ChIP-chip Analysis Research Articles

Deciphering the Transcriptional-Regulatory Network of Flocculation in Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, the transcriptional-regulatory network that governs flocculation remains poorly understood. Here, we systematically screened an array of transcription factor deletion and overexpression strains for flocculation and performed microarray expression profiling and ChIP–chip analysis to identify the flocculin target genes. We identified five transcription factors that displayed novel roles in the activation or inhibition of flocculation (Rfl1, Adn2, Adn3, Sre2, and Yox1), in addition to the previously-known Mbx2, Cbf11, and Cbf12 regulators. Overexpression of mbx2+ and deletion of rfl1+ resulted in strong flocculation and transcriptional upregulation of gsf2 +/pfl1+ and several other putative flocculin genes (pfl2+–pfl9+). Overexpression of the pfl+ genes singly was sufficient to trigger flocculation, and enhanced flocculation was observed in several combinations of double pfl+ overexpression. Among the pfl1+ genes, only loss of gsf2+ abrogated the flocculent phenotype of all the transcription factor mutants and prevented flocculation when cells were grown in inducing medium containing glycerol and ethanol as the carbon source, thereby indicating that Gsf2 is the dominant flocculin. In contrast, the mild flocculation of adn2+ or adn3+ overexpression was likely mediated by the transcriptional activation of cell wall–remodeling genes including gas2+, psu1+, and SPAC4H3.03c. We also discovered that Mbx2 and Cbf12 displayed transcriptional autoregulation, and Rfl1 repressed gsf2+ expression in an inhibitory feed-forward loop involving mbx2+. These results reveal that flocculation in S. pombe is regulated by a complex network of multiple transcription factors and target genes encoding flocculins and cell wall–remodeling enzymes. Moreover, comparisons between the flocculation transcriptional-regulatory networks of Saccharomyces cerevisiae and S. pombe indicate substantial rewiring of transcription factors and cis-regulatory sequences.

14-3-3ζ, a Novel Androgen-Responsive Gene, Is Upregulated in Prostate Cancer and Promotes Prostate Cancer Cell Proliferation and Survival

Androgen receptor is an essential transcriptional factor that contributes to the development and progression of prostate cancer. In this study, we investigated the androgen regulation and functional analysis of 14-3-3ζ in prostate cancer. Using chromatin immunoprecipitation (ChIP) combined with DNA microarray (ChIP-chip) analysis in LNCaP cells, we identified a functional androgen receptor-binding site in the downstream region of the 14-3-3ζ gene. Androgen regulation was examined by quantitative reverse transcription PCR and Western blot analysis. Prostate cancer cells stably expressing 14-3-3ζ and siRNA knockdown were used for functional analyses. We further examined 14-3-3ζ expression in clinical samples of prostate cancer by immunohistochemistry and quantitative reverse transcription PCR. Androgen-dependent upregulation of 14-3-3ζ was validated at the mRNA and protein levels. The 14-3-3ζ gene is favorable for cancer-cell survival, as its ectopic expression in LNCaP cells contributes to cell proliferation and the acquired resistance to etoposide-induced apoptosis. 14-3-3ζ expression was associated with androgen receptor transcriptional activity and prostate-specific antigen (PSA) mRNA expression. Immunoprecipitation indicated that 14-3-3ζ was associated with androgen receptor in the nucleus. Clinicopathologic studies further support the relevance of 14-3-3ζ in prostate cancers, as its higher expression is associated with malignancy and lymph node metastasis. 14-3-3ζ is a novel androgen-responsive gene that activates proliferation, cell survival, and androgen receptor transcriptional activity. 14-3-3ζ may facilitate the progression of prostate cancer.

Accurate identification of non-polyadenylated RNA using a custom human cytomegalovirus tiling array

Most eukaryotes regulate commonly translation by polyadenylation of messenger RNA. RNA sequences without 3' polyadenylation are usually quickly degraded, thus finely tuning the expression of gene transcripts. Some viruses are known to regulate mRNA levels via polyadenylation. A method is described that utilizes a customized tiling array to determine if any human cytomegalovirus (HCMV) transcripts are subject to regulation via de-polyadenylation and subsequent mRNA decay. The results of this study indicate that the highly expressed intron of large non-coding RNA (lncRNA5) that is not polyadenylated, as documented previously. This differential polyadenylation protocol can also be modified for other tiling array applications (such as ChIP-chip analysis).

