Methylation-Dependent and -Independent Genomic Targeting Principles of the MBD Protein Family

SummaryTo gain insight into the cellular readout of DNA methylation, we established a strategy for systematically profiling the genome-wide distribution of chromatin-interacting factors. This enabled us to create genomic maps for the methyl-CpG-binding domain (MBD) family of proteins, including disease-relevant mutants, deletions, and isoforms. In vivo binding of MBD proteins occurs predominantly as a linear function of local methylation density, requiring functional MBD domains and methyl-CPGs. This interaction directs specificity of MBD proteins to methylated, CpG-dense, and inactive regulatory regions. In contrast, binding to unmethylated sites varies between MBD proteins and is mediated via alternative domains or protein-protein interactions. Such targeting is exemplified by NuRD-complex-mediated tethering of MBD2 to a subset of unmethylated, active regulatory regions. Interestingly, MBD3 also occupies these sites, but like MBD2, binding is independent of the presence of hydroxymethylation. These functional binding maps reveal methylation-dependent and -independent binding modes and revise current models of DNA methylation readout through MBD proteins

Three-dimensional chromatin interactions remain stable upon CAG/CTG repeat expansion

Expanded CAG/CTG repeats underlie 13 neurological disorders, including myotonic dystrophy type 1 (DM1) and Huntington's disease (HD). Upon expansion, disease loci acquire heterochromatic characteristics, which may provoke changes to chromatin conformation and thereby affect both gene expression and repeat instability. Here, we tested this hypothesis by performing 4C sequencing at the DMPK and HTT loci from DM1 and HD-derived cells. We find that allele sizes ranging from 15 to 1700 repeats displayed similar chromatin interaction profiles. This was true for both loci and for alleles with different DNA methylation levels and CTCF binding. Moreover, the ectopic insertion of an expanded CAG repeat tract did not change the conformation of the surrounding chromatin. We conclude that CAG/CTG repeat expansions are not enough to alter chromatin conformation in cis. Therefore, it is unlikely that changes in chromatin interactions drive repeat instability or changes in gene expression in these disorders

Efficient pre-mRNA cleavage prevents replication-stress-associated genome instability

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability

ChromID identifies the protein interactome at chromatin marks

ISSN:1546-1696ISSN:1087-015

Lineage tracing of acute myeloid leukemia reveals the impact of hypomethylating agents on chemoresistance selection

Chemotherapy-resistant cancer recurrence is a major cause of mortality. In acute myeloid leukemia (AML), chemorefractory relapses result from the complex interplay between altered genetic, epigenetic and transcriptional states in leukemic cells. Here, we develop an experimental model system using in vitro lineage tracing coupled with exome, transcriptome and in vivo functional readouts to assess the AML population dynamics and associated molecular determinants underpinning chemoresistance development. We find that combining standard chemotherapeutic regimens with low doses of DNA methyltransferase inhibitors (DNMTi, hypomethylating drugs) prevents chemoresistant relapses. Mechanistically, DNMTi suppresses the outgrowth of a pre-determined set of chemoresistant AML clones with stemness properties, instead favoring the expansion of rarer and unfit chemosensitive clones. Importantly, we confirm the capacity of DNMTi combination to suppress stemness-dependent chemoresistance development in xenotransplantation models and primary AML patient samples. Together, these results support the potential of DNMTi combination treatment to circumvent the development of chemorefractory AML relapses

The histone methyltransferase SETD2 negatively regulates cell size

Cell size varies between cell types but is tightly regulated by cell intrinsic and extrinsic mechanisms. Cell size control is important for cell function, and changes in cell size are frequently observed in cancer. Here, we uncover a role for SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Using multiple independent perturbation strategies, we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that overexpression of the H3K36 demethylase KDM4A or the oncohistone H3.3K36M also increase cell size. In addition, ectopic overexpression of a decoy SRI domain increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development

DNA sequence and chromatin modifiers cooperate to confer epigenetic bistability at imprinting control regions

