The distinct effects of MEK and GSK3 inhibition upon the methylome and transcriptome of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) were first cultured in vitro in serum-containing medium with leukaemia inhibitory factor, in which they exhibit heterogeneous expression of both pluripotency and some early differentiation markers. Over the last decade, however, it has become commonplace to grow mESCs with inhibitors of MEK and GSK3 signalling, which together elicit a more homogeneously 'naive' state of pluripotency. Whilst 2i/L-cultured mESCs have been shown to be globally hypomethylated, a comprehensive understanding of the distinct effects of these signalling inhibitors upon the DNA methylome is still lacking. Here we carried out whole genome bisulphite and RNA sequencing of mESCs grown with MEK or GSK3 inhibition alone, including different time points and concentrations of MEK inhibitor treatment. This demonstrated that MEK inhibition causes a dose-dependent impairment of maintenance methylation via loss of UHRF1 protein, as well as rapid impairment of de novo methylation. In contrast, GSK3 inhibition triggers impairment of de novo methylation alone, and consequent hypomethylation is enriched at enhancers with a 2i/L-specific chromatin signature and coincides with upregulation of nearby genes. Our study provides detailed insights into the epigenetic and transcriptional impacts of inhibiting MEK or GSK3 signalling in mouse pluripotent cells.

Set1 Targets Genes with Essential Identity and Tumor-Suppressing Functions in Planarian Stem Cells

Tumor suppressor genes (TSGs) are essential for normal cellular function in multicellular organisms, but many TSGs and tumor-suppressing mechanisms remain unknown. Planarian flatworms exhibit particularly robust tumor suppression, yet the specific mechanisms underlying this trait remain unclear. Here, we analyze histone H3 lysine 4 trimethylation (H3K4me3) signal across the planarian genome to determine if the broad H3K4me3 chromatin signature that marks essential cell identity genes and TSGs in mammalian cells is conserved in this valuable model of in vivo stem cell function. We find that this signature is indeed conserved on the planarian genome and that the lysine methyltransferase Set1 is largely responsible for creating it at both cell identity and putative TSG loci. In addition, we show that depletion of set1 in planarians induces stem cell phenotypes that suggest loss of TSG function, including hyperproliferation and an abnormal DNA damage response (DDR). Importantly, this work establishes that Set1 targets specific gene loci in planarian stem cells and marks them with a conserved chromatin signature. Moreover, our data strongly suggest that Set1 activity at these genes has important functional consequences both during normal homeostasis and in response to genotoxic stress.

Chromatin Patterns Distinguish Breast Tumor Subtypes and Disease Progression in Association with ANP32E levels

Despite highly advanced diagnosis and treatment strategies, breast cancer patient outcomes vary extensively, even among individuals with the same diagnosis. Thus, a better understanding of the unique molecular characteristics that underlie tumor trajectories and responses to therapy remains a central goal. We report that chromatin patterns represent an important characteristic, capable of stratifying tumor identity and progression. We find that patterns of chromatin accessibility can be classified into 3 major groups, representing Basal-like tumors, hormone receptor (HR)-expressing tumors, and invasive lobular Luminal-A tumors. Major chromatin differences occur throughout the genome at motifs for the transcription factor FOXA1 in HR-positive tumors, and motifs for SOX9 in Basal-like tumors. A large portion of lobular Luminal-A tumors display a chromatin signature defined by accessibility at FOXA1 binding motifs, distinguishing them from others within this subtype. Expression of the histone chaperone ANP32E is inversely correlated with tumor progression and chromatin accessibility at FOXA1 binding sites. Tumors with high levels of ANP32E exhibit an immune response and proliferative gene expression signature, whereas tumors with low ANP32E levels appear programmed for differentiation. Our results indicate that ANP32E may function through chromatin state regulation to control breast cancer differentiation and tumor plasticity.

A coordinated function of lncRNA HOTTIP and miRNA-196b underpinning leukemogenesis by targeting Fas signaling

MicroRNAs (miRNAs) may modulate more than 60% of human coding genes and act as negative regulators, while long non-coding RNAs (lncRNAs) regulate gene expression on multiple levels by interacting with chromatin, functional proteins, and RNAs such as mRNAs and microRNAs. However, the crosstalk between lncRNA HOTTIP and miRNAs in leukemogenesis remains elusive. Using combined integrated analyses of global miRNA expression profiling and state-of-the-art genomic analyses of chromatin such as ChIRPseq., (genome wide HOTTIP binding analysis), ChIP-seq., and ATACseq., we found that miRNA genes are directly controlled by HOTTIP. Specifically, the HOX cluster miRNAs (miR-196a, miR-196b, miR-10a and miR-10b), located cis & trans, were most dramatically regulated and significantly decreased in HOTTIP knockout (KO) AML cells. HOTTIP bound to the miR-196b promoter, and HOTTIP deletion reduced chromatin accessibility and enrichment of active histone modifications at HOX cluster associated miRNAs in AML cells, while reactivation of HOTTIP restored miR gene expression and chromatin accessibility in the CTCF-boundary-attenuated AML cells. Inactivation of HOTTIP or miR-196b promotes apoptosis by altering the chromatin signature at the FAS promoter and increasing FAS expression. Transplantation of miR-196b knockdown MOLM13 cells in NSG mice increased overall survival compared to wild-type cells. Thus, HOTTIP remodels the chromatin architecture around miRNAs to promote their transcription, consequently repressing tumor suppressors and promoting leukemogenesis.

