Christel Krueger

Cohesin is required for higher-order chromatin conformation at the imprinted IGF2-H19 locus

Cohesin is a chromatin-associated protein complex that mediates sister chromatid cohesion by connecting replicated DNA molecules. Cohesin also has important roles in gene regulation, but the mechanistic basis of this function is poorly understood. In mammalian genomes, cohesin co-localizes with CCCTC binding factor (CTCF), a zinc finger protein implicated in multiple gene regulatory events. At the imprinted IGF2-H19 locus, CTCF plays an important role in organizing allele-specific higher-order chromatin conformation and functions as an enhancer blocking transcriptional insulator. Here we have used chromosome conformation capture (3C) assays and RNAi–mediated depletion of cohesin to address whether cohesin affects higher order chromatin conformation at the IGF2-H19 locus in human cells. Our data show that cohesin has a critical role in maintaining CTCF–mediated chromatin conformation at the locus and that disruption of this conformation coincides with changes in IGF2 expression. We show that the cohesin-dependent, higher-order chromatin conformation of the locus exists in both G1 and G2 phases of the cell cycle and is therefore independent of cohesins function in sister chromatid cohesion. We propose that cohesin can mediate interactions between DNA molecules in cis to insulate genes through the formation of chromatin loops, analogous to the cohesin mediated interaction with sister chromatids in trans to establish cohesion.

The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing

Long noncoding RNAs are implicated in a number of regulatory functions in eukaryotic genomes. The paternally expressed long noncoding RNA (ncRNA) Kcnq1ot1 regulates epigenetic gene silencing in an imprinted gene cluster in cis over a distance of 400 kb in the mouse embryo, whereas the silenced region extends over 780 kb in the placenta. Gene silencing by the Kcnq1ot1 RNA involves repressive histone modifications, including H3K9me2 and H3K27me3, which are partly brought about by the G9a and Ezh2 histone methyltransferases. Here, we show that Kcnq1ot1 is transcribed by RNA polymerase II, is unspliced, is relatively stable and is localised in the nucleus. Analysis of conditional Dicer mutants reveals that the RNAi pathway is not involved in gene silencing in the Kcnq1ot1 cluster. Instead, using RNA/DNA FISH we show that the Kcnq1ot1 RNA establishes a nuclear domain within which the genes that are epigenetically inactivated in cis are frequently found, whereas nearby genes that are not regulated by Kcnq1ot1 are localised outside of the domain. The Kcnq1ot1 RNA domain is larger in the placenta than in the embryo, consistent with more genes in the cluster being silenced in the placenta. Our results show for the first time that autosomal long ncRNAs can establish nuclear domains, which might create a repressive environment for epigenetic silencing of adjacent genes. Long ncRNAs in imprinting clusters and the Xist RNA on the inactive X chromosome thus appear to regulate epigenetic gene silencing by similar mechanisms.

MERVL/Zscan4 Network Activation Results in Transient Genome-wide DNA Demethylation of mESCs.

Summary Mouse embryonic stem cells are dynamic and heterogeneous. For example, rare cells cycle through a state characterized by decondensed chromatin and expression of transcripts, including the Zscan4 cluster and MERVL endogenous retrovirus, which are usually restricted to preimplantation embryos. Here, we further characterize the dynamics and consequences of this transient cell state. Single-cell transcriptomics identified the earliest upregulated transcripts as cells enter the MERVL/Zscan4 state. The MERVL/Zscan4 transcriptional network was also upregulated during induced pluripotent stem cell reprogramming. Genome-wide DNA methylation and chromatin analyses revealed global DNA hypomethylation accompanying increased chromatin accessibility. This transient DNA demethylation was driven by a loss of DNA methyltransferase proteins in the cells and occurred genome-wide. While methylation levels were restored once cells exit this state, genomic imprints remained hypomethylated, demonstrating a potential global and enduring influence of endogenous retroviral activation on the epigenome.

