ALDH1A3-acetaldehyde metabolism potentiates transcriptional heterogeneity in melanoma.

Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.

DAXX safeguards heterochromatin formation in embryonic stem cells.

Genomes comprise a large fraction of repetitive sequences folded into constitutive heterochromatin, which protect genome integrity and cell identity. De novo formation of heterochromatin during preimplantation development is an essential step for preserving the ground-state of pluripotency and the self-renewal capacity of embryonic stem cells (ESCs). However, the molecular mechanisms responsible for the remodeling of constitutive heterochromatin are largely unknown. Here, we identify that DAXX, an H3.3 chaperone essential for the maintenance of mouse ESCs in the ground state, accumulates in pericentromeric regions independently of DNA methylation. DAXX recruits PML and SETDB1 to promote the formation of heterochromatin, forming foci that are hallmarks of ground-state ESCs. In the absence of DAXX or PML, the three-dimensional (3D) architecture and physical properties of pericentric and peripheral heterochromatin are disrupted, resulting in de-repression of major satellite DNA, transposable elements and genes associated with the nuclear lamina. Using epigenome editing tools, we observe that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX is crucial for the maintenance and 3D organization of the heterochromatin compartment and protects ESC viability.

RINGs, DUBs and Abnormal Brain Growth-Histone H2A Ubiquitination in Brain Development and Disease.

During mammalian neurodevelopment, signaling pathways converge upon transcription factors (TFs) to establish appropriate gene expression programmes leading to the production of distinct neural and glial cell types. This process is partially regulated by the dynamic modulation of chromatin states by epigenetic systems, including the polycomb group (PcG) family of co-repressors. PcG proteins form multi-subunit assemblies that sub-divide into distinct, yet functionally related families. Polycomb repressive complexes 1 and 2 (PRC1 and 2) modify the chemical properties of chromatin by covalently modifying histone tails via H2A ubiquitination (H2AK119ub1) and H3 methylation, respectively. In contrast to the PRCs, the Polycomb repressive deubiquitinase (PR-DUB) complex removes H2AK119ub1 from chromatin through the action of the C-terminal hydrolase BAP1. Genetic screening has identified several PcG mutations that are causally associated with a range of congenital neuropathologies associated with both localised and/or systemic growth abnormalities. As PRC1 and PR-DUB hold opposing functions to control H2AK119ub1 levels across the genome, it is plausible that such neurodevelopmental disorders arise through a common mechanism. In this review, we will focus on advancements regarding the composition and opposing molecular functions of mammalian PRC1 and PR-DUB, and explore how their dysfunction contributes to the emergence of neurodevelopmental disorders.

PRL3-DDX21 Transcriptional Control of Endolysosomal Genes Restricts Melanocyte Stem Cell Differentiation.

Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer.

A central role for canonical PRC1 in shaping the 3D nuclear landscape.

Polycomb group (PcG) proteins silence gene expression by chemically and physically modifying chromatin. A subset of PcG target loci are compacted and cluster in the nucleus; a conformation that is thought to contribute to gene silencing. However, how these interactions influence gross nuclear organization and their relationship with transcription remains poorly understood. Here we examine the role of Polycomb-repressive complex 1 (PRC1) in shaping 3D genome organization in mouse embryonic stem cells (mESCs). Using a combination of imaging and Hi-C analyses, we show that PRC1-mediated long-range interactions are independent of CTCF and can bridge sites at a megabase scale. Impairment of PRC1 enzymatic activity does not directly disrupt these interactions. We demonstrate that PcG targets coalesce in vivo, and that developmentally induced expression of one of the target loci disrupts this spatial arrangement. Finally, we show that transcriptional activation and the loss of PRC1-mediated interactions are separable events. These findings provide important insights into the function of PRC1, while highlighting the complexity of this regulatory system.

Coolpup.py: versatile pile-up analysis of Hi-C data.

MOTIVATION: Hi-C is currently the method of choice to investigate the global 3D organization of the genome. A major limitation of Hi-C is the sequencing depth required to robustly detect loops in the data. A popular approach used to mitigate this issue, even in single-cell Hi-C data, is genome-wide averaging (piling-up) of peaks, or other features, annotated in high-resolution datasets, to measure their prominence in less deeply sequenced data. However, current tools do not provide a computationally efficient and versatile implementation of this approach. RESULTS: Here, we describe coolpup.py-a versatile tool to perform pile-up analysis on Hi-C data. We demonstrate its utility by replicating previously published findings regarding the role of cohesin and CTCF in 3D genome organization, as well as discovering novel details of Polycomb-driven interactions. We also present a novel variation of the pile-up approach that can aid the statistical analysis of looping interactions. We anticipate that coolpup.py will aid in Hi-C data analysis by allowing easy to use, versatile and efficient generation of pile-ups. AVAILABILITY AND IMPLEMENTATION: Coolpup.py is cross-platform, open-source and free (MIT licensed) software. Source code is available from https://github.com/Phlya/coolpuppy and it can be installed from the Python Packaging Index.

