CTCF deletion alters the pluripotency and DNA methylation profile of human iPSCs.

Pluripotent stem cells are characterized by their differentiation potential toward endoderm, mesoderm, and ectoderm. However, it is still largely unclear how these cell-fate decisions are mediated by epigenetic mechanisms. In this study, we explored the relevance of CCCTC-binding factor (CTCF), a zinc finger-containing DNA-binding protein, which mediates long-range chromatin organization, for directed cell-fate determination. We generated human induced pluripotent stem cell (iPSC) lines with deletions in the protein-coding region in exon 3 of CTCF, resulting in shorter transcripts and overall reduced protein expression. Chromatin immunoprecipitation showed a considerable loss of CTCF binding to target sites. The CTCF deletions resulted in slower growth and modest global changes in gene expression, with downregulation of a subset of pluripotency-associated genes and neuroectodermal genes. CTCF deletion also evoked DNA methylation changes, which were moderately associated with differential gene expression. Notably, CTCF-deletions lead to upregulation of endo-mesodermal associated marker genes and epigenetic signatures, whereas ectodermal differentiation was defective. These results indicate that CTCF plays an important role in the maintenance of pluripotency and differentiation, especially towards ectodermal lineages.

Targeted DNA Methylation Analysis Facilitates Leukocyte Counts in Dried Blood Samples.

BACKGROUND: Cell-type specific DNA methylation (DNAm) can be employed to determine the numbers of leukocyte subsets in blood. In contrast to conventional methods for leukocyte counts, which are based on cellular morphology or surface marker protein expression, the cellular deconvolution based on DNAm levels is applicable for frozen or dried blood. Here, we further enhanced targeted DNAm assays for leukocyte counts in clinical application. METHODS: DNAm profiles of 40 different studies were compiled to identify CG dinucleotides (CpGs) with cell-type specific DNAm using a computational framework, CimpleG. DNAm levels at these CpGs were then measured with digital droplet PCR in venous blood from 160 healthy donors and 150 patients with various hematological disorders. Deconvolution was further validated with venous blood (n = 75) and capillary blood (n = 31) that was dried on Whatman paper or on Mitra microsampling devices. RESULTS: In venous blood, automated cell counting or flow cytometry correlated well with epigenetic estimates of relative leukocyte counts for granulocytes (r = 0.95), lymphocytes (r = 0.97), monocytes (r = 0.82), CD4 T cells (r = 0.84), CD8 T cells (r = 0.94), B cells (r = 0.96), and NK cells (r = 0.72). Similar correlations and precisions were achieved for dried blood samples. Spike-in with a reference plasmid enabled accurate epigenetic estimation of absolute leukocyte counts from dried blood samples, correlating with conventional venous (r = 0.86) and capillary (r = 0.80) blood measurements. CONCLUSIONS: The advanced selection of cell-type specific CpGs and utilization of digital droplet PCR analysis provided accurate epigenetic blood counts. Analysis of dried blood facilitates self-sampling with a finger prick, thereby enabling easier accessibility to testing.

Epigenetic rejuvenation by partial reprogramming.

Rejuvenation of cells by reprogramming toward the pluripotent state raises increasing attention. In fact, generation of induced pluripotent stem cells (iPSCs) completely reverses age-associated molecular features, including elongation of telomeres, resetting of epigenetic clocks and age-associated transcriptomic changes, and even evasion of replicative senescence. However, reprogramming into iPSCs also entails complete de-differentiation with loss of cellular identity, as well as the risk of teratoma formation in anti-ageing treatment paradigms. Recent studies indicate that partial reprogramming by limited exposure to reprogramming factors can reset epigenetic ageing clocks while maintaining cellular identity. So far, there is no commonly accepted definition of partial reprogramming, which is alternatively called interrupted reprogramming, and it remains to be elucidated how the process can be controlled and if it resembles a stable intermediate state. In this review, we discuss if the rejuvenation program can be uncoupled from the pluripotency program or if ageing and cell fate determination are inextricably linked. Alternative rejuvenation approaches with reprogramming into a pluripotent state, partial reprogramming, transdifferentiation, and the possibility of selective resetting of cellular clocks are also discussed.

