Local Histone Acetylation Research Articles

A TLR4/TRAF6-dependent signaling pathway mediates NCoR coactivator complex formation for inflammatory gene activation.

The nuclear receptor corepressor (NCoR) forms a complex with histone deacetylase 3 (HDAC3) that mediates repressive functions of unliganded nuclear receptors and other transcriptional repressors by deacetylation of histone substrates. Recent studies provide evidence that NCoR/HDAC3 complexes can also exert coactivator functions in brown adipocytes by deacetylating and activating PPARγ coactivator 1α (PGC1α) and that signaling via receptor activator of nuclear factor kappa-B (RANK) promotes the formation of a stable NCoR/HDAC3/PGC1β complex that coactivates nuclear factor kappa-B (NFκB)- and activator protein 1 (AP-1)-dependent genes required for osteoclast differentiation. Here, we demonstrate that activation of Toll-like receptor (TLR) 4, but not TLR3, the interleukin 4 (IL4) receptor nor the Type I interferon receptor, also promotes assembly of an NCoR/HDAC3/PGC1β coactivator complex. Receptor-specific utilization of TNF receptor-associated factor 6 (TRAF6) and downstream activation of extracellular signal-regulated kinase 1 (ERK1) and TANK-binding kinase 1 (TBK1) accounts for the common ability of RANK and TLR4 to drive assembly of an NCoR/HDAC3/PGC1β complex in macrophages. ERK1, the p65 component of NFκB, and the p300 histone acetyltransferase (HAT) are also components of the induced complex and are associated with local histone acetylation and transcriptional activation of TLR4-dependent enhancers and promoters. These observations identify a TLR4/TRAF6-dependent signaling pathway that converts NCoR from a corepressor of nuclear receptors to a coactivator of NFκB and AP-1 that may be relevant to functions of NCoR in other developmental and homeostatic processes.

Epigenetic and Transcriptional Consequences of PIM Kinase Activity and Inhibition in Diffuse Large B-Cell Lymphoma (DLBCL)

The PIM kinase family consists of 3 proto-oncogenic proteins: PIM1, PIM2 and PIM3, expressed in numerous malignancies, including DLBCL. PIM kinases regulate crucial processes, such as proliferation, apoptosis, metabolism, or migration, therefore their inhibition is of great interest as a potential therapeutic strategy. Although recent studies have shed new light on the biological role of PIMs in lymphoid cancers, the details and mechanisms of PIM's oncogenic effects and the consequences of their inhibition in DLBCL remain insufficiently understood. Earlier studies have demonstrated a potential epigenetic role of PIM through histone H3S10 phosphorylation, however, described only at a single locus. The broad genomic role of PIM in modulating transcription in lymphoma cells remains unclear. We confirmed the effect of PIM inhibition on histone modifications using small molecule inhibitors as well as genetic silencing of PIM kinases in DLBCL cell lines. PIM kinase inhibition changed the global amounts of histone modifications (including histone H4 pan-acetylation), accompanied by lower phosphorylation of RNA polymerase II, suggesting an involvement of PIMs in the RNA polymerase II pause release/elongation phase of transcription. Local histone acetylation changes in the enhancer (H3K27ac) and promoter (H3K9ac) regions in response to treatment with the pan-PIM inhibitor were also identified, coupled with gene expression profiling in DLBCL cell lines. PIM inhibition decreased the expression of genes controlled by super-enhancers (SE), i.e. wide regulatory regions responsible for the high level of expression of the most important genes for the cell, including oncogenes. The integrated results of epigenomic and transcriptomic analyses explain the changes in expression of genes associated with many pathways, including transcription, inflammatory response, apoptosis, epigenetic mechanisms or DNA damage. The induction of the latter was further experimentally confirmed by the examination of DNA breaks using γH2AX, the activating phosphorylation of CHK2 or comet assays following PIM inhibition. The conducted studies document an epigenetic function of PIM kinases, and suggest that inhibition of PIM activity leads to alterations in the DLBCL epigenetic landscape and associated changes in transcription. They also indicate that PIM inhibitors can be a therapeutic option in DLBCL. Study supported aby Foundation For Polish Science TEAM Grant # POIR.04.04.00-00-5C84/17 and Polish National Science Centre Grant # 2018/29/B/NZ5/01471.

