Functional analysis of histones H3 and H4 in Tup1 repression and in GCN5 activation

Tup1 forms a complex with Ssn6 in yeast. Ssn6-Tup1 complex is recruited via direct interactions with specific DNA binding proteins to a specific promoter region and mediates repression of several sets of genes including a-cell specific genes (asg) in $\alpha$ cells. It has been shown that repression of asgs also requires histone H4 and that Tup1 can directly interact with H3 and H4 in vitro. To address whether histone H3 is required for the repression of asgs, I have examined the effect of H3 and H4 mutations on the expression of a $\alpha$2-controlled LacZ reporter. Assay of $\beta$-glactosidase shows that mutations in either H3 or H4 cause a weak derepression of the reporter gene. Some double mutations result in a stronger derepression, while others do not. The H3 N-terminal deletion also leads to a slightly decreased expression of the reporter gene in $\alpha$ cells. Our data suggest that the N-termini of both H3 and H4 are cooperatively involved in the repression of a-cell specific genes in $\alpha$ cells, possibly through their interaction with Tup1.^ GCN5 was originally identified as a transcriptional regulator required to activate a subset of genes in yeast. Recently, it has been shown that GCN5 encodes the catalytic subunit of a nuclear histone acetyltransferase, providing the first direct link between histone acetylation and gene transcription. Recombinant Gcn5p (rGcn5p) exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. In order to define the sites of histone acetylation mediated by Gcn5p in vivo and assess the significance of histone acetylation, more than 30 yeast strains have been constructed to bear specific H3 and/or H4 mutations in the presence or absence of GCN5 function. Our genetic data suggest that Gcn5p may have additional targets in vivo that are not identified as the targets of rGcn5p by previous studies. Western analysis using antibodies specifically recognizing particular acetylated isoforms of H3 and H4 led us to conclude that Gcn5p is necessary for full acetylation of multiple sites in both H3 and H4 in vivo. Consistent with these observations, rGcn5p still acetylates histones H3 and H4 bearing mutations either in H3 K14 or H4 K8,16, sites previously identified as the targets of acetylation by rGcn5p in H3 and H4. Our data also demonstrated that Gcn5p-mediated acetylation events are important for normal progression of the cell cycle and for transcriptional activation. Furthermore, a critical overall level of acetylation is essential for cell viability. ^

Involvement of histone H3 methylation in Tup1-Ssn6 repression

The Tup1-Ssn6 complex regulates the expression of diverse classes of genes in Saccharomyces cerevisiae including those regulated by mating type, DNA damage, glucose, and anaerobic stress. The complex is recruited to target genes by sequence-specific repressor proteins. Once recruited to particular promoters, it is not completely clear how it functions to block transcription. Repression probably occurs through interactions with both the basal transcriptional machinery and components of chromatin. Tup1 interactions with chromatin are strongly influenced by acetylation of histories H3 and H4. Tup1 binds to underacetylated histone tails and requires multiple histone deacetylases (HDACs) for its repressive functions. Like acetylation, histone methylation is involved in regulation of gene expression. The possible role of histone methylation in Tup1 repression is not known. Here we examined possible roles of histone methyltransferases in Tup1-Ssn6 functions. We found that like other genes, Tup1-Ssn6 target genes exhibit increases in the levels of histone H3 lysine 4 methylation upon activation. However, deletion of individual or multiple histone methyltransferases (HMTs) and other SET-domain containing genes has no apparent effect on Tup1-Ssn6 mediated repression of a number of well-defined targets. Interestingly, we discovered that Ssn6 interacts with Set2. Since deletion of SET2 does not affect Tup1-Ssn6 repression, Ssn6 may utilize Set2 in other contexts to regulate gene repression. In order examine if the two components of the Tup1-Ssn6 complex have independent functions in the cell, we identified genes differentially expressed in tup1Δ and ssn6Δ mutants using DNA microarrays. Our data indicate that ∼4% of genes in the cell are regulated by Ssn6 independently of Tup1. In addition, expression of genes regulated by Tup1-Ssn6 seems to be differently affected by deletion of Ssn6 and deletion of Tup1, which indicates that these components might have separate functions. Our data shed new light on the classical view of Tup1-Ssn6 functions, and indicate that Ssn6 might have repressive functions as well. ^

Antibodies targeting hepatoma-derived growth factor as a novel strategy in treating lung cancer.