Balance between autophagy and cell death is maintained by Polycomb-mediated regulation during stem cell differentiation.

Autophagy is a conserved cytoprotective process, aberrations in which lead to numerous degenerative disorders. While the cytoplasmic components of autophagy have been extensively studied, the epigenetic regulation of autophagy genes, especially in stem cells, is less understood. Deciphering the epigenetic regulation of autophagy genes becomes increasingly relevant given the therapeutic benefits of small-molecule epigenetic inhibitors in novel treatment modalities. We observe that, during retinoic acid-mediated differentiation of mouse embryonic stem cells (mESCs), autophagy is induced, and identify the Polycomb group histone methyl transferase EZH2 as a regulator of this process. In mESCs, EZH2 represses several autophagy genes, including the autophagy regulator DNA damage-regulated autophagy modulator protein 1 (Dram1). EZH2 facilitates the formation of a bivalent chromatin domain at the Dram1 promoter, allowing gene expression and autophagy induction during differentiation while retaining the repressive H3K27me3 mark. EZH2 inhibition leads to loss of the bivalent domain, with consequent 'hyper-expression' of Dram1, accompanied by extensive cell death. This study shows that Polycomb group proteins help maintain a balance between autophagy and cell death during stem cell differentiation, in part, by regulating the expression of the Dram1 gene.

Foxd3 controls heterochromatin-mediated repression of repeat elements and 2-cell state transcription.

Repeat element transcription plays a vital role in early embryonic development. The expression of repeats such as MERVL characterises mouse embryos at the 2-cell stage and defines a 2-cell-like cell (2CLC) population in a mouse embryonic stem cell culture. Repeat element sequences contain binding sites for numerous transcription factors. We identify the forkhead domain transcription factor FOXD3 as a regulator of major satellite repeats and MERVL transcription in mouse embryonic stem cells. FOXD3 binds to and recruits the histone methyltransferase SUV39H1 to MERVL and major satellite repeats, consequentially repressing the transcription of these repeats by the establishment of the H3K9me3 heterochromatin modification. Notably, depletion of FOXD3 leads to the de-repression of MERVL and major satellite repeats as well as a subset of genes expressed in the 2-cell state, shifting the balance between the stem cell and 2-cell-like population in culture. Thus, FOXD3 acts as a negative regulator of repeat transcription, ascribing a novel function to this transcription factor.

Understanding the role of Beclin1 in mouse embryonic stem cell differentiation through CRISPR-Cas9-mediated gene editing.

Autophagy is a vacuolar pathway for the regulated degradation and recycling of cellular components. Beclin1, a Bcl2-interacting protein, is a well-studied autophagy regulator. Homozygous loss of Beclin1 in mice leads to early embryonic lethality. However, the role of Beclin1 in regulating the pluripotency of embryonic stem cells and their differentiation remains poorly explored. To study this, we generated Beclin1-Knockout (KO) mouse embryonic stem cells (mESCs) using the CRISPR-Cas9 genome-editing tool. Interestingly, Beclin1-KO mESCs did not show any change in the expression of pluripotency marker genes. Beclin1-KO mESCs also displayed active autophagy, suggesting the presence of Beclin1-independent autophagy in mESCs. However, loss of Beclin1 resulted in compromised differentiation of mESCs in vitro and in vivo due to misregulated expression of transcription factors. Our results suggest that Beclin1 may play an autophagy-independent role in regulating the differentiation of mESCs.

Comparative nuclear matrix proteome analysis of skeletal muscle cells in different cellular states.