DNA Methylation Directs Polycomb-Dependent 3D Genome Re-organization in Naive Pluripotency.

The DNA hypomethylation that occurs when embryonic stem cells (ESCs) are directed to the ground state of naive pluripotency by culturing in two small molecule inhibitors (2i) results in redistribution of polycomb (H3K27me3) away from its target loci. Here, we demonstrate that 3D genome organization is also altered in 2i, with chromatin decompaction at polycomb target loci and a loss of long-range polycomb interactions. By preventing DNA hypomethylation during the transition to the ground state, we are able to restore to ESC in 2i the H3K27me3 distribution, as well as polycomb-mediated 3D genome organization that is characteristic of primed ESCs grown in serum. However, these cells retain the functional characteristics of 2i ground-state ESCs. Our findings demonstrate the central role of DNA methylation in shaping major aspects of 3D genome organization but caution against assuming causal roles for the epigenome and 3D genome in gene regulation and function in ESCs.

Decreased Enhancer-Promoter Proximity Accompanying Enhancer Activation.

Enhancers can regulate the promoters of their target genes over very large genomic distances. It is widely assumed that mechanisms of enhancer action involve the reorganization of three-dimensional chromatin architecture, but this is poorly understood. The predominant model involves physical enhancer-promoter interaction by looping out the intervening chromatin. However, studying the enhancer-driven activation of the Sonic hedgehog gene (Shh), we have identified a change in chromosome conformation that is incompatible with this simple looping model. Using super-resolution 3D-FISH and chromosome conformation capture, we observe a decreased spatial proximity between Shh and its enhancers during the differentiation of embryonic stem cells to neural progenitors. We show that this can be recapitulated by synthetic enhancer activation, is impeded by chromatin-bound proteins located between the enhancer and the promoter, and appears to involve the catalytic activity of poly (ADP-ribose) polymerase. Our data suggest that models of enhancer-promoter communication need to encompass chromatin conformations other than looping.

Chromatin folding and nuclear architecture: PRC1 function in 3D.

Embryonic development requires the intricate balance between the expansion and specialisation of defined cell types in time and space. The gene expression programmes that underpin this balance are regulated, in part, by modulating the chemical and structural state of chromatin. Polycomb repressive complexes (PRCs), a family of essential developmental regulators, operate at this level to stabilise or perpetuate a repressed but transcriptionally poised chromatin configuration. This dynamic state is required to control the timely initiation of productive gene transcription during embryonic development. The two major PRCs cooperate to target the genome, but it is PRC1 that appears to be the primary effector that controls gene expression. In this review I will discuss recent findings relating to how PRC1 alters chromatin accessibility, folding and global 3D nuclear organisation to control gene transcription.

Polycomb enables primitive endoderm lineage priming in embryonic stem cells.

Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver of these coordinated changes, known as lineage priming, in a process that exploits novel polycomb activities. We find that intragenic levels of the polycomb mark H3K27me3 anti-correlate with changes in transcription, irrespective of the gene's developmental trajectory or identity as a polycomb target. In contrast, promoter proximal H3K27me3 is markedly higher for PrEn priming genes. Consequently, depletion of this modification stimulates the degree to which ESCs are primed towards PrEn when challenged to differentiate, but has little effect on gene expression in self-renewing ESC culture. These observations link polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision.

Loss of Tet1-Associated 5-Hydroxymethylcytosine Is Concomitant with Aberrant Promoter Hypermethylation in Liver Cancer.

