Discovery of genes required for body axis and limb formation by global identification of retinoic acid-regulated epigenetic marks.
ABSTRACT: Identification of target genes that mediate required functions downstream of transcription factors is hampered by the large number of genes whose expression changes when the factor is removed from a specific tissue and the numerous binding sites for the factor in the genome. Retinoic acid (RA) regulates transcription via RA receptors bound to RA response elements (RAREs) of which there are thousands in vertebrate genomes. Here, we combined chromatin immunoprecipitation sequencing (ChIP-seq) for epigenetic marks and RNA-seq on trunk tissue from wild-type and Aldh1a2-/- embryos lacking RA synthesis that exhibit body axis and forelimb defects. We identified a relatively small number of genes with altered expression when RA is missing that also have nearby RA-regulated deposition of histone H3 K27 acetylation (H3K27ac) (gene activation mark) or histone H3 K27 trimethylation (H3K27me3) (gene repression mark) associated with conserved RAREs, suggesting these genes function downstream of RA. RA-regulated epigenetic marks were identified near RA target genes already known to be required for body axis and limb formation, thus validating our approach; plus, many other candidate RA target genes were found. Nuclear receptor 2f1 (Nr2f1) and nuclear receptor 2f2 (Nr2f2) in addition to Meis homeobox 1 (Meis1) and Meis homeobox 2 (Meis2) gene family members were identified by our approach, and double knockouts of each family demonstrated previously unknown requirements for body axis and/or limb formation. A similar epigenetic approach can be used to determine the target genes for any transcriptional regulator for which a knockout is available.
Project description:We have utilized retinoic acid receptor ? (gamma) knockout (RAR?(-/-)) embryonic stem (ES) cells as a model system to analyze RAR? mediated transcriptional regulation of stem cell differentiation. Most of the transcripts regulated by all-trans retinoic acid (RA) in ES cells are dependent upon functional RAR? signaling. Notably, many of these RA-RAR? target genes are implicated in retinoid uptake and metabolism. For instance, Lrat (lecithin:retinol acyltransferase), Stra6 (stimulated by retinoic acid 6), Crabp2 (cellular retinoic acid binding protein 2), and Cyp26a1 (cytochrome p450 26a1) transcripts are induced in wild type (WT), but not in RAR?(-/-) cells. Transcripts for the transcription factors Pbx1 (pre-B cell leukemia homeobox-1), Wt1 (Wilm's tumor gene-1), and Meis1 (myeloid ecotropic viral integration site-1) increase upon RA treatment of WT, but not RAR?(-/-) cells. In contrast, Stra8, Dleu7, Leftb, Pitx2, and Cdx1 mRNAs are induced by RA even in the absence of RAR?. Mapping of the epigenetic signature of Meis1 revealed that RA induces a rapid increase in the H3K9/K14ac epigenetic mark at the proximal promoter and at two sites downstream of the transcription start site in WT, but not in RAR?(-/-) cells. Thus, RA-associated increases in H3K9/K14ac epigenetic marks require RAR? and are associated with increased Meis1 transcript levels, whereas H3K4me3 is present at the Meis1 proximal promoter even in the absence of RAR?. In contrast, at the Lrat proximal promoter primarily the H3K4me3 mark, and not the H3K9/K14ac mark, increases in response to RA, independently of the presence of RAR?. Our data show major epigenetic changes associated with addition of the RAR? agonist RA in ES cells.
Project description:To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes.
Project description:Defects in the nuclear envelope or nuclear 'lamina' networks cause disease and can perturb histone posttranslational (epigenetic) regulation. Barrier-to-Autointegration Factor (BAF) is an essential but enigmatic lamina component that binds lamins, LEM-domain proteins, DNA and histone H3 directly. We report that BAF copurified with nuclease-digested mononucleosomes and associated with modified histones in vivo. BAF overexpression significantly reduced global histone H3 acetylation by 18%. In cells that stably overexpressed BAF 3-fold, silencing mark H3-K27-Me1/3 and active marks H4-K16-Ac and H4-Ac5 decreased significantly. Significant increases were also seen for silencing mark H3-K9-Me3, active marks H3-K4-Me2, H3-K9/K14-Ac and H4-K5-Ac and a mark (H3-K79-Me2) associated with both active and silent chromatin. Other increases (H3-S10-P, H3-S28-P and silencing mark H3-K9-Me2) did not reach statistical significance. BAF overexpression also significantly influenced cell cycle distribution. Moreover, BAF associated in vivo with SET/I2PP2A (protein phosphatase 2A inhibitor; blocks H3 dephosphorylation) and G9a (H3-K9 methyltransferase), but showed no detectable association with HDAC1 or HATs. These findings reveal BAF as a novel epigenetic regulator and are discussed in relation to BAF deficiency phenotypes, which include a hereditary progeria syndrome and loss of pluripotency in embryonic stem cells.