Sperm histone H3 lysine 4 trimethylation is altered in a genetic mouse model of transgenerational epigenetic inheritance

Advancing the molecular knowledge surrounding fertility and inheritance has become critical given the halving of sperm counts in the last 40 years, and the rise in complex disease which cannot be explained by genetics alone. The connection between both these trends may lie in alterations to the sperm epigenome and occur through environmental exposures. Changes to the sperm epigenome are also associated with health risks across generations such as metabolic disorders and cancer. Thus, it is imperative to identify the epigenetic modifications that escape reprogramming during spermatogenesis and embryogenesis. Here, we aimed to identify the chromatin signature(s) involved in transgenerational phenotypes in our genetic mouse model of epigenetic inheritance that overexpresses the histone demethylase KDM1A in their germ cells. We used sperm-specific chromatin immunoprecipitation followed by in depth sequencing (ChIP-seq), and computational analysis to identify whether differential enrichment of histone H3 lysine 4 trimethylation (H3K4me3), and histone H3 lysine 27 trimethylation (H3K27me3) serve as mechanisms for transgenerational epigenetic inheritance through the paternal germline. Our analysis on the sperm of KDM1A transgenic males revealed specific changes in H3K4me3 enrichment that predominantly occurred independently from bivalent H3K4me3/H3K27me3 regions. Many regions with altered H3K4me3 enrichment in sperm were identified on the paternal allele of the pre-implantation embryo. These findings suggest that sperm H3K4me3 functions in the transmission of non-genetic phenotypes transgenerationally.

Age-associated cryptic transcription in mammalian stem cells is linked to permissive chromatin at cryptic promoters

Suppressing spurious cryptic transcription by a repressive intragenic chromatin state featuring trimethylated lysine 36 on histone H3 (H3K36me3) and DNA methylation is critical for maintaining self-renewal capacity in mouse embryonic stem cells. In yeast and nematodes, such cryptic transcription is elevated with age, and reducing the levels of age-associated cryptic transcription extends yeast lifespan. Whether cryptic transcription is also increased during mammalian aging is unknown. We show for the first time an age-associated elevation in cryptic transcription in several stem cell populations, including murine hematopoietic stem cells (mHSCs) and neural stem cells (NSCs) and human mesenchymal stem cells (hMSCs). Using DECAP-seq, we mapped and quantified age-associated cryptic transcription in hMSCs aged in vitro. Regions with significant age-associated cryptic transcription have a unique chromatin signature: decreased H3K36me3 and increased H3K4me1, H3K4me3, and H3K27ac with age. Furthermore, genomic regions undergoing such age-dependent chromatin changes resemble known promoter sequences and are bound by the promoter-associated protein TBP even in young cells. Hence, the more permissive chromatin state at intragenic cryptic promoters likely underlies the increase of cryptic transcription in aged mammalian stem cells.

Establishment and function of chromatin modification at enhancers

How a single genome can give rise to distinct cell types remains a fundamental question in biology. Mammals are able to specify and maintain hundreds of cell fates by selectively activating unique subsets of their genome. This is achieved, in part, by enhancers—genetic elements that can increase transcription of both nearby and distal genes. Enhancers can be identified by their unique chromatin signature, including transcription factor binding and the enrichment of specific histone post-translational modifications, histone variants, and chromatin-associated cofactors. How each of these chromatin features contributes to enhancer function remains an area of intense study. In this review, we provide an overview of enhancer-associated chromatin states, and the proteins and enzymes involved in their establishment. We discuss recent insights into the effects of the enhancer chromatin state on ongoing transcription versus their role in the establishment of new transcription programmes, such as those that occur developmentally. Finally, we highlight the role of enhancer chromatin in new conceptual advances in gene regulation such as condensate formation.

STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells

Background Enhancers are distal regulators of gene expression that shape cell identity and control cell fate transitions. In mouse embryonic stem cells (mESCs), the pluripotency network is maintained by the function of a complex network of enhancers, that are drastically altered upon differentiation. Genome-wide chromatin accessibility and histone modification assays are commonly used as a proxy for identifying putative enhancers and for describing their activity levels and dynamics. Results Here, we applied STARR-seq, a genome-wide plasmid-based assay, as a read-out for the enhancer landscape in “ground-state” (2i+LIF; 2iL) and “metastable” (serum+LIF; SL) mESCs. This analysis reveals that active STARR-seq loci show modest overlap with enhancer locations derived from peak calling of ChIP-seq libraries for common enhancer marks. We unveil ZIC3-bound loci with significant STARR-seq activity in SL-ESCs. Knock-out of Zic3 removes STARR-seq activity only in SL-ESCs and increases their propensity to differentiate towards the endodermal fate. STARR-seq also reveals enhancers that are not accessible, masked by a repressive chromatin signature. We describe a class of dormant, p53 bound enhancers that gain H3K27ac under specific conditions, such as after treatment with Nocodazol, or transiently during reprogramming from fibroblasts to pluripotency. Conclusions In conclusion, loci identified as active by STARR-seq often overlap with those identified by chromatin accessibility and active epigenetic marking, yet a significant fraction is epigenetically repressed or display condition-specific enhancer activity.

H3K9me3 maintenance on a human artificial chromosome is required for segregation but not centromere epigenetic memory

ABSTRACTMost eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize de novo centromere formation, and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoidtetO human artificial chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase (also known as KDM4D), we removed heterochromatin mark H3K9me3 (histone 3 lysine 9 trimethylation) specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggered HAC mis-segregation. However, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low-H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A and CENP-C centromere levels, and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres.This article has an associated First Person interview with the first authors of the paper.