text:
MicroRNA-195 Inhibits the Proliferation of Human Glioma Cells by Directly Targeting Cyclin D1 and Cyclin E1

Glioma proliferation is a multistep process during which a sequence of genetic and epigenetic alterations randomly occur to affect the genes controlling cell proliferation, cell death and genetic stability. microRNAs are emerging as important epigenetic modulators of multiple target genes, leading to abnormal cellular signaling involving cellular proliferation in cancers.In the present study, we found that expression of miR-195 was markedly downregulated in glioma cell lines and human primary glioma tissues, compared to normal human astrocytes and matched non-tumor associated tissues. Upregulation of miR-195 dramatically reduced the proliferation of glioma cells. Flow cytometry analysis showed that ectopic expression of miR-195 significantly decreased the percentage of S phase cells and increased the percentage of G1/G0 phase cells. Overexpression of miR-195 dramatically reduced the anchorage-independent growth ability of glioma cells. Furthermore, overexpression of miR-195 downregulated the levels of phosphorylated retinoblastoma (pRb) and proliferating cell nuclear antigen (PCNA) in glioma cells. Conversely, inhibition of miR-195 promoted cell proliferation, increased the percentage of S phase cells, reduced the percentage of G1/G0 phase cells, enhanced anchorage-independent growth ability, upregulated the phosphorylation of pRb and PCNA in glioma cells. Moreover, we show that miR-195 inhibited glioma cell proliferation by downregulating expression of cyclin D1 and cyclin E1, via directly targeting the 3′-untranslated regions (3′-UTR) of cyclin D1 and cyclin E1 mRNA. Taken together, our results suggest that miR-195 plays an important role to inhibit the proliferation of glioma cells, and present a novel mechanism for direct miRNA-mediated suppression of cyclin D1 and cyclin E1 in glioma.

Introduction
The cyclins and their catalytic partners, the cyclin dependent kinases (CDKs), are cell cycle regulators. Cyclins act in concert with their CDKs to drive cells from one phase of the cell cycle to the next [1]. The first functions of cyclin D1 and cyclin E1 to be identified were related to control of G1-S phase cell cycle progression [2]. Cyclin D1 and cyclin E1 are thought to promote progression to the G1 phase of the cell cycle, on the basis of their cyclic pattern of mRNA expression, with maximal expression levels detected near the G1/S boundary [3][4][5]. During the G1 phase, the cyclin D1/CDK4 complex is phosphorylated by CDKactivating kinase (CAK). In turn, activated CDK4 is targeted by cyclin D1 and can hyperphosphorylate the tumor suppressor protein retinoblastoma (pRb) [6][7]. Phosphorylation of pRb leads to dissociation of the E2 promoter-binding protein dimerization partners (E2F) from the pRb/E2F complex, and dissociated E2F induces transcription of cyclin E1, which is required for entry to the S phase of the cell cycle [7].
The functions of cyclin D1 and cyclin E1 link the cell cycle to proliferation, apoptosis, invasion, differentiation and angiogenesis [8][9][10][11][12]. Therefore, cyclin D1 and cyclin E1 are considered to be key oncogenes. In agreement with their roles as oncogenes, cyclin D1 and cyclin E1 are overexpressed in breast, liver, lung and brain cancers [13][14][15][16]. However, the mechanisms by which cyclin D1 and cyclin E1 are upregulated in cancer cells remain to be fully elucidated.
MicroRNAs (miRNA) are small, non-coding 21-23 nucleotide RNAs which regulate gene expression by binding to the 39unstranslated regions of their target mRNA molecules, to repress transcription or induce mRNA degradation [17][18]. miRNAs have been demonstrated to play important roles in a wide variety of oncogenic activities, such as proliferation, angiogenesis, apoptosis, invasion and metastasis [19][20][21][22]. While the molecular mechanisms of miRNA-mediated gene regulation are still under investigation, recent studies have suggested that miRNA expression signatures are diagnostically and/or prognostically useful in human cancers.
Glioma, arising from glial cells, remains one of the most aggressive primary central nervous system (CNS) tumors. In spite of significant improvements in neurosurgery, radiotherapy and chemotherapy, the median survival time of high-grade glioma patients has remained at 12-15 months over the past decade, and the cumulative 1-year survival rate remains lower than 30% [23][24][25][26][27][28]. The poor prognosis of gliomas is largely attributed to their rapid growth, invasive/migratory nature and high rate of recurrence [29][30][31]. Although both genetic and environmental factors are considered to be major causes, the miRNA-based pathogenic mechanisms for glioma remains incompletely understood. Therefore, idenfication of microRNAs, whose deregulation would lead to development and progression of gliomas, is the the key to develop prognostic markers and effective therapeutic strategies.
In the present study, we report that miR-195 was significantly downregulated in glioma cells and clinical glioma tissues, compared to normal human astrocytes (NHA) and non-tumor associated tissues. We demonstrated that miR-195 promotes glioma cell proliferation by directly targeting the 39-UTRs of cyclin D1 and cyclin E1, consequently reducing phosphorylation of pRb and downregulating the proliferative marker PCNA. Our results suggest that downregulation of miR-195 plays an important role in enhancing the proliferation of glioma cells.

Ethics Statement
For the use of clinical materials for research purposes, samples were obtained with prior written informed consents from the patients and approval from the Institutional Research Ethics Committees of Sun Yat-sen University and its Third Affiliated Hospital. Glioma cell lines A172, LN340, U118MG, LN464, SNB19, LN18, T98G, U251MG and LN235 were purchased from American Type Culture Collection (Manassas, VA).

Cell Culture
Primary normal human astrocytes (NHA) were purchased from the Sciencell Research Laboratories (Carlsbad, CA) and cultured under the conditions as instructed by the manufacturer. Glioma cell lines A172, LN340, U118MG, LN464, SNB19, LN18, T98G, U251MG and LN235 were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS). Cells were maintained in a humidified atmosphere at 37uC with 5% CO 2 .

RNA Extraction and Real-Time Quantitative PCR
Total miRNA from cultured cells and fresh surgical glioma tissues was extracted using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. cDNA was synthesized from 5 ng of total RNA using the TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA), and the expression levels of miR-195 were quantified using miRNA-specific TaqMan MiRNA Assay Kit (Applied Biosystems) on the Applied Biosystems 7500 Sequence Detection system. The expression of miRNA was defined based on the threshold cycle (Ct), and relative expression levels were calculated as 2 2[(Ct of miR2195) -(Ct of U6)] after normalization with reference to expression of U6 small nuclear RNA.

Anchorage-independent Growth Assay
Cells were trypsinized and 500 cells were resuspended in 2 ml complete medium plus 0.3% agar (Sigma, St Louis, MO). The agar-cell mixture was plated on top of a bottom layer consisting of 1% agar in complete medium. After 10 days, colony size was measured using an ocular micrometer and colonies larger than 0.1 mm in diameter were counted. The experiment was performed three times for each cell line.

Bromodeoxyuridine Labeling and Immunofluorescence
Cells grown on coverslips (Fisher, Pittsburgh, PA) were incubated with bromodeoxyuridine (BrdUrd) for 1 h and then stained with an anti-BrdUrd antibody (Upstate, Temecula, CA) according to the manufacturer's instructions. Gray level images were acquired using a laser scanning microscope (Axioskop 2 plus, Carl Zeiss Co. Ltd., Jena, Germany).

Luciferase Assays
Cells (3.5610 4 ) were seeded in triplicate in 24-well plates and allowed to settle for 24 h. 100 ng of pGL3-Cyclin D1-39UTR (wt/mut), pGL3-Cyclin E1-39UTR (wt/mut), or control-luciferase plasmid plus 1 ng pRL-TK renilla plasmid (Promega, Madison, WI) were transfected into cells using Lipofectamine 2000 (Invitrogen Co., Carlsbad, CA) according to the manufacturer's recommendations. Luciferase and renilla signals were measured 48 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI) according to the manufacturer's protocol. Three independent experiments were performed and the data are presented as the mean 6 SD.

Flow Cytometry Analysis
Cells were harvested by trypsinization, washed in ice-cold PBS, and fixed in 80% ice-cold ethanol in PBS. Before staining, cells were sedimented in a chilled centrifuge and resuspended in cold PBS. Bovine pancreatic RNase (Sigma-Aldrich) was added to a final concentration of 2 mg/ml, and cells were incubated at 37uC for 30 min, followed by incubation with 20 mg/ml of propidium iodide (Sigma-Aldrich) for 20 min at room temperature. Cell cycle profiles of 5610 4 cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences).

Xenografted Tumor Model
BALB/c-nu mice (4-5 weeks of age, 18-20 g) were purchased from the Center of Experimental Animal of Guangzhou University of Chinese Medicine, and were housed in barrier facilities on a 12 h light/dark cycle. The BALB/c nude mice were inoculated subcutaneously with LN18-Vector (5610 6 ) in the left dorsal flank and with LN18-miR-195 (5610 6 ) in the right dorsal flank per mouse. Tumors were examined weekly; length, width, and thickness measurements were obtained with calipers and tumor volumes were calculated. Tumor volume was calculated using the equation (L*W 2 )/2. On day 35, then animals were euthanized, tumors were excised and weighed.