Hepatoma-derived growth factor (HDGF) is overexpressed in lung cancer and the overexpression correlates with aggressive biological behaviors and poor clinical outcomes. We developed anti-HDGF monoclonal antibodies and tested their antitumor activity in lung cancer xenograft models. We also determined biological effects in tumors treated with the antibody alone or in combination with bevacizumab/avastin (an anti-vascular endothelial growth factor antibody) and/or gemcitabine (a chemotherapeutic agent). We found the anti-HDGF was effective to inhibit tumor growth in non-small cell lung cancer xenograft models. In the A549 model, compared with control IgG, tumor growth was substantially inhibited in animals treated with anti-HDGF antibodies, particularly HDGF-C1 (P = 0.002) and HDGF-H3 (P = 0.005). When HDGF-H3 was combined with either bevacizumab or gemcitabine, we observed enhanced tumor growth inhibition, particularly when the three agents were used together. HDGF-H3-treated tumors exhibited significant reduction of microvessel density with a pattern distinctive from the microvessel reduction pattern observed in bevacizumab-treated tumors. HDGF-H3-treated but not bevacizumab-treated tumors also showed a significant increase of apoptosis. Interestingly, many of the apoptotic cells in HDGF-H3-treated tumors are stroma cells, suggesting that the mechanism of the antitumor activity is, at least in part, through disrupting formation of tumor-stroma structures. Our results show that HDGF is a novel therapeutic target for lung cancer and can be effectively targeted by an antibody-based approach.

Role of NonO-histone interaction in TNFalpha-suppressed prolyl-4-hydroxylase alpha1.

Inflammation is a key process in cardiovascular diseases. The extracellular matrix (ECM) of the vasculature is a major target of inflammatory cytokines, and TNFalpha regulates ECM metabolism by affecting collagen production. In this study, we have examined the pathways mediating TNFalpha-induced suppression of prolyl-4 hydroxylase alpha1 (P4Halpha1), the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Using human aortic smooth muscle cells, we found that TNFalpha activated the MKK4-JNK1 pathway, which induced histone (H) 4 lysine 12 acetylation within the TNFalpha response element in the P4Halpha1 promoter. The acetylated-H4 then recruited a transcription factor, NonO, which, in turn, recruited HDACs and induced H3 lysine 9 deacetylation, thereby inhibiting transcription of the P4Halpha1 promoter. Furthermore, we found that TNFalpha oxidized DJ-1, which may be essential for the NonO-P4Halpha1 interaction because treatment with gene specific siRNA to knockout DJ-1 eliminated the TNFalpha-induced NonO-P4Halpha1 interaction and its suppression. Our findings may be relevant to aortic aneurysm and dissection and the stability of the fibrous cap of atherosclerotic plaque in which collagen metabolism is important in arterial remodeling. Defining this cytokine-mediated regulatory pathway may provide novel molecular targets for therapeutic intervention in preventing plaque rupture and acute coronary occlusion.

Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha.