Project description:Retinoic acid (RA) generated in the mesoderm of vertebrate embryos controls body axis extension by downregulating Fgf8 expression in cells exiting the caudal progenitor zone. RA activates transcription by binding to nuclear RA receptors (RARs) at RA response elements (RAREs), but it is unknown whether RA can directly repress transcription. Here, we analyzed a conserved RARE upstream of Fgf8 that binds RAR isoforms in mouse embryos. Transgenic embryos carrying Fgf8 fused to lacZ exhibited expression similar to caudal Fgf8, but deletion of the RARE resulted in ectopic trunk expression extending into somites and neuroectoderm. Epigenetic analysis using chromatin immunoprecipitation of trunk tissues from E8.25 wild-type and Raldh2(-/-) embryos lacking RA synthesis revealed RA-dependent recruitment of the repressive histone marker H3K27me3 and polycomb repressive complex 2 (PRC2) near the Fgf8 RARE. The co-regulator RERE, the loss of which results in ectopic Fgf8 expression and somite defects, was recruited near the RARb RARE by RA, but was released from the Fgf8 RARE by RA. Our findings demonstrate that RA directly represses Fgf8 through a RARE-mediated mechanism that promotes repressive chromatin, thus providing valuable insight into the mechanism of RA-FGF antagonism during progenitor cell differentiation.
Project description:BACKGROUND: Homeobox genes murine Rhox5 and human RHOXF1 are expressed in early embryonic stages and then mostly restricted to germline tissues in normal adult, yet they are aberrantly expressed in cancer cells in vitro and in vivo . Here we study the epigenetic regulation and potential functions of Rhox5 gene. FINDINGS: In Rhox5-silenced or extremely low expresser cells, we observed low levels of active histone epigenetic marks (H3ac, H4ac and H3K4me2) and high levels of repressive mark H3K9me2 along with DNA hypermethylation in the promoter. In Rhox5 low expresser cells, we typically observed modest levels of both active and repressive histone marks along with moderate DNA methylation. In Rhox5 highly expressed CT26 cancer cells, we observed DNA hypomethylation along with high levels of both active and repressive histone marks. Epigenetic drugs (retinoic acid and MS-275) induced F9 cell differentiation with enhanced Rhox5 expression and dynamic changes of epigenetic marks. Finally, Rhox5 knockdown by small hairpin RNA (shRNA) in CT26 colon cancer decreased cell proliferation and migration in vitro and tumor growth in vivo . CONCLUSIONS: Both DNA methylation and histone methylation/acetylation play key roles in modulating Rhox5 expression in various cell types. The stem cell-like "bivalent domain", an epigenetic feature originally identified in key differentiation genes within stem cells, exists in the Rhox5 gene promoter in not only embryonic stem cells but also cancer cells, cancer stem cells, and differentiated Sertoli cells. As Ras signaling-dependent Rhox5 expression promotes tumor growth, Rhox5 may be an ideal target for therapeutic intervention in cancer.
Project description:Heart diseases are a leading cause of death. While the link between early exposure to nutritional excess and heart disease risk is clear, the molecular mechanisms involved are poorly understood. In the developmental programming field, increasing evidence is pointing out the critical role of epigenetic mechanisms. Among them, polycomb repressive complex 2 (PRC2) and DNA methylation play a critical role in heart development and pathogenesis. In this context, we aimed at evaluating the role of these epigenetic marks in the long-term cardiac alterations induced by early dietary challenge. Using a model of rats exposed to maternal high-fat diet during gestation and lactation, we evaluated cardiac alterations at adulthood. Expression levels of PRC2 components, its histone marks di- and trimethylated histone H3 (H3K27me2/3), associated histone mark (ubiquitinated histone H2A, H2AK119ub1) and target genes were measured by Western blot. Global DNA methylation level and DNA methyl transferase 3B (DNMT3B) protein levels were measured. Maternal high-fat diet decreased H3K27me3, H2Ak119ub1 and DNA methylation levels, down-regulated the enhancer of zeste homolog 2 (EZH2), and DNMT3B expression. The levels of the target genes, isl lim homeobox 1 (Isl1), six homeobox 1 (Six1) and mads box transcription enhancer factor 2, polypeptide C (Mef2c), involved in cardiac pathogenesis were up regulated. Overall, our data suggest that the programming of cardiac alterations by maternal exposure to high-fat diet involves the derepression of pro-fibrotic and pro-hypertrophic genes through the induction of EZH2 and DNMT3B deficiency.