Aberrant hypermethylation of CpG islands (CGI) in human tumors occurs predominantly at repressed genes in the host tissue, but the preceding events driving this phenomenon are poorly understood. In this study, we temporally tracked epigenetic and transcriptomic perturbations that occur in a mouse model of liver carcinogenesis. Hypermethylated CGI events in the model were predicted by enrichment of the DNA modification 5-hydroxymethylcytosine (5hmC) and the histone H3 modification H3K27me3 at silenced promoters in the host tissue. During cancer progression, selected CGIs underwent hypo-hydroxymethylation prior to hypermethylation, while retaining H3K27me3. In livers from mice deficient in Tet1, a tumor suppressor involved in cytosine demethylation, we observed a similar loss of promoter core 5hmC, suggesting that reduced Tet1 activity at CGI may contribute to epigenetic dysregulation during hepatocarcinogenesis. Consistent with this possibility, mouse liver tumors exhibited reduced Tet1 protein levels. Similar to humans, DNA methylation changes at CGI in mice did not appear to be direct drivers of hepatocellular carcinoma progression, rather, dynamic changes in H3K27me3 promoter deposition correlated strongly with tumor-specific activation and repression of transcription. Overall, our results suggest that loss of promoter-associated 5hmC in liver tumors licenses reprograming of DNA methylation at silent CGI during progression. Cancer Res; 76(10); 3097-108. ©2016 AACR.

Polycomb-mediated chromatin compaction weathers the STORM.

A recent super-resolution imaging study by Boettiger et al. elegantly demonstrates that three epigenetically defined, and functionally disparate, chromatin states have distinct folding characteristics in Drosophila nuclei.

The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development.

Polycomb-repressive complex 1 (PRC1) and PRC2 maintain repression at many developmental genes in mouse embryonic stem cells and are required for early development. However, it is still unclear how they are targeted and how they function. We show that the ability of RING1B, a core component of PRC1, to ubiquitinate histone H2A is dispensable for early mouse embryonic development and much of the gene repression activity of PRC1. Our data support a model in which PRC1 and PRC2 reinforce each other's binding but suggest that the key functions of PRC1 lie beyond the enzymatic capabilities of RING1B.

Inter-individual variability contrasts with regional homogeneity in the human brain DNA methylome.

The possibility that alterations in DNA methylation are mechanistic drivers of development, aging and susceptibility to disease is widely acknowledged, but evidence remains patchy or inconclusive. Of particular interest in this regard is the brain, where it has been reported that DNA methylation impacts on neuronal activity, learning and memory, drug addiction and neurodegeneration. Until recently, however, little was known about the 'landscape' of the human brain methylome. Here we assay 1.9 million CpGs in each of 43 brain samples representing different individuals and brain regions. The cerebellum was a consistent outlier compared to all other regions, and showed over 16 000 differentially methylated regions (DMRs). Unexpectedly, the sequence characteristics of hypo- and hypermethylated domains in cerebellum were distinct. In contrast, very few DMRs distinguished regions of the cortex, limbic system and brain stem. Inter-individual DMRs were readily detectable in these regions. These results lead to the surprising conclusion that, with the exception of cerebellum, DNA methylation patterns are more homogeneous between different brain regions from the same individual, than they are for a single brain region between different individuals. This finding suggests that DNA sequence composition, not developmental status, is the principal determinant of the human brain DNA methylome.

Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization.

Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods.

Chromatin decondensation is sufficient to alter nuclear organization in embryonic stem cells.

During differentiation, thousands of genes are repositioned toward or away from the nuclear envelope. These movements correlate with changes in transcription and replication timing. Using synthetic (TALE) transcription factors, we found that transcriptional activation of endogenous genes by a viral trans-activator is sufficient to induce gene repositioning toward the nuclear interior in embryonic stem cells. However, gene relocation was also induced by recruitment of an acidic peptide that decondenses chromatin without affecting transcription, indicating that nuclear reorganization is driven by chromatin remodeling rather than transcription. We identified an epigenetic inheritance of chromatin decondensation that maintained central nuclear positioning through mitosis even after the TALE transcription factor was lost. Our results also demonstrate that transcriptional activation, but not chromatin decondensation, is sufficient to change replication timing.

Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells.

Trimethylation of histone H3 Lys 4 (H3K4me3) is a mark of active and poised promoters. The Set1 complex is responsible for most somatic H3K4me3 and contains the conserved subunit CxxC finger protein 1 (Cfp1), which binds to unmethylated CpGs and links H3K4me3 with CpG islands (CGIs). Here we report that Cfp1 plays unanticipated roles in organizing genome-wide H3K4me3 in embryonic stem cells. Cfp1 deficiency caused two contrasting phenotypes: drastic loss of H3K4me3 at expressed CGI-associated genes, with minimal consequences for transcription, and creation of "ectopic" H3K4me3 peaks at numerous regulatory regions. DNA binding by Cfp1 was dispensable for targeting H3K4me3 to active genes but was required to prevent ectopic H3K4me3 peaks. The presence of ectopic peaks at enhancers often coincided with increased expression of nearby genes. This suggests that CpG targeting prevents "leakage" of H3K4me3 to inappropriate chromatin compartments. Our results demonstrate that Cfp1 is a specificity factor that integrates multiple signals, including promoter CpG content and gene activity, to regulate genome-wide patterns of H3K4me3.