Aldosterone plays a major role in the regulation of salt balance and the pathophysiology of cardiovascular and renal diseases. Many aldosterone-regulated genes--including that encoding the epithelial Na+ channel (ENaC), a key arbiter of Na+ transport in the kidney and other epithelia--have been identified, but the mechanisms by which the hormone modifies chromatin structure and thus transcription remain unknown. We previously described the basal repression of ENaCalpha by a complex containing the histone H3 Lys79 methyltransferase disruptor of telomeric silencing alternative splice variant a (Dot1a) and the putative transcription factor ALL1-fused gene from chromosome 9 (Af9) as well as the release of this repression by aldosterone treatment. Here we provide evidence from renal collecting duct cells and serum- and glucocorticoid-induced kinase-1 (Sgk1) WT and knockout mice that Sgk1 phosphorylated Af9, thereby impairing the Dot1a-Af9 interaction and leading to targeted histone H3 Lys79 hypomethylation at the ENaCalpha promoter and derepression of ENaCalpha transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCalpha and likely other aldosterone-induced genes.

Regulation of Set1-mediated methylation of Dam1

Eukaryotic genomes exist within a dynamic structure named chromatin in which DNA is wrapped around an octamer of histones forming the nucleosome. Histones are modified by a range of posttranslational modifications including methylation, phosphorylation, and ubiquitination, which are integral to a range of DNA-templated processes including transcriptional regulation. A hallmark for transcriptional activity is methylation of histone H3 on lysine (K) 4 within active gene promoters. In S. cerevisiae, H3K4 methylation is mediated by Set1 within the COMPASS complex. Methylation requires prior ubiquitination of histone H2BK123 by the E2-E3 ligases Rad6 and Bre1, as well as the Paf1 transcriptional elongation complex. This regulatory pathway exemplifies cross-talk in trans between posttranslational modifications on distinct histone molecules. Set1 has an additional substrate in the kinetochore protein Dam1, which is methylated on K233. This methylation antagonizes phosphorylation of adjacent serines by the Ipl1 Aurora kinase. The discovery of a second Set1 substrate raised the question of how Set1 function is regulated at the kinetochore. I hypothesized that transcriptional regulatory factors essential for H3K4 methylation at gene promoters might also regulate Set1-mediated methylation of Dam1K233. Here I show that the regulatory factors essential for COMPASS activity at gene promoters is also indispensable for the methylation of Dam1K233. Deletion of members of the COMPASS complex leads to loss of Dam1K233 methylation. In addition, deletion of Rad6, Bre1, or members of the Paf1 complex abolishes Dam1 methylation. The role of Rad6 and Bre1 in Dam1 methylation is dependent on H2BK123 ubiquitination, as mutation of K123 within H2B results in complete loss of Dam1 methylation. Importantly, methylation of Dam1K233 is independent of transcription and occurs at the kinetochore. My results demonstrate that Set1-mediated methylation is regulated by a general pathway regardless of substrate that is composed of transcriptional regulatory factors functioning independently of transcription at the kinetochore. My data provide the first example of cross-talk in trans between modifications on a histone and a non-histone protein. Additionally, my results indicate that several factors previously thought to be required for Set1 function at gene promoters are more generally required for the catalytic activity of the COMPASS complex regardless of substrate or cellular process.

ROLE OF SYNAPSIN IN LONG-TERM SYNAPTIC FACILITATION IN APLYSIA

Enhanced expression of the presynaptic protein synapsin has been correlated with certain forms of long-term plasticity and learning and memory. However, the regulation and requirement for enhanced synapsin expression in long-term memory remains unknown. In the present study the technical advantages of the marine mollusc Aplysia were exploited in order to address this issue. In Aplysia, learning-induced enhancement in synaptic strength is modulated by serotonin (5-HT) and treatment with 5-HT in vitro of the sensorimotor synapse induces long-term facilitation (LTF) of synaptic transmission, which lasts for days, as well as the formation of new connections between the sensory and motor neuron. Results from immunofluorescence analysis indicated that 5-HT treatment upregulates synapsin protein levels within sensory neuron varicosities, the presumed site of neurotransmitter release. To investigate the mechanisms underlying increased synapsin expression, the promoter region of the Aplysia synapsin gene was cloned and a cAMP response element (CRE) was identified, raising the possibility that the transcriptional activator cAMP response element-binding protein-1 (CREB1) mediates the 5-HT-induced regulation of synapsin. Results from Chromatin Immunoprecipitation (ChIP) assays indicated that 5-HT treatment enhanced association of CREB1 surrounding the CRE site in the synapsin promoter and led to increased acetylation of histones H3 and H4 and decreased association of histone deacetylase 5 surrounding the CRE site in the synapsin promoter, a sign of transcriptional activation. In addition, sensory neurons injected with an enhanced green fluorescent protein (EGFP) reporter vector driven by the synapsin promoter exhibited a significant increase in EGFP expression following treatment with 5-HT. These results suggest that synapsin expression is regulated by 5-HT in part through transcriptional activation of the synapsin gene and through CREB1 association with the synapsin promoter. Furthermore, RNA interference that blocks 5-HT-induced elevation of synapsin expression also blocked long-term synaptic facilitation. These results indicate that 5-HT-induced regulation of synapsin is necessary for LTF and that synapsin is part of the cascade of synaptic events involved in the consolidation of memory.