Statistical Analysis
The two-tailed Student's t-test was used to evaluate the significance of the differences between two groups of data in all pertinent experiments; A P values ,0.05 was considered significant.

miR-195 is Downregulated in Glioma Cell Lines and Glioma Tissues
Real-time PCR analyses showed that expression of miR-195 was markedly lower in all eight analyzed glioma cell lines, including A172, LN340, U118MG, LN464, SNB19, LN18, T98G, U251MG and LN235, as compared with that in normal human astrocytes (NHA) ( Figure 1A). To determine whether the miR-195 downregulation in glioma cell lines is also clinical relevant, we further examined the miR-195 expression in eight paired glioma tissues and adjacent nontumor tissues from the same patients. As shown in Figure 1B, comparative analysis showed that the expression level of miR-195 was also reduced in all 8 examined tumor tissues as compared to that in paired adjacent nontumor tissues. Collectively, our results indicate that miR-195 is downregulated in gliomas.

miR-195 Overexpression Inhibits Proliferation of Glioma Cells
To investigate the biological role of miR-195 in glioma progression, we first examined the effect of overexpressing miR-195 in glioma cells and relative miR-195 expression were analyzed using realtime PCR (Supplemental Figure 1). Strikingly, MTT assay showed that miR-195 overexpressing LN18 and T98G glioma cells exhibited significantly lower growth rates (approximately 2-fold lower) than control cells at day 4 after plating ( Figure 2A). Furthermore, we found that ectopic expression of miR-195 dramatically reduced the anchorage-independent growth ability of LN18 and T98G glioma cells ( Figure 2B), as indicated by reduced colony numbers and colony sizes. These results suggest that miR-195 upregulation inhibits proliferation of glioma cells.
To investigate the anti-proliferative effect of overexpressing miR-195 in glioma cells, BrdUrd incorporation assay was performed. As shown in Figure 2C, overexpression of miR-195 significantly decreased the percentage of BrdUrd-incorporating cells (21.3% vs 36.5% for LN18 cells, and 18.7% vs 34.2% for T98G cells), indicating that upregulation of miR-195 in LN18 and T98G glioma cells inhibits the DNA synthesis. Consistently, flow cytometry analysis showed that enforced expression of miR-195 not only significantly decreased the percentage of cells in the S peak but also increased the percentage of cells in the G1/G0 peak ( Figure 2D), suggesting that miR-195 upregulation inhibits proliferation of glioma cells through induction of G1-S arrest.

Inhibition of miR-195 Promotes Proliferation of Glioma Cells
To further demonstrate the significance of the anti-proliferative function of miR-195 in glioma cells, the growth rate was examined in LN18 and T98G glioma cells transfected with miR-195 inhibitor. As shown in Figure 3A, inhibition of miR-195 dramatically increased the growth of LN18 and T98G glioma cells. Consistent with these results, the anchorage-independent growth ability of LN18 and T98G glioma cells transfected with the miR-195 inhibitor significantly increased, as indicated by increased colony number and size on soft agar ( Figure 3B). Together, these results suggest that inhibition of miR-195 plays an important role in the proliferation and tumorigenic phenotype of glioma cells. Furthermore, we found that the percentage of S phase glioma cells dramatically increased in the miR-195 inhibited-glioma cells as compared with that in the control cells ( Figure 3C). Moreover, flow cytometry assay displayed that the percentage of cells in the G1/G0 peak significantly decreased in response to miR-195 inhibition ( Figure 3D). Collectively, our results suggest that inhibition of miR-195 promotes proliferation and the G1/S cell cycle transition in glioma cells.

miR-195 Downregulates Cyclin D1 and Cyclin E1 by Directly Targeting their-39UTRs
Western blotting analysis revealed that the phosphorylated pRb (p-pRb) and proliferative marker PCNA were also decreased in the miR-195-transfected cells and increased in the miR-195-inhibited cells, further demonstrating that miR-195 plays an important role in the proliferation of glioma cells ( Figure 4B). Importantly, analysis using publicly available algorithms (TargetScan, Pictar, miRANDA) indicates that cyclin D1 and cyclin E1, key regulators of cell cycle, are the predicted targets of miR-195 ( Figure 4A). As predicted, upregulation of miR-195 decreased, but inhibition of miR-195 increased, the expression levels of cyclin D1 and cyclin E1 in LN18 and T98G glioma cells ( Figure 4B). However, dysregulation of miR-195 did not result in altered expression of another two cell cycle regulators, CDK2 and p21, which are not the predicted targets of miR-195, further demonstrating the specific effect of miR-195 on proliferation through targeting cyclin D1 and cyclin E1 ( Figure 4B). Importantly, As shown in Figure 4C, ectopically expressing miR-195 only decreased the expression of GFP, which containing the 39UTR of cyclin D1 or cyclin E1, but did not decreased the expression of GFP-c-tubulin, indicating that miR-195 specifically affected the 39UTR of cyclin D1 or cyclin E1 ( Figure 4C). Furthermore, we found that overexpression of miR-195 reduced, but inhibition of miR-195 increased, the luciferase activity of cyclin D1-39UTR or cyclin E1-39UTR in a consistent and dose-dependent manner ( Figure 4D). However, transfection of the miR-195-mut, containing mutations in the miR-195 seed region, did not decreased the luciferase activity of cyclin D1-39UTR or cyclin E1-39UTR ( Figure 4D). Moreover, point mutations in the tentative miR-195-binding seed region in cyclinD1 39-UTR and cyclinE1 39-UTR abrogated the aforementioned repressive effect of miR-195 (Supplemental Figure 2A and 2B). Taken together, our results demonstrate that cyclin D1 and cyclin E1 are bona fide targets of miR-195.
To further investigate the role of Cyclin D1 and Cyclin E1 repression in miR-195-induced glioma cell growth arrest, we first examined the effects of Cyclin D1 and Cyclin E1 re-introduction on glioma cell proliferation. As predicted, re-introduction of the Cyclin D1 or Cyclin E1 ORF (without 39UTR) in miR-195transfected cells abrogated, at least partially, the miR-195mediated glioma cell growth arrest, as analyzed by the BrdUrd incorporation assay. Importantly, co-expression of Cyclin D1 and Cyclin E1 indeed could further potentiate the proliferation of glioma cells, compared to that of Cyclin D1 or Cyclin E1 expressed alone (Supplemental Figure 2C). On the other hand, miR-195 inhibition-induced glioma cell growth promotion could be antagonized by individually silencing these two targets, and to further extent by co-silencing the two targets (Supplemental Figure 2D). Taken together, these results suggest that both Cyclin D1 and Cyclin E1 downregulations contribute to miR-195induced glioma cell growth arrest.

miR-195 Inhibits Glioma Proliferation in vivo
To further investigate the effect of miR-195 reduction on glioma proliferation, the LN18 glioma cell lines, established to stably overexpress miR-195, were implanted into the right dorsal flank and with LN18-Vector cells in the right dorsal flank per mouse. As expected, the modified cells displayed upregulated miR-195, reduced Cyclin D1 and Cyclin E1 expression and presented lower growth rate in vitro. (Figure 5A-C). Importantly, consistent with the observed effects of the miR-195 in vitro, miR-195-overexpressing LN18 glioma cells formed much smaller tumors, about 0.27 fold of the control, indicating a dramatic inhibition of glioma cell proliferation by miR-195 in vivo ( Figure 5D-F). Meanwhile, western blotting analysis confirmed that the expression levels of Cyclin D1 and Cyclin E1 in the tumors derived from LN18/miR-195 were significantly lower than those in the tumors derived from LN18/Vector (Supplemental Figure 3). Taken together, our results suggest that miR-195 inhibit glioma proliferation both in vitro and in vivo by repressing Cyclin D1 and Cyclin E1 expression.
Finally, we examined whether the low miR-195-induced Cyclin D1/Cyclin E1 upregulation identified by our study is clinically relevant. As shown in Figure 6, miR-195 levels in 10 freshly collected glioma samples inversely correlated with the expression of Cyclin D1 (r = 20.709, P = 0.022), and Cyclin E1 (r = 20.783, P = 0.002). Moreover, as shown in Supplemental Figure 4, the levels of Cyclin D1 and Cyclin E1 proteins were upregulated in glioma cell lines relative to normal astrocytes, and in tumors relative to adjacent normal tissue, which further confirm the notion that downregulation of miR-195 could lead to upregulation of Cyclin D1 and Cyclin E1 expression in glioma. Taken together, these results suggest that miR-195 can inhibit proliferation in gliomas, and that downregulation of miR-195 plays an important role in the progression of gliomas.

