Structural dynamics and epigenetic modifications of Hoxc loci along the anteroposterior body axis in developing mouse embryos

Hox genes are organized as clusters and specify regional identity along the anteroposterior body axis by sequential expression at a specific time and region during embryogenesis. However, the precise mechanisms underlying the sequential spatio-temporal, collinear expression pattern of Hox genes are not fully understood. Since epigenetic modifications such as chromatin architecture and histone modifications have become crucial mechanisms for highly coordinated gene expressions, we examined such modifications. E14.5 mouse embryos were dissected into three parts along the anteroposterior axis: brain, trunk-anterior, and trunk-posterior. Then, structural changes and epigenetic modifications were analyzed along the Hoxc cluster using chromosome conformation capture and chromatin immunoprecipitation-PCR methods. Hox non-expressing brain tissues had more compact, heterochromatin-like structures together with the strong repressive mark H3K27me3 than trunk tissues. In the trunk, however, a more loose euchromatin-like topology with a reduced amount of H3K27me3 modifications were observed along the whole cluster, regardless of their potency in gene activation. The active mark H3K4me3 was rather closely associated with the collinear expression of Hoxc genes; at trunk-anterior tissues, only 3' anterior Hoxc genes were marked by H3K4me3 upon gene activation, whereas whole Hoxc genes were marked by H3K4me3 and showed expression in trunk-posterior tissues. Altogether, these results indicated that loosening of the chromatin architecture and removing H3K27me3 were not sufficient for, but rather the concomitant acquisition of H3K4me3 drove the collinear expression of Hoxc genes.     

Epigenomic Landscape of Human Fetal Brain,Heart, and Liver

The epigenetic regulation of spatiotemporal gene expression is crucial for human development. Here, we present whole-genome chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-seq) analyses of a wide variety of histone markers in the brain, heart, and liver of early human embryos shortly after their formation. We identified 40,181 active enhancers, with a large portion showing tissue-specific and developmental stage-specific patterns, pointing to their roles in controlling the ordered spatiotemporal expression of the developmental genes in early human embryos. Moreover, using sequential ChIP-seq, we showed that all three organs have hundreds to thousands of bivalent domains that are marked by both H3K4me3 and H3K27me3, probably to keep the progenitor cells in these organs ready for immediate differentiation into diverse cell types during subsequent developmental processes. Our work illustrates the potentially critical roles of tissue-specific and developmental stage-specific epigenomes in regulating the spatiotemporal expression of developmental genes during early human embryonic development.     

Should I stay or should I go: Protection and maintenance of DNA methylation at imprinted genes

Recent findings shed light on the coordination of two fundamental, yet mechanistically opposing, processes in the early mammalian embryo. During the oocyte-to-embryo transition and early preimplantation development nuclear reprogramming occurs. This resetting of the epigenome in maternal and paternal pronuclei to a ground state is the essential step ensuring totipotency in the zygote, the first embryonic stage. Radical, global DNA demethylation, which occurs actively in the paternal and passively in the maternal genome, is a prominent feature of nuclear reprogramming; yet, this process poses a danger to a subset of methylated sequences that must be preserved for their germline to soma inheritance. Genomic imprinting and its importance were demonstrated three decades ago by a series of experiments generating non-viable mammalian uniparental embryos. Indeed, imprinted loci, gene clusters with parent-of-origin specific gene expression patterns, must retain their differential methylation status acquired during gametogenesis throughout embryogenesis and in adult tissues. It is just recently that the molecular players that protect/maintain imprinting marks during reprogramming in preimplantation embryos have been identified, in particular, an epigenetic modifier complex formed by ZFP57 and TRIM28/KAP1. The interaction of these and other molecules with the newly formed embryonic chromatin and imprinted genes is discussed and highlighted herein.     

