. Cell transplantations were performed under a stereomicroscope using a glass micropipette needle. Spermatogonia were transplanted into the abdominal cavity under the swim bladder close to the gonads according to a published method [19]. Two weeks after transplantation, the recipients were examined by fluorescence microscopy and the potential germline chimeras were identified based on the presence of DsRed-positive cells in the gonadal region. To determine if the transplanted cells were able to generate functional gametes, the recipient fish were raised to sexual maturity and paired with wild-type zebrafish mates.

Statistical Analysis
Data obtained from the growth assays were presented as the mean and standard deviation. For statistical analysis Student t-tests or one-way ANOVA was applied followed by Bonferroni-Dunn tests using SAS program. The significance was accepted at p,0.05.

Derivation of Spermatogonial Cell Cultures from
Tg(piwil1:neo);Tg(piwil1:DsRed) Zebrafish Immunocytochemical analysis of testicular tissue dissected from Tg(piwil1:neo) fish confirmed that Neo expression overlapped with Vasa in the spermatogonial cells (Fig. 1A-C) indicating that a drug selection approach could be used to isolate the germ cells in culture. To initiate spermatogonial cell cultures, an enriched fraction of DsRed-positive testicular germ cells was obtained by Percoll separation of dissociated testes dissected from Tg(piwil1: neo);Tg(piwil1:DsRed) fish. The germ cell fraction was seeded onto confluent growth-arrested OFC3 feeder cells that were engineered to express zebrafish Lif and Fgf2 (OFC3LF) or Lif and Gdnf (OFC3LG) [7] and G418 was added to eliminate any remaining somatic cells. Following 3 weeks of drug selection colonies of 4 or more DsRed-positive spermatogonia were observed in the culture ( Fig. 2A-D). The small (about 10 mm-diameter) DsRed-positive cells possessed morphological characteristics of spermatogonia in the testis [19] with round nucleus and a high nucleus-to-cytoplasm ratio ( Fig. 2A,B). Cell counts performed on 3 week-old cultures revealed that the number of DsRed-positive, G418-resistant spermatogonia was significantly greater on OFC3LF and OFC3LG feeder layers compared to the parent OFC3 feeder line (Fig. 2E). After 3 weeks the cell number began to dramatically decrease in all of the cultures resulting in only 5-10% of the cells remaining in cultures maintained for 6 weeks on OFC3LF and OFC3LG feeder layers (Fig. 2E).

Addition of Dorsomorphin Prolonged Spermatogonial Cell Growth but Inhibited FGSC Survival in Culture
The decrease in cell number observed in the spermatogonial cell cultures after 3 weeks was most likely due to differentiation of the spermatogonia since the DsRed-expressing cells became smaller and formed aggregates that detached from the feeder layer as described in previous studies of zebrafish spermatogonial cell cultures [13]. Analysis of the feeder cells by RT-PCR revealed that bmp2a, bmp2b and bmp4 were all expressed by the OFC3LF and OFC3LG cells (Fig. 3A) raising the possibility that Bmp signaling may be involved in inducing spermatogonial differentiation. To test this hypothesis, spermatogonial cell cultures were initiated and maintained in the presence of dorsomorphin, an inhibitor of the Bmp type I receptor. Results from growth assays revealed that the addition of dorsomorphin significantly enhanced the proliferation of spermatogonia in 3 week-old cultures (Fig. 3B) with the greatest mitogenic effect observed when the inhibitor was added at a concentration of 2 mM. At this concentration of dorsomorphin, the growth rate of DsRed-expressing spermatogonia increased 80% compared to the control with several large colonies containing more than 60 cells present in the cultures (Fig. 3C). In 6 week-old cultures the presence of dorsomorphin increased the number of spermatogonia by 30-fold compared to the control (Fig. 4A) and DsRed-positive colonies containing more than 100 cells were observed (Fig. 4B)  cultured spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 [24,25] for at least 6 weeks in the presence of dorsomorphin (Fig. 4C). In contrast, when dorsomorphin was added to drug-selected FGSC cultures [7] cell growth was significantly inhibited compared to controls maintained in the absence of dorsomorphin (Fig. 5A-D). Also, the expression of germ cell markers was significantly reduced or completely lost by 3 weeks in the dorsomorphin-treated FGSC cultures (Fig. 5E).

Dorsomorphin Enhanced the Capacity of Spermatogonial Cell Cultures to Colonize the Gonads and Produce Functional Gametes Following Transplantation to Recipient Fish
Cell transplantation experiments were performed to evaluate the germline competency of the spermatogonial cell cultures. Spermatogonia obtained from 3-or 6-week-old cultures were transplanted into recipient larvae that were treated as embryos with a MO nucleotides against dnd to block endogenous germ cell formation [23]. For each experiment approximately 20 to 60 spermatogonia were transplanted into the recipient larva. Due to the small number of cells available, only approximately 10 or less DsRed-expressing cells were transplanted in experiments involving spermatogonia cultured for 6 weeks in the absence of dorsomorphin. Two weeks after transplantation, the recipients were screened by fluorescence microscopy to identify those that possessed DsRed-positive cells in the gonadal region (Fig. 6A). Survivors were raised to sexual maturity and successful germline transmission of the transplanted cells was confirmed by pairing the chimeric fish with wild-type zebrafish mates. All of the surviving adult recipient fish that were obtained from the transplantation experiments were male. A total of 36 recipient male fish were obtained from 2 independent cell transplantations using 3 weekold spermatogonia cultured in the absence of dorsomorphin. Five of the fish (13.8%) were fertile and generated normal offspring (Table 1). When spermatogonia that were cultured for 3 weeks in the presence of dorsomorphin were transplanted, 10 of the 52 (19.2%) adult recipients obtained were fertile ( Table 2). The presence of dorsomorphin had a dramatic effect on promoting the germline competency of 6 week spermatogonial cell cultures. None of the 28 adult recipients obtained by transplanting 6-week-old spermatogonia cultured without dorsomorphin were fertile while 9 of 50 (18%) recipients (Table 3) produced from the 6 week dorsomorphin-treated spermatogonia were able to reproduce. Genomic PCR analysis revealed 100% inheritance of piwil1-neo confirming that all of the offspring were derived from the transplanted spermatogonia (Fig. 6B). All of the fertile recipient fish obtained were able to produce multiple batches of normal offspring for at least 6 months. Dissection of three fertile recipients that were produced from transplanted 6-week-old spermatogonial cell cultures revealed that two of the fish developed only a single testis on one side of the body and the third fish developed two testes of unequal size (Fig. 6C). All of the testes examined contained DsRed-positive cells (Fig. 6C1). Histological examination of the fully-developed testes revealed the presence of a significant number of mature sperm along with spermatogonial cells at different developmental stages present in individual tissue sections (Fig. 6D). Results from cell transplantation experiments demonstrated the presence of SSCs in the spermatogonial cell cultures derived from the testes of Tg(piwil1:neo);Tg(piwil1:DsRed) zebrafish.

Discussion
In adult mammals, the SSC population in the testis is represented by the type A undifferentiated (A und ) spermatogonia that are present as single (A s ), paired (A pr ) or multiple aligned (A al ) cells [26,27]. The type A pr and A al spermatogonia are able to dissociate to generate type A s cells [28]. In contrast, the zebrafish testis possesses two subtypes of undifferentiated type A spermatogonia, A und and A und* , that possess stem cell characteristics based on cell transplantation experiments [29,30]. In this study we showed that Neo is expressed in the spermatogonia of the Tg(piwil1:neo) fish enabling us to use a drug selection approach to derive spermatogonial cell cultures initiated from the testes of Tg(piwil1:neo);Tg(piwil1:DsRed) fish. Using G418 selection to eliminate the somatic cells that were present in the testicular primary cultures enabled us to avoid the problem of the spermatogonia becoming overgrown in culture which was encountered in previous studies [13,31]. Another advantage of using the Tg(piwil1:neo);Tg(piwil1:DsRed) fish was that the expression of piwil1:DsRed made it possible to visually evaluate the growth of the spermatogonia in culture by fluorescence microscopy. The zebrafish spermatogonial cell cultures were derived in conditions that were similar to those previously used to propagate zebrafish FGSCs in culture [7]. A unique requirement of the spermatogonial cell culture system, however, was to block Bmp signaling by the addition of dorsomorphin to the medium. Previous studies have shown that Bmp functions to promote spermatogonial differentiation both in vivo and in spermatogonial cultures. Mutation of the zebrafish Bmp type I receptor resulted in an impairment of spermatogonial differentiation in vivo while addition of BMP4 to mouse SSC cultures promoted the production of differentiated spermatids [14,15,16]. Since our analysis of the OFC3 feeder cells, used to derive our spermatogonial cell cultures, showed that the cells expressed bmp2a, bmp2b and bmp4, we decided to block Bmp signaling by adding dorsomorphin to the culture medium. Dorsomorphin, also known as compound C, is a small molecular weight inhibitor of the Bmp type I receptor [32]. The compound has been shown to disrupt dorsoventral patterning when it is administered to zebrafish embryos [33]. We found that in cultures maintained for 3 weeks in the absence of dorsomorphin the zebrafish spermatogonia became smaller and formed aggregates that detached from the feeder cells indicating that the cells had differentiated to non-proliferating spermatids [11]. Addition of dorsomorphin to the cultures inhibited differentiation and enabled the spermatogonia to proliferate and express appropriate markers for more than 6 weeks in vitro. Further evidence that dorsomorphin maintained the SSC characteristics in culture was obtained from the cell transplantation experiments showing that the presence of dorsomorphin extended the time that the cultured SSCs could be transplanted and colonize the gonads of recipient larvae and produce functional gametes in the adult chimeric fish. In contrast, when dorsomorphin was added to zebrafish FGSC cultures, the cells stopped proliferating and lost expression of the germ cell markers within 3 weeks, demonstrating a requirement for Bmp signaling by the female germ cells in culture. These results showing that Bmp signaling has the opposite effect on zebrafish male and female germline stem cells in culture may be useful in optimizing conditions that support the growth of germline stem cells from other species. The OFC3 feeder cell line used to initiate the spermatogonial cell cultures was derived from ovaries obtained from Tg(gsdf:neo) zebrafish [7]. Gsdf is produced in somatic cells of the fish ovary and testis and functions to promote the growth of germ cells [34]. To derive OFC3, G418 selection was applied to Tg(gsdf:neo) ovarian primary cultures to isolate the Gsdf-expressing cells. Since OFC3 cells express Gsdf, it was expected that they may also produce other unknown factors that promote the growth and survival of germ cells in culture and therefore serve as an ideal feeder layer for spermatogonia. To optimize the spermatogonial cell culture conditions, OFC3 was transfected to express zebrafish Lif and Fgf2 (OFC3LF) or Gdnf (OFC3LG). Recombinant LIF, FGF2 and GDNF have all been shown to be important factors in establishing mouse SSC cultures [22,35]. Previously, we demonstrated that optimal growth of zebrafish FGSCs was achieved using the OFC3LF and OFC3LG feeder layers indicating that the cells produce factors that support zebrafish germ cells in culture [7]. A previously reported zebrafish spermatogonial cell culture system employed a feeder cell line that was derived from a spontaneously arising zebrafish testicular tumor [11]. Although we made several attempts to establish a continuously-growing G418 resistant feeder cell line initiated from healthy Tg(gsdf:neo) testicular tissue, we were not successful.
Using the drug selection strategy and culture conditions that were developed in this study, spermatogonia could be propagated for several weeks in vitro and then transplanted into infertile recipient larvae where the cells colonized the gonads and generated fertile adult fish at a high frequency. Since recipient larvae were treated as embryos with a MO that blocked endogenous germ cell formation and gonad development resulting exclusively in the production of infertile male [23], the transplanted spermatogonia were able to direct the formation of a functional gonad and rescue fertility of the host fish. Although only approximately 60 spermatogonia were transplanted into each recipient, the fertile adult fish that resulted from the transplantations were able to produce multiple batches of offspring over a 6 month period. These results demonstrate that the transplanted cells re-established the entire spermatogonial cell lineage in the host testis and therefore possessed germline stem cell qualities that are characteristics of SSCs. Only male fertile recipients were produced from successful germline transmission of transplanted spermatogonial cells. Using germ cell ablation, a recent zebrafish study has suggested that signals from female germ cells, particularly the oocytes, may be needed to promote primary female sex determination and maintain female development throughout adulthood [36]. It is most likely that the cultured spermatogonial cells did not produce these signals that are required for female development. As such, all the fertile recipients completely developed into males. The spermatogonial culture system described in this study will provide an in vitro model for studies of extracellular factors controlling zebrafish spermatogonial differentiation and may form the basis of gene transfer approach.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts*