Discussion
The key finding of the current study is that miR-195 expression is markedly downregulated in glioma cells and clinical glioma tissues as compared to NHA and normal brain tissues. Ectopic expression of miR-195 could decreased the proliferation and anchorage-independent growth of glioma cells, while inhibition of miR-195 reversed these effects. Moreover, we demonstrated that upregulation of miR-195 in glioma cells led to the downregulation of phosphorylated pRb and proliferative marker PCNA through As members of the cell cycle regulator family, cyclin D1 and cyclin E1 have been recognized as oncogenes. It has been reported that the expression levels of cyclin D1 and cyclin E1 are related to the malignant degree of cancers, including glioma, breast cancer, bladder cancer, etc. [33][34][35][36][37][38]. Cyclin D1, cyclin E1 and pRb are known to orchestrate cell cycle progression, and functional interaction between cyclin D1, cyclin E1 and pRb plays an important role in cancer cell growth and tumorigenesis [39][40]. Cyclins form complexes with and function as regulatory subunits of the cyclin-dependent kinases (CDK), and cyclin/CDK complex activity is required for the G1/S cell cycle transition [39][40].
Cyclin/CDKs complexes phosphorylate and inactivate the tumor suppressor protein pRb, resulting in the transcriptional initiation of E2F-regulated genes, which are important for entering the S phase and for DNA replication [41][42][43]. Consequently, upregulation of cyclin D1 and cyclin E1 have been demonstrated to induce a rapid cell cycle transition and increase cell proliferation rate.
MicroRNAs, a class small regulatory RNA molecules that represse gene expression of their mRNA targets in a sequencespecific manner, have been identified as key regulators in a wide variety of oncogenic processes, such as cell proliferation, angiogenesis, cellular differentiation, invasion and metastasis, and can function as either tumor suppressors or oncogenes [26][27][28][29][30][31]. This study demonstrates that miR-195 was significantly downregulated in human glioma cell lines and glioma tissues, compared to the adjacent non-cancerous tissues. Furthermore, overexpression of miR-195 reduced glioma proliferation, tumorigenicity and cell cycle progression, whereas suppression of miR-195 enhanced proliferation, tumorigenicity and cell cycle progression. These results indicate that miR-195 may function as a tumor suppressor miRNA and downregulation of miR-195 may correlate with clinical progression in glioma. Consistent with these findings, miR-195 has been reported to be downregulated in a variety of different tumor types including hepatocellular carcinoma, gastric cancer and breast cancer [44][45][46]. Analysis of the GSE13030 dataset also revealed low expression of miR-195 in glioma tissues. Coincidently, Zhang QQ, et al. recently demonstrated that microRNA-195 could play a tumor-suppressor role in human glioblastoma cells by targeting E2F3 and CCND3 [47]. Combining Zhang's work and ours, both two groups demonstrated that miR-195 downregulation promotes glioma cell proliferation. Strikingly, we both highlighted the point that one microRNA has multiple targets to perform its biological function, as found that miR-195 exhibited proliferationinhibiting role by targeting Cyclin D1, Cyclin E1, E2F3 and CCND3.
Recently, accumulating evidence has indicated that expression of the cyclin family can be regulated by miRNAs. It was reported that miR-17, miR-20a and miR-106b coordinately regulate the expression of cyclin D1 by directly targeting its 39-UTR, to affect cell cycle progression in unrestricted somatic stem cells [48]. Zhang W, et al. found that miR-520b is able to inhibit the growth of hepatoma cells by targeting MEKK2 or cyclin D1 both in vitro and in vivo [49]. Additionally, Ding and colleagues reported that upregulation of miR-29c downregulated cyclin E and suppressed  its oncogenic activity in esophageal squamous cell carcinoma, without affecting other G1 phase-related proteins level, such as cyclin D1, cyclin D2 [50].
In this study, cyclin D1 and cyclin E1 were identified as theoretical targets of miR-195 using bioinformatic analysis. We were able to demonstrate that cyclin D1 and cyclin E1 are bona fide targets of miR-195 using different methods. Western blotting analysis confirmed that overexpression of miR-195 downregulated cyclin D1 and cyclin E1, and luciferase reporter assays demonstrated that miR-195 targeted cyclin D1 and cyclin E1 via the miR-195 binding sites in their 39-UTRs. Furthermore, ectopic overexpression of miR-195 did not affect other cell cycle regulators  (CDK2 and p21) which were not predicted to be targets of miR-195, further demonstrating that miR-195 affects proliferation by specifically targeting cyclin D1 and cyclin E1. Similar to our results in glioma, it was previously reported that miR-195 inhibited the hepatocellular carcinoma cells and hepatic stellate cells proliferation, respectively through targeting CyclinD1and CyclinE1 [51][52], indicating that the miR-195-downregulation-induced Cyclin D1/Cyclin E1 expression might be Universal among different types of tumors.
In summary, the current study provides the first evidence of an important link between miR-195 and proliferation in human glioma cells. Our findings suggest that miR-195 directly targets the 39-UTR of the oncogenes cyclin D1 and cyclin E1 to inhibit proliferation by inducing G1-S arrest. Understanding the precise role played by miR-195 in the progression of glioma will increase our knowledge of tumor biology and explore the potential of miR-195 as a diagnostic/prognostic marker and novel therapeutic target for glioma.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Role of Antilipopolysaccharide Antibodies in Serum Bactericidal Activity against Salmonella enterica Serovar Typhimurium in Healthy Adults and Children in the United States

ABSTRACT Recent observations from Africa have rekindled interest in the role of serum bactericidal antibodies in protecting against systemic infection with Salmonella enterica serovar Typhimurium. To determine whether the findings are applicable to other populations, we analyzed serum samples collected from healthy individuals in the United States. We found that all but 1 of the 49 adult samples tested had robust bactericidal activity against S. Typhimurium in a standard in vitro assay. The activity was dependent on complement and could be reproduced by immunoglobulin G (IgG) purified from the sera. The bactericidal activity was inhibited by competition with soluble lipopolysaccharide (LPS) from S. Typhimurium but not from Escherichia coli, consistent with recognition of a determinant in the O-antigen polysaccharide. Sera from healthy children aged 10 to 48 months also had bactericidal activity, although it was significantly less than in the adults, correlating with lower levels of LPS-specific IgM and IgG. The lone sample in our collection that lacked bactericidal activity was able to inhibit killing of S. Typhimurium by the other sera. The inhibition correlated with the presence of an LPS-specific IgM and was associated with decreased complement deposition on the bacterial surface. Our results indicate that healthy individuals can have circulating antibodies to LPS that either mediate or inhibit killing of S. Typhimurium. The findings contrast with the observations from Africa, which linked bactericidal activity to antibodies against an S. Typhimurium outer membrane protein and correlated the presence of inhibitory anti-LPS antibodies with human immunodeficiency virus infection.

G astroenteritis caused by nontyphoidal Salmonella (NTS) infection is a major public health problem in Africa, with an estimated disease burden of 2.5 million cases and 4,100 deaths per year (1)(2)(3). Moreover, invasive NTS disease, specifically, septicemia caused by Salmonella enterica serovar Typhimurium, is an important cause of infant morbidity and mortality in sub-Saharan Africa, with case fatality rates as high as 24% (1,2). Understanding the immunological mechanisms that protect against invasive disease caused by NTS is essential to developing effective vaccines against this potentially devastating infection. Although protection is usually thought to depend mainly on cell-mediated immunity (4), recent clinical data have rekindled interest in the role played by antibodies. Serum antibodies that are able to mediate in vitro, complement-dependent killing of various bacteria, including S. Typhimurium, have been studied for a long time, but evidence of their importance in protection against in vivo infection has been scanty (5). This issue has received renewed interest as a result of recent studies from Malawi. These studies demonstrated that most healthy individuals older than 16 months had serum IgM and IgG antibodies that killed S. Typhimurium in vitro in a complement-dependent fashion but that healthy children below this age lacked such bactericidal antibodies, observations that correlated with the declining occurrence of NTS bacteremia in patients above the age of 12 months (6). The anti-Salmonella antibodies also facilitated bacterial killing by promoting the oxidative burst activity of circulating phagocytes (7). Further evidence of the importance of serum bactericidal antibodies in protection against S. Typhimurium was provided by data showing that the effect of these antibodies was inhibited in human immunodeficiency virus (HIV)-infected African adults, a population that is unusually susceptible to NTS bacteremia (3,8). The inhibition was shown to be mediated by high levels of IgG antibodies to the O antigen of S.
Typhimurium lipopolysaccharide (LPS) present in the sera of the HIV-infected individuals, which were proposed to prevent the action of bactericidal anti-outer membrane protein antibodies present in HIV-uninfected adults (8,9).
Although there have been many studies carried out to characterize the molecular basis of the susceptibility and resistance of S. Typhimurium to killing by human serum, most have made use of pooled sera and have focused on the role of complement (10). One of the few analyses of individual sera, apart from the recent ones from Malawi, compared bactericidal activities against S. Typhimurium in healthy controls and patients with sickle cell anemia but did not examine the antigenic target of the bactericidal antibodies (11). Thus, it is not clear whether the features of the anti-Salmonella bactericidal activity delineated in Malawi-its age-dependent increase, its mediation by anti-outer membrane protein antibodies in non-HIV-infected adults, and its inhibition by high levels of anti-LPS antibodies in HIV-infected adults (6,8)-are peculiar to that country or whether they are applicable to other populations. It is of particular interest and importance to determine if the findings from Africa can be extended to a high-income country such as the United States, where the burden of NTS disease is considerably lower (about 1.4 million infections and 400 deaths per year) (12). Accordingly, we carried out a study to char-acterize serum bactericidal activity against S. Typhimurium in healthy individuals from the United States. Our results confirmed some of the observations from Africa, including the wide prevalence of the bactericidal activity and its increase with maturation from child to adult, but also revealed unexpected differences regarding the roles of anti-LPS antibodies in both mediating and inhibiting the killing of S. Typhimurium.