The nuclear matrix (NuMat) serves as the structural framework for organizing and maintaining nuclear architecture, however, the mechanisms by which this non-chromatin compartment is constructed and regulated are poorly understood. This study presents a proteomic analysis of the NuMat isolated from cultured skeletal muscle cells in three distinct cellular states- proliferating myoblasts (MBs), terminally differentiated myotubes (MTs), and mitotically quiescent (G0) myoblasts. About 40% of the proteins identified were found to be common in the NuMat proteome of these morphologically and functionally distinct cell states. These proteins, termed as the "core NuMat," define the stable, conserved, structural constituent of the nucleus, with functions such as RNA splicing, cytoskeletal organization, and chromatin modification, while the remaining NuMat proteins showed cell-state specificity, consistent with a more dynamic and potentially regulatory function. Specifically, myoblast NuMat was enriched in cell cycle, DNA replication and repair proteins, myotube NuMat in muscle differentiation and muscle function proteins, while G0 NuMat was enriched in metabolic, transcription, and transport proteins. These findings offer a new perspective for a cell-state-specific role of nuclear architecture and spatial organization, integrated with diverse cellular processes, and implicate NuMat proteins in the control of the cell cycle, lineage commitment, and differentiation.

Stress - (self) eating: Epigenetic regulation of autophagy in response to psychological stress.

Autophagy is a constitutive and cytoprotective catabolic process. Aberrations in autophagy lead to a multitude of degenerative disorders, with neurodegeneration being one of the most widely studied autophagy-related disorders. While the field has largely been focusing on the cytosolic constituents and processes of autophagy, recent studies are increasingly appreciating the role of chromatin modifications and epigenetic regulation in autophagy maintenance. Autophagy has been implicated in the regulation of neurogenesis, and disruption of neurogenesis in response to psychological stress is a proximal risk factor for development of neuropsychiatric disorders such as major depressive disorder (MDD). In this review, we will discuss the regulation of autophagy in normal neurogenesis as well as during chronic psychological stress, focusing on the epigenetic control of autophagy in these contexts, and also highlight the lacunae in our understanding of this process. The systematic study of these regulatory mechanisms will provide a novel therapeutic strategy, based on the use epigenetic regulators of autophagy to enhance neurogenesis and potentially alleviate stress-related behavioral disorders.

Identification of PRDM2 regulated genes in quiescent C2C12 myoblasts.

Quiescent stem cells contribute to tissue homeostasis and repair in adult mammals. We identified a tumor suppressor PRDM2, as an epigenetic regulator induced in quiescent muscle stem cells as well as in cultured quiescent myoblasts. To delineate the functions of PRDM2 in muscle cells, we compared the gene expression profiles of control and PRDM2 knockdown myoblasts in growing, differentiating and quiescent conditions (GEO accession number: GSE 58676). To identify the direct targets of PRDM2 and the promoters co-associated with H3K9me2 (mark catalyzed by PRDM2), ChIP-Chip analysis was performed (GSE58748). In this report we discuss in detail the methodology used to identify PRDM2 regulated genes and classify them into potential direct and indirect targets.

A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene.

Adult stem cell quiescence is critical to ensure regeneration while minimizing tumorigenesis. Epigenetic regulation contributes to cell cycle control and differentiation, but few regulators of the chromatin state in quiescent cells are known. Here we report that the tumor suppressor PRDM2/RIZ, an H3K9 methyltransferase, is enriched in quiescent muscle stem cells in vivo and controls reversible quiescence in cultured myoblasts. We find that PRDM2 associates with >4400 promoters in G0 myoblasts, 55% of which are also marked with H3K9me2 and enriched for myogenic, cell cycle and developmental regulators. Knockdown of PRDM2 alters histone methylation at key promoters such as Myogenin and CyclinA2 (CCNA2), and subverts the quiescence program via global de-repression of myogenesis, and hyper-repression of the cell cycle. Further, PRDM2 acts upstream of the repressive PRC2 complex in G0. We identify a novel G0-specific bivalent chromatin domain in the CCNA2 locus. PRDM2 protein interacts with the PRC2 protein EZH2 and regulates its association with the bivalent domain in the CCNA2 gene. Our results suggest that induction of PRDM2 in G0 ensures that two antagonistic programs-myogenesis and the cell cycle-while stalled, are poised for reactivation. Together, these results indicate that epigenetic regulation by PRDM2 preserves key functions of the quiescent state, with implications for stem cell self-renewal.

High-wire act: the poised genome and cellular memory.