The SWI/SNF chromatin remodeling factor DPF3 regulates metastasis of ccRCC by modulating TGF-β signaling

DPF3, a component of the SWI/SNF chromatin remodeling complex, has been associated with clear cell renal cell carcinoma (ccRCC) in a genome-wide association study. However, the functional role of DPF3 in ccRCC development and progression remains unknown. In this study, we demonstrate that DPF3a, the short isoform of DPF3, promotes kidney cancer cell migration both in vitro and in vivo, consistent with the clinical observation that DPF3a is significantly upregulated in ccRCC patients with metastases. Mechanistically, DPF3a specifically interacts with SNIP1, via which it forms a complex with SMAD4 and p300 histone acetyltransferase (HAT), the major transcriptional regulators of TGF-β signaling pathway. Moreover, the binding of DPF3a releases the repressive effect of SNIP1 on p300 HAT activity, leading to the increase in local histone acetylation and the activation of cell movement related genes. Overall, our findings reveal a metastasis-promoting function of DPF3, and further establish the link between SWI/SNF components and ccRCC.

Systematic analysis of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs.

Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq (chromatin immunoprecipitation sequencing). We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPβ binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPβ at these locations tolerated changes in spacing ranging from 5 bp increase to >30 bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation.

Differential Activity and Utilization of Glucocorticoid Receptor Binding Sites Yields Transcriptional Heterogeneity

Synthetic glucocorticoids such as Dexamethasone (Dex) are widely prescribed drugs used to treat a variety of human diseases including auto-immune disorders, asthma, cancer, and COVID-19. The transcriptional response to glucocorticoids is elicited by the Glucocorticoid Receptor (GR), which enters the nucleus upon Dex treatment and interacts with thousands of enhancer elements throughout the genome. We recently demonstrated that the Dex response in human breast cancer cells is highly heterogeneous and that individual cells have unique transcriptional responses to Dex. To examine whether this heterogeneity arises from differential utilization of distinct GR-bound enhancers, we focused on the Dex response at the DNA Damage Inducible Transcript 4 (DDIT4) gene. Using a variety of genomic techniques, we identified four GR binding sites (GBSs) 18-30kb upstream of the DDIT4 TSS with differential patterns of chromatin accessibility, histone acetylation, SWI/SNF recruitment, and enhancer RNA (eRNA) transcription. To determine whether these GBSs had unique requirements for DDIT4 transcription, we used CRISPR-CAS9 to generate homozygous deletions of each site. Using ChIP-seq, 4C-seq, single molecule fluorescent in situ hybridization (smFISH), and RT-PCR, we demonstrated GR binding to these GBSs was independent and each GBS deletion had unique effects on DDIT4 and eRNA transcription, local histone acetylation, and chromatin looping. Deletion of any of the first three GBSs resulted in delayed and/or decreased induction of DDIT4 transcription whereas deletion of the fourth GBS resulted in significant upregulation of both DDIT4 and eRNA transcription. Thus, three of the GBSs acted as enhancers of DDIT4 expression while the fourth functioned as a suppressor. Strikingly, smFISH also revealed that these enhancers contributed to cellular heterogeneity, as deleting the GBSs altered the frequency and amplitude of DDIT4 transcription across cell populations. Taken together, these results demonstrate that individual GBSs uniquely contribute to cell-to-cell heterogeneity within the transcriptional response of DDIT4 to Dex. Furthermore, they underscore the possibility that targeted modification of individual GBSs could be utilized to tailor custom, patient-specific strategies for the treatment of human diseases.

Epigenomes of Human Hearts Reveal New Genetic Variants Relevant for Cardiac Disease and Phenotype.