CHROMOSOMAL MAPPING, LINKAGE RELATIONSHIPS, AND EVOLUTIONARY STUDIES OF THE HUMAN ANONYMOUS DNA CLONE D1S1 (IN SITU HYBRIDIZATION, PRIMATES, GENE MAPPING, AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA, GENE DUPLICATION)

D1S1, an anonymous human DNA clone originally called (lamda)Ch4-H3 or (lamda)H3, was the first single copy mapped to a human chromosome (1p36) by in situ hybridization. The chromosomal assignment has been confirmed in other laboratories by repeating the in situ hybridization but not by another method. In the present study, hybridization to a panel of hamster-human somatic cell hybrids revealed copies of D1S1 on both chromosomes 1 and 3. Subcloning D1S1 showed that the D1S1 clone itself is from chromosome 3, and the sequence detected by in situ hybridization is at least two copies of part of the chromosome 3 copy. This finding demonstrates the importance of verifying gene mapping with two methods and questions the accuracy of in situ hybridization mapping.^ Non-human mammals have only one copy of D1S1, and the non-human primate D1S1 map closely resembles the human chromosome 3 copy. Thus, the human chromosome 1 copies appear to be part of a very recent duplication that occurred after the divergence between humans and the other great apes.^ A moderately informative HindIII D1S1 RFLP was mapped to chromosome 3. This marker and 12 protein markers were applied to a linkage study of autosomal dominant retinitis pigmentosa (ADRP). None of the markers proved linkage, but adding the three families examined to previously published data raises the ADRP:Rh lod score to 1.92 at (THETA) = 0.30. ^

Functional requirements of histone deacetylases in Tup1-Ssn6 repression

The corepressor complex Tup1-Ssn6 regulates many classes of genes in yeast including cell type specific, glucose repressible, and DNA damage inducible. Tup1 and Ssn6 are recruited to target promoters through their interactions with specific DNA binding proteins such as α2, Mig1, and Crt1. Most promoters that are repressed by this corepressor complex exhibit a high degree of nucleosomal organization. This chromatin domain occludes transcription factor access to the promoter element resulting in gene repression. Previous work indicated that Tup1 interacts with underacetylated isoforms of H3 and H4, and that mutation of these histones synergistically compromises repression. These studies predict that Tup1-hypoacetyalted histone interaction is important to the repression mechanism, and in vivo hyperacetylation might compromise the corepressors ability to repress target genes. ^ One way to alter histone acetylation levels in vivo is to alter the balance between histone acetyltransferases and histone deacetylases. To date five histone deacetylases (HDACs) have been identified in yeast Rpd3, Hos1, Hos2, Hos3 and Hda1. Deletion of single or double HDAC genes had little to no effect on Tup1-Ssn6 repression, but simultaneous deletion of three specific activities Rpd3, Hos1, and Hos2 abolished repression in vivo. Promoter regions of Tup1-Ssn6 target genes in these triple deacetylase mutant cells are dramatically hyperacetylated in both H3 and H4. Examination of bulk histone acetylation levels showed that this specific HDAC triple mutant combination (rpd3 hos1 hos2) caused a dramatic and concomitant hyperacetylation of both H3 and H4. The loss of repression in the rpd3 hos1 hos2 cells, but not in other mutants, is consistent with previous observations, which indicate that histones provide redundant functions in the repression mechanism and that high levels of acetylation are required to prevent Tup1 binding. Investigation into a potential direct interaction between the Tup1-Ssn6 corepressor complex and one or more HDAC activities showed that both Rpd3 and Hos2 interact with the corepressor complex in vivo. These findings indicate that Tup1-Ssn6 repression involves the recruitment of histone deacetylase activities to target promoters, where they locally deacetylate histone residues promoting Tup1-histone tail interaction to initiate and/or maintain the repressed state. ^