Enrichment of HP1a on Drosophila Chromosome 4 Genes Creates an Alternate Chromatin Structure Critical for Regulation in this Heterochromatic Domain

Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP–chip) analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs), HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low Tm downstream of the TSS, characteristics that could contribute to a low incidence of RNA polymerase pausing. Our data also indicate that EGG and POF jointly regulate H3K9 methylation and promote HP1a binding over gene bodies, while HP1a targeting and H3K9 methylation are maintained at the repeats by an independent mechanism. The HP1a-enriched, POF-associated chromatin structure over the gene bodies may represent one type of adaptation for genes embedded in repetitive DNA.

Global genome analysis of the downstream binding targets of testis determining factor SRY and SOX9.

A major event in mammalian male sex determination is the induction of the testis determining factor Sry and its downstream gene Sox9. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. A modified ChIP-Chip analysis using a comparative hybridization was used to identify 71 direct downstream binding targets for SRY and 109 binding targets for SOX9. Interestingly, only 5 gene targets overlapped between SRY and SOX9. In addition to the direct response element binding gene targets, a large number of atypical binding gene targets were identified for both SRY and SOX9. Bioinformatic analysis of the downstream binding targets identified gene networks and cellular pathways potentially involved in the induction of Sertoli cell differentiation and testis development. The specific DNA sequence binding site motifs for both SRY and SOX9 were identified. Observations provide insights into the molecular control of male gonadal sex determination.

Negative Regulation of JAK2 by H3K9 Methyltransferase G9a in Leukemia

Histone methylation at specific lysine residues is a crucial regulatory process in transcriptional regulation. Using chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis, we found that the H3K9-me2 target gene JAK2 was an important factor during differentiation of the HL-60 promyelocytic leukemia cell line by all-trans-retinoic acid (ATRA) treatment. Here, we report that the H3K9 methyltransferase G9a negatively regulated JAK2 transcription in histone methyltransferase activity and in a YY1-dependent manner during ATRA-mediated leukemia cell differentiation. We found that G9a knockdown repressed ATRA-mediated HL-60 cell differentiation. We demonstrated that G9a interacts with YY1 and is recruited to the JAK2 promoter along with corepressors, including histone deacetylase, that induced H3K9-me2. Repression of JAK2 transcription by G9a decreased H3Y41 phosphorylation and promoted inhibition of the recently identified JAK2-H3Y41P-HP1α pathway-mediated leukemogenesis.

High-Temperature Requirement Protein A4 (HtrA4) Suppresses the Fusogenic Activity of Syncytin-1 and Promotes Trophoblast Invasion

Cell-cell fusion and cell invasion are essential for placental development. Human cytotrophoblasts in the chorionic villi may undergo cell-cell fusion to form syncytiotrophoblasts to facilitate nutrient-gas exchange or differentiate into extravillous trophoblasts (EVTs) to facilitate maternal-fetal circulation. The placental transcription factor glial cells missing 1 (GCM1) regulates syncytin-1 and -2 expression to mediate trophoblast fusion. Interestingly, GCM1 and syncytin-1 are also expressed in EVTs with unknown physiological functions. In this study, we performed chromatin immunoprecipitation-on-chip (ChIP-chip) analysis and identified the gene for high-temperature requirement protein A4 (HtrA4) as a GCM1 target gene, which encodes a serine protease facilitating cleavage of fibronectin and invasion of placental cells. Importantly, HtrA4 is immunolocalized in EVTs at the maternal-fetal interface, and its expression is decreased by hypoxia and in preeclampsia, a pregnancy complication associated with placental hypoxia and shallow trophoblast invasion. We further demonstrate that HtrA4 interacts with syncytin-1 and suppresses cell-cell fusion. Therefore, HtrA4 may be crucial for EVT differentiation by playing a dual role in prevention of cell-cell fusion of EVTs and promotion of their invasion into the uterus. Our study reveals a novel function of GCM1 and HtrA4 in regulation of trophoblast invasion and that abnormal HrtA4 expression may contribute to shallow trophoblast invasion in preeclampsia.