Background: The phosphatidic acid (PtdOH) binding site of TGD4 is not yet known. Results: Amino acids 1–80 and 110–145 of TGD4 represent two PtdOH interacting sequences and TGD4 forms a homodimer in vitro and in vivo. Conclusion: TGD4 N terminus binds PtdOH while its C terminus interacts with a second TGD4 protein. Significance: This work reveals the structural functional relationship of an essential chloroplast β-barrel lipid transfer protein. Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus.

In land plants, glycolipids, such as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG) comprise ϳ64% of total membrane lipids (1). Glycolipids are synthesized through two independent pathways: the prokaryotic and the eukaryotic pathways (2). Plastid specific acyltransferases of the prokaryotic pathway are associated with the inner plastid envelope membrane and transfer the acyl groups, which are de novo synthesized in the chloroplast and attached to acyl carrier proteins (ACPs), to glycerol-3-phosphate producing PtdOH. This PtdOH is then dephosphorylated to diacylglycerol (DAG), 3 the direct substrate for glycolipid synthesis. In the eukaryotic pathway, endoplasmic reticulum (ER)-associated acyltransferases use acyl-CoAs derived from plastid-exported acyl groups and glycerol-3-phosphate as substrates, providing PtdOH for the synthesis of extraplastidic phospholipids, such as phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). A certain portion of lipid precursors returns to the chloroplast, where ER-assembled DAG moieties are incorporated into glycolipids (3). Glycolipids originating from the prokaryotic pathway have a 16-carbon acyl chain in the sn-2 position of the glyceryl backbone, while glycolipids derived from the eukaryotic pathway have an acyl chain of 18 carbons in the same position. This difference has been attributed to the different substrate preferences of acyltransferases in the chloroplast and the ER (4,5). Plants that utilize both pathways, such as Arabidopsis thaliana, have 16:3 fatty acids in the sn-2 position of their thylakoid lipids, and thus are referred to as 16:3 plants. Other plants, such as maize or castor bean, rely exclusively on the eukaryotic pathway and have mostly 18:3 fatty acids in the sn-2 position of their thylakoid lipids; these plants are designated 18:3 plants (6).
Four Arabidopsis proteins, TGD1-4, are currently known to be involved in the ER-to-chloroplast lipid trafficking process (7)(8)(9)(10). Plants with mutations in any of the TGD genes are impaired in the eukaryotic pathway and accumulate an additional lipid, trigalactosyldiacylglycerol (TGDG). The TGD1, 2, and 3 proteins are proposed to form a bacterial-type ABC transporter complex (11), in which TGD1 is a membrane permease, TGD2 a substrate-binding protein, which specifically binds to PtdOH, and TGD3 an ATPase. TGD4 is a predicted ␤-barrel membrane protein (12,13) embedded in the chloroplast outer envelope and, like TGD2, specifically binds PtdOH (14). Based on the current findings, it seems likely that the TGD proteins form a lipid transfer conduit to import PtdOH from the ER through the plastid envelopes. Here we describe the identification of two PtdOH binding sequences of TGD4, the lipid bind- ing properties of TGD4 proteins in 18:3 plants, and the composition of the TGD4 complex.

EXPERIMENTAL PROCEDURES
Expression and Purification of DsRED-TGD4 Proteins-The truncation mutants T1-T21 were generated starting with construct pLW01/DsRED-TGD4 as the template for polymerase chain reaction (PCR) (14). The PCR products were subsequently inserted into the pLW01 vector using SacI and NotI restriction sites. M1-M9 point mutations were generated by site-directed mutagenesis (Stratagene, La Jolla, CA) using pLW01/DsRED-TGD4 T14 (AA 110 -145) as the template and M10-M11 were commercially synthesized (IDT, San Jose, CA). To isolate TGD4 homologs from Ricinus communis (RcTGD4) and Zea mays (ZmTGD4), cDNAs obtained by reverse transcription from isolated total RNA were used as the template for PCR. RcTGD4 and ZmTGD4 were inserted into the BamHI/ NotI and EcoRI/NotI restriction sites of the pLW01/DsRED-His vector, respectively. Primer sequences used in this study are summarized in supplemental Table S1. Sequencing was performed by the Research Technology Support Facility (RTSF) at Michigan State University to verify the accuracy of cloned fragments. Recombinant proteins were produced in Escherichia coli strain BL21 (DE3) and purified as previously described (14). Briefly, all proteins were produced at 16°C overnight after adding 100 M isopropyl ␤-D-1-thiogalactopyranoside (IPTG) to the cell culture at an A 600 ϭ 0.6 except for T1, T3, T17-T20, M10, M11, which were produced by incubation at 28°C for 3 h. Cells were resuspended in lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, protease inhibitor mixture (Roche, Indianapolis, IN), 1% forskolin-12 (Affymetrix, Santa Clara, CA), pH 8.0) and lysed by incubating with lysozyme (Sigma) at a final concentration of 0.2 mg/ml for 30 min followed by sonication using the sonicator 3000 with a microprobe (Misonix, Farmingdale, NY) for 2 min with 10 s pulses. Recombinant proteins were purified to homogeneity by Ni 2ϩ -nitrilotriacetic acid (NTA)-agarose (Qiagen, Valencia, CA) in the presence of 0.1% foscholine-12 according to the manufacturer's instruction. The purified proteins were dialyzed with Tris-buffered saline (TBS: 50 mM Tris-HCl, pH 7.0, 0.1 M NaCl) with 2 M choline chloride using Amicon centrifugal filter devices with 10 kDa cutoff (Millipore, Billerica, MA). Purified proteins were quantified by RC DC protein assay (Bio-Rad, Hercules, CA).
Liposome Association Assay and Data Quantification-Multi-lamellar liposomes composed of dioleoyl-PtdCho and dioleoyl-PtdOH with different w/w ratios as mentioned in the figure legends were prepared as previously described (14). Briefly, 250 g of total lipids (Avanti, Alabaster, AL; Larodan, Sweden) dried under a stream of nitrogen were hydrated by adding 500 l of TBS with 0.2 M choline chloride and incubating at 37°C for 1 h. Multi-lamellar liposomes were obtained by vortexing for 2 min at the highest speed. Purified proteins (0.25 M) were incubated with liposomes on ice for 30 min. Unbound proteins were separated from liposome-bound proteins by centrifugation at 13,000 ϫ g for 10 min at 4°C followed by two washes with TBS containing 0.2 M choline chloride. The liposome-protein pellet was examined on a SDS-PAGE gel (15) followed by Coomassie Brilliant Blue R-250 (Sigma) staining.
Quantitative analysis was based on densitometry using ImageJ software (16) with three technical repeats. The amount of liposome-bound full-length DsRED-TGD4 was set at 100% and the final data were normalized to individual loading controls. The results in Fig. 6C were fitted to Hill's equation (Equation 1) using Origin software (OriginLab, Northampton, MA), where "n " is Hill's number, "L" is ligand concentration, and K d is the dissociation constant.
Protein Lipid Overlay Assay-Lipid strips were prepared as previously described (14). Briefly, 1 nmol of lipids in 1 l of buffer (25% v/v chloroform, 50% v/v methanol, 10 M HCl, 1% Ponseau S) were spotted onto a nitrocellulose membrane (GE Healthcare, Piscataway, NJ) and dried in a fume hood for 1 h. Finished membranes were blocked in TBST (TBS with 0.1% Tween-20) buffer plus 3% Bovine Serum Albumin (BSA) for 2 h at room temperature. Purified proteins were added to the blocking solution at 1 g/ml final concentration and incubated with lipid strips at 4°C overnight followed by washing in TBST buffer 3 ϫ 10 min and immunoblotting with anti-His (C-term) antibody (Invitrogen, Grand Island, NY) at a dilution of 1:5000. Secondary antibody anti-mouse horseradish peroxidase (Bio-Rad) (dilution: 1:20,000) was added for 0.5 h followed by washing in TBST buffer 6 ϫ 10 min. Signal was detected using a chemiluminescence kit from Sigma.
Chloroplast Preparation-Arabidopsis chloroplasts were prepared as previously described with modifications (19). Briefly, 4-week-old seedlings harvested from MS solid medium were ground in grinding buffer (330 mM sorbitol, 50 mM HEPES-KOH, pH 8.0, 2 mM EGTA, 0.1% w/v BSA with 5 mM MgCl 2 ) and separated on a 40 and 80% discontinuous Percoll (Sigma) gradient by centrifugation at 4,000 ϫ rpm for 10 min. The intact chloroplasts collecting at the interface were isolated and washed once with buffer (330 mM Sorbitol, 50 mM HEPES-KOH, pH 8.0, 5 mM MgCl 2 ) followed by centrifugation at 700 ϫ g for 5 min.
Accession Number-The sequence of Arabidopsis thaliana TGD4 can be found in The Arabidopsis Information Resource under the name At3g06960. The sequence of Ricinus communis and Zea mays TGD4 can be found in GenBank TM under the accession numbers XM_002519286.1 and XM_002519286.1, respectively.