MATERIALS AND METHODS
Blood samples. Deidentified serum and plasma samples from the United States were provided by the Partners Biorepository under protocol number 2012-P-000484/1. The samples were collected from healthy adults (18 to 75 years of age) and children (6 months to 5 years of age) during routine medical checkups by their primary care providers at Massachusetts General Hospital. Aliquots of excess serum that otherwise would have been discarded were selected by the staff of the biorepository using International Statistical Classification of Diseases and Related Health Problems (ICD-9) codes V70.0 ("routine general medical examination at a health care facility") and V20.2 ("routine infant or child health check"). Based on the time required for identification and selection, the samples spent 12 to 16 h at 4°C before being stored at Ϫ80°C until use. The protocol was reviewed by the Human Research Committee of Massachusetts General Hospital and deemed not to meet the definition of human subject research.
Bacterial strains. The S. Typhimurium strains SL1344 (wild type, mouse and calf virulent), SL3201 (wild type, mouse virulent), F98 (wild type, chicken isolate), JS107 (wild-type derivative of strain 14028, a chicken isolate), JS93 (the isogenic oafA-deficient derivative of JS107), and the S. enterica serovar Dublin strain 2229 have all been described previously (13)(14)(15)(16)(17). SL1344, SL3201, and 2229 were originally obtained from Beth McCormick, University of Massachusetts Medical Center, Worcester, MA, and F98 was obtained from Edouard Galyov, Institute for Animal Health, Berkshire, United Kingdom. JS107 and JS93 were provided for this study by James Slauch, University of Illinois, Urbana, IL. In addition, a human isolate of S. Typhimurium DT104, strain ATCC BAA-189, was obtained from the American Type Culture Collection, Manassas, VA (18). Bacteria were grown overnight in Luria-Bertani (LB) broth at 37°C with vigorous agitation on a rotary shaker. The saturated cultures were back-diluted 1/100 into fresh LB broth and then grown to an optical density at 600 nm of 0.1 to 0.15. The bacteria were harvested by centrifugation, washed with phosphate-buffered saline (PBS), and resuspended in PBS at a density of approximately 2 ϫ 10 6 CFU per ml based on preliminary titering experiments.
Bactericidal assay. Killing of Salmonella by serum or plasma was assessed essentially as described by MacLennan et al. (6). In brief, 5 l of bacterial suspension prepared as described above (containing approximately 10 4 CFU) was incubated with 22.5 l of serum or plasma and 22.5 l of PBS at 37°C for 1 h. The number of bacteria surviving at the end of that period was determined by serial dilution of the reaction mix and plating on LB agar and are displayed as log 10 numbers of CFU in the figures. LPS competition was carried out as described previously (8) by preincubating sera or plasma for 30 to 60 min at room temperature with 100 g/ml of purified LPS from either S. Typhimurium or Escherichia coli (O111:B4) before they were tested for bactericidal activity. Both LPS preparations were obtained from List Biological Laboratories, Inc., Campbell, CA (catalog number 225 or 201, respectively). To test the role of antibody in the bactericidal activity, IgG was purified from 100-l aliquots of individual sera using the Melon Gel IgG purification kit (Thermo Scientific, Rockford, IL) according to the manufacturer's directions. Approximately 90% pure IgG was obtained as indicated by protein staining (not shown). The purified IgGs were used in the bactericidal assay at concentrations comparable to those of the parental sera along with a source of human complement (nonbactericidal serum depleted of anti-Salmonella antibodies as described below). To test for inhibitory activity, 22.5 l of bactericidal serum or plasma was mixed with an equal volume of the nonbac-tericidal sample and then incubated at 37°C for 1 h with 5 l of bacterial suspension. The number of surviving bacteria was determined as described above.
Hemolytic complement assay. We estimated hemolytic complement activity in our samples using a published microassay that has been shown to have good correlation with the traditional CH50 titration method (19). In brief, 50-l aliquots of serum or plasma were incubated with 150 l of antibody-coated sheep erythrocytes suspended at 5 ϫ 10 8 cells per ml in gelatin-veronal buffer (Complement Technology Inc., Tyler, TX) for 30 min at 37°C in a microtiter plate. In addition, 50 l of human serum complement (Sigma-Aldrich, St. Louis, MO; derived from pooled normal human plasma; CH50 of 41 units/ml) was assayed in parallel to provide a reference hemolytic activity. The negative control consisted of 50 l of gelatin-veronal buffer instead of serum or plasma, while 50 l of serum plus 150 l of buffer was used as the blank. At the end of 30 min, the plate was vortexed briefly and the absorbance at 700 nm (A 700 ) read as an indicator of the number of nonhemolyzed erythrocytes left in the reaction. The difference in A 700 between the negative control and the test or reference sample provides a measure of hemolytic activity. The hemolytic activity of the individual serum or plasma samples was calculated as a percentage of the activity of the reference sample as follows: A 700 of negative control -A 700 of test sample/A 700 of negative control -A 700 of reference sample ϫ 100.
Absorption of anti-Salmonella antibodies from serum. One and one-half milliliter of an overnight saturated culture of either SL1344 or 2229 was spun down, and the bacterial pellet was washed with PBS and resuspended in 0.5 ml of the serum sample. The samples were incubated for 1 h at 4°C with gentle rotation. After the bacteria were spun down, the supernatant serum was filter sterilized by passage through a 0.22-m filter and stored at Ϫ80°C until use.
Enzyme-linked immunosorbent assay (ELISA) for serum antibodies. To detect S. Typhimurium LPS-specific antibodies, we used procedures that we have described in detail earlier (20). Immulon II microtiter plates were coated with S. Typhimurium LPS diluted in PBS at a concentration of 10 g/ml, washed, blocked, and then incubated overnight at 4°C with triplicate aliquots of sera diluted 500-fold in PBS. The plates were developed with horseradish peroxidase-conjugated secondary antibodies specific for human IgM or human IgG (BD Biosciences, San Jose, CA), followed by an o-phenylenediamine substrate. The intensity of color change was measured at 490 nm in a plate reader.
Total serum IgM, IgG, and IgA were measured using ELISA kits from eBioscience (San Diego, CA) and by following the manufacturer's guidelines.
Flow cytometry of bacteria. To examine IgM binding to bacteria, 5 l of PBS containing 10 7 CFU of mid-log-phase SL1344 was incubated for 30 min on ice with 12.5 l of undiluted serum and 12.5 l of PBS. After the bacteria were washed, bound IgM was detected by a biotinylated antihuman IgM antibody followed by Cy5-conjugated streptavidin (BD Biosciences, San Jose, CA). Analysis of fluorescence was carried out on an Accuri C6 flow cytometer. Examination of complement deposition on the bacterial surface was carried out similarly except that the initial incubation with serum was at 37°C for 15 min. The presence of activated complement on the bacteria was detected using a mouse antibody specific for the activated terminal complement components C5b to 9 (aE11; Abcam, Cambridge, MA) followed by a fluorescein-conjugated anti-mouse IgG antibody and flow cytometric analysis.
Statistical analysis. Results from groups of serum or plasma samples are displayed as scattergrams, with each symbol representing an individual sample and a horizontal line the median of the group. Intergroup differences were analyzed by the Mann-Whitney U test using Prism v3.0 (GraphPad Software, Inc., La Jolla, CA). A P value of Ͻ0.05 was considered to be significant. Specific P values corresponding to the asterisks in the figures are provided in the figure legends.

RESULTS
Deidentified serum or plasma samples from healthy adults (18 to 75 years old), obtained during routine health maintenance visits at a Boston hospital, were used to assess bactericidal activity against the S. Typhimurium strain SL1344 as described in Materials and Methods. Although SL1344 has been reported to be resistant to the bactericidal activity of human serum (21), the results from the first 32 of our samples clearly demonstrated that incubation of the bacteria with the serum or plasma for 1 h resulted in a statistically significant 1-to 2-log drop in the number of CFU (relative to the starting inoculum of 10 4 CFU), whereas incubation with PBS alone or human complement alone (serum depleted of anti-Salmonella antibodies by preabsorption) did not lead to a decrease in bacterial number (Fig. 1A and data not shown). Similar bactericidal activity was obtained with an additional 16 out of 17 samples examined and was abolished when the sample was heated to 56°C for 30 min (data not shown). To confirm that the handling and storage of our samples had not compromised their complement activity, we assayed their ability to lyse antibody-coated sheep erythrocytes. Most of the samples tested had hemolytic complement activity that was equal to or greater than the activity of the reference complement used in the assay (pooled human plasma), while the remainder had activity that ranged from 84 to 99% of the reference activity (Fig. 2). This distribution of activities is very similar to results obtained by others using randomly selected human serum samples (12). Taken together, our findings indicate that complement is functionally intact in our samples. Sera from 10 healthy Boston children aged 10 to 48 months were also able to kill S. Typhimurium, but the reduction in bacterial numbers was significantly less than that achieved by the adult sera (Fig. 1A). The reduced bactericidal activity of the pediatric samples was not related to a deficiency of complement, since the hemolytic complement activities of these samples did not differ significantly from those of the adults (Fig. 2) (P ϭ 0.39). We also tested the sera against 3 additional strains of S. Typhimurium, JS107, SL3201, and F98, that have origins and virulence characteristics distinct from SL1344's (13)(14)(15)(16)(17) and obtained similar results (data for JS107 are shown in Fig. 5; SL3201 and F98 results are not shown). Moreover, when we tested 30 of the adult sera against a human isolate of S. Typhimurium (BAA-189 [18]), the bacteria were killed as effectively as were the laboratory strains by all of the samples (Fig. 1B). Adult sera and SL1344 were used in most of the experiments described here except where specifically indicated. To evaluate the role of antibody in the bactericidal activity, IgG was purified from 4 of the sera and tested in the presence of a source of complement (nonbactericidal human serum depleted of anti-Salmonella antibodies by preabsorption with SL1344 as described in Materials and Methods). All 4 purified IgGs were able to kill Salmonella in the presence of this source of complement ( Fig.  3) but not when complement was inactivated by heating it to 56°C (data not shown).
We considered the possibility that the antigenic target of the bactericidal activity was LPS since both IgM and IgG antibodies to S. Typhimurium LPS were readily detectable in the adult sera and were appreciably reduced in the pediatric samples (Fig. 4A). Consistent with this possibility, preincubation of the adult sera with an excess of purified S. Typhimurium LPS completely inhibited the bactericidal activity (Fig. 4B). LPS preincubation also inhibited the bactericidal activity of the pediatric samples against SL1344, as well as of the adult sera against the human isolate BAA-189 (data not shown). Interestingly, LPS from E. coli did not inhibit serummediated killing of Salmonella (Fig. 4B), suggesting that the bactericidal antibodies are specific for an epitope in the outer polysaccharide (O antigen), the region that structurally distinguishes different types of Gram-negative LPSs (22).  S. Typhimurium belongs to serogroup B and has an O antigen made up of repeats of the tetrasaccharide abequose-mannoserhamnose-galactose (23). In some strains of S. Typhimurium, including SL1344, the 2-hydroxyl group of the abequose moiety in the O antigen is acetylated, causing conformational changes that can affect antibody binding (16,24). To determine if the serum bactericidal activity might be affected by such acetylation, we tested adult sera against the wild-type S. Typhimurium strain JS107 and its isogenic mutant derivative JS93, which is deficient in the oafA locus required for abequose acetylation (16). All the tested sera were effective against JS107, whereas 2 of them had clearly reduced or no activity against JS93 (Fig. 5). The results indicate that abequose acetylation affected the bactericidal activity of only a small proportion of the sera.
One sample in our collection (designated NB for "nonbactericidal") completely lacked bactericidal activity against SL1344 (Fig.  6A) despite having S. Typhimurium LPS-specific IgM and IgG levels comparable to those in the bactericidal sera (Fig. 6B). This sample also had hemolytic complement activity that was similar to that of the bactericidal samples, as well as levels of complement that were sufficient to restore the killing of S. Typhimurium by heat-inactivated bactericidal sera ( Fig. 2 and data not shown). Interestingly, NB was able to inhibit the activity of the bactericidal samples when equal volumes of the 2 types were mixed together (Fig. 6A). The inhibitory effect was abrogated when NB was depleted of anti-Salmonella antibodies by preabsorption with the S. Typhimurium strain SL1344 but not with the S. Dublin strain 2229 (Fig. 6A). We tested if the preabsorption with the 2 strains might have differential effects on the presence of antibodies to S. Typhimurium LPS, since one of the differences between S. Typhimurium and S. Dublin is in the compositions of their LPS O antigens. As mentioned above, S. Typhimurium has an O antigen made up of repeats of the tetrasaccharide abequose-mannoserhamnose-galactose (Kaufmann-White O-antigen designation 1,4,5,12), whereas S. Dublin (serogroup D) has an O antigen with repeats of the tetrasaccharide tyvelose-mannose-rhamnose-galactose (Kaufmann-White O-antigen designation 1,9,12) (23). Indeed, we found by ELISA that preabsorption of NB with SL1344 produced a dramatic decrease in the levels of S. Typhimurium LPS-specific IgM but that preincubation with 2229 had little or no effect in this regard (Fig. 7A). Consistent with this result, we found corresponding differences in the amounts of IgM bound to the surface of SL1344 by flow cytometry (Fig. 7B). Equivalent reductions in S. Typhimurium LPS-specific IgG levels were observed after preabsorption with either of the 2 strains (Fig. 7A). Taken together, these findings suggest that it is the S. Typhimurium LPSspecific IgM that mediates the inhibitory effect of NB.
Finally, to elucidate the mechanism of the inhibitory effect, we   used flow cytometry to examine the amount of complement deposited on the bacterial surface by bactericidal sera in the presence or absence of NB. The results for one of the bactericidal sera is shown in Fig. 8. They demonstrate clearly that, compared to PBS, the bactericidal serum alone induced the deposition of an appreciable amount of activated complement on the bacterial surface. This effect was almost completely lost when the serum was heat inactivated. NB alone did not induce deposition of complement and also inhibited complement deposition by the bactericidal serum. Similar results were obtained with 3 other bactericidal sera (data not shown). These findings suggest that the inhibitory effect of NB on bactericidal activity is the result of reduced deposition of activated complement on the bacterial surface.