KDM3B Is the H3K9 Demethylase Involved in Transcriptional Activation of lmo2 in Leukemia

Histone lysine methylation and demethylation are considered critical steps in transcriptional regulation. In this report, we performed chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis to examine the genome-wide occupancy of H3K9-me2 during all-trans-retinoic acid (ATRA)-induced differentiation of HL-60 promyelocytic leukemia cells. Using this approach, we found that KDM3B, which contains a JmjC domain, was downregulated during differentiation through the recruitment of a corepressor complex. Furthermore, KDM3B displayed histone H3K9-me1/2 demethylase activity and induced leukemogenic oncogene lmo2 expression via a synergistic interaction with CBP. Here, we found that KDM3B repressed leukemia cell differentiation and was upregulated in blood cells from acute lymphoblastic leukemia (ALL)-type leukemia patients. The combined results of this study provide evidence that the H3K9-me1/2 demethylase KDM3B might play a role in leukemogenesis via activation of lmo2 through interdependent actions with the histone acetyltransferase (HAT) complex containing CBP.

HP1a Targets the Drosophila KDM4A Demethylase to a Subset of Heterochromatic Genes to Regulate H3K36me3 Levels

The KDM4 subfamily of JmjC domain-containing demethylases mediates demethylation of histone H3K36me3/me2 and H3K9me3/me2. Several studies have shown that human and yeast KDM4 proteins bind to specific gene promoters and regulate gene expression. However, the genome-wide distribution of KDM4 proteins and the mechanism of genomic-targeting remain elusive. We have previously identified Drosophila KDM4A (dKDM4A) as a histone H3K36me3 demethylase that directly interacts with HP1a. Here, we performed H3K36me3 ChIP-chip analysis in wild type and dkdm4a mutant embryos to identify genes regulated by dKDM4A demethylase activity in vivo. A subset of heterochromatic genes that show increased H3K36me3 levels in dkdm4a mutant embryos overlap with HP1a target genes. More importantly, binding to HP1a is required for dKDM4A-mediated H3K36me3 demethylation at a subset of heterochromatic genes. Collectively, these results show that HP1a functions to target the H3K36 demethylase dKDM4A to heterochromatic genes in Drosophila.

Structure and regulon of Campylobacter jejuni ferric uptake regulator Fur define apo-Fur regulation

The full regulatory potential of the ferric uptake regulator (Fur) family of proteins remains undefined despite over 20 years of study. We report herein an integrated approach that combines both genome-wide technologies and structural studies to define the role of Fur in Campylobacter jejuni (Cj). CjFur ChIP-chip assays identified 95 genomic loci bound by CjFur associated with functions as diverse as iron acquisition, flagellar biogenesis, and non-iron ion transport. Comparative analysis with transcriptomic data revealed that CjFur regulation extends beyond solely repression and also includes both gene activation and iron-independent regulation. Computational analysis revealed the presence of an elongated holo-Fur repression motif along with a divergent holo-Fur activation motif. This diversity of CjFur DNA-binding elements is supported by the crystal structure of CjFur, which revealed a unique conformation of its DNA-binding domain and the absence of metal in the regulatory site. Strikingly, our results indicate that the apo-CjFur structure retains the canonical V-shaped dimer reminiscent of previously characterized holo-Fur proteins enabling DNA interaction. This conformation stems from a structurally unique hinge domain that is poised to further contribute to CjFur's regulatory functions by modulating the orientation of the DNA-binding domain upon binding of iron. The unique features of the CjFur crystal structure rationalize the binding sequence diversity that was uncovered during ChIP-chip analysis and defines apo-Fur regulation.

Dietary Fat Is a Lipid Source in 2,3,7,8-Tetrachlorodibenzo-ρ-Dioxin (TCDD)-Elicited Hepatic Steatosis in C57BL/6 Mice