Identifying genetic markers for heterogeneous complex diseases such as heart failure is challenging and requires prohibitively large cohort sizes in genome-wide association studies to meet the stringent threshold of genome-wide statistical significance. On the other hand, chromatin quantitative trait loci, elucidated by direct epigenetic profiling of specific human tissues, may contribute toward prioritizing subthreshold variants for disease association. Here, we captured noncoding genetic variants by performing epigenetic profiling for enhancer H3K27ac chromatin immunoprecipitation followed by sequencing in 70 human control and end-stage failing hearts. We have mapped a comprehensive catalog of 47 321 putative human heart enhancers and promoters. Three thousand eight hundred ninety-seven differential acetylation peaks (FDR [false discovery rate], 5%) pointed to pathways altered in heart failure. To identify cardiac histone acetylation quantitative trait loci (haQTLs), we regressed out confounding factors including heart failure disease status and used the G-SCI (Genotype-independent Signal Correlation and Imbalance) test1 to call out 1680 haQTLs (FDR, 10%). RNA sequencing performed on the same heart samples proved a subset of haQTLs to have significant association also to gene expression (expression quantitative trait loci), either in cis (180) or through long-range interactions (81), identified by Hi-C (high-throughput chromatin conformation assay) and HiChIP (high-throughput protein centric chromatin) performed on a subset of hearts. Furthermore, a concordant relationship between the gain or disruption of TF (transcription factor)-binding motifs, inferred from alternative alleles at the haQTLs, implied a surprising direct association between these specific TF and local histone acetylation in human hearts. Finally, 62 unique loci were identified by colocalization of haQTLs with the subthreshold loci of heart-related genome-wide association studies datasets. Disease and phenotype association for 62 unique loci are now implicated. These loci may indeed mediate their effect through modification of enhancer H3K27 acetylation enrichment and their corresponding gene expression differences (bioRxiv: https://doi.org/10.1101/536763). Graphical Abstract: A graphical abstract is available for this article.

Abstract 368: Disease and Phenotype-relevant Genetic Variants Identified From Histone Acetylomes in Human Hearts

Identifying genetic markers for heterogeneous complex diseases such as heart failure has been challenging, and may require prohibitively large cohort sizes in genome-wide association studies (GWAS) in order to meet genome-wide statistical significance. On the other hand, chromatin quantitative trait loci (QTL), elucidated by direct epigenetic profiling of specific human tissues, may contribute towards prioritising sub-threshold variants for disease-association. Here, we captured non-coding genetic variants by performing enhancer H3K27ac ChIP-seq in 70 human control and end-stage failing hearts, mapping out a comprehensive catalogue of 47,321 putative human heart enhancers. 3,897 differential acetylation peaks (FDR 5%) pointed to pathways altered in heart failure (HF). To identify cardiac histone acetylation QTLs (haQTLs), we regressed out confounding factors including HF disease status, and employed the G-SCI test to call out 1,680 haQTLs (FDR 10%). RNA-seq performed on the same heart samples proved a subset of haQTLs to have significant association also to gene expression (expression QTLs), either in cis (180), or through long range interactions (81), identified by Hi-C and Hi-ChIP performed on a subset of hearts. We validated 2 of the haQTLs through base editing to show that the presence of those SNPs indeed affects gene expression in human embryonic stem cells-derived cardiomyocytes. Furthermore, a concordant relationship between the gain or disruption of transcription factor (TF) binding motifs, inferred from alternative alleles at the haQTLs, implied a surprising direct association between these specific TF and local histone acetylation in human hearts. Finally, 62 unique loci were identified by colocalisation of haQTLs with heart-related GWAS datasets. Disease-association for these new loci may indeed be mediated through modification of H3K27-acetylation enrichment and their corresponding gene expression differences.

Role of Histone Acetylation in Cell Cycle Regulation.

Core histone acetylation is a key prerequisite for chromatin decondensation and plays a pivotal role in regulation of chromatin structure, function and dynamics. The addition of acetyl groups disturbs histone/DNA interactions in the nucleosome and alters histone/histone interactions in the same or adjacent nucleosomes. Acetyl groups can also provide binding sites for recruitment of bromodomain (BRD)-containing non-histone readers and regulatory complexes to chromatin allowing them to perform distinct downstream functions. The presence of a particular acetylation pattern influences appearance of other histone modifications in the immediate vicinity forming the "histone code". Although the roles of the acetylation of particular lysine residues for the ongoing chromatin functions is largely studied, the epigenetic inheritance of histone acetylation is a debated issue. The dynamics of local or global histone acetylation is associated with fundamental cellular processes such as gene transcription, DNA replication, DNA repair or chromatin condensation. Therefore, it is an essential part of the epigenetic cell response to processes related to internal and external signals.