Amino acids 110 -145 Are Necessary and Sufficient for
PtdOH Binding by TGD4-To locate the PtdOH binding site(s) within the TGD4 primary sequence, partially overlapping trun-cation mutants T1-T5 were constructed (Fig. 1A). The N termini of the respective truncations were fused to DsRED to enhance solubility, and the resulting recombinant proteins were produced in E. coli and purified to homogeneity using a C-terminal 6ϫ His tag. The proteins were then tested for PtdOH binding in a liposome association assay, wherein proteins interacting with the PtdOH-containing liposome precip-FIGURE 1. Coarse mapping of the TGD4 PtdOH binding domain. A, schematic representation of TGD4 truncation mutants T1-T5 fused with DsRED on the N terminus. White bar, DsRED; black bar, TGD4 fragment; gray bar, 6ϫ His tag; numbers below indicate amino acid positions in the full-length, wild-type sequence. B, multi-lameller liposomes consisting of 40% (w/w) PtdOH and 60% (w/w) PtdCho were incubated with purified proteins. Proteins bound to the liposome co-precipitated after centrifugation at 13,000 ϫ g. Protein content of the pellet was analyzed by SDS-PAGE and stained by Coomassie Brilliant Blue. Numbers indicate molecular masses in kDa. T, total protein used in this assay. C, images were quantified by densitometry. Data were normalized with bound DsRED-TGD4 as 100%. Averages and standard deviations were calculated from three repeats. White bar, DsRED; black bar, TGD4 fragment; gray bar: 6ϫ His tag; numbers below indicate amino acids. B, same liposome binding assay was performed for truncation mutants T7-T12 as in described in the legend of Fig. 1B. Numbers indicate molecular masses in kDa. T, total protein used in this assay. Images were quantified and data were normalized with liposome-bound T2 protein as 100%. Averages and standard deviations were calculated from 3 repeats. C, T14 (AA 100 -150) secondary structure predicted by PROF (Predict-Protein). Y-axis represents the likelihood of each structural signature. Note that amino acids 110 -145 formed a putative soluble loop.
itate with the liposomes following centrifugation. The protein content of the liposome pellet was then examined by SDS-PAGE, and the protein signals were quantified by densitometric scanning. As shown in Fig. 1, B and C, the N-terminal 1-300 amino acids accounted for the PtdOH binding activity of TGD4, while the C-terminal portion of the protein showed greatly reduced PtdOH binding.
Additional mutants were constructed to test this hypothesis: T13 (AA 125-145), T14 (AA 110 -145), and T15, which is a truncated version of TGD4 that contains the entire TGD4 protein sequence, except that the loop was substituted with a 6ϫ His linker sequence (Fig. 3A). While T13 only retained partial PtdOH binding activity, T14 still showed full PtdOH binding (Fig. 3B). To test if these truncations were still specific to PtdOH, a lipid overlay assay was used. The purified proteins were incubated with a hydrophobic membrane onto which different plant lipids were spotted. The proteins were then detected with an anti-His antibody (Fig. 3C). Both T14 and full-length TGD4 bound exclusively to PtdOH, and with similar strength. T15 bound specifically to PtdOH as well, but the signal was attenuated suggesting weakened PtdOH binding. In the liposome association assay, T15 lost about 50% of the total PtdOH binding activity, suggesting the existence of an additional PtdOH binding sequence (Fig. 3D). As positively charged amino acids are usually essential for PtdOH binding through electrostatic interactions with the negatively charged PtdOH below indicate amino acids. B, same liposome binding assay was performed for truncation mutants T13-T15 as in Fig. 1B. Numbers indicate molecular masses in kDa. T, total protein. Images were similarly quantified as in Fig. 2B and normalized with liposome-bound T11 as 100%. C, membranes spotted with various lipids were incubated with purified proteins. Proteins bound to lipids were detected using anti-His antibody. DAG, dioleoyl-diacylglycerol; DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PtdCho, dioleoyl-phosphatidylcholine; PtdEtn, dioleoyl-phosphatidylethanolamine; PtdGly, phosphatidylglycerol; PtdIns, dioleoyl-phosphatidylinositol; PtdOH, dioleoyl-phosphatidic acid; PtdSer, dioleoyl-phosphatidylserine; SQDG, sulfoquinovosyldiacylglycerol; TAG, trioleoyl-triacylglycerol. D, same liposome binding assay as in B, except that the PtdOH concentration was 30% (w/w). Data were normalized with liposome-bound DsRED-AtTGD4 as 100%. Numbers indicate molecular masses in kDa. T, total protein. E, alignment of TGD4 amino acids 110 -145 within three species. Black boxes, identical amino acids; gray boxes, similar amino acids; M1-M9, T14 point mutants with indicated amino acid changed to alanine. F, liposome binding assays with 30% (w/w) PtdOH were conducted with different point mutants. T, total proteins. Numbers indicate molecular masses in kDa. The data were quantified by densitometry with three repeats and normalized with liposome-bound T14 as 100%.
head group (25), we hypothesized that mutating those positive residues in the minimal PtdOH binding domain of TGD4 would affect PtdOH binding. Thus, we changed each positively charged amino acids between residues 110 and 145 to alanine (Fig. 3E) and tested the point mutants' PtdOH binding activity except for M6 (AA 110 -145, K140A), which we were unable to produce in E. coli. We also included a point mutant targeting a conserved serine, M1 (AA 110 -145, S128A). while no single point mutant abolished PtdOH binding, mutants M1-to-M4 retained only about 60% of PtdOH binding activity compared with T14 (AA 110 -145) (Fig. 3F).
A Second PtdOH Binding Domain Was Present in the First 80 Amino Acids of TGD4-As shown in Fig. 1C, T5 (AA 1-100) retained wild-type PtdOH binding activity despite lacking the aforementioned, PtdOH-binding sequence (AA 110 -145). We investigated which sequence in this most N-terminal portion of the protein represented by T5 (AA 1-100) is responsible for PtdOH binding. Additional truncation mutants T17-T19 were constructed (Fig. 4A) and their PtdOH binding activity was tested by liposome association assay. Only T19 (AA 1-80) bound to PtdOH while T18 (AA 1-50) and T17 (AA 1-25) did not, indicating that amino acids 50 -80 were necessary for PtdOH binding by this fragment (Fig. 4B). This region was also predicted to have a loop structure and was highly conserved among different species (Fig. 4, A and E). Additional mutants were tested, which included a truncation spanning residues 50 -80 (T20) and two point mutants, M10 and M11, in the 50 -80 AA region that had an alanine in place of a positively charged arginine (Fig. 4A). In contrast to T14 (AA 110 -145), T20 (AA 50 -80) was not sufficient for PtdOH binding. The truncation mutant T21 that lacked both PtdOH binding sequences (AA 110 -145 and AA 1-80) abolished the PtdOH binding activity, indicating that both sequences are necessary in order for TGD4 to bind PtdOH and that these two sequences are the only PtdOH binding sequences in the TGD4 protein.  binding assay with 40% (w/w) PtdOH was conducted for truncation mutants T17-T19. T: total proteins; Numbers indicate protein masses in kDa. Data were normalized and quantified as Fig. 1C. C, liposome binding assay with 30% (w/w) PtdOH was performed for T20, M10, M11, and T21. The results were quantified as above. Note that T20 was not sufficient for PtdOH binding. D, lipid overlay assay testing the lipid binding specificity of T19 (AA 1-80). Lipid abbreviations are the same as in Fig. 3C. E, T19 (AA 1-80) secondary structure predicted by PROF (PredictProtein). Y-axis represents the likelihood of each structural signature. FEBRUARY 15, 2013 • VOLUME 288 • NUMBER 7 plants (4,5) leading to the suggestion that DAG instead of PtdOH is the lipid transferred from the ER to the chloroplast in 18:3 plants.

TGD4 Phosphatidic Acid Binding Site
To test this hypothesis, cDNAs of TGD4 orthologs of the 18:3 plants Ricinus comunis (RcTGD4) and Zea mays (ZmTGD4) were isolated and expressed in E. coli to produce fusion proteins with DsRED at the TGD4 N terminus. AtTGD4 shares about 50% amino acid identity with RcTGD4 and ZmTGD4 (supplemental Fig. S1). The lipid overlay assay showed that, similar to AtTGD4, both RcTGD4 and ZmTGD4 bound only to PtdOH and not to DAG (Fig. 5A). To estimate the relative PtdOH binding affinities, the liposome association assay was employed. Both TGD4 orthologs from the 18:3 plants, especially RcTGD4, showed much stronger PtdOH binding than AtTGD4 (Fig. 5B).
TGD4 Forms a Functional Homodimer in Vitro and in Vivo-Proteins in a cell often form large complexes with other proteins involved in the same pathway to efficiently carry out their biological functions. These proteins may not be identified through genetic screening due to gene redundancy or mutant lethality. To identify possible additional components involved in lipid transfer from the ER to the plastid as part of the eukaryotic pathway, we searched for TGD4-interacting proteins in vivo using immunoprecipitation followed by mass spectrometry. However, no new proteins were found to interact with TGD4, and only TGD4 was identified repeatedly with high confidence (supplemental Table S2). The recombinant DsRED-TGD4 proteins migrated as oligomers, mainly dimers and pentamers, on a Blue-Native PAGE gel, while DsRED alone behaved as a monomer (Fig. 6, A and B). This oligomerization activity was mainly attributable to the C-terminal portion of TGD4 (DsRED-TGD4C), the region that was not responsible for PtdOH binding (Fig. 6, A and B). Liposome association experiments with increasing percentages of PtdOH showed a sigmoidal binding curve reminiscent of positive cooperativity characteristic of allosteric soluble proteins with a Hill number equal to 3 (Fig. 6C). It should be noted that the TGD4 protein is in a membrane-bound complex accepting a lipohilic substrate, and that the increasing percentage of PtdOH may alter the physical properties of the multi-lamellar liposome, which is a scenario in which the Hill equation is not strictly applicable. However, while the quantitative relationship between substrate concentration and TGD4 complex behavior in the membrane remains unresolved, qualitatively the observed sigmoidal relationship would be consistent with a protein complex containing multiple substrate-binding sites that cooperatively interact upon ligand binding.
To study the TGD4 complex in its native environment, and to employ an approach independent of DsRED, which represents a relatively large tag that might affect oligomerization of the fusion protein even though an engineered monomeric version of DsRED was used, chloroplasts from HA-TGD4 transgenic plants were isolated and their protein content was analyzed by Blue-Native PAGE. The TGD4 complex was specifically detected by immunoblotting using an HA antibody. The size of the complex fell between protein markers of 66 and 132 kDa under both resting and lipid-importing conditions (Fig. 6D). Under resting condition, isolated chloroplasts were incubated in an iso-osmotic buffer while in the lipid-importing condition liposomes composed of PtdOH and PtdCho were added to the chloroplast to allow lipid import as was previously shown (17). Both conditions were chosen to account for the possibility that TGD4 may exist in different complexes depending on the conditions. To better estimate the size of the complex, protein marker sizes were plotted against their migration distances. The approximate size of the complex was 115 kDa, which is approximately twice the size of the TGD4 monomer of 53 kDa. Since no further interacting partners were identified by mass spectrometry, and TGD4 did not interact with other known TGD proteins by co-immunoprecipitation, we concluded that TGD4 forms homodimers in vivo.