2,3,7,8-Tetrachlorodibenzo-ρ-dioxin (TCDD) increases fatty acid (FA) transport and FA levels resulting in hepatic steatosis in mice. Diet as a source of lipids was investigated using customized diets, stearoyl-CoA desaturase 1 (Scd1) null mice, and (14)C-oleate (18:1n9) uptake studies. C57BL/6 mice fed with 5, 10, or 15% fat or 50, 60 or 70% carbohydrate diets exhibited increased relative liver weight following gavage with 30 µg/kg TCDD for 168 h. Hepatic lipid extract analysis from mice fed with 5, 10, and 15% fat diets identified a dose-dependent increase in total FAs induced by TCDD. Mice fed with fat diet also exhibited a dose-dependent increase in the dietary essential linoleic (18:2n6) and α-linolenic (18:3n3) acids. No dose-dependent FA increase was detected on carbohydrate diets, suggesting dietary fat as a source of lipids in TCDD-induced steatosis as opposed to de novo lipogenesis. TCDD also induced oleate levels threefold in Scd1 null mice that are incapable of desaturating stearate (18:0). This is consistent with oleate representing > 90% of all monounsaturated FAs in rodent chow. Moreover, TCDD increased hepatic (14)C-oleate levels twofold in wild type and 2.4-fold in Scd1 null mice concurrent with the induction of intestinal and hepatic lipid transport genes (Slc27a, Fabp, Ldlr, Cd36, and Apob). In addition, computational scanning identified putative dioxin response elements and in vivo ChIP-chip analysis revealed regions of aryl hydrocarbon receptor (AhR) enrichment in lipid transport genes differentially regulated by TCDD. Collectively, these results suggest the AhR mediates increased uptake of dietary fats that contribute to TCDD-elicited hepatic steatosis.

Abstract 2219: Mechanism of trancriptional repression by EWS/FLI in Ewing's sarcoma

Abstract Objective: Ewing's sarcoma is a highly aggressive bone-associated malignancy affecting children and young adults with most cases harboring a somatic translocation that encodes the EWS/FLI oncoprotein. EWS/FLI is an aberrant transcription factor. Most studies indicate that EWS/FLI functions as a transcriptional activator. However, recent genome-wide transcriptional studies have raised the possibility that EWS/FLI may also repress target genes, although the mechanistic basis for this repression is unknown. We sought to test the hypothesis that EWS/FLI functions as a direct transcriptional repressor at some genetic loci, and that transcriptional repression is required for the oncogenic function of EWS/FLI. Furthermore, we sought to define relevant domains and corepressors that are involved in this activity. Methods: RNAi based microarray combined with genome-wide ChIP-Chip analyses were used to identify direct repressed target genes of EWS/FLI. Soft agar and xenograft tumor formation assays were performed to assess oncogenic transformation. Deletion mutants of EWS/FLI were constructed and used to map the repression domain. Co-immunoprecipitations were performed to validate the interaction of EWS/FLI with transcriptional co-repressors. Luciferase reporter assays and gel shift assays were utilized to characterize the binding site for EWS/FLI at repressed gene promoters. Results: We identified a subset of EWS/FLI-repressed target genes that are directly bound by the protein. Using these target genes, we identified domains in EWS/FLI necessary and sufficient for repression. Interestingly, these repression domains are also necessary for oncogenic transformation. We identified two HDACs that are involved in this repression, and identified a likely candidate transcriptional repression complex that binds EWS/FLI and contributes to its repressive function. Finally, we found that transcriptional repression is absolutely required for the oncogenic function of EWS/FLI, as two downregulated targets function as suppressors of Ewing's sarcoma oncogenesis. Conclusion: Transcriptional deregulation by EWS/FLI is a key oncogenic event in Ewing's sarcoma pathogenesis. Our data demonstrated that, in addition to transcriptional activation, EWS/FLI mediated transcriptional repression is also critical in Ewing's sarcoma oncogenesis. This is the first time that a detailed molecular mechanism of EWS/FLI mediated direct repression is reported, and has important implications for therapeutic targeting of EWS/FLI in Ewing's sarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2219. doi:1538-7445.AM2012-2219

Genome‐wide identification of SOC1 and SVP targets during the floral transition in Arabidopsis