DISCUSSION
Like the findings from Africa (6,8), our results indicate the widespread occurrence of complement-dependent, antibody-mediated bactericidal activity against S. Typhimurium in sera from healthy adults in the Boston area, as well as an age-dependent increase in this activity during maturation from child to adult. However, in contrast to the results from Africa, the bactericidal antibodies in our study appear to be directed against S. Typhimurium LPS rather than outer membrane proteins. Our data also indicate that the bactericidal antibodies discriminate between LPS from S. Typhimurium and E. coli, suggesting that the antibodies recognize a determinant in the outer polysaccharide of LPS (22,23). The bactericidal activities of 2 of our samples appeared to be affected by acetylation of the abequose residue in the LPS outer polysaccharide, suggesting fine differences in antigen specificity within our collection. IgG purified from our samples was sufficient to recapitulate the bactericidal activity in the presence of complement, but it is likely that IgM also contributes to the activity, since LPS-specific antibodies of both isotypes were detected.
Our results, as well as those from Malawi, suggest that an agedependent increase in exposure to a ubiquitous environmental antigen induces serum antibodies that react with S. Typhimurium. In developing countries such as Malawi, where the population may have limited access to protected food and water sources, such exposure may well correspond to subclinical or clinical infection with S. Typhimurium itself. Whether asymptomatic or symptomatic infection with S. Typhimurium accounts for the al-  adult population is open to question, but it is a possibility that needs to be considered. Certainly, there are multiple animal reservoirs of NTS, including pet reptiles and amphibians, that may act as sources of human infection, particularly in children (25,26), and both epidemic and sporadic gastroenteritis caused by S. Typhimurium is not uncommon in the United States (1). Asymptomatic carriage of Salmonella is also a potential outcome of infection, as has been documented in Sweden, although the exact prevalence of subclinical infection is not known (27,28). An alternative possibility is that the bactericidal antibodies detected in our study develop as a result of exposure to a cross-reactive LPS. LPS is present widely in the external environment and is also a component of the commensal microflora present within the gas-trointestinal tract (29,30). Exposure and immunological sensitization to LPS is thus likely to be a characteristic of most populations. Indeed, serum IgM, IgA, and IgG antibodies to surface antigens of a number of different gut commensal bacteria have been observed in healthy individuals (31). Taking these facts into consideration, it seems plausible that the bactericidal antibodies detected in our study represent a response to some source of LPS present in either the external or the internal (commensal) environment and that these antibodies fortuitously cross-react with S. Typhimurium LPS. Evidence in support of this idea is provided by observations showing that human volunteers fed a commensal E. coli strain developed cross-reacting serum bactericidal anticapsular antibodies against Haemophilus influenzae type b (32,33).
There may be a number of explanations, not mutually exclu- sive, for the intriguing differences between our findings and the data from Africa with respect to the antigen targeted by the bactericidal antibodies (LPS in the case of the United States and outer membrane proteins in Malawi). First, the S. Typhimurium strains circulating in Africa are genetically different from the ones circulating in the United States (34,35). Second, as discussed above, it is possible that the bactericidal antibodies in the United States develop as a result of sensitization by environmental LPS but that they may develop as a result of subclinical infection with S. Typhimurium in Malawi. These circumstances may account for the difference in the antigen specificities of the bactericidal antibodies detected in the 2 locations. It should also be noted that different S. Typhimurium strains were used to test bactericidal activity in the 2 sets of studies. Microheterogeneities in the polysaccharide compositions of LPSs have been described even within the same serotype (36), and it is possible that such variations affect antibody binding and function, especially since the exact epitope to which antibody binds may alter bactericidal activity (5,8). In this context, it may be relevant that recent experiments have shown that antibody-mediated killing of the S. Typhimurium isolate used to assess bactericidal activity in the Malawi study required unusually large amounts of complement (37). This characteristic may also contribute to the differences from our results. The importance of serum bactericidal antibodies in protection against NTS sepsis in humans is supported by circumstantial evidence. Studies in the mouse model of infection indicate that both humoral and cell-mediated immunity contribute to defense against S. Typhimurium, although the role of T cells is generally considered to be more crucial (4). The strong correlation between the presence of serum bactericidal activity and resistance to NTS sepsis observed in the studies from Malawi certainly strengthens the idea that antibodies have an important role to play in protection against invasive S. Typhimurium infection (6)(7)(8). The agedependent increase in bactericidal activity observed in our study is consistent with the results from Malawi and provides further support for this idea.
Only a single sample in our collection, NB, lacked bactericidal activity. This sample was able to abolish the killing of S. Typhimurium by the bactericidal sera, similar to the inhibitory effect observed with sera from HIV-infected African adults (8). However, our collection, including NB, was derived from healthy individuals without known acute or chronic illnesses. Moreover, NB did not have the hypergammaglobulinemia seen in the inhibitory sera from HIV-infected African adults (8). The IgM, IgG, and IgA concentrations of NB were 1.83 mg/ml, 3.04 mg/ml, and 1.87 mg/ml, respectively, which were comparable to the values for 7 randomly selected bactericidal samples from our collection (IgM, 2.11 Ϯ 1.05 mg/ml; IgG, 5.28 Ϯ 2.61 mg/ml; IgA, 2.19 Ϯ 1.60 mg/ml) and were not above the normal range for adults (38). These findings suggest that the presence of the inhibitory activity may not be unique to the HIV-infected population and may simply represent a normal, albeit relatively rare, variation. Based on preabsorption experiments and the analysis of complement deposition on the bacterial surface, the inhibitory effect of our sample appears to be mediated by LPS-specific IgM antibodies that are unable to induce complement activation. These antibodies are probably directed against an LPS O-antigen epitope that is expressed specifically by S. Typhimurium, since they are removed by preabsorption with S. Typhimurium (Kaufmann-White O-antigen designation 1,4,5,12) but not S. Dublin (Kaufmann-White O-antigen designa-tion 1,9,12) (23). However, we cannot definitively exclude the involvement of antibodies against S. Typhimurium-specific surface antigens other than LPS. Presumably, the binding of these inhibitory antibodies blocks the binding of the complement-activating bactericidal antibodies to the same region (if not the same epitope or overlapping epitopes). This presumptive mechanism of inhibition appears to be different from that operating in the sera of HIV-infected African adults, since complement activation and deposition on the Salmonella surface occurred normally in that population (8).
Since IgM is normally a robust activator of complement, it is not clear why the LPS-specific IgM in our nonbactericidal sample fails to activate complement. Glycosylation of the IgM heavychain constant region is known to affect complement activation (39)(40)(41), so it is possible that peculiarities of the oligosaccharides attached to the LPS-specific IgM in the nonbactericidal sample may be responsible for its failure to activate complement. Such variations, as well as the other differences in bactericidal activity revealed by our study and the studies from Africa, may contribute to interindividual and interpopulation differences in susceptibility to S. Typhimurium. Clarifying these issues will increase our understanding of the pathogenesis of NTS sepsis and facilitate the development of strategies to prevent this disease.