Emerging evidence aided by genome-wide analysis of chromatin and transcriptional states has shed light on the mechanisms by which stem cells achieve cellular memory. The epigenetic and transcriptional plasticity governing stem cell behavior is highlighted by the identification of 'poised' genes, which permit cells to maintain readiness to undertake alternate developmental fates. This review focuses on two crucial mechanisms of gene poising: bivalent chromatin marks and RNA polymerase II stalling. We provide the context for these mechanisms by exploring the current consensus on the regulation of chromatin states, especially in quiescent adult stem cells, where poised genes are critical for recapitulating developmental choices, leading to regenerative function.

A ChIP-on-chip tiling array approach detects functional histone-free regions associated with boundaries at vertebrate HOX genes.

Hox genes impart segment identity to body structures along the anterior-posterior axis and are crucial for proper development. A unique feature of the Hox loci is the collinearity between the gene position within the cluster and its spatial expression pattern along the body axis. However, the mechanisms that regulate collinear patterns of Hox gene expression remain unclear, especially in higher vertebrates. We recently identified novel histone-free regions (HFRs) that can act as chromatin boundary elements demarcating successive murine Hox genes and help regulate their precise expression domains (Srivastava et al., 2013). In this report, we describe in detail the ChIP-chip analysis strategy associated with the identification of these HFRs. We also provide the Perl scripts for HFR extraction and quality control analysis for this custom designed tiling array dataset.

Vertebrate GAGA factor associated insulator elements demarcate homeotic genes in the HOX clusters.

BACKGROUND: Hox genes impart segment identity to body structures along the anterior-posterior axis and are crucial for the proper development of all organisms. Multiple regulatory elements, best defined in Drosophila melanogaster, ensure that Hox expression patterns follow the spatial and temporal colinearity reflected in their tight genomic organization. However, the precise mechanisms that regulate colinear patterns of Hox gene expression remain unclear, especially in higher vertebrates where it is not fully determined how the distinct activation domains of the tightly clustered Hox genes are defined independently of each other. Here, we report the identification of a large number of novel cis-elements at mammalian Hox clusters that can help in regulating their precise expression pattern. RESULTS: We have identified DNA elements at all four murine Hox clusters that show poor association with histone H3 in chromatin immunoprecipitation (ChIP)-chip tiling arrays. The majority of these elements lie in the intergenic regions segregating adjacent Hox genes; we demonstrate that they possess efficient enhancer-blocking activity in mammalian cells. Further, we find that these histone-free intergenic regions bear GA repeat motifs and associate with the vertebrate homolog of the GAGA binding boundary factor. This suggests that they can act as GAGA factor-dependent chromatin boundaries that create independent domains, insulating each Hox gene from the influence of neighboring regulatory elements. CONCLUSIONS: Our results reveal a large number of potential regulatory elements throughout the murine Hox clusters. We further demarcate the precise location of several novel cis-elements bearing chromatin boundary activity that appear to segregate successive Hox genes. This reflects a pattern reminiscent of the organization of homeotic genes in Drosophila, where such regulatory elements have been characterized. Our findings thus provide new insights into the regulatory processes and evolutionarily conserved epigenetic mechanisms that control homeotic gene expression.

The paternal hidden agenda: Epigenetic inheritance through sperm chromatin.

Epigenetic modifications play a crucial role in developmental gene regulation. These modifications, being reversible, provide a layer of information over and above the DNA sequence, that has plasticity and leads to the generation of cell type-specific epigenomes during cellular differentiation. In almost all higher eukaryotes, the oocyte provides not only its cytoplasm, mitochondria, maternally deposited RNA and proteins but also an epigenetic component in the form of DNA and histone-modifications. During spermeiogenesis however, most of the histones are replaced by protamines, leading to a loss of the epigenetic component. The sperm is, therefore, viewed as a passive carrier of the paternal genome with a disproportionate, lower epigenetic contribution except for DNA methylation, to the next generation. A recent study overturns this view by demonstrating a locus-specific retention of histones, with specific modifications in the sperm chromatin at the promoters of developmentally important genes. This programmed retention of epigenetic marks with a role in embryonic development is suggested to offset, in some measure, the dominant maternal effect. This new finding helps in addressing the question of epigenetic transmission of environmental and 'lifestyle' experiences across generations and raises the question of 'parental conflict' at the loci that may be differentially marked.