Floral transition in Arabidopsis is tightly controlled by complex genetic regulatory networks in response to endogenous and environmental flowering signals. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and SHORT VEGETATIVE PHASE (SVP), two key MADS-domain transcription factors, perceive these signals and function as antagonistic flowering regulators. To understand how these factors mediate floral transition, we mapped in vivo binding sites of SOC1 and SVP using chromatin immunoprecipitation followed by hybridization to whole-genome tiling arrays (ChIP-chip). Genes that encoded proteins with transcription regulator activity and transcription factor activity were the most enriched groups of genes of those bound by SOC1 and SVP, which indicates their central roles in flowering regulatory networks. In combination with gene expression microarray studies, we further identified the genes whose expression was controlled directly by SOC1 or SVP. Among the common direct targets identified, APETALA2 (AP2)-like genes that repress FT and SOC1 expression were down-regulated by SOC1, but up-regulated by SVP, revealing a complex feedback regulation among the key genes that determine the integration of flowering signals. SOC1 regulatory regions were also accessed by SOC1 itself and SVP, suggesting that self-activation and repression by SVP contribute to the control of SOC1 expression. In addition, ChIP-chip analysis demonstrated that miR156e and miR172a, which are involved in the regulation of AP2-like genes, were direct targets of SOC1 and SVP, respectively. Taken together, these findings revealed that feedback regulatory loops mediated by SOC1 and SVP are essential components of the gene regulatory networks that underpin the integration of flowering signals during floral transition.

Chromatin modifications induced by the AML1-ETO fusion protein reversibly silence its genomic targets through AML1 and Sp1 binding motifs

The AML1-ETO fusion protein, which is present in 10-15% of cases of acute myeloid leukemia, is known to repress myeloid differentiation genes through DNA binding and recruitment of chromatin-modifying proteins and transcription factors in target genes. ChIP-chip analysis of human hematopoietic stem/progenitor cells transduced with the AML1-ETO fusion gene enabled us to identify 1168 AML1-ETO target genes, 103 of which were co-occupied by histone deacetylase 1 (HDAC1) and had lost the hyperacetylation mark at histone H4, and 264 showed a K9 trimethylation at histone H3. Enrichment of genes involved in hematopoietic differentiation and in specific signaling pathways was observed in the presence of these epigenetic modifications associated with an 'inactive' chromatin status. Furthermore, AML1-ETO target genes had a significant correlation between the chromatin marks studied and transcriptional silencing. Interestingly, AML1 binding sites were absent on a large number of selected AML1-ETO promoters and an Sp1 binding site was found in over 50% of them. Reversible silencing induced by the fusion protein in the presence of AML1 and/or Sp1 transcription factor binding site was confirmed. Therefore, this study provides a global analysis of AML1-ETO functional chromatin modifications and identifies the important role of Sp1 in the DNA binding pattern of AML1-ETO, suggesting a role for Sp1-targeted therapy in this leukemia subtype.

The bZIP Transcription Factor Rca1p Is a Central Regulator of a Novel CO2 Sensing Pathway in Yeast

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO2 concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO2 and identify the bZIP transcription factor Rca1p as the first CO2 regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO2 build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO2 sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO2 availability in environments as diverse as the phagosome, yeast communities or liquid culture.

H3K4me3 inversely correlates with DNA methylation at a large class of non-CpG-island-containing start sites.

BackgroundIn addition to mutations, epigenetic silencing of genes has been recognized as a fundamental mechanism that promotes human carcinogenesis. To date, characterization of epigenetic gene silencing has largely focused on genes in which silencing is mediated by hypermethylation of promoter-associated CpG islands, associated with loss of the H3K4me3 chromatin mark. Far less is known about promoters lacking CpG-islands or genes that are repressed by alternative mechanisms.MethodsWe performed integrative ChIP-chip, DNase-seq, and global gene expression analyses in colon cancer cells and normal colon mucosa to characterize chromatin features of both CpG-rich and CpG-poor promoters of genes that undergo silencing in colon cancer.ResultsEpigenetically repressed genes in colon cancer separate into two classes based on retention or loss of H3K4me3 at transcription start sites. Quantitatively, of transcriptionally repressed genes that lose H3K4me3 in colon cancer (K4-dependent genes), a large fraction actually lacks CpG islands. Nonetheless, similar to CpG-island containing genes, cytosines located near the start sites of K4-dependent genes become DNA hypermethylated, and repressed K4-dependent genes can be reactivated with 5-azacytidine. Moreover, we also show that when the H3K4me3 mark is retained, silencing of CpG island-associated genes can proceed through an alternative mechanism in which repressive chromatin marks are recruited.ConclusionsH3K4me3 equally protects from DNA methylation at both CpG-island and non-CpG island start sites in colon cancer. Moreover, the results suggest that CpG-rich genes repressed by loss of H3K4me3 and DNA methylation represent special instances of a more general epigenetic mechanism of gene silencing, one in which gene silencing is mediated by loss of H3K4me3 and methylation of non-CpG island promoter-associated cytosines.