DISCUSSION
TGD4 was identified as a protein involved in ER-to-chloroplast lipid trafficking in a genetic screen (9). However, its molecular function was unknown until recently. TGD4 is a PtdOH binding protein that forms a ␤-barrel and is embedded in the chloroplast outer envelope membrane (14). Here we provide a detailed molecular and biochemical analysis of TGD4, in particular its PtdOH binding activity. Among the best characterized PtdOH binding proteins in plants are ABI1 (abscisic acid insensitive-1), AtPDK1 (3Ј-phosphoinositide-dependent kinase-1), PEPC (phosphoenolpyruvate carboxylase), ACBP1 (acyl-coenzyme A-binding protein-1), TGD2, TGD4, and MGD1 (monogalactosyldiacylglycerol synthase 1), of which a few have a characterized PtdOH binding region (7, 26 -31). Comparing characterized peptide sequences involved in PtdOH binding from diverse organisms, a few generalized conclusions can be drawn: 1) PtdOH-interacting, primary amino acid sequences do not appear to be conserved and are not easily recognizable, requiring that each be mapped individually; 2) positively charged amino acids are essential for PtdOH binding; 3) most PtdOH interacting sequences are located in solventexposed peptide loops with helix or coiled structures; and 4) peptide sequences mediating PtdOH binding can be relatively short. The latter point is important for the interpretation of our data, because a prerequisite for the validity of the systematic deletion approach chosen here is that PtdOH binding is fairly localized within the protein sequence and that the engineered mutations do not fundamentally affect the overall protein fold. As we observed binding to relatively small peptides of TGD4 in our experiments, we believe that the chosen approach is valid. However, knowing the full extent of the PtdOH binding site(s) of TGD4 will ultimately require a crystal structure of the TGD4-lipid substrate complex.
Concurring with the general features of PtdOH binding sites summarized above, we propose that TGD4 likely binds PtdOH through its aqueous solvent-exposed loops. The ␤-barrel core is suggested to be embedded in the membrane, and might serve as the conduit for lipid transfer. In support of this hypothesis, both 110 -145 AA and 50 -80 AA PtdOH binding sequences of TGD4 were predicted by BOCTOPUS (32) to face the cytosol. This would place the PtdOH binding site(s) in a position able to accept PtdOH from an extraplastidic membrane such as the ER, as shown in Fig. 7.
It should be noted that binding of PtdOH to TGD4 does not necessarily imply that it is also the transferred lipid substrate. Rather, binding of PtdOH by cytosol-exposed loops of TGD4 otherwise embedded into the outer chloroplast envelope could establish contact to an adjacent membrane such as the ER to provide a possible lipid transfer conduit. In the absence of an assay to directly measure lipid transfer by TGD4, we tried to obtain indirect clues regarding the transported lipid species. Toward this end, we determined the lipid binding properties of TGD4 from two 18:3 plant species. Based on the observation that plants lacking the prokaryotic pathway show reduced PtdOH phosphatase activity associated with the plastid envelope membranes (4,5), one would assume that a different lipid than PtdOH, namely DAG, should be imported from the ER to serve as precursors for thylakoid lipid biosynthesis. However, just like TGD4 of Arabidopsis, the two TGD4 proteins from maize and castor bean specifically bound to PtdOH and not to DAG (Fig. 5). In fact, they appeared to bind PtdOH more read-  FEBRUARY 15, 2013 • VOLUME 288 • NUMBER 7 ily, or with higher affinity than TGD4 of Arabidopsis. Because 18:3 plants lack the prokaryotic pathway for glycolipid synthesis, there must be increased metabolite flux through the eukaryotic pathway and proteins involved might have higher efficiency in transport or turnover of the lipid precursor derived from the ER. Thus our observation on the TGD4 proteins from maize and castor bean would support the hypothesis that PtdOH is indeed the transported lipid species. At this time we only have an incomplete knowledge of the molecular nature of plastid PtdOH phosphatase(s) (33), and further analysis of this enzyme and its location within the chloroplast envelope membranes in 18:3 and 16:3 plants will be required to more clearly understand the molecular basis for the differences in thylakoid lipid biosynthesis in these two plant types.

TGD4 Phosphatidic Acid Binding Site
Probing for additional proteins that might interact directly with TGD4 in a possible complex we came to the conclusion that TGD4 likely forms a homodimer in vivo. In contrast, most studied ␤-barrel membrane proteins are monomers (34). Within the limitation of the experimental approach discussed above, homodimer formation could explain the apparent cooperativity of PtdOH binding indicating a dynamic interaction between the two subunits. The C-terminal portion of TGD4 was necessary for this dimerization, while the PtdOH binding activity resided in the N-terminal sequences of the TGD4 subunits. We did not observe a TGD4 complex containing also the proteins of the TGD1, 2, 3 transporter of the inner envelope membrane of the chloroplast. Thus one has to assume that interactions between the TGD4 and the TGD1, -2, -3 complexes are transient or that they work independently.
In summary, our current analysis of TGD protein function supports the hypothesis that a TGD4 homodimer binds to PtdOH in the ER through its N-terminal PtdOH binding sequences (Fig. 7) and presumably transfers it across the outer envelope. The TGD2 protein of the TGD1, -2, -3 transporter complex extracts PtdOH from the inner leaflet of the outer envelope and presents it to the TGD1 permease, which transports PtdOH across the inner envelope at the expense of ATP hydrolyzed by TGD3. This hypothesis predicts that PtdOH has to be converted to DAG on the stroma side of the inner envelope membrane as DAG is the direct substrate for the bulk synthesis of chloroplast glycolipids. Further corroboration of this hypothesis would be aided by crystal structures of lipid substrate complexes, reconstitution of functional lipid transfer complexes in vitro, and a better understanding of location and substrate specificity of the PtdOH phosphatases predicted to be involved in the process.


text:
Msi1-Like (MSIL) Proteins in Fungi

Msi1-like (MSIL) proteins, which are eukaryote-specific and contain a series of WD40 repeats, have pleiotropic roles in chromatin assembly, DNA damage repair, and regulation of nutrient/stress-sensing signaling pathways. In the fungal kingdom, the functions of MSIL proteins have been studied most intensively in the budding yeast model Saccharomyces cerevisiae, an ascomycete. Yet their functions are largely unknown in other fungi. Recently, an MSIL protein, Msl1, was discovered and functionally characterized in the pathogenic yeast Cryptococcus neoformans, a basidiomycete. Interestingly, MSIL proteins appear to have redundant and unique roles in both fungi, suggesting that MSIL proteins may have evolutionarily divergent roles in different parts of the fungal kingdom. In this review, we will describe the current findings regarding the role of MSIL proteins in fungi and discuss future directions for research on this topic.

MSIL proteins have multifunctional roles in histone binding, nucleosome/chromatin assembly and modification, DNA damage repairs, and regulation of signaling pathways. Because MSIL proteins have no catalytic functions, their roles are mainly mediated as adaptors or regulators. Although MSIL proteins have crucial roles in basic cellular functions and genome architecture in mammalian cells, they do not appear to be essential for cell growth in fungi. There are some exceptions, however, including one of the two MSIL proteins in Schizosaccharomyces pombe, Mis16, which is essential for viability because it is required for kinetochore assembly during mitosis [3].
In humans, two MSIL proteins, RbAp46/48 (retinoblastoma protein [pRB]-associated proteins 46/48), alone are able to interact directly with helix 1 of histone H4 not yet assembled to the chromatin [4]. By interacting with two larger components, an MSIL protein forms a heterotrimeric complex called chromatin assembly factor 1 (CAF-1), which assists in assembling the tetramers of two histone H3-H4 dimers on newly synthesized DNA to form nucleosomes. In addition, the MSIL proteins interact with histone modification enzymes, such as histone deacetylases (HDACs), acetyltransferases, and methyltransferases (see review [2]). Additionally, the mammalian MSIL proteins serve as a co-repressor for the pRB, a well-known tumor suppressor, by repressing the transcription of E2F target genes [5]. pRB can carry out transcriptional repression by recruiting HDACs [6][7][8][9][10][11].
MSIL proteins also have been studied extensively in plants, particularly Arabidopsis thaliana. Among the five Arabidopsis MSIL proteins [12][13][14], MSI2 and MSI3 are related genes, although their functions are not yet known. MSI4 and MSI5 are also related genes, and MSI4 is an allelic gene of FVE, which functions in the autonomous 2 Yang et al. floral inductive pathway [15,16] responsible for suppressing the floral repressor Flowering Locus C (FLC) [17]. Therefore, fve mutants exhibit delayed flowering behavior by increasing FLC expression [17]. Like other MSIL proteins, Arabidopsis MSI1 can be a part of several protein complexes. In particular, MSI1 is a subunit of the fertilization independent seed (FIS)-Polycomb repressive complex 2 (PRC2) complex consisting of FIS1 (MEDEA), FIS2, and FIS3 (FIE), which controls seed development and parental imprinting [18]. Additionally, MSI1 physically interacts with the CUL4-DDB1 complex, which regulates embryo formation and has a role in maintaining parental imprinting of MEDEA [19]. Thus, MSI1 is also associated with Arabidopsis CAF-1 [20,21] and the retinoblastoma-related protein RBR1 [12,22]. Furthermore, transcriptome profiling of msi1-cs (cosuppression line) plants reveals that abscisic acid-responsive genes (osmotic and salt stress-related genes) are upregulated in msi1-cs, suggesting a novel role of MSI1 for negative regulation in response to drought stress [23].
Despite their pleiotropic roles in multicellular, complex eukaryotes, knowledge of fungal MSIL proteins has largely stemmed from studies of MSIL proteins in the budding yeast model Saccharomyces cerevisiae, an ascomycete. Information about the functions of MSIL proteins in other fungi is quite limited, except for a recent publication about the role of MSIL protein Msl1 in the human fungal pathogen Cryptococcus neoformans, a basidiomycete [24]. Interestingly, this report showed that the MSIL protein in C. neoformans also has pleiotropic roles and yet appears to have a rather different regulatory mechanism from that of the MSIL proteins in S. cerevisiae. This finding raises the possibility that the functions and regulatory mechanisms of MSIL proteins are diverse throughout the fungal kingdom despite their evolutionary conservation. Several excellent reviews of MSIL proteins or WD40-repeat proteins are available [1,2,[25][26][27][28][29]. In this review, we focus mainly on describing the functions of MSIL proteins in model yeasts and pathogenic fungi to discuss their potential as novel therapeutic targets.