The Set1 methyltransferase opposes Ipl1 aurora kinase functions in chromosome segregation

A phosphorylation balance governed by Ipl1 Aurora kinase and the Glc7 phosphatase is essential for normal chromosome segregation in S. cerevisiae . Deletion of SET1, a histone K4 methyltransferase, suppresses the temperature sensitive phenotype of ipl1-2, and loss the catalytic activity of Set1 is important for this suppression. SET1 deletion also suppresses chromosome loss in ipl1-2 cells. Deletion of other Set1 complex components suppresses the temperature sensitivity of ipl1-2 as well. In contrast, SET1 deletion is synthetic lethal combined with glc7-127. Strikingly, these effects are independent of previously defined functions for Set1 in transcription initiation and histone H3 methylation. I find that Set1 methylates conserved lysines in a kinetochore protein, Dam1, a key mitotic substrate of Ipl1/Glc7. Biochemical and genetic experiments indicate that Dam1 methylation inhibits Ipl1-mediated phosphorylation of flanking serines. My studies demonstrate that Set1 has important, unexpected functions in mitosis through modulating the phosphorylation balance regulated by Ipl1/Glc7. Moreover, my findings suggest that antagonism between lysine methylation and serine phosphorylation is a fundamental mechanism for controlling protein function. ^

Targeting dysregulated nuclear proteins androgen receptor and enhancer of zeste homolog 2: Clinical implication and mechanism underline

Cancer is the most devastating disease that has tremendous impacts on public health. Many efforts have been devoted to fighting cancer through either translational or basic researches for years. Nowadays, it emerges the importance to converge these two research directions and complement to each other for battling with cancer. Thus, our study aims at both translational and basic research directions. The first goal of our study is focus on translational research to search for new agents targeting prevention and therapy of advanced prostate cancer. Hormone refractory prostate cancer is incurable and lethal. Androgen receptor (AR) mediates androgen's effect not only on the tumor initiation but also plays the major role in the relapse transition of prostate cancer. Here we demonstrate that emodin, a natural compound, can directly target AR to suppress prostate cancer cell growth in vitro and prolong the survival of C3(1)/SV40 transgenic mice in vivo. Emodin treatment resulted in repressing androgen-dependent transactivation of AR by inhibiting AR nuclear translocation. Emodin decreased the association of AR and heat shock protein 90 and increased the association of AR and MDM2, which in turn, induces AR degradation through a proteasome-mediated pathway in a ligand independent manner. Our work indicates a new mechanism for the emodin-mediated anticancer effect and justifies further investigation of emodin as a therapeutic and preventive agent for prostate cancer. The second goal of our study is try to elucidate the fundamental tumor biology of cancer progression then provide the rationale to develop more efficient therapeutic strategy. Enhancer of zeste homologue 2 (EZH2) plays an important role in many biological processes through its intrinsic methyltransferase activity to trimethylate lysine 27 in histone H3. Although overexpression of EZH2 has been shown to be involved in cancer progression, the detailed mechanisms are elusive. Here, we show that Akt phosphorylates EZH2 at serine 21 and suppresses its methyltransferase activity by impeding the binding to its substrate histone H3, resulting in a decrease of lysine 27 trimethylation and derepression of silenced genes, thus promotes cell proliferation and tumorigenicity. Our results also show that histone methylation is not permanent but regulated in a dynamic manner and that the Akt signaling pathway is involved in the regulation of this epigenetic modification through phosphorylation of EZH2, thus contributing to oncogenic processes. ^