A histone modification identifies a DNA element controlling slo BK channel gene expression in muscle

The slo gene encodes the BK-type Ca2+-activated K+ channels. In Drosophila, expression of slo is induced by organic solvent sedation (benzyl alcohol and ethanol), and this increase in neural slo expression contributes to the production of functional behavioral tolerance (inducible resistance) to these drugs. Within the slo promoter region, we observed that benzyl alcohol sedation produces a localized spike of histone acetylation over a 65-nucleotide (65-n) conserved DNA element called 55b. Changes in histone acetylation are commonly the consequence of transcription factor activity, and previously, a localized histone acetylation spike was used to successfully map a DNA element involved in benzyl alcohol-induced slo expression. To determine whether the 55b element was also involved in benzyl alcohol-induced neural expression of slo, we deleted it from the endogenous slo gene by homologous recombination. Flies lacking the 55b element were normal with respect to basal and benzyl alcohol-induced neural slo expression, the capacity to acquire and maintain functional tolerance, their threshold for electrically-induced seizures, and most slo-related behaviors. Removal of the 55b element did however increase the level of basal expression from the muscle/tracheal cell-specific slo core promoter and produced a slight increase in overall locomotor activity. We conclude that the 55b element is involved in control of slo expression from the muscle and tracheal-cell promoter but is not involved in the production of functional benzyl alcohol tolerance.

H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress.

The selectivity of transcriptional responses to extracellular cues is reflected by the deposition of stimulus-specific chromatin marks. Although histone H3 phosphorylation is a target of numerous signaling pathways, its role in transcriptional regulation remains poorly understood. Here, for the first time, we report a genome-wide analysis of H3S28 phosphorylation in a mammalian system in the context of stress signaling. We found that this mark targets as many as 50% of all stress-induced genes, underlining its importance in signal-induced transcription. By combining ChIP-seq, RNA-seq, and mass spectrometry we identified the factors involved in the biological interpretation of this histone modification. We found that MSK1/2-mediated phosphorylation of H3S28 at stress-responsive promoters contributes to the dissociation of HDAC corepressor complexes and thereby to enhanced local histone acetylation and subsequent transcriptional activation of stress-induced genes. Our data reveal a novel function of the H3S28ph mark in the activation of mammalian genes in response to MAP kinase pathway activation.

Nuclear receptor co-repressors are required for the histone-deacetylase activity of HDAC3 in vivo

Histone deacetylase 3 (HDAC3) is an epigenome-modifying enzyme that is required for normal mouse development and tissue-specific functions. In vitro, HDAC3 protein itself has minimal enzyme activity, but gains its histone deacetylation function from stable association with the conserved deacetylase activation domain (DAD) contained in nuclear receptor corepressors NCOR1 and SMRT. Here we show that HDAC3 enzyme activity is undetectable in mice bearing point mutations in the DAD of both NCOR1 and SMRT (NS-DADm), despite normal levels of HDAC3 protein. Local histone acetylation is increased, and genomic HDAC3 recruitment is reduced though not abrogated. Remarkably, the NS-DADm mice are born and live to adulthood, whereas genetic deletion of HDAC3 is embryonic lethal. These findings demonstrate that nuclear receptor corepressors are required for HDAC3 enzyme activity in vivo, and suggest that a deacetylase-independent function of HDAC3 may be required for life.

Oxidative Damage Targets Complexes Containing DNA Methyltransferases, SIRT1, and Polycomb Members to Promoter CpG Islands

Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an invivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.

Nucleotide supply, not local histone acetylation, sets replication origin usage in transcribed regions

In eukaryotes, only a fraction of replication origins fire at each S phase. Local histone acetylation was proposed to control firing efficiency of origins, but conflicting results were obtained. We report that local histone acetylation does not reflect origin efficiencies along the adenosine monophosphate deaminase 2 locus in mammalian fibroblasts. Reciprocally, modulation of origin efficiency does not affect acetylation. However, treatment with a deacetylase inhibitor changes the initiation pattern. We demonstrate that this treatment alters pyrimidine biosynthesis and decreases fork speed, which recruits latent origins. Our findings reconcile results that seemed inconsistent and reveal an unsuspected effect of deacetylase inhibitors on replication dynamics.

A Role for Gcn5 in Replication-Coupled Nucleosome Assembly

Acetylation of lysine residues at the H3 N terminus is proposed to function in replication-coupled (RC) nucleosome assembly, a process critical for the inheritance of epigenetic information and maintenance of genome stability. However, the role of H3 N-terminal lysine acetylation and the corresponding lysine acetyltransferase (KAT) in RC nucleosome assembly are not known. Here we show that Gcn5, a KAT that functions in transcription, works in parallel with Rtt109, the H3 lysine 56 KAT, to promote RC nucleosome assembly. Cells lacking both Gcn5 and Rtt109 are highly sensitive to DNA damaging agents. Moreover, cells lacking GCN5 or those that express N-terminal H3 mutants are compromised for deposition of new H3 onto replicating DNA and also show reduced binding of H3 to CAF-1, a histone chaperone involved in RC nucleosome assembly. These results demonstrate that Gcn5 regulates RC nucleosome assembly, in part, by promoting H3 association with CAF-1 via H3 acetylation.