text:
Toward an Understanding of the Gene-Specific and Global Logic of Inducible Gene Transcription

Virtually all living organisms have evolved mechanisms to adapt to their environment by sensing environmental stresses and inducing the transcription of appropriate sets of response genes in acoordinated fashion. In the vertebrate immune system, the highlyselectiveresponsetoanenvironmentalstimulus,oftenaninvadingmicroorganism,playsanespeciallyimportantroleinregulatingtheactivitiesof,andinteractionsamong,themanycelltypesinvolvedininnateandadaptiveimmunity.Itisnowwidelyappreciatedthattheselectiveresponsetoastimulusrequirestheconcertedactionofsignaltransductionpathways, transcription factors, and chromatin structure. Many proteins and pathways that help to regulate a response have been characterized. However, our understanding of the gene-specific and global logic through which a highly selective response is elicited has only recently begun to emerge.

Humans and all other living organisms have acquired the ability to respond to a broad range of environmental stresses. The responses are initiated by the sensing or recognition of the stress, usually leading to the activation of signal transduction pathways that culminate in the altered expression of genes encoding proteins that help the organism to tolerate the stress. Changes in transcription are of central importance for the gene expression changes.
The responses to environmental stresses possess special significance in the context of the vertebrate innate and adaptive immune systems that comprise a large number of cell types that coordinate their response to microbial pathogens and other environmental stresses through the induction of innate effector pathways and/or a wide range of highly specific B-and T-cell responses (Akira et al. 2006). Because of the complexity of the immune system and the diversity of responses that can be elicited, exquisite specificity is needed. As one example, the specific cytokines expressed by cells of the innate immune system in response to microbial infection help to regulate the differentiation of naive T cells into one of the several T-helper-cell types, with different T helper subsets essential for the control of different microbial pathogens.
The exquisite specificity of transcriptional responses is readily observed by measuring mRNA levels following exposure of cells to different stimuli. For example, in response to microbial signaling through most Toll-like receptors (TLRs), macrophages and dendritic cells potently induce transcription of the Il12b gene that encodes the p40 subunit of the IL-12 and IL-23 cytokines that play critical roles in Th1 and Th17 differentiation, respectively (Trinchieri 2003). In contrast, in response to tumor necrosis factor (TNF) stimulation through the TNF recep-tors, the Il12b gene remains silent, despite the common induction of hundreds of other genes by TLRs and the TNF receptors. A major goal is to understand how this exquisite selectivity is achieved, not only at the level of an individual gene such as Il12b, but also at a broader systems level. That is, what is the gene-specific and global logic through which highly specific responses to diverse stimuli are regulated?
In the simplest scenario, each stimulus induces a different set of signal transduction pathways, each of which activates one or more transcription factors that bind the control regions of the potential target genes; transcriptional induction then occurs at genes bound by an appropriate combination of induced transcription factors ). Although the signal transduction pathways induced by a stimulus play a central role in dictating the transcriptional response, and although combinatorial mechanisms have long been known to be essential for the diverse gene regulation patterns needed in complex organisms (Britten and Davidson 1969;Georgiev 1969;Gierer 1973), it is now widely appreciated that other factors, such as the developmental history of the cells and chromatin features of candidate genes, collaborate with signaling pathways and inducible transcription factors to regulate the response. This collaboration has been studied extensively and has been the topic of several recent reviews Smale 2010;Fowler et al. 2011;Natoli et al. 2011;Nechaev and Adelman 2011;Smale et al. 2014). This article focuses on the experimental strategies used in our laboratory to pursue a better understanding of the logic through which transcription factors and chromatin structure collaborate with signaling pathways to induce a selective response to a microbial stimulus.

BASIC STUDIES OF Il12B TRANSCRIPTIONAL INDUCTION
Our studies of stimulus-induced transcription began with an emphasis on the molecular mechanisms regulating induction of the Il12b gene in mouse macrophages in its native chromosomal environment. These studies began soon after the critical role of chromatin in regulated transcription became widely appreciated, due to compelling evidence that histone acetyltransferases and ATP-dependent nucleosome remodeling complexes help to regulate transcription (Côté et al. 1994;Imbalzano et al. 1994;Kwon et al. 1994;Brownell et al. 1996). Until recently, a preferred approach toward understanding gene regulation in a specific biological setting was to examine the regulation of a specific model gene, with the expectation that regulatory principles emerging from studies of the model gene would be broadly relevant. The Il12b gene was chosen as the model for our studies because of its potent induction in mouse macrophages in response to microbial stimuli and because the critical role of IL-12 (and later IL-23) in bridging innate and adaptive immune responses had recently been described (Trinchieri 2003).
Although our objective was to understand Il12b regulation in the context of chromatin, it was first important to identify DNA elements and transcription factors that contribute to Il12b regulation. In the 1980s and 1990s, the most common strategy toward this goal was to develop a functional assay in which the activity of the promoter could be examined; if proper regulation of the wildtype promoter was observed, mutations could be introduced systematically into promoter sequences, allowing the identification of DNA elements that are important for promoter activity ). Electrophoretic mobility shift assays, DNase I footprinting, and other assays could then be used to identify transcription factors that bind the important elements. Using a functional assay in which macrophage cell lines were transfected with plasmids containing the Il12b promoter fused to a luciferase or chloramphenicol acetyltransferase gene, followed by lipopolysaccharide (LPS) stimulation of the transfected cells, putative binding sites for NF-kB, C/EBP, AP-1, and NFAT were identified (Plevy et al. 1997;Zhu et al. 2001Zhu et al. , 2003. Subsequent studies led to the identification of an Il12b enhancer located 10 kb upstream of the transcription start site that also appears to contribute to Il12b induction (Zhou et al. 2007).
Importantly, none of the transcription-factor-binding sites identified in the Il12b promoter or enhancer provided an obvious explanation for the selectivity of induction (e.g., its induction by TLRs but not by the TNF receptor). That is, all of the factors identified were known to be induced by a broad range of stimuli. It is important to emphasize, however, the inherent challenges in evaluating data obtained in conventional gene regulation analyses. For example, all of the transcription factors implicated in Il12b regulation are members of multiprotein families, with different family members often recognizing similar sequences but potentially contributing different functions. Redundancy among family members and other technical limitations make it difficult to identify the family member that contributes to regulation and rigorously dissect regulatory mechanisms. Furthermore, transfection assays often do not accurately reflect the regulation of an endogenous gene; DNA elements and transcription factors that are critical for regulation of the endogenous gene may play no role in the regulation of a transfected promoter-reporter plasmid; one reason is that transfected plasmids do not assemble into a chromatin structure that resembles the chromatin structure found at the endogenous gene in its native chromosomal location (Smith and Hager 1997;Carey et al. 2009).

NUCLEOSOME REMODELING AT Il12B
REGULATORY REGIONS To determine whether chromatin contributes to Il12b regulation, we first used micrococcal nuclease to map the locations of nucleosomes in the vicinity of the Il12b promoter (Weinmann et al. 1999). The data suggested that, in unstimulated macrophages, a nucleosome encompasses all of the key promoter elements that had been identified. A restriction enzyme accessibility assay was then used to determine whether chromatin structure was altered in response to LPS stimulation (Weinmann et al. 1999). In this assay, nuclei from unstimulated or LPS-stimulated macrophages are incubated with a restriction endonuclease that cleaves in the genomic region of interest. Because the genome remains assembled into chromatin in the nuclei, most genomic regions will be largely resistant to restriction enzyme cleavage. However, the cleavage efficiency is generally higher at regions where nucleosomes have been removed or physically remodeled.
Using the restriction enzyme accessibility assay, the Il12b promoter region was found to be largely resistant to cleavage in unstimulated cells, but cleavage efficiency was substantially higher in nuclei from LPS-stimulated cells ( Fig. 1) (Weinmann et al. 1999;Ramirez-Carrozzi et al. 2006). Similar results were later obtained with the Il12b enhancer ( Fig. 1) (Zhou et al. 2007). These results suggested that nucleosome-remodeling events at the Il12b promoter and enhancer accompany transcriptional activation. These findings led to speculation that the regulation of nucleosome remodeling may be of central importance for the selective regulation of transcriptional induction. For example, TLR4 and TNF receptors both induce NF-kB, AP-1, and other common inducible transcription factors. However, according to our hypothesis, most of these factors cannot gain access to their binding sites in the promoter and enhancer unless a prior event promotes nucleosome remodeling. Consistent with this hypothesis, nucleosome remodeling at the Il12b promoter and enhancer was not observed in TNF-stimulated cells.
On the basis of the above results and hypothesis, a critical goal is to identify the inducible factor or events that regulate inducible remodeling at the Il12b promoter and enhancer. Nucleosome remodeling at the promoter and enhancer, like Il12b transcription, is known to be strongly inhibited by the protein synthesis inhibitor, cy-cloheximide (Weinmann et al. 1999;Zhou et al. 2007). This result suggests that one or more proteins synthesized during the primary response to TLR4 signaling plays a critical role in the inducible remodeling. Unfortunately, the identity of this key inducible factor remains unknown.