INO80 promotes H2A.Z occupancy to regulate cell fate transition in pluripotent stem cells

The INO80 chromatin remodeler is involved in many chromatin-dependent cellular functions. However, its role in pluripotency and cell fate transition is not fully defined. We examined the impact of Ino80 deletion in the naïve and primed pluripotent stem cells. We found that Ino80 deletion had minimal effect on self-renewal and gene expression in the naïve state, but led to cellular differentiation and de-repression of developmental genes in the transition toward and maintenance of the primed state. In the naïve state, INO80 pre-marked gene promoters that would adopt bivalent histone modifications by H3K4me3 and H3K27me3 upon transition into the primed state. In the primed state, in contrast to its known role in H2A.Z exchange, INO80 promoted H2A.Z occupancy at these bivalent promoters and facilitated H3K27me3 installation and maintenance as well as downstream gene repression. Together, our results identified an unexpected function of INO80 in H2A.Z deposition and gene regulation. We showed that INO80-dependent H2A.Z occupancy is a critical licensing step for the bivalent domains, and thereby uncovered an epigenetic mechanism by which chromatin remodeling, histone variant deposition and histone modification coordinately control cell fate.     

Sequence and epigenetic determinants in the regulation of the Math6 gene by Neurogenin3

The bHLH factor Neurogenin3 initiates the differentiation program that leads to formation of pancreatic endocrine cells. Math6 is a closely related bHLH factor transiently activated downstream of Neurogenin3 in endocrine progenitors. Here we characterize the Math6 promoter and locate the Neurogenin3 binding site, thus confirming that Math6 is a genuine Neurogenin3 target. We also show that Math6 activation rates are largely controlled by epigenetic mechanisms involving the balance between activating H3K4 and repressive H3K27 methylation marks. High Math6 expression in the embryonic pancreas associates with an H3K4me3-only state, whereas low Math6 expression in differentiated endocrine cells correlates with chromatin dually marked with H3K4me3/H3K27me3, a feature originally associated with developmental genes that are repressed but poised for activation in ES cells. Importantly, we show that Neurogenin3 can trigger the conversion of Math6 from a poorly transcribed bivalent to an active monovalent state in vitro, hence providing a mechanism whereby Neurogenin3 may activate Math6 in endocrine progenitors. Finally, because Neurogenin3-induced changes in histone methylation are observed at other endocrine gene promoters, we propose that this mechanism may contribute to the determination of endocrine cell fate by Neurogenin3 in the pancreas.     

Imprinting of the murine MAS protooncogene is restricted to its antisense RNA.

The Mas protooncogene encodes a G protein-coupled receptor with the common seven transmembrane domains and may be involved in the actions of angiotensins. The gene is located in close proximity to the paternally imprinted Igf2r gene and its maternal imprinting has been reported but remained controversial. We used mice carrying a targeted deletion of the Mas protooncogene on the maternal or paternal chromosome to clarify this issue. In all Mas-expressing organs of adult mice such as heart, kidney, testis or brain, no Mas mRNA was missing in heterozygous animals inheriting the deleted allele from the father excluding mono-allelic paternal expression. However, we show exclusive paternal expression of a Mas antisense RNA, confirming the maternal imprinting of this antisense RNA in all investigated adult tissues and in embryos. Our results strongly suggest that Mas is not imprinted in mice but that an antisense RNA probably starting in the neighboring Igf2r gene is maternally imprinted in both embryos and adult organs.     

Maternal and paternal alleles exhibit differential histone methylation and acetylation at maize imprinted genes

Imprinting is an epigenetically controlled form of gene regulation in which the expression of a gene is based on its parent of origin. This epigenetic regulation is likely to involve allele-specific DNA or histone modifications. The relative abundance of eight different histone modifications was tested at various regions in several imprinted maize (Zea mays) genes using a chromatin immunoprecipitation protocol coupled with quantitative allele-specific single nucleotide polymorphism assays. Histone H3 lysine-27 di- and tri-methylation are paternally enriched at the imprinted loci Mez1, ZmFie1 and Nrp1. In contrast, acetylation of histones H3 and H4 and H3K4 dimethylation are enriched at the maternal alleles of these genes. Di- and tri-methylation of H3 lysine-9, which is generally associated with constitutively silenced chromatin, was not enriched at either allele of imprinted loci. These patterns of enrichment were specific to tissues that exhibit imprinting. In addition, the enrichment of these modifications was dependent upon the parental origin of an allele and not sequence differences between the alleles, as demonstrated by reciprocal crosses. This study presents a detailed view of the chromatin modifications that are associated with the maternal and paternal alleles at imprinted loci and provides evidence for common histone modifications at multiple imprinted loci.     