Project description:Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function.
Project description:Meis1, which belongs to TALE-type class of homeobox gene family, appeared as one of the key regulators of hematopoietic stem cell (HSC) self-renewal and a potential therapeutical target. However, small molecule inhibitors of MEIS1 remained unknown. This led us to develop inhibitors of MEIS1 that could modulate HSC activity. To this end, we have established a library of relevant homeobox family inhibitors and developed a high-throughput in silico screening strategy against homeodomain of MEIS proteins using the AutoDock Vina and PaDEL-ADV platform. We have screened over a million druggable small molecules in silico and selected putative MEIS inhibitors (MEISi) with no predicted cytotoxicity or cardiotoxicity. This was followed by in vitro validation of putative MEIS inhibitors using MEIS dependent luciferase reporter assays and analysis in the ex vivo HSC assays. We have shown that small molecules named MEISi-1 and MEISi-2 significantly inhibit MEIS-luciferase reporters in vitro and induce murine (LSKCD34l°w cells) and human (CD34+, CD133+, and ALDHhi cells) HSC self-renewal ex vivo. In addition, inhibition of MEIS proteins results in downregulation of Meis1 and MEIS1 target gene expression including Hif-1?, Hif-2? and HSC quiescence modulators. MEIS inhibitors are effective in vivo as evident by induced HSC content in the murine bone marrow and downregulation of expression of MEIS target genes. These studies warrant identification of first-in-class MEIS inhibitors as potential pharmaceuticals to be utilized in modulation of HSC activity and bone marrow transplantation studies.
Project description:Cyp26A1 is a major enzyme that controls retinoic acid (RA) homeostasis by metabolizing RA into bio-inactive metabolites. Previous research revealed that the mouse Cyp26A1 promoter has two canonical RA response elements (RAREs) that underlie the regulation of the gene by RA. Analyzing the 2,533-base pairs (2.5 k) genomic sequence upstream of zebrafish cyp26a1 start codon, we report that the two RAREs are conserved in zebrafish cyp26a1 promoter. Mutagenesis demonstrated that the two RAREs work synergistically in RA inducibility of cyp26a1. Fusing the 2.5 k (kilobase pairs) fragment to the enhanced yellow fluorescent protein (eYFP) reporter gene, we have generated two transgenic lines of zebrafish [Tg(cyp26a1:eYFP)]. The transgenic zebrafish display expression patterns similar to that of cyp26a1 gene in vivo. Consistent with the in vitro results, the reporter activity is RA inducible in embryos. Taken together, our results demonstrate that the 2.5 k fragment underlies the regulation of the zebrafish cyp26a1 gene by RA.
Project description:Polycomb proteins play key roles in mediating epigenetic modifications that occur during cell differentiation. The Polycomb repressive complex 2 (PRC2) mediates the tri-methylation of histone H3 lysine 27 (H3K27me3). In this study, we identify a distinguishing feature of two classes of PRC2 target genes, represented by the Nr2F1 (Coup-TF1) and the Hoxa5 gene, respectively. Both genes are transcriptionally activated by all-trans retinoic acid (RA) and display increased levels of the permissive H3K9/K14ac and tri-methylated histone H3 lysine 4 epigenetic marks in response to RA. However, while in response to RA the PRC2 and H3K27me3 marks are greatly decreased at the Hoxa5 promoter, these marks are initially increased at the Nr2F1 promoter. Functional depletion of the essential PRC2 protein Suz12 by short hairpin RNA (shRNA) technology enhanced the RA-associated transcription of Nr2F1, Nr2F2, Meis1, Sox9 and BMP2, but had no effect on the Hoxa5, Hoxa1, Cyp26a1, Cyp26b1 and RAR?2 transcript levels in wild-type embryonic stem cells. We propose that PRC2 recruitment attenuates the RA-associated transcriptional activation of a subset of genes. Such a mechanism would permit the fine-tuning of transcriptional networks during differentiation.