P4-01-21: An Estrogen-Inducible Transcription Factor FOXP1 Promotes Estrogen-Dependent Cell Proliferation of Breast Cancer Cells and Is Associated with 5-Year Disease-Free Survival in Patients with Tamoxifen-Treated Breast Cancer.

Abstract Estrogen signaling pathways are involved in the growth and development of breast tumors through the activation of estrogen receptor a (ERα). ERα is expressed in most breast cancers and involves in estrogen-dependent proliferation by acting as transcription factors activating the expression of target genes. Various coregulators and transcription factors are associated with ERα-mediated transcriptional control of target genes. Therefore, a comprehensive understanding of estrogen signaling pathways in breast cancer is required for both treatment and diagnosis of the disease. Forkhead box P1 (FOXP1) is a member of the forkhead box transcription factor family and has been reported to be associated with various types of tumors. Here, we investigated the expression pattern of FOXP1 by immunohistochemistry in a series of 133 invasive breast cancers and compared it with clinicopathological factors. The expression of FOXP1 was detected in nuclei in 89 cases (67%) and correlated positively with tumor grade and hormone receptor status, including ERα and progesterone receptor (PgR), and negatively with pathological tumor size (pT). And in ERα-positive MCF-7 breast cancer cells, we demonstrated that FOXP1 mRNA was upregulated by estrogen and that ERα recruitment to ER binding sites within the FOXP1 gene region identified by ChIP-chip analysis was increased. We also demonstrated that proliferation of MCF-7 cells was increased by exogenously transfected FOXP1 and decreased by FOXP1-specific siRNA. Moreover, in MCF-7 cells, FOXP1 enhanced estrogen response element (ERE)-driven transcription. Finally, FOXP1 immunoreactivity was significantly more elevated in relapse-free breast cancer patients treated with tamoxifen than in relapse patients treated with it. Taken together, these results suggest that FOXP1 plays an important role in proliferation of breast cancer cells by modulating estrogen signaling and that FOXP1 immunoreactivity might be associated with the estrogen dependency of breast cancer clinically, which may predict favorable prognosis in the patients treated with tamoxifen. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-21.

Chromatin Modifications Induced by the AML1/ETO Fusion Protein Reversibly Silence Its Genomic Targets Through AML1 and Sp1 Binding Motifs