Chromatin remodeling and bivalent histone modifications in embryonic stem cells

Pluripotent embryonic stem cells (ESCs) are characterized by distinct epigenetic features including a relative enrichment of histone modifications related to active chromatin. Among these is tri‐methylation of lysine 4 on histone H3 (H3K4me3). Several thousands of the H3K4me3‐enriched promoters in pluripotent cells also contain a repressive histone mark, namely H3K27me3, a situation referred to as “bivalency”. While bivalent promoters are not unique to pluripotent cells, they are relatively enriched in these cell types, largely marking developmental and lineage‐specific genes which are silent but poised for immediate action. The H3K4me3 and H3K27me3 modifications are catalyzed by lysine methyltransferases which are usually found within, although not entirely limited to, the Trithorax group (TrxG) and Polycomb group (PcG) protein complexes, respectively, but these do not provide selective bivalent specificity. Recent studies highlight the family of ATP‐dependent chromatin remodeling proteins as regulators of bivalent domains. Here, we discuss bivalency in general, describe the machineries that catalyze bivalent chromatin domains, and portray the emerging connection between bivalency and the action of different families of chromatin remodelers, namely INO80, esBAF, and NuRD, in pluripotent cells. We posit that chromatin remodeling proteins may enable “bivalent specificity”, often selectively acting on, or selectively depleted from, bivalent domains.     

Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver

Post‐translational modifications of histone tails play a crucial role in gene regulation. Here, we performed chromatin profiling by quantitative targeted mass spectrometry to assess all possible modifications of the core histones. We identified a bivalent combination, a dually marked H3K9me3/H3K14ac modification in the liver, that is significantly decreased in old hepatocytes. Subsequent sequential ChIP‐Seq identified dually marked single nucleosome regions, with reduced number of sites and decreased signal in old livers, confirming mass spectrometry results. We detected H3K9me3 and H3K14ac bulk ChIP‐Seq signal in reChIP nucleosome regions, suggesting a correlation between H3K9me3/H3K14ac bulk bivalent genomic regions and dually marked single nucleosomes. Histone H3K9 deacetylase Hdac3, as well as H3K9 methyltransferase Setdb1, found in complex Kap1, occupied both bulk and single nucleosome bivalent regions in both young and old livers, correlating to presence of H3K9me3. Expression of genes associated with bivalent regions in young liver, including those regulating cholesterol secretion and triglyceride synthesis, is upregulated in old liver once the bivalency is lost. Hence, H3K9me3/H3K14ac dually marked regions define a poised inactive state that is resolved with loss of one or both of the chromatin marks, which subsequently leads to change in gene expression.     

The 3' portion of the mouse H19 Imprinting-Control Region is required for proper tissue-specific expression of the Igf2 gene

Genomic imprinting at the H19/Igf2 locus is governed by a cis-acting Imprinting-Control Region (ICR), located 2 kb upstream of the H19 gene. This region possesses an insulator function which is activated on the unmethylated maternal allele through the binding of the CTCF factor. It has been previously reported that paternal transmission of the H19(SilK) deletion, which removes the 3' portion of H19 ICR, leads to the loss of H19 imprinting. Here we show that, in the liver, this reactivation of the paternal H19 gene is concomitant to a dramatic decrease in Igf2 mRNA levels. This deletion alters higher-order chromatin architecture, Igf2 promoter usage and tissue-specific expression. Therefore, when methylated, the 3' portion of the H19 ICR is a bi-functional regulatory element involved not only in H19 imprinting but also in 'formatting' the higher-order chromatin structure for proper tissue-specific expression of both H19 and Igf2 genes.