Two New Pimelic Diphenylamide HDAC Inhibitors Induce Sustained Frataxin Upregulation in Cells from Friedreich's Ataxia Patients and in a Mouse Model

BackgroundFriedreich's ataxia (FRDA), the most common recessive ataxia in Caucasians, is due to severely reduced levels of frataxin, a highly conserved protein, that result from a large GAA triplet repeat expansion within the first intron of the frataxin gene (FXN). Typical marks of heterochromatin are found near the expanded GAA repeat in FRDA patient cells and mouse models. Histone deacetylase inhibitors (HDACIs) with a pimelic diphenylamide structure and HDAC3 specificity can decondense the chromatin structure at the FXN gene and restore frataxin levels in cells from FRDA patients and in a GAA repeat based FRDA mouse model, KIKI, providing an appealing approach for FRDA therapeutics.Methodology/Principal FindingsIn an effort to further improve the pharmacological profile of pimelic diphenylamide HDACIs as potential therapeutics for FRDA, we synthesized additional compounds with this basic structure and screened them for HDAC3 specificity. We characterized two of these compounds, 136 and 109, in FRDA patients' peripheral blood lymphocytes and in the KIKI mouse model. We tested their ability to upregulate frataxin at a range of concentrations in order to determine a minimal effective dose. We then determined in both systems the duration of effect of these drugs on frataxin mRNA and protein, and on total and local histone acetylation. The effects of these compounds exceeded the time of direct exposure in both systems.Conclusions/SignificanceOur results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACIs for FRDA and provide information for the design of future human trials of these drugs, suggesting an intermittent administration of the drug.

The Reelin and GAD67 Promoters Are Activated by Epigenetic Drugs That Facilitate the Disruption of Local Repressor Complexes

The epigenetic down-regulation of genes is emerging as a possible underlying mechanism of the GABAergic neuron dysfunction in schizophrenia. For example, evidence has been presented to show that the promoters associated with reelin and GAD67 are down-regulated as a consequence of DNA methyltransferase (DNMT)-mediated hypermethylation. Using neuronal progenitor cells to study this regulation, we have previously demonstrated that DNMT inhibitors coordinately increase reelin and GAD67 mRNAs. Here, we report that another group of epigenetic drugs, histone deacetylase (HDAC) inhibitors, activate these two genes with dose and time dependence comparable with that of DNMT inhibitors. In parallel, both groups of drugs decrease DNMT1, DNMT3A, and DNMT3B protein levels and reduce DNMT enzyme activity. Furthermore, induction of the reelin and GAD67 mRNAs is accompanied by the dissociation of repressor complexes containing all three DNMTs, MeCP2, and HDAC1 from the corresponding promoters and by increased local histone acetylation. Our data imply that drug-induced promoter demethylation is relevant for maximal activation of reelin and GAD67 transcription. The results suggest that HDAC and DNMT inhibitors activate reelin and GAD67 expression through similar mechanisms. Both classes of drugs attenuate, directly or indirectly, the enzymatic and transcriptional repressor activities of DNMTs and HDACs. These data provide a mechanistic rationale for the use of epigenetic drugs, individually or in combination, as a potential novel therapeutic strategy to alleviate deficits associated with schizophrenia.

Estrogen Induces Estrogen-related Receptor α Gene Expression and Chromatin Structural Changes in Estrogen Receptor (ER)-positive and ER-negative Breast Cancer Cells