NUCLEOSOME REMODELING BY SWI/SNF COMPLEXES
Although the factor responsible for the inducibility of nucleosome remodeling at the Il12b promoter and enhancer remains elusive, the enzyme that catalyzes nucleosome remodeling appears to have been identified. Importantly, further analysis of this enzyme's role in TLR4-induced transcription provided a starting point toward elucidation of the global logic through which transcriptional responses to microbial and environmental stimuli are regulated.
As described above, the increased access to restriction endonuclease cleavage upon macrophage activation was the most pronounced chromatin change observed at the Il12b promoter and enhancer. (Increases in histone modifications have also been observed [Weinmann et al. 1999;Ramirez-Carrozzi et al. 2006], but the magnitudes of these changes are relatively small.) The protein complexes that have been implicated most frequently in physical changes to nuclease cleavage are ATP-dependent nucleosome-remodeling complexes that use the energy of ATP hydrolysis to disrupt interactions between the histone octamer and DNA, resulting in nucleosome eviction, sliding, or simply increased accessibility of the nucleosomal DNA (Clapier and Cairns 2009).
To determine whether an ATP-dependent nucleosome remodeling complex might be responsible for the increased accessibility of the Il12b promoter and enhancer, different complexes were depleted in a macrophage cell line by retroviral delivery of small hairpin RNAs (shRNAs) directed against the mRNAs encoding the catalytic subunits of the complexes (Ramirez-Carrozzi et al. 2006). Transcriptional induction of the Il12b gene was greatly reduced only upon expression of an shRNA target- Cleavage sites for the restriction enzyme SpeI are shown. Restriction enzyme accessibility quantified by genomic Southern blot is shown at the bottom for both the enhancer and promoter in unstimulated and LPS-stimulated bone-marrow-derived macrophages. The band labeled "Uncut" corresponds to a restriction fragment generated by complete cleavage of the purified genomic DNA with a restriction enzyme whose sites flank the critical SpeI cleavage site at the enhancer or promoter. The fraction of this fragment cleaved by SpeI in the intact nuclei before purification of the genomic DNA is labeled as "Cut." The absence of the "Cut" band when the cells were stimulated in the presence of cycloheximide (CHX) shows the protein synthesis dependence of the inducible nucleosome remodeling. (Adapted from Ramirez-Carrozzi et al. 2006.) ing the two catalytic subunits of the SWI/SNF remodeling complex, Brg1 and Brm. These two proteins act with partial redundancy through their association with a similar set of Brg1-associated factors (Clapier and Cairns 2009). Simultaneous depletion of both catalytic subunits was achieved through the use of an shRNA that targets a homologous region of their mRNAs.
In addition to the reduced Il12b transcription observed following knockdown of Brg1 and Brm, LPS-induced restriction enzyme accessibility at both the Il12b promoter and enhancer was reduced. Inducible Brg1 recruitment to the Il12b promoter and enhancer, which was observed by chromatin immunoprecipitation (ChIP), was also reduced following Brg1 knockdown (Ramirez-Carrozzi et al. 2006). Together, these results strongly suggest that Il12b induction requires nucleosome remodeling at the promoter and enhancers by the SWI/SNF-remodeling complexes following their specific recruitment by a recruitment mechanism that remains unknown. Interesting, a study by Chi et al. has suggested that, following its recruitment, SWI/SNF activity requires an additional calmodulin-dependent signaling event (Lai et al. 2009).

VARIABLE ROLE OF SWI/SNF COMPLEXES AT INDUCIBLE GENES
The evidence that inducible nucleosome remodeling is needed for transcriptional induction of Il12b was initially difficult to reconcile with results emerging from the study of other inducible genes. Specifically, classic studies of heat-shock genes in Drosophila had revealed that their promoters are assembled into constitutively accessible chromatin (Tsukiyama et al. 1994;Shopland et al. 1995;Nechaev and Adelman 2011). Not only are their promoters accessible, but RNA polymerase II appears to have initiated transcription, although it remains paused slightly downstream from the transcription start site. Heat-shock induction then promotes elongation of the paused polymerase, with its release serving as a major regulator of transcriptional induction (Nechaev and Adelman 2011). Initiation of transcription and pausing before cell stimulation was also reported at the inducible human immunodeficiency virus long-terminal repeat promoter (Verdin et al. 1993). In contrast, similar to the inducible nucleosome remodeling observed at the Il12b promoter, inducible nucleosome remodeling at the promoter appeared to be important for induction of the yeast PHO5 gene, the mouse mammary tumor virus long-terminal repeat, and the human IFN-b gene (Almer et al. 1986;Fryer and Archer 1998;Agalioti et al. 2000).
These findings raised the question of why different chromatin properties are present at different inducible genes before their induction. One possibility is that the variability simply reflects the evolution of diverse mechanisms to regulate inducible transcription, with no biological significance to the variability. A more interesting possibility is that the variability is an important feature of the global logic through which selective responses to diverse stimuli are elicited. A closer examination of the impact of Brg1/Brm knockdown in macrophages provided an opportunity to explore this important question.
Specifically, an examination of the mRNA levels for several LPS-induced genes in mouse macrophages revealed that these genes, upon their induction in a single cell type by a single stimulus, could be grouped into different categories on the basis of their sensitivity to Brg1/ Brm knockdown (Ramirez-Carrozzi et al. 2006. Many genes, including a substantial fraction of primary response genes (genes induced in the presence of cycloheximide), were induced in a Brg1/Brm-independent manner. In contrast, the remaining primary response genes and most secondary response genes were induced at greatly reduced levels following Brg1/Brm knockdown.

MECHANISTIC AND BIOLOGICAL INSIGHTS INTO VARIABLE NUCLEOSOME REMODELING REQUIREMENTS
To examine the significance of the variable SWI/SNF dependence of inducible genes in LPS-stimulated macrophages, the impact of Brg1/Brm knockdown on the expression of 67 inducible genes was examined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) (Fig. 2) (Ramirez-Carrozzi et al. 2009). An evaluation of the chromatin properties and biological properties of the SWI/SNF-dependent and SWI/SNF-independent genes suggested models to explain the variability at both the mechanistic and biological levels.
Mechanistically, the promoters of genes activated in a SWI/SNF-independent manner were generally assembled into a chromatin structure in unstimulated macrophages resembling that found at transcriptionally active genes. Specifically, the promoters were accessible to restriction enzymes in unstimulated cells, with little change upon induction (Fig. 2, Tnf ). ChIP experiments further revealed that the promoters of most of these genes contained high levels of histone H3 lysine 4 trimethylation (H3K4me3) in unstimulated macrophages, with relatively little increase following LPS stimulation (Ramirez-Carrozzi et al. 2009). In addition, RNA polymerase II was associated with the promoters of many SWI/SNF-independent genes in the unstimulated cells; initiation and pausing appears to occur at some of these genes before stimulation, as observed at the Drosophila heat-shock genes (Adelman et al. 2009;Hargreaves et al. 2009). Together, these results suggest that inducible remodeling by SWI/SNF complexes is not needed at these genes because their promoters are pre-assembled into an active chromatin state in unstimulated cells.
Examination of the promoter sequences of the SWI/ SNF-independent genes revealed a high prevalence of CpG-island promoters (Hargreaves et al. 2009;Ramirez-Carrozzi et al. 2009). CpG islands are found at 70% of promoters in mammalian cells, but their functional significance is not fully understood (Deaton and Bird 2011). Early biochemical studies revealed that DNA sequences with an extremely high content of G/C base pairs assemble into unstable nucleosomes (Drew and Travers 1985), raising the possibility that the absence of a remodeling requirement at CpG-island promoters may be due, at least in part, to an intrinsic instability of nucleosomes assembled at these promoters. Consistent with this possibility, intrinsic instability was observed at many CpG-island promoters in nucleosome assembly experiments performed in vitro (Ramirez-Carrozzi et al. 2009). The relevance of this intrinsic instability for SWI/SNF-independent transcription awaits further analysis.
In contrast to the properties of SWI/SNF-independent genes, SWI/SNF-dependent genes were usually found to contain low CpG promoters with low histone H3K4me3 in unstimulated cells (Ramirez-Carrozzi et al. 2009). The promoters of representative genes in this class showed low accessibility to restriction enzyme cleavage in unstimulated cells, with increased accessibility following stimulation (Fig. 2, Ccl5, and Il6). Thus, mechanistically, the variable dependence of inducible genes on SWI/SNF complexes correlated closely with the variable chromatin properties observed at their promoters in unstimulated cells.
At a biological level, SWI/SNF-independent induction was generally observed at genes that are known to be induced by a broad range of stimuli, such as the Fos, Jun, Egr1, Egr2, Egr3, and Nfikba genes. This finding suggests a hypothesis in which genes induced by a broad Figure 2. Variable sensitivities of LPS-induced genes to Brg1/Brm knockdown in mouse macrophages. At left, the effect of Brg1/ Brm knockdown on mRNA levels for 67 LPS-induced genes (55 primary response genes and 12 secondary response genes) in mouse bone-marrow-derived macrophages is shown. mRNA levels are presented as a percentage of the levels observed in control cells stimulated with LPS. Genes that are induced at nearly normal or enhanced levels following Brg1/Brm knockdown are colored red, whereas genes whose expression is reduced to the greatest extent are colored green. Genes colored brown or yellow showed intermediate effects. At the right, restriction enzyme accessibility results are shown for the promoters of three representative genes: Tnf, representative of a Brg1/Brm-independent primary response gene; Ccl5, representative of a Brg1/Brm-dependent primary response gene; and Il6, representative of a Brg1/Brm-dependent secondary response gene. (Adapted from Ramirez-Carrozzi et al. 2006 range of stimuli have little need for a nucleosome barrier to activation. In contrast, SWI/SNF dependence was observed at cytokine and chemokine genes that are known to be selectively regulated, such as Il12b, Il6, and Ifnb. These findings are consistent with the model described above, in which nucleosome remodeling provides a critical barrier to activation that contributes to selectivity of the response to a stimulus. It is important to emphasize that, although the results described above provide an initial framework, a number of findings during the course of these studies have revealed additional complexity that will need to be examined further to fully understand how chromatin contributes to transcriptional induction and the selectivity of a transcriptional response. For example, although most SWI/ SNF-independent genes contain CpG-island promoters that are assembled into constitutively active chromatin, a subset of the SWI/SNF-independent genes were found to contain low CpG promoters assembled into inaccessible chromatin in unstimulated cells (Ramirez-Carrozzi et al. 2009). One possibility is that nucleosomes at these promoters require remodeling by an ATP-dependent nucleosome remodeling complex other than the SWI/SNF complexes. In addition, the Il6 gene, which is an SWI/ SNF-dependent secondary response gene in macrophages, was found to be an SWI/SNF-independent primary response gene with a constitutively accessible promoter in fibroblasts (Ramirez-Carrozzi et al. 2009). Presumably, a factor expressed during fibroblast development helps to catalyze the assembly of a constitutively accessible chromatin structure at the low CpG Il6 promoter, thereby allowing rapid activation in the absence of nucleosome remodeling. Although the significance of this finding is unknown, one possibility is that it reflects the different biological functions of the IL-6 cytokine in the two cell types. In macrophages, IL-6 is thought to be a major regulator of T-helper-cell differentiation, a function that may require highly selective regulation by different stimuli. In contrast, IL-6 appears to function as a broad regulator of inflammation when expressed by fibroblasts, perhaps suggesting a lesser requirement for tight regulation.