Hypoxia-induced transcriptional regulation through chromatin modifications and NC2 function

In response to tumor hypoxia, specific genes that promote angiogenesis, proliferation, and survival are induced. Globally, I find that hypoxia induces a mixed pattern of histone modifications that are typically associated with either transcriptional activation or repression. Furthermore, I find that selective activation of hypoxia-inducible genes occurs simultaneously with widespread repression of transcription. I analyzed histone modifications at the core promoters of hypoxia-repressed and -activated genes and find that distinct patterns of histone modifications correlate with transcriptional activity. Additionally, I discovered that trimethylated H3-K4, a modification generally associated with transcriptional activation, is induced at both hypoxia-activated and repressed genes, suggesting a novel pattern of histone modifications induced during hypoxia. ^ In order to determine the mechanism of hypoxia-induced widespread repression of transcription, I focused my studies on negative cofactor 2 (NC2). Previously, we found that hypoxia-induced repression of the alpha-fetoprotein (AFP) gene occurs during preinitiation complex (PIC) assembly, and I find that NC2, an inhibitor of PIC assembly, is induced during hypoxia. Moreover, I find that the beta subunit of NC2 is essential for hypoxia-mediated repression of AFP, as well as the widespread repression of transcription observed during hypoxia. Previous data in Drosophila and S. cerevisiae indicate that NC2 functions as either an activator or a repressor of transcription. The mechanism of NC2-mediated activation remains unclear; although, Drosophila NC2 function correlates with specific core promoter elements. I tested if NC2 activates transcription in mammalian cells using this core promoter-specific model as a guide. Utilizing site-specific mutagenesis, I find that NC2 function in mammalian cells is not dependent upon specific core promoter elements; however, I do find that mammalian NC2 does function in a gene-specific manner as either an activator or repressor of transcription during hypoxia. Furthermore, I find that binding of the alpha subunit of NC2 specifically correlates with NC2-mediated transcriptional activation. NC2α and NC2β are both required for NC2-mediated transcriptional activation; whereas, NC2β alone is required for hypoxia-induced transcriptional repression. Together, these data indicate that hypoxia mediates changes in gene expression through both chromatin modifications and NC2 function. ^

Maximizing efficacy of the hypomethylating drug 5-aza-2'-deoxycytidine in human leukemia

5-aza-2'-deoxycytidine (DAC) is a cytidine analogue that strongly inhibits DNA methylation, and was recently approved for the treatment of myelodysplastic syndromes (MDS). To maximize clinical results with DAC, we investigated its use as an anti-cancer drug. We also investigated mechanisms of resistance to DAC in vitro in cancer cell lines and in vivo in MDS patients after relapse. We found DAC sensitized cells to the effect of 1-β-D-Arabinofuranosylcytosine (Ara-C). The combination of DAC and Ara-C or Ara-C following DAC showed additive or synergistic effects on cell death in four human leukemia cell lines in vitro, but antagonism in terms of global methylation. RIL gene activation and H3 lys-9 acetylation of short interspersed elements (Alu). One possible explanation is that hypomethylated cells are sensitized to cell killing by Ara-C. Turning to resistance, we found that the IC50 of DAC differed 1000 fold among and was correlated with the dose of DAC that induced peak hypomethylation of long interspersed nuclear elements (LINE) (r=0.94, P<0.001), but not with LINE methylation at baseline (r=0.05, P=0.97). Sensitivity to DAC did not significantly correlate with sensitivity to another hypomethylating agent 5-azacytidine (AZA) (r=0.44, P=0.11). The cell lines most resistant to DAC had low dCK, hENT1, and hENT2 transporters and high cytosine deaminase (CDA). In an HL60 leukemia cell line, resistance to DAC could be rapidly induced by drug exposure, and was related to a switch from monoallelic to biallelic mutation of dCK or a loss of wild type DCK allele. Furthermore, we showed that DAC induced DNA breaks evidenced by histone H2AX phosphorylation and increased homologous recombination rates 7-10 folds. Finally, we found there were no dCK mutations in MDS patients after relapse. Cytogenetics showed that three of the patients acquired new abnormalities at relapse. These data suggest that in vitro spontaneous and acquired resistance to DAC can be explained by insufficient incorporation of drug into DNA. In vivo resistance to DAC is likely due to methylation-independent pathways such as chromosome changes. The lack of cross resistance between DAC and AZA is of potential clinical relevance, as is the combination of DAC and Ara-C. ^