FUNGAL MSIL PROTEINS AS COMPONENTS FOR THE CAF-1 AND HISTONE-MODIFYING COMPLEX
The primary function of two MSIL proteins, Cac3 (also known as Msi1) and Hat2, in the non-pathogenic model yeast S. cerevisiae is the assembly and modification of histones ( Fig. 1) [30]. During cell division, newly replicated DNA is rapidly assembled into the chromatin, which consists of repeating units of nucleosomes. A single nucleosome is deposited with a tetramer of histone H3 and H4 followed by the addition of two dimers of the histones H2A and H2B. S. cerevisiae contains three CAF-1 proteins, designated Cac1 (also known as Rlf2; Rap1p localization factor 2), Cac2, and Cac3/Msi1, which correspond to the human CAF-1 proteins p150, p60, and p48, respectively.
CAF-1 helps in the binding of histone H3 and H4 onto newly synthesized DNA during chromosome replication or onto recently repaired DNA sites after DNA damage by directly interacting with proliferating cell nuclear antigen, which is a DNA polymerase processivity factor [31][32][33]. Therefore, deletion of any of the CAC genes causes increased susceptibility to DNA damaging agents, such as UV irradiation, methyl methanesulfonate (MMS), and hydroxyurea (HU) [34]. Despite the seemingly essential role of the CAF-1 complex, deletion of any or all of these genes does not cause lethality, indicating that other chromatin assembly factor(s) exists [31,35]. In fact, another chaperone for histones H3 and H4 is the replication-coupling assembly factor (RCAF) complex, which consists of anti-silencing function 1 (Asf1) and acetylated H3 and H4 histones [36]. It is known that RCAF and CAF-1 act synergistically for chromatin assembly by interacting with newly synthesized DNA during both DNA replication and DNA damage repair [36]. Expectedly, the asf1∆ cac1∆ double mutant grows much more poorly than does either mutant alone [36]. After DNA damages are repaired, CAF-1 and Asf1 work together to recover the normal chromatin structure at the repair sites and deactivate the checkpoint proteins to stop the double-strand break response [37]. In addition, CAF-1 is required to maintain heterochromatin gene silencing at telomeric sites and at the mating type locus (Fig. 1) [38]. For this position-dependent gene silencing, Asf1 also has been implicated because double deletion of CAC1 and ASF1 synergistically reduces heterochromatin gene silencing [36]. Asf1 requires interaction with histone regulatory (Hir) proteins, which are involved in nucleosome formation and heterochromatin gene silencing [39]. Therefore, loss of both the functional CAF-1 complex and the Hir proteins leads to increased chromosome missegregation, increased mutation in kinetochore protein genes, and altered centromeric chromatin structures [40].
Compared with CAC1 and CAC2, whose expression is differentially regulated during cell cycle progress, CAC3 expression is largely constant during the cell cycle [41]. In CAF-1, Cac1 physically interacts with Cac2 and Cac3 separately, whereas Cac2 and Cac3 do not interact with each other [42]. Interestingly, Cac1 is usually co-purified with Cac2 and Cac3, but Cac3 is not always purified with Cac1 or Cac2, suggesting that Cac3 has other cellular roles or interaction partners [43]. Cac1 was independently identified as one of the Cac3/Msi1-interacting proteins through yeast two-hybrid screening with Cac3 as the bait protein [44]. Interaction between Cac1 and Cac3 does not require Cac2 [44]. Furthermore, Cac3 localizes to both the nucleus and the cytoplasm, whereas Cac1 primarily localizes to the nucleus [45].
S. cerevisiae contains a second MSIL protein, Hat2, which is a core component of histone acetyltransferases (HATs). Hat2 is the regulatory subunit of the yeast HAT-B (type B HAT) complex composed of Hat1 and Hat2 in the cytoplasm that acetylates newly synthesized histones. In the nucleus, the HAT-B complex further interacts with Hat1p-interacting factor 1 (Hif1), causing the Nub4 (nuclear type B HAT; Hat1-Hat2-Hif1) complex to mediate the DNA repair-linked chromatin reassembly process ( Fig. 1) [46,47]. In contrast, another HAT family, the HAT-A (type A HAT) complex, localizes in the nucleus, where the complex acetylates , which binds to and acetylates newly synthesized histone H4. Then, the HAT-B complex transfers the histone H3/H4 tetramer into the nucleus. In the nucleus, the HAT-B complex forms a NuB4 complex, which delivers the H3/H4 tetramer to anti-silencing function 1 (Asf1) by interaction with the Hat1p-interacting factor 1 (Hif1) histone chaperone. The histone H3 in the delivered H3/H4 tetramer is acetylated by another nuclear histone, acetyltransferase Rtt109, associated with Asf1. As a histone chaperone, Cac3/Msi1 is the smallest subunit of the chromatin assembly factor 1 (CAF-1) complex, which additionally contains Cac1/Rlf2 (the largest subunit) and Cac2. CAF-1 is involved in chromatin assembly as a histone-deposition factor during DNA replication as well as DNA damage repair. The replication-coupled assembly factor (RCAF), composed of Asf1 and an acetylated tetramer of H3/H4, serves as a histone donor and also plays overlapping roles with CAF-1 in chromatin assembly during DNA replication. Histon regulatory (Hir) 1 and Hir2 are also histone-deposition factors, which act independently from DNA replication. Asf1 can interact with the Cac2 of CAF-1 and Hir1/2. CAF-1 assembles histone H3/H4 onto newly replicated DNA by targeting proliferating cell nuclear antigen. Two dimers of histones H2A and H2B are transferred by the nucleasome assembly protein 1 (Nap-1) which shuttles between the cytoplasm and the nucleus. Histone deacetylation by histone deacetylases (HDACs) facilitates aggregation of the nucleosome and results in chromatin assembly. This complex, compact structure of chromatin provides the basis for heterochromatin gene silencing at telomeric regions and mating type locus. When DNA damage occurs, such as double-strand DNA breaks, histones in the chromatin structure are dissociated and a series of DNA-repair signaling cascades, including Mec1, Rad53, Chk1, Pds1, Dun1, and Crt1, are activated to induce the expression of genes involved in DNA damage repair and to block the progression of cell-cycle. Notably, Asf1 physically interacts with Rad53 under normal conditions, and upon DNA damage, Asf1 and Rad53 phosphorylated by Mec1 are released, serving as a histone donor and activator of the DNA damage repair system, respectively. PCNA, proliferating cell nuclear antigen. For more detailed information, please refer to the following review [30].
histones in a chromatin context to facilitate transcriptional activation [48]. Hat1 is the catalytic subunit of the HAT-B complex and acetylates H4 on lysine residues 5 and 12 [49]. Hat1, which is not an MSIL protein, is involved in histone deposition, nucleosome formation, and chromatin assembly during the progression of DNA replication, and it is required for histone modifications during DNA repair [50,51]. Hat2 has an adaptor role between the Hat1 and Hif1 proteins and is responsible for the high affinity binding of Hat1 to histone H4, while Hif1p is a histone chaperone [47]. In the absence of either Hat1 or Hat2, no growth deficiencies have been reported [51]. Hat1 and Hat2 are involved in telomeric silencing [50]. Notably, however, in transcriptome analysis, hat1∆ and hat2∆ mutants exhibit distinct transcriptional patterns in yeast cells [52]. In addition, deletion of HAT2, but not HAT1, expands the chronological lifespan of yeast [52], providing further support for the suggestion that Hat1 and Hat2 do not have completely overlapping functions in the cell. Recently, the genetic interaction of the Hat1-Hat2 complex with origin-recognition complex, which is required for the initiation of chromosomal DNA replication, has been identified [53].
Besides studies in S. cerevisiae, a single study by Yang et al. [24] has investigated the role of an MSIL protein in C. neoformans, which is a basidiomycetous fungus that causes fatal meningoencephalitis in both immunocompromised and immunocompetent individuals. A single gene (CNAG_ 03297.2) called MSI1-like gene 1 (MSL1) was identified as the closest Cryptococcus ortholog to both the yeast Msi1 and Hat2. Similar to the yeast Cac3/Msi1, Msl1 also appears to promote resistance to DNA damaging agents, such as UV irradiation, HU, and MMS, indicating that Msl1 may also have a role as a component of the CAF-1like complex in C. neoformans ( Fig. 1) [24]. As further evidence in support of this idea, Msl1 localizes to both the cytoplasm and nucleus but is more enriched in the nucleus [24]. Furthermore, the Cac2 ortholog of the CAF-1 complex was identified in C. neoformans, and its deletion mutant (cac2∆) shows hypersensitivity to genotoxic agents such as the msl1∆ mutant [24]. Despite the presence of the conserved Msl1 (Cac3 ortholog) and Cac2, rather unique features have been discovered in the CAF-1 components in C. neoformans. First, MSL1 does not functionally complement the yeast CAC3 (unpublished data by Y.-S. Bahn). Second, Cryptococcus does not contain an obvious Cac1 ortholog, which is a key CAF-1 protein that interacts with both Cac2 and Msi1/Cac3. It is conceivable that Cryptococcus may have a structurally distinct Rlf2/Cac1like protein, which might have unique interfaces that interact separately with Msl1 and Cac2. This may explain why Msl1 cannot be substituted for Msi1 in S. cerevisiae. To investigate the function of CAF-1 in Cryptococcus, an Rlf2/Cac1-like protein must be identified and functionally characterized with relation to Cac2 and Msl1 in C. neoformans.