RNA-SEQUENCING ANALYSES OF INDUCIBLE TRANSCRIPTION
A limitation of the studies described above is that they involved an analysis of only 67 inducible genes, with the expression of each gene monitored by quantitative RT-PCR in all experiments. In other words, a true genomescale analysis was not performed. Efforts to extend the analysis using microarrays were unsuccessful due to the limited quantitation and accuracy of microarray platforms. However, the emergence of RNA-sequencing (RNA-seq) as a more accurate and quantitative alternative to microarrays has provided an opportunity to perform truly genome-scale analyses of inducible gene expression cascades.
An additional advantage of RNA-seq is that it has been used with considerable success to examine RNA popula-tions other than mature mRNA. Of particular relevance to the goal of dissecting transcriptional cascades, RNA-seq can be used to monitor nascent transcripts for genes that are being actively transcribed. A major benefit of analyzing nascent transcripts when studying transcriptional cascades is that changes in nascent transcript levels following cell stimulation provide a more accurate measure of the kinetics of transcriptional activation and inactivation. After transcription of a gene is maximally induced, its mRNA will continue to accumulate until it reaches its steady state, which is dictated by both its rate of synthesis and degradation. Thus, two genes that are transcriptionally induced at the same time after stimulation will show different mRNA kinetics if their stabilities differ. Similarly, transcriptional inactivation is not accurately measured by mRNA analyses because a stable mRNA can persist for several hours after transcription of the gene is turned off. To understand the global logic through which transcriptional cascades are regulated, it is important to identify groups of genes that are turned on and off with similar kinetics in response to a stimulus; nascent transcript analyses provide a much more accurate measure of transcriptional kinetics than do mRNA analyses.
Several RNA-seq methods have been developed to monitor nascent transcripts, including GRO-seq (genome-wide nuclear runon) (Core et al. 2008), NET-Seq (analysis of RNA polymerase/DNA/RNA ternary complexes) (Churchman and Weissman 2011), and metabolic labeling (Friedel and Dölken 2009;Rabani et al. 2011). Our laboratory has relied extensively on the fourth method, in which chromatin is first separated from the cytoplasm and nucleoplasm of a cell population by biochemical fractionation (Bhatt et al. 2012). RNAs that purify with the chromatin fraction include recently transcribed RNAs and RNAs being actively transcribed. Notably, the chromatin fraction also includes noncoding RNAs that may function in association with the chromatin.
An analysis of chromatin-associated RNAs in LPSstimulated macrophages allowed groups of genes to be identified that are expressed with similar kinetics (Bhatt et al. 2012). To determine whether coregulated genes might be regulated by similar mechanisms, computational methods were used to determine whether binding sites for specific transcription factors are enriched in the promoters of each coregulated gene cluster. This analysis revealed strong overrepresentation of known regulators of LPS-induced transcription, such as NF-kB, AP-1, serum response factor, interferon response factor 3 in a limited number of clusters. Binding sites for signal transduction and transcription (STAT) proteins were also found to be overrepresented in specific kinetic clusters, presumably due to the autocrine activation of interferon (IFN) receptors following the earlier induction of IFN-b.
The strong enrichment of transcription-factor-binding sites in genes within specific kinetic clusters provides strong evidence that the global analysis of nascent transcripts by RNA-seq of chromatin-associates transcripts provides an important step toward an understanding of the global logic through which a transcriptional cascade is regulated. However, it is important to emphasize that much remains to be learned. In particular, although binding sites for transcription factors, such as NF-kB, were found in several promoters within specific coregulated clusters, several other promoters in these same clusters lacked the overrepresented binding site. This observation, combined with the fact that the genes within each cluster are not perfectly coregulated, suggests that the clusters are not homogenous with respect to regulatory mechanisms. In fact, it seems unlikely that any coregulated cluster presented in published reports is homogenous.
From the perspective of chromatin structure, the global analysis of chromatin-associated transcripts by RNA-seq confirmed some of our previous findings from analysis of a limited set of genes, but other early models required revision. Specifically, as observed in the earlier studies (Ramirez-Carrozzi et al. 2009), a high percentage of rapidly induced primary response genes was found to contain CpG-island promoters assembled into constitutively active chromatin (Bhatt et al. 2012). Furthermore, the most potently induced late genes contained low CpG promoters that lacked active chromatin in unstimulated cells. Also consistent with our earlier studies, a smaller group of genes containing low CpG promoters and lacking active chromatin was observed among the most rapidly induced genes.
However, in the RNA-seq analysis, the presence of a CpG island correlated most closely with genes that are not only rapidly activated but also rapidly inactivated after their rapid activation (Bhatt et al. 2012). Almost every transiently induced gene was found to contain a CpGisland promoter, whereas a much larger number of genes that show rapid but sustained induction contain low CpG promoters. This finding suggests that a CpG-island promoter may be particularly important for the rapid inactivation of transcription, perhaps because inactivation of these classes of promoters, in which chromatin may play a lesser role, is easier to achieve.
Another new finding from the RNA-seq analysis was the presence of a large number of CpG-island promoters among genes that are activated with relatively slow kinetics during the transcriptional cascade (Bhatt et al. 2012). This contrasts with the finding from the analysis of 67 individual genes that the vast majority of genes induced with slow kinetics contain low CpG promoters (Ramirez-Carrozzi et al. 2009). An examination of possible reasons for this difference revealed that it is due to the inclusion of a broader range of induction magnitudes in the RNA-seq studies. The CpG-island-containing genes induced with slow kinetics are mostly genes induced by only 10-to 20fold; as with other CpG-islands, the CpG islands at these promoters are assembled into constitutively active chromatin. In contrast, the genes that are most potently induced with slow kinetics contain low CpG promoters assembled into inactive chromatin in unstimulated cells.
These findings suggest that CpG-island promoters are suitable at genes that require transcriptional fluctuations of relatively modest magnitudes, whereas most genes requiring potent induction have evolved low CpG promoters. One possible explanation for this difference was that genes in the latter group are transcribed at a lower basal level in unstimulated cells, leading to larger fold-induction magnitudes. However, basal transcription levels were not consistently lower in the low CpG genes (Bhatt et al. 2012). We therefore favor a slightly different model, in which the nucleosome barrier present at low CpG promoters helps to reduce the basal transcription levels of genes that have evolved to support extremely high levels of transcription, thereby contributing to the large dynamic range of these genes (not necessarily by conferring a low basal transcription level in comparison to other genes but by conferring a lower basal level than might otherwise be found at a gene capable of supporting extremely high levels of transcription).

CONCLUSIONS
Since the discovery of the first inducible transcription factors (Sen and Baltimore 1986;Bohmann et al. 1987), much has been learned about the mechanisms used to regulate inducible transcription at both a gene-specific and global level. A large number of transcription factors that contribute to inducible transcription have been discovered, signaling pathways that lead to the activation of many of these factors have been characterized, and initial insights into the role of chromatin structure in inducible transcription have been described. However, it can be argued that the field remains in its infancy. Growing evidence suggests that many and possible all transcription factors will be subject to multiple layers of regulation, with our current knowledge limited to the most basic regulatory layers. Furthermore, our limited understanding of the in vivo functions of the dozens of histone modifications that have been described, combined with our limited knowledge of the chromatin structure into which eukaryotic genes are assembled in their native chromosomal environment, have presented formidable barriers to efforts to develop a clear understanding of how chromatin contributes to the regulation of inducible transcription and the selectivity of a transcriptional response to a stimulus. Fortunately, the development of increasingly sophisticated experimental methods for the analysis of transcriptional regulation in cell populations and in single cells continue to suggest novel strategies toward the study of inducible transcription, paving the way for continuing progress toward an understanding of the logic underlying the selective responses to microbial and environmental stimuli.