CTCF binding site classes exhibit distinct evolutionary, genomic, epigenomic and transcriptomic features

CTCF DNA binding sites are classified into distinct functional classes, with distinct biological properties, shedding light on the differing functional roles of CTCF binding

Maintenance of Paternal Methylation and Repression of the Imprinted H19 Gene Requires MBD3

Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division

A Hox-Embedded Long Noncoding RNA: Is It All Hot Air?

Over 20 years ago, the discovery of Xist as a critical component of X chromosome inactivation revealed a fundamental role for long noncoding RNAs (lncRNAs) in epigenetic regulation during mammalian development and foreshadowed a fascinating connection between RNA and chromatin modification [1–3]. In the last decade, the field has exploded, heralded in part by a 2007 landmark paper from the group of Howard Chang [4] describing that knockdown of a lncRNA (Hox Antisense Intergenic RNA [HOTAIR]) was associated with loss of transcriptional repression from a locus on another chromosome in trans. HOTAIR lncRNA—encoded within the HOXC locus, although its expression seemed to be required for normal epigenetic silencing of HOXD genes—became one of the most well-known examples of functional lncRNAs in the field of developmental epigenetics. Interest intensified when a subsequent paper from the Chang lab [5] reported that targeted deletion of the orthologous locus in the mouse (Hotair) caused homeotic transformations underpinned by derepression of HoxD gene transcription in vivo. Discovery of new lncRNAs and exploration of their potential actions and effects during development and disease is a continued source of excitement [6,7]. But questions about the effects and actions of Hotair have been controversial, raised in part by work from the group of Denis Duboule [8]. This debate is addressed directly in the current issue of PLOS Genetics in a manuscript from Duboule’s laboratory [9] that reanalyzes Hotair mutant mice generated by the Chang lab, a formal comment in response to that manuscript from the Chang lab [10], and this perspective

Humanized H19/Igf2 locus reveals diverged imprinting mechanism between mouse and human and reflects Silver–Russell syndrome phenotypes

Genomic imprinting is essential for mammalian development. Curiously, elements that regulate genomic imprinting, the imprinting control regions (ICRs), often diverge across species. To understand whether the diverged ICR sequence plays a species-specific role at the H19/insulin-like growth factor 2 (Igf2) imprinted locus, we generated a mouse in which the human ICR (hIC1) sequence replaced the endogenous mouse ICR. We show that the imprinting mechanism has partially diverged between mouse and human, depending on the parental origin of the hIC1 in mouse. We also suggest that our mouse model is optimal for studying the imprinting disorders Beckwith–Wiedemann syndrome when hIC1 is maternally transmitted, and Silver–Russell syndrome when hIC1 is paternally transmitted

Characterization of BRD4 during mammalian post-meiotic sperm development

During spermiogenesis, the post-meiotic phase of mammalian spermatogenesis, transcription is progressively repressed as nuclei of haploid spermatids are compacted through a dramatic chromatin reorganization involving hyper-acetylation and replacement of most histones with protamines. Although BRDT functions in transcription and histone removal in spermatids, it is unknown whether other BET family proteins play a role. Immunofluorescence of spermatogenic cells revealed BRD4 in a ring around the nuclei of spermatids containing hyper-acetylated histones. The ring lies directly adjacent to the acroplaxome, the cytoskeletal base of the acrosome, previously linked to chromatin reorganization. The BRD4 ring does not form in acrosomal mutant mice. ChIP sequencing in spermatids revealed enrichment of BRD4 and acetylated histones at the promoters of active genes. BRD4 and BRDT show distinct and synergistic binding patterns, with a pronounced enrichment of BRD4 at spermatogenesis-specific genes. Direct association of BRD4 with acetylated H4 decreases in late spermatids as acetylated histones are removed from the condensing nucleus in a wave following the progressing acrosome. These data provide evidence for a prominent transcriptional role of BRD4 and suggest a possible removal mechanism for chromatin components from the genome via the progressing acrosome as transcription is repressed in response to chromatin condensation during spermiogenesis

Human oocyte-derived methylation differences persist in the placenta revealing widespread transient imprinting

Thousands of regions in gametes have opposing methylation profiles that are largely resolved during the post-fertilization epigenetic reprogramming. However some specific sequences associated with imprinted loci survive this demethylation process. Here we present the data describing the fate of germline-derived methylation in humans. With the exception of a few known paternally methylated germline differentially methylated regions (DMRs) associated with known imprinted domains, we demonstrate that sperm-derived methylation is reprogrammed by the blastocyst stage of development. In contrast a large number of oocyte-derived methylation differences survive to the blastocyst stage and uniquely persist as transiently methylated DMRs only in the placenta. Furthermore, we demonstrate that this phenomenon is exclusive to primates, since no placenta-specific maternal methylation was observed in mouse. Utilizing single cell RNA-seq datasets from human preimplantation embryos we show that following embryonic genome activation the maternally methylated transient DMRs can orchestrate imprinted expression. However despite showing widespread imprinted expression of genes in placenta, allele-specific transcriptional profiling revealed that not all placenta-specific DMRs coordinate imprinted expression and that this maternal methylation may be absent in a minority of samples, suggestive of polymorphic imprinted methylation

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat-/- mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation. © 2013 McGraw et al