FUNGAL MSIL PROTEINS AS REGULATORS OF THE NUTRIENT-AND STRESS-SENSING SIGNALING PATHWAYS
Between the two MSIL proteins, Msi1/Cac3 appears to have additional cellular roles besides its function in histone and chromatin assembly and modification. In fact, Msi1 was first identified as a negative regulator of the Ras/ cAMP pathway in S. cerevisiae because it was screened as multicopy suppressors of the ira1∆ mutation [54]. The Ras/cAMP pathway is a nutrient-sensing signaling pathway required for the growth, stress response, and differentiation of S. cerevisiae (Fig. 2) [55]. Under certain environmental stresses, the Cyr1/Cdc35 adenylyl cyclase produces cAMP, which binds to Bcy1, the regulatory subunit of protein kinase A (PKA), and releases the catalytic subunit PKA (Tpk1/Tpk2/Tpk3) from Bcy1. The released Tpk proteins regulate downstream transcription factors to respond to environmental stresses [56]. The cAMP pathway is negatively controlled by feedback regulation from phosphodiesterase (Pde) 1 and Pde2 [57,58]. Upstream of Cyr1, two bifurcated signaling branches exist: a heterotrimeric GTP-binding protein (G-protein) and small G-protein Ras. Ras1 and Ras2 are activated by Cdc25 guanine nucleotide exchange factor, which enhances the replacement of GDP with GTP [59]. Ira1 and Ira2, which are GTPase-activating proteins, negatively control Ras activation, in contrast to Cdc25 [60]. Alternatively, a G-protein coupled receptor (GPCR) associated with a heterotrimeric G-protein complex (Gα, Gβ, and Gγ) receives signals, such as glucose addition, and undergoes conformational changes with the release of the active GTP-binding form of the Gα subunit [61,62]. Activated Ras or Gα subunit activates Cyr1/Cdc35 adenylyl cyclase directly and subsequently controls PKA (Fig. 2) [55,63]. The multicopy expression of MSI1 inhibits the phenotypes of ira1 mutation-mediated Ras over-activation, such as heat-shock sensitivity [54]. Interestingly, overexpression of human RbAp48 can also suppress the over-activated Ras/ cAMP pathway in S. cerevisiae [64]. The mechanism of how Msi1 regulates the Ras/cAMP pathway, however, has not been completely elucidated (Fig. 2). The step at which Msi1 works in the cAMP-signaling pathway was originally proposed to be upstream of PKA because MSI1 overexpression suppresses heat-shock sensitive phenotypes caused by the ira1∆ or RAS2 G19V mutation, but not by the bcy1∆ mutation [54]. Furthermore, the overexpression of MSI1 decreases the intracellular cAMP levels induced by glucose addition, suggesting that Msi1 may regulate Ras1, like Ira1 or Cyr1 [54]. Due to the similar β-propeller structures, Ruggieri et al. [54] proposed that Msi1 is similar to the β-subunit of the G-protein. Nevertheless, the msl1∆ deletion mutant does not show any cAMP-related phenotypes, suggesting that other MSI1-like genes may exist [44,54]. The role of Msi1/Cac3 as a negative regulator of the Ras/cAMP-signaling pathway is independent from the function of the CAF-1 complex because overexpression of MSI1/CAC3 can still suppress the overactive phenotypes of the RAS2 G19V mutant in the cac1∆ or cac2∆ mutant backgrounds [45]. Furthermore, functional Gpr1 (GPCR)/Gpa1 (Gα-subunit) signaling is dispensable for phenotypic recovery of the RAS2 G19V mutant by MSI1/CAC3 overexpression [45]. to certain environmental cues (such as glucose addition), adenylyl cyclase (Cyr1/Cdc35) is activated by either activated GTP-bound Ras proteins or activated GTP-bound Gpa2 Ga subunit through the Gpr1 G-protein coupled receptor (GPCR) to produce cAMP, which is a secondary messenger. Synthesized cAMP either is degraded to AMP by phosphodiesterases (Pde1/2) as a negative feedback regulation or binds to the regulatory subunit (Bcy1) of PKA, which normally suppresses the activation of the catalytic subunits (Tpk1/2/3) of PKA. Released and activated Tpks, which have redundant and unique roles, phosphorylate or activate diverse target genes. Downstream of PKA, nitrogen permease reactivator 1 (Npr1) activates downstream effector proteins, mediating heat shock sensitivity or stabilization of nutrient transporters, which otherwise are degraded by ubiquitination. Cac3/Msi1 normally sequesters Npr1 and inhibits its activation. At this step, it is unclear how Bcy1 acts on the Cac3/Msi1 (depicted as dotted arrow). Activated PKA also phosphorylates and inhibits yet another kinase 1 (Yak1), which antagonizes the Ras/cAMP signaling, although the YAK1 transcription itself is also activated by PKA, perhaps as an autoregulatory loop. Interestingly, Yak1 stabilizes the interaction between Cac1 and Cac3/Msi1 in the nucleus without affecting other CAF-1 interactions. Under non-fermentable carbon sources, Yak1 inhibits the nuclear localization of Cac3/Msi1 without phosphorylation, whereas Yak1 phosphorylates a nuclear-localized Bcy1 and facilitates its export to the cytoplasm. Conversely, Msi1 can also control Yak1. Overexpression of YAK1 confers growth arrest only in the presence of Msi1. In Cryptococcus neoformans, however, the regulatory mechanism of the Msl1 MSIL protein in terms of the Ras and cAMP/PKA pathways is rather different from that in S. cerevisiae. First, the Ras signaling pathway is less functionally connected to the cAMP pathway and instead governs thermotolerance and other stress-related phenotypes by controlling an additional GTPase protein Cdc42 and a Cdc24 guanine nucleotide exchange factor protein. All of the Msl1-related phenotypes appear to be independent from the Ras-signaling pathways.
Second, there is no evidence showing that Msl1 acts downstream of PKA in C. neoformans. In fact, the negative role of Msl1 in melanin and mating efficiency is abolished by the loss of Cac1, indicating that Msl1 may act on Cac1 or its upstream factors (depicted dotted lines). In C. neoformans, Yak1-like or Npr1-like kinases have not been identified or functionally characterized. Therefore, physical and functional relationships between Msl1 and other PKA downstream kinases remain to be elucidated in future studies (in gray symbols and dotted arrows). CAF-1, chromatin assembly factor 1. Zds1, zillion different screens 1.
Nevertheless, a series of studies have been reported to show that Msi1 may work downstream of Cyr1. Overexpression of MSI1 does not significantly affect intracellular cAMP levels or PKA activity in itself [44]. In addition, the heat-shock sensitivity caused by the deletion of two phosphodiesterase genes, PDE1 and PDE2, can be suppressed by the overexpression of MSI1 even in the presence of increased intracellular cAMP levels, but not in the bcy1∆ mutant [44]. These data suggest that Msi1 may work downstream of PKA, but in a Bcy1-dependent manner. In efforts to identify the Cac3/Msi1-interacting proteins, Johnston et al. [45] performed yeast two-hybrid screening with CAC3/MSI1 as the bait and identified the nitrogen permease reactivator 1 (NPR1) gene, which encodes a Ser/Thr protein kinase. It appears that the Npr1 kinase, which is one of the positive PKA targets, is normally sequestered and inactivated by Msi1. Upon release from Msi1mediated inhibition, the activated Npr1 can phosphorylate and stabilize a number of nutrient transporters, such as Gap1 (a general amino acid permease) [65] and Mep2 (an ammonium permease) [66], which otherwise is normally degraded by the ubiquitin-mediated proteasome (Fig. 2) [44,45]. In a supportive finding, the phenotypic restoration caused by MSI1 overexpression in the RAS2 G19V mutant is inhibited by the overexpression of NPR1, and ubiquitin overexpression can suppress the RAS2 G19V mutant [45]. Another piece of supporting evidence for this working model is that the expression of the target genes downstream of the Ras/cAMP pathway, such as TPS1 and HSP104, is not affected by MSI1 overexpression, which indicates that Msi1 may negatively regulate the Ras/cAMP pathway by controlling protein degradation, possibly through Npr1 and ubiquitination rather than through transcriptional regulation of target genes.
In addition, Msi1 has mutual interdependence with yet another kinase 1 (Yak1), which is a dual-specificity tyrosineregulated protein kinase that antagonizes the Ras/cAMP pathway of S. cerevisiae (Fig. 2) [42]. YAK1 was initially identified as a gene whose deletion rescues the growth defect caused by the loss of the cAMP-dependent PKA function or Ras function [67]. In other reports, it was proposed that PKA directly phosphorylates the Yak1 kinase in vitro [68] and indirectly activates transcription of the YAK1 gene, suggesting that Yak1 antagonizes the Ras/ cAMP signaling with an autoregulatory loop [69]. Yak1 has additional functions in regulating several cellular processes. In the absence of YAK1, strong pseudohyphal growth induced by either constitutive over-activation of Ras or catalytic subunits of PKA appeared to be blocked [70]. Indeed, the yak1∆ mutant shows 50-to 100-fold enhanced sensitivity in response to heat-shock, which was also observed in strains with low PKA activity [71]. This heat shock sensitivity is suppressed by overexpression of MSI1 or deletion of NPR1, which implies that Npr1 sequestration by Msi1 regulates the downstream targets of the Ras/cAMP pathway in a Yak1-dependent manner [42]. Additionally, the interaction between Msi1 and Cac1 for the formation of the CAF-1 complex requires Yak1 [42]. Therefore, it is possible that the deletion of YAK1 could release Msi1 from CAF-1, which subsequently sequesters Npr1 to inactivate the Ras/cAMP signaling (Fig. 2). However, the interaction of either Cac1-Cac2 or Msi1-Npr1 occurs in a Yak1independent manner. Yak1 affects the subcellular location of Msi1 as well as the interaction of Msi1 with Cac1. Msi1 is normally localized in both the cytoplasm and nucleus when the cells were grown with a fermentable carbon source (e.g., glucose), and this localization is Yak1-independent. Under the growth condition with non-fermentable carbon sources (e.g., glycerol or ethanol), however, Msi1 accumulates in the nucleus, and this nuclear accumulation is blocked by overexpression of YAK1 [42]. In contrast to the Yak1dependent regulation of Msi1, Yak1 itself also appears to be controlled by Msi1. Yak1 negatively regulates the cell growth of S. cerevisiae, and the overproduction of YAK1 thereby arrests the cell cycle. The cell growth defect caused by an excess of YAK1 is diminished in the msi1∆ mutant, whereas deletion of CAC1 does not restore the growth defect by YAK1 overexpression; this indicates that Yak1 has a role in cell growth in an Msi1-dependent manner but in a CAF-1-independent manner [42]. Therefore, Msi1 and Yak1 mutually control each other.
The functions and regulatory mechanisms of Msl1 in the Ras and cAMP/PKA signaling pathways of C. neoformans are somewhat different from those of the yeast Msi1 (Fig.  2). In fact, the Ras and cAMP/PKA-signaling pathways are not strongly connected in C. neoformans, as they control distinct cellular features. The cAMP/PKA pathway positively controls two major virulence factors of C. neoformans, the polysaccharide cell surface capsule and antioxidant melanin pigment and sexual differentiation (see reviews [72,73]). In contrast, the Ras-signaling pathway does not significantly control capsule and melanin biosynthesis, but it affects sexual differentiation (see reviews [72,73]). Instead, the Ras-signaling pathway has a major role in controlling growth at high temperatures, including the host physiological temperature (37 o C) [74]. In a supportive finding, transcriptome profiles between ras1∆ and cAMP-signaling mutants have been found to be quite different in C. neoformans [75]. Msl1 mainly acts independently from the Ras signaling pathway in C. neoformans. Msl1 has pleiotropic roles in diverse stress responses. Similar to the ras1∆ mutant, the msl1∆ mutant shows increased susceptibility to high temperature and membrane destabilizers, such as sodium dodecyl sulfate, and the msl1∆ ras1∆ mutant shows even greater susceptibility [24]. In contrast, Msl1 is involved in other stress responses, such as oxidative and heavy metal stress responses, unlike Ras2. Therefore, it is likely that the Msl1 and Ras signaling pathways mainly control the stress response of C. neoformans independently. In contrast to the Ras-signaling pathway, the cAMP/PKA pathway is partly related to the Msl1-signaling pathway. Msl1 appears to negatively regulate the cAMP-signaling pathway in melanin production and sexual reproduction. Distinct from yeast Msi1, however, deletion of the adenylyl cyclase gene (CAC1) can completely abolish the effect of MSL1 deletion, indicating that Msl1 may act upstream of Cac1 in the cAMP pathway [24]. Strangely, capsule production, which is known to be positively controlled by the cAMP pathway, is not affected by the msl1∆ mutation [24]. In conclusion, the role of the Msi1/Cac3-like protein as regulators of the nutrition-and stress-signaling pathways appears to be divergent among fungi.

