Achim Breiling

Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development

Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in various embryonic and adult tissues. Mice mutant for either Tet1 or Tet2 are viable, raising the question of whether these enzymes have overlapping roles in development. Here we have generated Tet1 and Tet2 double-knockout (DKO) embryonic stem cells (ESCs) and mice. DKO ESCs remained pluripotent but were depleted of 5hmC and caused developmental defects in chimeric embryos. While a fraction of double-mutant embryos exhibited midgestation abnormalities with perinatal lethality, viable and overtly normal Tet1/Tet2-deficient mice were also obtained. DKO mice had reduced 5hmC and increased 5mC levels and abnormal methylation at various imprinted loci. Nevertheless, animals of both sexes were fertile, with females having smaller ovaries and reduced fertility. Our data show that loss of both enzymes is compatible with development but promotes hypermethylation and compromises imprinting. The data also suggest a significant contribution of Tet3 to hydroxylation of 5mC during development.

General transcription factors bind promoters repressed by Polycomb group proteins

To maintain cell identity during development and differentiation, mechanisms of cellular memory have evolved that preserve transcription patterns in an epigenetic manner. The proteins of the Polycomb group (PcG) are part of such a mechanism, maintaining gene silencing. They act as repressive multiprotein complexes that may render target genes inaccessible to the transcriptional machinery, inhibit chromatin remodelling, influence chromosome domain topology and recruit histone deacetylases (HDACs). PcG proteins have also been found to bind to core promoter regions, but the mechanism by which they regulate transcription remains unknown. To address this, we used formaldehyde-crosslinked chromatin immunoprecipitation (X-ChIP) to map TATA-binding protein (TBP), transcription initiation factor IIB (TFIIB) and IIF (TFIIF), and dHDAC1 (RPD3) across several Drosophila promoter regions. Here we show that binding of PcG proteins to repressed promoters does not exclude general transcription factors (GTFs) and that depletion of PcG proteins by double-stranded RNA interference leads to de-repression of developmentally regulated genes. We further show that PcG proteins interact in vitro with GTFs. We suggest that PcG complexes maintain silencing by inhibiting GTF-mediated activation of transcription.

Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila

RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21–30 nucleotide) RNAs. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive. Here we show that the key RNAi components Dicer 2 (DCR2) and Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact with the core transcription machinery. Notably, loss of function of DCR2 or AGO2 showed that transcriptional defects are accompanied by the perturbation of RNA polymerase II positioning on promoters. Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, as well as missense mutations that compromise the RNAi activity, impaired the global dynamics of RNA polymerase II. Finally, the deep sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq) revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and other genetic loci on both the sense and antisense DNA strands, but with a strong bias for the antisense strand, particularly after heat shock. Taken together, our results show that DCR2 and AGO2 are globally associated with transcriptionally active loci and may have a pivotal role in shaping the transcriptome by controlling the processivity of RNA polymerase II.

Genome-wide promoter DNA methylation dynamics of human hematopoietic progenitor cells during differentiation and aging

DNA methylation plays an important role in the self-renewal of hematopoietic stem cells and in the commitment to the lymphoid or myeloid lineages. Using purified CD34⁺ hematopoietic progenitor cells and differentiated myeloid cell populations from the same human samples, we obtained detailed methylation profiles at distinct stages of hematopoiesis. We identified a defined set of differentiation-related genes that are methylated in CD34⁺ hematopoietic progenitor cells but show pronounced DNA hypomethylation in monocytes and in granulocytes. In addition, by comparing hematopoietic progenitor cells from umbilical cord blood to hematopoietic progenitor cells from peripheral blood of adult donors we were also able to analyze age-related methylation changes in CD34⁺ cells. Interestingly, the methylation changes observed in older progenitor cells showed a bimodal pattern with hypomethylation of differentiation-associated genes and de novo methylation events resembling epigenetic mutations. Our results thus provide detailed insight into the methylation dynamics during differentiation and suggest that epigenetic changes contribute to hematopoietic progenitor cell aging.

Loss of Tet enzymes compromises proper differentiation of embryonic stem cells.

Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and are dynamically expressed during development. Whereas loss of individual Tet enzymes or combined deficiency of Tet1/2 allows for embryogenesis, the effect of complete loss of Tet activity and 5hmC marks in development is not established. We have generated Tet1/2/3 triple-knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential. Combined deficiency of all three Tets depleted 5hmC and impaired ESC differentiation, as seen in poorly differentiated TKO embryoid bodies (EBs) and teratomas. Consistent with impaired differentiation, TKO ESCs contributed poorly to chimeric embryos, a defect rescued by Tet1 reexpression, and could not support embryonic development. Global gene-expression and methylome analyses of TKO EBs revealed promoter hypermethylation and deregulation of genes implicated in embryonic development and differentiation. These findings suggest a requirement for Tet- and 5hmC-mediated DNA demethylation in proper regulation of gene expression during ESC differentiation and development.