Pairing of Homologous Regions in the Mouse Genome Is Associated with Transcription but Not Imprinting Status

Although somatic homologous pairing is common in Drosophila it is not generally observed in mammalian cells. However, a number of regions have recently been shown to come into close proximity with their homologous allele, and it has been proposed that pairing might be involved in the establishment or maintenance of monoallelic expression. Here, we investigate the pairing properties of various imprinted and non-imprinted regions in mouse tissues and ES cells. We find by allele-specific 4C-Seq and DNA FISH that the Kcnq1 imprinted region displays frequent pairing but that this is not dependent on monoallelic expression. We demonstrate that pairing involves larger chromosomal regions and that the two chromosome territories come close together. Frequent pairing is not associated with imprinted status or DNA repair, but is influenced by chromosomal location and transcription. We propose that homologous pairing is not exclusive to specialised regions or specific functional events, and speculate that it provides the cell with the opportunity of trans-allelic effects on gene regulation.

Random monoallelic expression: making a choice

Monoallelic gene expression exposes an organism to the risks associated with the unmasking of recessive mutations. A recent study by Gimelbrant and colleagues, supported by results from two methodologically different studies, demonstrated that random monoallelic expression is surprisingly widespread among autosomal genes. This raises important questions about why, when and how cells choose and tolerate monoallelism and whether functional hemizygosity might provide an unappreciated advantage.

Robust 3D DNA FISH using directly labeled probes.

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

A non-catalytic role of TET3 promotes open chromatin and enhances global transcription

The methylcytosine dioxygenase Tet3 is highly expressed as a specific isoform in oocytes and zygotes but essentially absent from later stages of mouse preimplantation development. Here, we show that Tet3 expression promotes transdifferentiation of embryonic stem cells to trophoblast-like stem cells. By genome-wide analyses we demonstrate that TET3 associates with and co-occupies chromatin with RNA Polymerase II. Tet3 expression induces a global increase of transcription and total RNA levels, and its presence further enhances chromatin accessibility in regions open for transcription. This novel function of TET3 is not specific to the oocyte isoform, independent of its catalytic activity, the CXXC domain, or its interaction with OGT, and is localised in its highly conserved exon 4. We propose a more general role for TET3 promoting open chromatin and enhancing global transcription during changes of cellular identity, separate from its catalytic function.

Tri-methylation of histone H3 lysine 4 facilitates gene expression in ageing cells

Transcription of protein coding genes is accompanied by recruitment of COMPASS to promoter-proximal chromatin, which methylates histone H3 lysine 4 (H3K4) to form H3K4me1, H3K4me2 and H3K4me3. Here, we determine the importance of COMPASS in maintaining gene expression across lifespan in budding yeast. We find that COMPASS mutations reduce replicative lifespan and cause expression defects in almost 500 genes. Although H3K4 methylation is reported to act primarily in gene repression, particularly in yeast, repressive functions are progressively lost with age while hundreds of genes become dependent on H3K4me3 for full expression. Basal and inducible expression of these genes is also impaired in young cells lacking COMPASS components Swd1 or Spp1. Gene induction during ageing is associated with increasing promoter H3K4me3, but H3K4me3 also accumulates in non-promoter regions and the ribosomal DNA. Our results provide clear evidence that H3K4me3 is required to maintain normal expression of many genes across organismal lifespan.

Dppa2 and Dppa4 directly regulate the Dux driven zygotic transcriptional programme

The molecular regulation of zygotic genome activation (ZGA) in mammals remains poorly understood. Primed mouse embryonic stem cells contain a rare subset of “2C-like” cells that are epigenetically and transcriptionally similar to the two cell embryo and thus represent an ideal system for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed exclusively in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA either in a Dux dependent or independent manner. Here we performed a candidate-based overexpression screen, identifying, amongst others, Developmental Pluripotency Associated 2 (Dppa2) and 4 (Dppa4) as positive regulators of 2C-like cells and ZGA transcription. In the germ line, promoter DNA demethylation coincides with upregulation of Dppa2 and Dppa4 which remain expressed until E7.5 when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during iPSC reprogramming at the time 2C-like ZGA transcription transiently peaks. Through a combination of overexpression, knockdown, knockout and rescue experiments, together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we tease apart the molecular hierarchy in which the 2C-like transcriptional programme is initiated and stabilised. Dppa2 and Dppa4 require Dux to initiate 2C-like ZGA transcription, suggesting they act upstream by directly regulating Dux. Supporting this, ChIP-seq analysis revealed Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild type cells, but, in contrast to Dux, can no longer do so in Dppa2/4 double knockout cells, suggesting it may act to stabilise rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux upregulation and activation of the 2C-like transcriptional programme which is subsequently reinforced by Zscan4c.