Genomic imprinting is regulated by parental-specific DNA methylation of imprinting control regions (ICRs). Despite an identical DNA sequence, ICRs can exist in two distinct epigenetic states that are memorized throughout unlimited cell divisions and reset during germline formation. Here, we systematically study the genetic and epigenetic determinants of this epigenetic bistability. By iterative integration of ICRs and related DNA sequences to an ectopic location in the mouse genome, we first identify the DNA sequence features required for maintenance of epigenetic states in embryonic stem cells. The autonomous regulatory properties of ICRs further enabled us to create DNA-methylation-sensitive reporters and to screen for key components involved in regulating their epigenetic memory. Besides DNMT1, UHRF1 and ZFP57, we identify factors that prevent switching from methylated to unmethylated states and show that two of these candidates, ATF7IP and ZMYM2, are important for the stability of DNA and H3K9 methylation at ICRs in embryonic stem cells

Genomic Prevalence of Heterochromatic H3K9me2 and Transcription Do Not Discriminate Pluripotent from Terminally Differentiated Cells

Cellular differentiation entails reprogramming of the transcriptome from a pluripotent to a unipotent fate. This process was suggested to coincide with a global increase of repressive heterochromatin, which results in a reduction of transcriptional plasticity and potential. Here we report the dynamics of the transcriptome and an abundant heterochromatic histone modification, dimethylation of histone H3 at lysine 9 (H3K9me2), during neuronal differentiation of embryonic stem cells. In contrast to the prevailing model, we find H3K9me2 to occupy over 50% of chromosomal regions already in stem cells. Marked are most genomic regions that are devoid of transcription and a subgroup of histone modifications. Importantly, no global increase occurs during differentiation, but discrete local changes of H3K9me2 particularly at genic regions can be detected. Mirroring the cell fate change, many genes show altered expression upon differentiation. Quantitative sequencing of transcripts demonstrates however that the total number of active genes is equal between stem cells and several tested differentiated cell types. Together, these findings reveal high prevalence of a heterochromatic mark in stem cells and challenge the model of low abundance of epigenetic repression and resulting global basal level transcription in stem cells. This suggests that cellular differentiation entails local rather than global changes in epigenetic repression and transcriptional activity

Regulatory mechanisms governing chromatin organization and function

Nucleosomes, the basic structures used to package genetic information into chromatin, are subject to a diverse array of chemical modifications. A large number of these marks serve as interaction hubs for many nuclear proteins and provide critical structural features for protein recruitment. Dynamic deposition and removal of chromatin modifications by regulatory proteins ensure their correct deposition to the genome, which is essential for DNA replication, transcription, chromatin compaction, or DNA damage repair. The spatiotemporal regulation and maintenance of chromatin marks relies on coordinated activities of writer, eraser, and reader enzymes and often depends on complex multicomponent regulatory circuits. In recent years, the field has made enormous advances in uncovering the mechanisms that regulate chromatin modifications. Here, we discuss well-established and emerging concepts in chromatin biology ranging from cooperativity and multivalent interactions to regulatory feedback loops and increased local concentration of chromatin-modifying enzymes

DNA methyltransferases hitchhiking on chromatin

DNA methylation is an epigenetic modification that plays a central regulatory role in various biological processes. Methyl groups are coupled to cytosines by the family of DNA methyltransferases (DNMTs), where DNMT1 is the main maintenance enzyme and the DNMT3 branch of the family is mostly responsible for de novo methylation. The regulation and function of DNA methylation are dependent on the genomic and chromatin context, such as binding sites for transcription factors or the presence of histone marks. Yet how local context, especially chromatin marks, influences the recruitment of the different DNMTs to their genomic target sites remains to be completely revealed. Elucidating the crosstalk between different histone modifications and DNA methylation, and their combined effect on the genome-wide epigenetic landscape, is of particular interest. Aberrant distribution of chromatin marks that guide DNMT activity or DNMT mutations that influence their correct recruitment to the genome have a profound impact on the deposition of DNA methylation, with consequences for genome function and gene activity. In this review, we describe the current state of knowledge on this topic and provide an overview on how chromatin marks can guide DNMT recruitment in healthy and diseased cells