Estrogen-related receptor alpha (ERRalpha), a member of the nuclear receptor superfamily, is closely related to the estrogen receptors (ERalpha and ERbeta). The ERRalpha gene is estrogen-responsive in several mouse tissues and cell lines, and a multiple hormone-response element (MHRE) in the promoter is an important regulatory region for estrogen-induced ERRalpha gene expression. ERRalpha was recently shown to be a negative prognostic factor for breast cancer survival, with its expression being highest in cancer cells lacking functional ERalpha. The contribution of ERRalpha in breast cancer progression remains unknown but may have important clinical implications. In this study, we investigated ERRalpha gene expression and chromatin structural changes under the influence of 17beta-estradiol in both ER-positive MCF-7 and ER-negative SKBR3 breast cancer cells. We mapped the nucleosome positions of the ERRalpha promoter around the MHRE region and found that the MHRE resides within a single nucleosome. Local chromatin structure of the MHRE exhibited increased restriction enzyme hypersensitivity and enhanced histone H3 and H4 acetylation upon estrogen treatment. Interestingly, estrogen-induced chromatin structural changes could be repressed by estrogen antagonist ICI 182 780 in MCF-7 cells yet were enhanced in SKBR3 cells. We demonstrated, using chromatin immunoprecipitation assays, that 17beta-estradiol induces ERRalpha gene expression in MCF-7 cells through active recruitment of co-activators and release of co-repressors when ERRalpha and AP1 bind and ERalpha is tethered to the MHRE. We also found that this estrogen effect requires the MAPK signaling pathway in both cell lines.

Krüppel-like Factor 4 Is Acetylated by p300 and Regulates Gene Transcription via Modulation of Histone Acetylation

Colon cancer is the second leading cause of cancer death in the United States. Krüppel-like factor 4 (KLF4) is a transcription factor involved in both proliferation and differentiation in the colon. It is down-regulated in both mouse and human colonic adenomas and has been implicated as a tumor suppressor in the gut, whereas in breast cancer, KLF4 is an oncogene. KLF4 is also involved in reprogramming differentiated cells into pluripotent stem cells. KLF4 can act as a transcriptional activator or repressor, but the underlying mechanisms are poorly understood. We found that p300, a CREB-binding protein-related protein, interacts with KLF4 both in vitro and in vivo and activates transcription. We further made the novel observation that endogenous KLF4 is acetylated by p300/CBP in vivo and that mutations of the acetylated lysines resulted in a decreased ability of KLF4 to activate target genes, suggesting that acetylation is important for KLF4-mediated transactivation. Furthermore, we found that KLF4 differentially modulates histone H4 acetylation at the promoters of target genes. Co-transfection of KLF4 and HDAC3 resulted in a synergistic repression of a cyclin B(1) reporter construct. Our results suggest that KLF4 might function as an activator or repressor of transcription depending on whether it interacts with co-activators such as p300 and CREB-binding protein or co-repressors such as HDAC3.

Identification of a HoxA10 Activation Domain Necessary for Transcription of the Gene Encoding β3 Integrin during Myeloid Differentiation

Transcription of the ITGB3 gene, which encodes beta3 integrin, increases during myeloid differentiation. alphavbeta3 integrin mediates adhesion to fibronectin or vitronectin and regulates various aspects of the inflammatory response in mature phagocytes. In these studies, we found that the homeodomain transcription factor HoxA10 interacted with a specific ITGB3 cis element and activated transcription of this gene during myeloid differentiation. We also found that increased fibronectin adhesion in differentiating myeloid cells was dependent upon this HoxA10-induced increase in beta3 integrin expression. We determined that activation of ITGB3 transcription required a HoxA10 domain that was not identical to the "hexapeptide" that mediates interaction of Hox and Pbx proteins. This activation domain was also not identical to a previously identified HoxA10 repression domain that mediates interaction with transcriptional co-repressors. Instead, this HoxA10 activation domain had homology to "PQ" protein-protein interaction domains that have been described previously in other transcription factors. Consistent with this, we found that the HoxA10 PQ-like domain recruited the CREB-binding protein (CBP) to the ITGB3 promoter. This was associated with an increase in local histone acetylation in vivo. In immature myeloid cells, we previously determined that HoxA10 repressed transcription of the CYBB and NCF2 genes, which encode the phagocyte oxidase proteins gp91(PHOX) and p67(PHOX), respectively. Therefore, our studies indicated that HoxA10 either activates or represses gene transcription at various points during myelopoiesis. Our studies also suggested that HoxA10 is a bifunctional protein that is involved in dynamic regulation of multiple aspects of phagocyte phenotype and function.

Role of DNA Methylation in Stable Gene Repression

A large fraction of the animal genome is maintained in a transcriptionally repressed state throughout development. By generating viable Dnmt1(-)(/)(-) mouse cells we have been able to study the effect of DNA methylation on both gene expression and chromatin structure. Our results confirm that the underlying methylation pattern has a profound effect on histone acetylation and is the major effector of me-H3(K4) in the animal genome. We demonstrate that many methylated genes are subject to additional repression mechanisms that also impact on histone acetylation, and the data suggest that late replication timing may play an important role in this process.