Abstract 2422The AML1/ETO fusion protein, which is present in 10% to 15% of cases of acute myeloid leukemia (AML), is known to repress myeloid differentiation genes through DNA binding and recruitment of chromatin-modifying proteins and transcription factors in target genes. Previous studies have shown that both histone H4 deacetylation and K9 trimethylation of histone H3 (H3K9me3), respectively catalyzed by enzymes such as histone deacetylase 1 (HDAC1) and lysine methyltransferase enzyme suppressor of variegation 3–9 homolog 1(SUV39H1), are direct AML1/ETO effects on specific locus, but to date no global analysis of the AML1/ETO functional repressive chromatin modifications have been performed. ChIP-chip analysis of human hematopoietic stem/progenitor cells transduced with the AML1/ETO fusion gene (HSPC-AE) enabled us to identify 1168 AML1/ETO target genes, 103 of which were co-occupied by HDAC1 with a lost of hyperacetylation at histone H4, and 264 showed an increase H3K9me3 (p(X)<0.001; Normalized Log Ratio>0.7). We found a significant enrichment of genes involved in hematopoietic differentiation and in specific signaling pathways (i.e. TGF-β and Wnt/β-catenin) in AML1/ETO epigenetically modified target gene sets. Among them we identified novel genes (i.e. CTCF, MAPK1, SIRT1, YES1, AML1, MLLT3, and RPS19) previously described to be involved on the hematopoietic development, which epigenetic repression could implicate relevant steps in AML development induced by t(8;21). Gene Set Enrichment Analysis (GSEA) showed that expression of the 103 AML1/ETO-HDAC1 and 264 AML1/ETO-H3K9me3 target genes is highly diminished on HSCP-AE cells compared to the normal HSPC (p<0.0001 and p<0.001 respectively), suggesting that transduction with AML1/ETO directly causes this transcriptional silencing by the induction of chromatin modifications. Individual analysis confirmed a significant downregulation (p< 0.0001) of 6 out of 7 selected candidate genes. Furthermore, by using a Cre-LoxP AML1/ETO system we showed that this transcriptional repression is reversible, as the expression levels of SIRT1, AML1, CTCF, RPS19, YES1, GSK3A and MAPK1 genes were significantly restored upon AML1/ETO knockout. The clinical importance of these target genes was further analyzed in a set of 15 t(8;21) AML primary samples. All genes were found downregulated on the primary samples compared to the control HSCP, although different levels of significance were observed between them. Interestingly, AML1 binding sites were absent on a large number of the 1168 AML1/ETO target genes identified, and a Sp1 binding site was found in over 50% of them. AML1/ETO association with other motifs (e.g. E-Box motifs) has been already described and, in fact, Sp1 transcription factor is essential in hematopoietic differentiation and is known to interact with AML1/ETO. A significant enrichment of Sp1 presence in all the promoters studied was confirmed in the HSPC-AE samples compared to normal HSPC (p<0.1).Therefore, this study provides a global analysis of AML1/ETO functional chromatin modifications and identifies the important role of Sp1 in the DNA-binding pattern of AML1/ETO, suggesting a role for Sp1 targeted therapy in this leukemia subtype. Disclosures:No relevant conflicts of interest to declare.

IRF8 Governs Expression of Genes Involved in Innate and Adaptive Immunity in Human and Mouse Germinal Center B Cells

IRF8 (Interferon Regulatory Factor 8) is a transcription factor expressed throughout B cell differentiation except for mature plasma cells. Previous studies showed it is part of the transcriptional network governing B cell specification and commitment in the bone marrow, regulates the distribution of mature B cells into the splenic follicular and marginal zone compartments, and is expressed at highest levels in germinal center (GC) B cells. Here, we investigated the transcriptional programs and signaling pathways affected by IRF8 in human and mouse GC B cells as defined by ChIP-chip analyses and transcriptional profiling. We show that IRF8 binds a large number of genes by targeting two distinct motifs, half of which are also targeted by PU.1. Over 70% of the binding sites localized to proximal and distal promoter regions with ∼25% being intragenic. There was significant enrichment among targeted genes for those involved in innate and adaptive immunity with over 30% previously defined as interferon stimulated genes. We also showed that IRF8 target genes contributes to multiple aspects of the biology of mature B cells including critical components of the molecular crosstalk among GC B cells, T follicular helper cells, and follicular dendritic cells.

Myc and Miz-1 have coordinate genomic functions including targeting Hox genes in human embryonic stem cells

BackgroundA proposed role for Myc in maintaining mouse embryonic stem (ES) cell pluripotency is transcriptional repression of key differentiation-promoting genes, but detail of the mechanism has remained an important open topic.ResultsTo test the hypothesis that the zinc finger protein Miz-1 plays a central role, in the present work we conducted chromatin immunoprecipitation/microarray (ChIP-chip) analysis of Myc and Miz-1 in human ES cells, finding homeobox (Hox) genes as the most significant functional class of Miz-1 direct targets. Miz-1 differentiation-associated target genes specifically lack acetylated lysine 9 and trimethylated lysine 4 of histone H3 (AcH3K9 and H3K4me3) 9 histone marks, consistent with a repressed transcriptional state. Almost 30% of Miz-1 targets are also bound by Myc and these cobound genes are mostly factors that promote differentiation including Hox genes. Knockdown of Myc increased expression of differentiation genes directly bound by Myc and Miz-1, while a subset of the same genes is downregulated by Miz-1 loss-of-function. Myc and Miz-1 proteins interact with each other and associate with several corepressor factors in ES cells, suggesting a mechanism of repression of differentiation genes.ConclusionsTaken together our data indicate that Miz-1 and Myc maintain human ES cell pluripotency by coordinately suppressing differentiation genes, particularly Hox genes. These data also support a new model of how Myc and Miz-1 function on chromatin.