FUNGAL MSIL PROTEINS AS DEVELOPMENTAL REGULATORS
In plants, MSIL proteins are known to repress genes involved in the developmental process and sexual reproduction. In Drosophila, the MSIL protein p55 is required for the repression of developmentally regulated dE2F2/RBF-target genes but not for proliferation-regulated E2F target genes [76]. Furthermore, expression of p55 is considerably higher in actively differentiating cells than in non-differentiating ones [77]. In fungi, also, several lines of evidence strongly suggest that MSIL proteins could be involved in the developmental process and morphological differentiation. In S. cerevisiae, expression of MSI1 is greatly increased in meiotic division during sporulation [78], although the physiological meaning of this finding is not known. Msi1 serves as a co-repressor for pRB and helps recruit Rpd3 HDAC to pRB [79]. pRB controls differentiation as well as proliferation by repressing the target genes of the E2F transcription factor [79]. The role of Msi1 in the transcriptional repression of pRB is independent of its function as a component of the CAF-1 complex. In C. neoformans, deletion of MSL1 significantly increases mating efficiency [24]. The repressing role of Msl1 in mating requires the functional components in the Ras or cAMP pathway because deletion of genes in these signaling pathways abolishes the enhanced mating effect by the msl1∆ mutation [24]. Taken together, the role of MSIL proteins in the developmental program and differentiation appears to be highly conserved in fungi.

MSIL PROTEINS AS TRANSCRIPTIONAL ACTIVATORS IN THE PRESENCE OF NON-FERMENTABLE CARBON SOURCES
Pratt and colleagues reported an additional interesting function of Msi1: a cryptic transcriptional activation activity in non-fermentable carbon sources in S. cerevisiae [42]. Surprisingly, Msi1 fused to the Gal4 DNA-binding domain exhibits one-hybrid activity (the ability of transcriptional activation) only in the presence of non-fermentable carbon sources (e.g., glycerol, ethanol, acetate, lactase, etc.), but not in the presence of fermentable carbon sources (e.g., glucose, fructose, etc.) [42]. This activity requres only Yak1 and not CAF-1 and Ras/cAMP/Npr1-signaling, whereas Yak1 alone is not sufficient for this Msi1 function. During growth of S. cerevisia on non-fermentable carbon sources, the total protein accumulation of Msi1 (not by transcriptional induction) and its nuclear localization both increase, whereas Msi1 localizes to both the cytoplasm and the nucleus during growth on glucose [42]. The accumulation of Msi1 protein does not require Yak1. Instead, Yak1 inhibits nuclear localization of Msi1, although Yak1 does not phosphorylate Msi1 [42]. The in vivo target genes regulated by Msi1 and other unknown co-factors under non-fermentable carbon conditions have not been discovered. Furthermore, it is still not known whether this carbonsource-regulated function of the MSIL protein is conserved in C. neoformans and other fungi.

MSIL PROTEINS IN OTHER FUNGAL PATHOGENS
So far, the functions and regulatory mechanisms of MSIL proteins in fungi have been studied only in non-pathogenic S. cerevisiae and pathogenic C. neoformans. Most of the animal and plant fungal pathogens appear to have at least one MSIL protein, containing four to six intact WD40 repeats (Fig. 3). In S. cerevisiae, Msi1 has seven WD40 repeats (six intact WD40), and the N-terminal and Cterminal domains have separate functions. The first four WD40 repeats are required for sequestration of Npr1 and suppression of Ras/cAMP signaling, whereas the last three WD40 repeats are required for interaction with Cac1 of CAF-1 and for the cryptic ability to activate transcription in the presence of non-fermentable carbon sources (Fig. 3) [42]. Particularly, the latter functions of the Msi1 Cterminus require Yak1 [42]. Most MSIL-like proteins share certain features in common, such as the length of the amino acid sequences and the locus of the conserved WD40 repeats. Generally, the C-terminal WD40 domain structure is more conserved than the N-terminal WD40 domain structure, where less conserved WD40 motifs are often discovered. In addition, C. glabrata XP_448393 and A. gossypii NP_984338 have an extra-extended N-terminus compared with the others. This finding may indicate that the CAF-1 function of MSIL proteins could be more conserved among other fungi than their Npr1-sequestration function for suppressing Ras/cAMP signaling. When the structural organization of the WD40 repeats of MSIL proteins in fungal pathogens were further compared, the following common features were discovered. First, all of the MSIL proteins in the fungal pathogens contain a starting WD40-like domain without a conserved terminal WD dipeptide at the N-terminus, unlike the S. cerevisiae Msi1 and human MSIL proteins (Fig. 3). Second, the terminal WD40 domains always contain the conserved Trp or random amino acid (WX) motif (Fig. 3). The MSIL proteins in fungal pathogens show a greater than 40% sequence identity to C. neoformans Msl1 (AEX_58661). Therefore, it seems that the general structural features of

POTENTIAL FOR MSIL PROTEINS AS NOVEL ANTIFUNGAL DRUG TARGETS
The potential to exploit MSIL proteins as novel antifungal drug targets has been suggested by studies in C. neoformans [24]. A strain with a MSL1 deletion exhibits attenuated virulence [24]. Interestingly, however, the msl1∆ mutant was found to enhance azole resistance, suggesting that a potential inhibitor of Msl1 or its related signaling components may have antagonistic effects with azole drugs. Using direct inhibitors of MSIL proteins as antifungal drugs is not likely to be a good approach because MSIL proteins are evolutionarily conserved throughout eukaryotes. Instead, however, fungal-specific regulators downstream of MSIL or the manipulation of specific interactions between the MSIL protein and its binding partner could be alternative therapeutic methods. For example, if there is an Rlf2/Cac1like protein in the CAF-1 complex of C. neoformans, it could be quite divergent from its counterpart in yeast or humans. Transcriptome analysis performed in C. neoformans uncovered several Msl1-regulated genes, which could potentially contribute to the virulence of the pathogen [24]. In line with a previous finding that the expression of some target genes of cAMP-signaling is not affected by msi1 mutation in S. cerevisiae [44,45], only a small number of genes (17 genes) are differentially regulated by msl1 mutation in C. neoformans. This fact indicates that an MSIL protein alone may not be sufficient to negatively regulate the Ras/cAMP-signaling pathway at full scale. Interestingly, a significant portion of Msl1-regulated genes are stress-related, including Hsp12 (small heat shock proteins) and Hsp78 (an oligomeric mitochondrial matrix chaperone), further supporting the role of Msl1 in stress response and adaptation in C. neoformans. Because MSIL proteins appear to play physiological roles by interacting with other proteins, such as the CAF-1 components, pRB, Npr1, and Yak1 in yeast, Msl1-interacting proteins must be screened in C. neoformans and other pathogens. For example, an ortholog for one of the yeast Msi1-interacting kinases, Npr1, does not seem to be found in the C. neoformans genome, suggesting the possibility that novel MSIL-interacting proteins may exist in fungal pathogens.

CONCLUSION AND FUTURE RESEARCH DIRECTIONS
MSIL proteins with WD40 repeats have pleiotropic roles as regulators of stress-signaling pathways such as Ras/cAMP and other pathways, as a component of the chromatin-assembly factor and as a developmental regulator in pathogenic and non-pathogenic model yeasts. In particular, in pathogenic fungi such as C. neoformans, MSIL proteins govern the production of virulence factors and in vivo virulence, suggesting that MSIL-related signaling pathways could be developed as novel antifungal drug targets. Despite their evolutionary conservation and multi-functionality, the functions of MSIL in other pathogenic yeasts and filamentous fungi, such as Candida albicans, Candida glabrata, and Aspergillus fumigatus, remain completely unknown. Therefore, the following questions need to be further addressed in future studies.
1. Are such pleiotropic functions of MSIL proteins conserved in other ascomycete fungi, such as S. pombe and Neurospora crassa, similar to those in S. cerevisiae? 2. How divergent among fungi is the chromatin assembly factor, functionally and structurally? 3. Are Msi1-interacting proteins discovered in S. cerevisiae, such as the CAF-1 complex, Npr1, and Yak1, evolutionarily and functionally conserved in C. neoformans and other fungi? 4. Is the role of MSIL proteins in virulence conserved in other pathogenic fungi? 5. If MSIL proteins are conserved in other pathogenic fungi, are they functionally connected to the Ras and/or cAMP/PKA signaling pathways? 6. Is the role of MSIL proteins in development and morphological differentiation conserved in filamentous fungi? 7. Is the function of MSIL proteins in sensing and responding to non-fermentable carbon sources conserved in C. neoformans and other fungi?


s2fieldsofstudy: Biology Medicine

text:
MicroRNAs networks in thyroid cancers: focus on miRNAs related to the fascin

miRNAs are non coding ribonucleic acids which are protected with respect to evolution, and have a length of 18–25 nucleotides. microRNAs control the gene expression after transcription, through mRNA destruction or translation processing, and therefore participate in arrangement of the physiologic and pathologic cellular processes; They also may act as oncogene or tumor suppressors. Altered expression of a number of microRNAs is reported in process of progression and metastasis of thyroid cancers. Therefore, identification of these microRNAs may shed a light to oncogenesis pathway of thyroid cancers and their metastasis. In addition, microRNAs might apply as potential biological markers in diagnosis and treatment of thyroid cancers. The changes made in miRNAs profile of thyroid cancers are reviewed in this paper.