Molecular mechanisms by which p53/LSD1 and ERalpha/Trim24 complexes mediate gene regulation

In this dissertation, I identify two molecular mechanisms by which transcription factors cooperate with their co-regulators to mediate gene regulation. In the first part, I demonstrate that p53 directly recruits LSD1, a histone demethylase, to AFP chromatin to demethylate methylated H3K4 and actively mediate transcription repression. Loss of p53 and LSD1 interaction at chromatin leads to derepression of AFP in hepatic cells. In the second part, I reveal that Trim24 functions as an important co-activator in ERα-mediated gene activation in response to estrogen stimulation. Trim24 is recruited by ligand-bound ERα to chromatin and stabilizes ERα-chromatin interactions by binding to histone H3 via its PHD finger, which preferentially recognizes unmethylated H3K4. ^

Regulation of chromatin structure, Smad binding and AFP expression by the Forkhead box transcription factor: Foxa1

Transcription factors must be able to access their DNA binding sites to either activate or repress transcription. However, DNA wrapping and compaction into chromatin occludes most binding sites from ready access by proteins. Pioneer transcription factors are capable of binding their DNA elements within a condensed chromatin context and then reducing the level of nucleosome occupancy so that the chromatin structure is more accessible. This altered accessibility increases the probability of other transcription factors binding to their own DNA binding elements. My hypothesis is that Foxa1, a ‘pioneer’ transcription factor, activates alpha-fetoprotein (AFP) expression by binding DNA in a chromatinized environment, reducing the nucleosome occupancy and facilitating binding of additional transcription factors.^ Using retinoic-acid differentiated mouse embryonic stem cells, we illustrate a mechanism for activation of the tumor marker AFP by the pioneer transcription factor Foxa1 and TGF-β downstream effector transcription factors Smad2 and Smad4. In differentiating embryonic stem cells, binding of the Foxa1 forkhead box transcription factor to chromatin reduces nucleosome occupancy and levels of linker histone H1 at the AFP distal promoter. The more accessible DNA is subsequently bound by the Smad2 and Smad4 transcription factors, concurrent with activation of transcription. Chromatin immunoprecipitation analyses combined with siRNA-mediated knockdown indicate that Smad protein binding and the reduction of nucleosome occupancy at the AFP distal promoter is dependent on Foxa1. In addition to facilitating transcription factor binding, Foxa1 is also associated with histone modifications related to active gene expression. Acetylation of lysine 9 on histone H3, a mark that is associated active transcription, is dependent on Foxa1, while methylation of H3K4, also associated with active transcription, is independent of Foxa1. I propose that Foxa1 potentiates a region of chromatin to respond to Smad proteins, leading to active expression of AFP.^ These studies demonstrate one mechanism whereby a transcription factor can alter the accessibility of additional transcription factors to chromatin, by altering nucleosome positions. Specifically, Foxa1 exposes DNA so that Smad4 can bind to its regulatory element and activate transcription of the tumor-marker gene AFP.^