PRC1 and PRC2 are not required for targeting of H2A.Z to developmental genes in embryonic stem cells.

The essential histone variant H2A.Z localises to both active and silent chromatin sites. In embryonic stem cells (ESCs), H2A.Z is also reported to co-localise with polycomb repressive complex 2 (PRC2) at developmentally silenced genes. The mechanism of H2A.Z targeting is not clear, but a role for the PRC2 component Suz12 has been suggested. Given this association, we wished to determine if polycomb functionally directs H2A.Z incorporation in ESCs. We demonstrate that the PRC1 component Ring1B interacts with multiple complexes in ESCs. Moreover, we show that although the genomic distribution of H2A.Z co-localises with PRC2, Ring1B and with the presence of CpG islands, H2A.Z still blankets polycomb target loci in the absence of Suz12, Eed (PRC2) or Ring1B (PRC1). Therefore we conclude that H2A.Z accumulates at developmentally silenced genes in ESCs in a polycomb independent manner.

Cell type-specific DNA methylation at intragenic CpG islands in the immune system.

Human and mouse genomes contain a similar number of CpG islands (CGIs), which are discrete CpG-rich DNA sequences associated with transcription start sites. In both species, ∼50% of all CGIs are remote from annotated promoters but, nevertheless, often have promoter-like features. To determine the role of CGI methylation in cell differentiation, we analyzed DNA methylation at a comprehensive CGI set in cells of the mouse hematopoietic lineage. Using a method that potentially detects ∼33% of genomic CpGs in the methylated state, we found that large differences in gene expression were accompanied by surprisingly few DNA methylation changes. There were, however, many DNA methylation differences between hematopoietic cells and a distantly related tissue, brain. Altered DNA methylation in the immune system occurred predominantly at CGIs within gene bodies, which have the properties of cell type-restricted promoters, but infrequently at annotated gene promoters or CGI flanking sequences (CGI "shores"). Unexpectedly, elevated intragenic CGI methylation correlated with silencing of the associated gene. Differentially methylated intragenic CGIs tended to lack H3K4me3 and associate with a transcriptionally repressive environment regardless of methylation state. Our results indicate that DNA methylation changes play a relatively minor role in the late stages of differentiation and suggest that intragenic CGIs represent regulatory sites of differential gene expression during the early stages of lineage specification.

Immunostaining of modified histones defines high-level features of the human metaphase epigenome.

BACKGROUND: Immunolabeling of metaphase chromosome spreads can map components of the human epigenome at the single cell level. Previously, there has been no systematic attempt to explore the potential of this approach for epigenomic mapping and thereby to complement approaches based on chromatin immunoprecipitation (ChIP) and sequencing technologies. RESULTS: By immunostaining and immunofluorescence microscopy, we have defined the distribution of selected histone modifications across metaphase chromosomes from normal human lymphoblastoid cells and constructed immunostained karyotypes. Histone modifications H3K9ac, H3K27ac and H3K4me3 are all located in the same set of sharply defined immunofluorescent bands, corresponding to 10- to 50-Mb genomic segments. Primary fibroblasts gave broadly the same banding pattern. Bands co-localize with regions relatively rich in genes and CpG islands. Staining intensity usually correlates with gene/CpG island content, but occasional exceptions suggest that other factors, such as transcription or SINE density, also contribute. H3K27me3, a mark associated with gene silencing, defines a set of bands that only occasionally overlap with gene-rich regions. Comparison of metaphase bands with histone modification levels across the interphase genome (ENCODE, ChIP-seq) shows a close correspondence for H3K4me3 and H3K27ac, but major differences for H3K27me3. CONCLUSIONS: At metaphase the human genome is packaged as chromatin in which combinations of histone modifications distinguish distinct regions along the euchromatic chromosome arms. These regions reflect the high-level interphase distributions of some histone modifications, and may be involved in heritability of epigenetic states, but we also find evidence for extensive remodeling of the epigenome at mitosis.