Introduction
Despite the fact that thyroid malignances are ranked 14th among all kinds of cancers, and affect only 1-2% of all cancer sufferers, but they are considered as the most prevalent cancer of the endocrinology system. The annual occurrence of such cancer ranges from 0.5 to 10% among 100000 persons in different parts of the world [1][2][3]; women are believed to be 2-4 times more likely to develop the condition [4]. Various studies show that about 3-5% of the affected subjects have positive family histories of the cancer [5,6]. In addition to the effect of sex and genetic factors, the size of the body [7,8], race [9], geographical distribution [10] and the amount of iodine intake [11] affect the rate of developing thyroid cancers. Thyroid malignances may break out at any age; nevertheless, the majority of the cases are aged over 30. It is specified that with aging, the invasion rate of such cancers increase [2,12].
Pathological analysis of thyroid cancer demonstrates that four types of the condition are more prevalent: papillary, follicular, anaplastic and medullary thyroid carcinoma. The first three categories origin from follicular cells, whereas medullary carcinoma derives from parafollicular cells (C-cells) [3,13]. Recent studies conducted in this field revealed that the gradual evolution of natural cells to neoplastic ones in the process of tumor development is the result of sequential genetic events, from among which changes in miRNAs profile is the main focus of the present study.

miRNAs
In 1993, the first miRNA, also known as Lin-4 (Lineage-4), was discovered in C. Elegans; it was, however, not earlier than 2001 that Let-7 was discovered. At that time, the importance of such molecules as a biological regulator had not been determined [14].
Micro-RNAs (miRNAs) are single strand 18-25nucleotide RNAs, which are transcribed by RNA Polymerase II from miRNA genes. They are however not translated into proteins. As the negative regulators of gene expression, they bind to 3'UTR of the Target mRNA, and prevent the translation process through destructing or blocking the mRNA. miRNAs are non-encoder endogenous RNAs, that apart from negative regulation of encoder protein genes, regulate various cellular processes such as reproduction, proliferation, difference, cell survival and carcinogenesis mechanisms [15]. Mechanism of miRNAs in the regulation of gene expression is the same as that of siRNAs, although, they are completely different. It is estimated that miRNA genes account for 2-5% of the human genome [16]. Up to now, more than 1800 types of miRNAs are recognized in plants, animals and even in viruses. It is estimated that human's genome includes 800 to 1000 miRNAs, most of which are only found in human beings [17]. Most of the miRNAgenes, located within the intron and exon of the encoding genes of various proteins and may be transcribed the polycistron. For instance, miR-221 and miR-222 are located on chromosome X and their deregulated expression is shown in some carcinomas such as thyroid cancer [18].
Approximately, each miRNA controls the expression of more than 200 Genes. Many miRNAs have special tissue expression, which may be higher or lower than that of normal tissues [19]. In other words, altered expression of certain miRNAs is linked with some cancers, including various kinds of thyroid cancer [18]. Although, the exact function of many miRNAs is not well distinguished, many believe determination of important miRNAs in cancerous tissues may help improve the understanding of the regulation of gene expression in various types of cancer. Cancer occurs when the regulation, proliferation and differentiation process is altered. The cancers that are insensitive to growth stimulating and suppressing signals, have unlimited proliferation and angiogenesis potential, are preserved from apoptosis, escape the immune system, have genome instability and metastasis ability may become malignant [20][21][22][23] (Table 1).
Altered expression of miRNAs through decreased expression of certain genes involved in cell proliferation and survival may result in cancer. This however does not suggest that miRNAs are directly involved in tumorigenesis and the development of cancer as the available studies have failed to show whether altered expression of mRNAs is the consequence of cancer or malignancies are secondary to altered expression. Nonetheless, certain changes that occur in cancerous cells can affect miRNAs expression directly or indirectly [25].
In some types of cancers, such as thyroid neoplasia, miRNAs are deregulated. Altered expression of miRNAs plays an important role in tumorigenesis. Special subsets of miRNAs in certain tumors have increased or reduced expression. Increased expression of miRNAs may result in reduced expression of tumor suppressor genes, and their decreased expression may activate proto-oncogenes. The number of genes, considered as the target of miRNAs and involved in cancer, is increasing very fast [19]. For instance, let-7 is a negative regulator of RAS [25], whereas miR-221 and miR-222 are the negative regulators of KIT Receptor [26]. CDKN1B, an important control factor in cell cycle, is linked with cell development in S Phase of the cycle) [27]. miR-16-1 and miR-15a may cause reduced expression of BCL2, which is involved in apoptosis [28]. Such miRNAs, also known as oncomirs, are present in various types of cancer. Their genes are located on the loci on which deletion, duplication and point mutation has occurred [29,30]. Some of microRNAs act through reducing the expression of tumor suppressing genes, cell differentiation regulatory genes and apoptosis. Others, on the other hand, act through targeting proto-oncogenic mRNAs and silencing such mRNAs, thus, lower the risk of these cells becoming cancerous [31]. Altered expression of some miRNAs may cause cells to become cancerous, affect cell growth through interfering with the regulation of cell cycle. miRNAs are the most important factors in the regulation of programmed cell death during tumorigenesis; the survival of cancerous cells, on the other hand, are influenced by changing the expression of such micro-RNAs.
Cancerous cells become immortal by protecting their telomeres through positive regulation of telomerase and telomere preservation. Any change in the expression of  [32]. Deregulatory mechanisms of miRNAs in tumor tissues are not completely determined [33,34].
Recently, adding other molecular markers such as miRNAs to the mutant panels has improved the sensitivity of cancer diagnosis. miRNAs are involved in the pathogenesis of certain cancers, such as thyroid neoplasia. Not much is known about the clinical and pathological properties of the disease and expression of special miRNAs; however altered miRNA expression is reported in cancerous thyroid tissues compared with healthy cells. In other words, increased miRNA expression is reported in 32% of thyroid malignances, whereas 38% of them present reduced expression [35] (Table 2).
Such information suggests the role of deregulated expression of miRNAs in the transformation of thyroid cells. For instance, increased miR-187 expression is reported in tumors with RET/PTC gene rearrangement, whereas that of miR-221 and miR-22 is observed in tumors positive for BRAF and RAS mutations as well as papillary carcinomas with unclear mutation. Increased miR-146b expression is seen in tumors with RAS mutation. Such information can help facilitate detecting malignant tumors in FNA (Fine Needle Aspiration) samples with high sensitivity even when molecular tests have failed to report any mutations [35][36][37][38][39][40].

microRNAs in Epithelial Mesenchymal Transition (EMT)
Metastasis is a complicated dynamic biological event, subject to the separation of cancerous cells from adjacent tissue. Indeed, metastasis increases the risk of death secondary to cancer. There are different special mechanisms for the metastatic processin various cancerous cells. Shifting the focus to the studies aiming to recognize the reason behind the changes noted in the expression of suppressor genes or metastasis activators can pave the way to better understand different aspects of such phenomenon, and thus develop effective ways to prevent cancer. EMT is one of the most important way of the metastatic process [41]. The miRNAs-200 family islately considered as strong regulators of EMT, as they affect the balance between EMT and MET its reversed process [42]. This family consists of five members divided into two categories of 200a/200b/429 and 200c/ 141 [43]. miRNA-200c regulates EMT through suppressing ZEB1/2 (transcription suppressors of E-cadherin). This miRNAcan suppress Bmi-1, which is a polycomb protein responsible for the preservation of its essential characteristics, in healthy and cancerous stem cells. Recent studies confirm the role of miRNA-200c in the regulation of EMT [44].
The invasion and metastasis of tumor cells is a major cause of mortality in cancer patients. Fascin is another protein involved in EMT process. Fascins are globular proteins of approximately 55 kDa composed of four tandem fascin domains, each of which corresponds structurally to a β-trefoil fold. Fascin1, an actin-bundling protein, has been demonstrated to be critical for filopodia formation and thus is believed to be vital in movement including and cell migration. The formation of such structures is associated with increased risk of invasion and metastasis [45]. Immunohistochemistry (IHC), tissue microarray (TMA) study and qRT-PCR of various cancers confirms increased expression of Fascin-1,especially in metastatic lung and breast neoplasia [46]. miRNAs are important factors involved in the regulation of fascin expression rate. Different studies have revealed the role of 145, 133a, 133b and miRNA-146a in increased expression of fascin [47].

Conclusion
More research is needed to clarify the regulatory mechanisms of microRNAs and their role in the development of thyroid cancers. The study of target microRNA molecules along with their effects on signaling pathways and metastasis may cause better understanding of such mechanisms. Furthermore, microRNAs can pave the way for the development of novel treatment modalities for thyroid cancers; however, these options are associated with certain impediments and problems. For instance, microRNAs control the expression of certain target genes; therefore, change in any of such genes may target other genes rather than the main ones. On the other hand, several microRNAs may control a single gene by and thus, any change in the expression of even one of this microRNA may not be an effective treatment in this regard. Also, transfer of antisense oligonucleotides for decreased expression of microRNAs, which are involved in cancer, may affect non-targeted microRNAs, and therefore result in undesirable effects in Table 2 Some of the miRNAs involved in various kinds of thyroid cancers Anaplastic Carcinoma miR-302c,-205,-137,-187,-214,-155,-224,-222,-221 miR-30d,-125b,-26a,-30a,-5p [35,36] Medullary Carcinoma miR-323,-370,-129,-137,-10a,-124a,-224,-127,-9,-154 [35] the process of the disease. Considering the above mentioned facts, in order to benefit from microRNAs, increase their usefulness, and minimize the undesirable effects arising from interconnection of antisense oligonucleotides to the non-target microRNAs, further studies are needed.