Drosophila Chromosome Condensation Proteins Topoisomerase II and Barren Colocalize with Polycomb and Maintain Fab-7 PRE Silencing

Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin structure. We have investigated whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. We show that Topoisomerase II (TOPOII) and Barren (BARR) interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, we find that the PcG protein Polyhomeotic (PH) interacts physically with TOPOII and BARR and that BARR is required for Fab-7-regulated homeotic gene expression. Conversely, we find defects in chromosome segregation associated with ph mutations. We propose that chromatin condensation proteins are involved in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the maintenance of gene expression.

Epigenetic regulatory functions of DNA modifications: 5 -methylcytosine and beyond

The chemical modification of DNA bases plays a key role in epigenetic gene regulation. While much attention has been focused on the classical epigenetic mark, 5-methylcytosine, the field garnered increased interest through the recent discovery of additional modifications. In this review, we focus on the epigenetic regulatory roles of DNA modifications in animals. We present the symmetric modification of 5-methylcytosine on CpG dinucleotide as a key feature, because it permits the inheritance of methylation patterns through DNA replication. However, the distribution patterns of cytosine methylation are not conserved in animals and independent molecular functions will likely be identified. Furthermore, the discovery of enzymes that catalyse the hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine not only identified an active demethylation pathway, but also a candidate for a new epigenetic mark associated with activated transcription. Most recently, N6-methyladenine was described as an additional eukaryotic DNA modification with epigenetic regulatory potential. Interestingly, this modification is also present in genomes that lack canonical cytosine methylation patterns, suggesting independent functions. This newfound diversity of DNA modifications and their potential for combinatorial interactions indicates that the epigenetic DNA code is substantially more complex than previously thought.

Hydroxylation of 5-methylcytosine by TET2 maintains the active state of the mammalian HOXA cluster

Differentiation is accompanied by extensive epigenomic reprogramming, leading to the repression of stemness factors and the transcriptional maintenance of activated lineage-specific genes. Here we use the mammalian Hoxa cluster of developmental genes as a model system to follow changes in DNA modification patterns during retinoic acid-induced differentiation. We find the inactive cluster to be marked by defined patterns of 5-methylcytosine (5mC). Upon the induction of differentiation, the active anterior part of the cluster becomes increasingly enriched in 5-hydroxymethylcytosine (5hmC), following closely the colinear activation pattern of the gene array, which is paralleled by the reduction of 5mC. Depletion of the 5hmC generating dioxygenase Tet2 impairs the maintenance of Hoxa activity and partially restores 5mC levels. Our results indicate that gene-specific 5mC-5hmC conversion by Tet2 is crucial for the maintenance of active chromatin states at lineage-specific loci.

Epigenome changes in active and inactive polycomb-group-controlled regions.

The Polycomb group (PcG) of proteins conveys epigenetic inheritance of repressed transcriptional states. In Drosophila, the Polycomb repressive complex 1 (PRC1) maintains the silent state by inhibiting the transcription machinery and chromatin remodelling at core promoters. Using immunoprecipitation of in vivo formaldehyde‐fixed chromatin in phenotypically diverse cultured cell lines, we have mapped PRC1 components, the histone methyl transferase (HMT) Enhancer of zeste (E(z)) and histone H3 modifications in active and inactive PcG‐controlled regions. We show that PRC1 components are present in both cases, but at different levels. In particular, active target promoters are nearly devoid of E(z) and Polycomb. Moreover, repressed regions are trimethylated at lysines 9 and 27, suggesting that these histone modifications represent a mark for inactive PcG‐controlled regions. These PcG‐specific repressive marks are maintained by the action of the E(z) HMT, an enzyme that has an important role not only in establishing but also in maintaining PcG repression.

The Drosophila polycomb protein interacts with nucleosomal core particles In vitro via its repression domain.

ABSTRACT The proteins of the Polycomb group (PcG) are required for maintaining regulator genes, such as the homeotic selectors, stably and heritably repressed in appropriate developmental domains. It has been suggested that PcG proteins silence genes by creating higher-order chromatin structures at their chromosomal targets, thus preventing the interaction of components of the transcriptional machinery with theircis-regulatory elements. An unresolved issue is how higher order-structures are anchored at the chromatin base, the nucleosomal fiber. Here we show a direct biochemical interaction of a PcG protein—the Polycomb (PC) protein—with nucleosomal core particles in vitro. The main nucleosome-binding domain coincides with a region in the C-terminal part of PC previously identified as the repression domain. Our results suggest that PC, by binding to the core particle, recruits other PcG proteins to chromatin. This interaction could provide a key step in the establishment or regulation of higher-order chromatin structures.