Distinct Histone Modifications Research Articles

Epigenetic Dynamics in Meniscus Cell Migration and its Zonal Dependency in Response to Inflammatory Conditions: Implications for Regeneration Strategies.

Meniscus injuries pose significant challenges in clinical settings, primarily due to the intrinsic heterogeneity of the tissue and the limited efficacy of current treatments. Endogenous cell migration is crucial for the healing process, yet the regulatory mechanisms of meniscus cell migration and its zonal dependency within the meniscus are not fully understood. Thus, this study investigates the role of epigenetic mechanisms in governing meniscus cell migration under inflammatory conditions, with a focus on their implications for injury healing and regeneration. Here, we discovered that a proinflammatory cytokine, TNF-α treatment significantly impedes the migration speed of inner meniscus cells, while outer meniscus cells are unaffected, underscoring a zonal-dependent response within the meniscus. Our analysis identified distinct histone modification patterns and chromatin dynamics between inner and outer meniscus cells during migration, highlighting the necessity to consider these zonal-dependent properties in devising repair strategies. Specifically, we found that TNF-α differentially influences histone modifications, particularly H3K27me3, between the two cell types. Transcriptome analysis further revealed that TNF-α treatment induces substantial gene expression changes, with inner meniscus cells exhibiting more pronounced alterations than outer cells. Gene cluster analysis pointed to distinct responses in chromatin remodeling, extracellular matrix assembly, and wound healing processes between the zonal cell populations. Moreover, we identified potential therapeutic targets by employing existing epigenetic drugs, GSKJ4 (a histone demethylase inhibitor) and C646 (a histone acetyltransferase inhibitor), to successfully restore the migration speed of inner meniscus cells under inflammatory conditions. This highlights their potential utility in treating meniscus tear injuries. Overall, our findings elucidate the intricate interplay between epigenetic mechanisms and meniscus cell migration, along with its meniscus zonal dependency. This study provides insights into potential targets for enhancing meniscus repair and regeneration, which may lead to improved clinical outcomes for patients with meniscus injuries and osteoarthritis.

Gastric Cancer in the Era of Epigenetics.

Gastric cancer (GC) remains a significant contributor to cancer-related mortality. Novel high-throughput techniques have enlightened the epigenetic mechanisms governing gene-expression regulation. Epigenetic characteristics contribute to molecular taxonomy and give rise to cancer-specific epigenetic patterns. Helicobacter pylori (Hp) infection has an impact on aberrant DNA methylation either through its pathogenic CagA protein or by inducing chronic inflammation. The hypomethylation of specific repetitive elements generates an epigenetic field effect early in tumorigenesis. Epstein-Barr virus (EBV) infection triggers DNA methylation by dysregulating DNA methyltransferases (DNMT) enzyme activity, while persistent Hp-EBV co-infection leads to aggressive tumor behavior. Distinct histone modifications are also responsible for oncogene upregulation and tumor-suppressor gene silencing in gastric carcinomas. While histone methylation and acetylation processes have been extensively studied, other less prevalent alterations contribute to the development and migration of gastric cancer via a complex network of interactions. Enzymes, such as Nicotinamide N-methyltransferase (NNMT), which is involved in tumor's metabolic reprogramming, interact with methyltransferases and modify gene expression. Non-coding RNA molecules, including long non-coding RNAs, circular RNAs, and miRNAs serve as epigenetic regulators contributing to GC development, metastasis, poor outcomes and therapy resistance. Serum RNA molecules hold the potential to serve as non-invasive biomarkers for diagnostic, prognostic or therapeutic applications. Gastric fluids represent a valuable source to identify potential biomarkers with diagnostic use in terms of liquid biopsy. Ongoing clinical trials are currently evaluating the efficacy of next-generation epigenetic drugs, displaying promising outcomes. Various approaches including multiple miRNA inhibitors or targeted nanoparticles carrying epigenetic drugs are being designed to enhance existing treatment efficacy and overcome treatment resistance.

Dynamic and distinct histone modifications facilitate human trophoblast lineage differentiation

The placenta serves as an essential organ for fetal growth throughout pregnancy. Histone modification is a crucial regulatory mechanism involved in numerous biological processes and development. Nevertheless, there remains a significant gap in our understanding regarding the epigenetic regulations that influence trophoblast lineage differentiation, a fundamental aspect of placental development. Here, through comprehensive mapping of H3K4me3, H3K27me3, H3K9me3, and H3K27ac loci during the differentiation of trophoblast stem cells (TSCs) into syncytiotrophoblasts (STs) and extravillous trophoblasts (EVTs), we reveal dynamic reconfiguration in H3K4me3 and H3K27ac patterns that establish an epigenetic landscape conducive to proper trophoblast lineage differentiation. We observe that broad H3K4me3 domains are associated with trophoblast lineage-specific gene expression. Unlike embryonic stem cells, TSCs lack robust bivalent domains. Notably, the repression of ST- and EVT-active genes in TSCs is primarily attributed to the weak H3K4me3 signal rather than bivalent domains. We also unveil the inactivation of TSC enhancers precedes the activation of ST enhancers during ST formation. Our results provide a comprehensive global map of diverse histone modifications, elucidating the dynamic histone modifications during trophoblast lineage differentiation.

Abstract PR008: The SLC1A1/EAAT3 dicarboxylic amino acid transporter: An actionable, HIF-independent, hub that links epigenetic dysregulation to nucleotide metabolism in kidney cancer

Abstract Significance: The clear cell renal cell carcinomas (ccRCCs), which harbor the hallmark inactivation of the von Hippel Lindau tumor suppressor protein (pVHL), represent the majority of adult kidney cancers. Although, early stage/localized ccRCC is effectively managed by surgery and surveillance, therapeutic options for advanced stage disease are limited, highlighting the unmet need for novel therapies. Chromatin dysregulation is routinely observed in ccRCC and our previous work identified a distinct histone modification signature associated with pVHL-deficient ccRCCs, versus the pVHL-proficient chromophobe and papillary sub-types, which included elevated levels of bulk Histone H3 K27 acetylation (H3K27ac). H3K27ac marks transcriptionally activated regions in the genome. We hypothesized that, as in other biological contexts, H3K27ac accumulation in pVHL-deficient ccRCCs drives the expression of key oncogenes. Here, we functionally probed this dysregulated epigenetic program to identify actionable targets in kidney cancer. Experimental Approach and Results: We identified genes marked by elevated H3K27ac (ChIP-Seq) and higher expression (RNA-Seq), specifically in pVHL-deficient cells and prioritized the top 100 genes that encoded druggable products. Using a library representing these ORFs, we did an in vivo ‘sufficiency’ screen, to identify candidates whose expression promoted tumor growth in otherwise poorly tumorigenic pVHL-proficient cells. Our screen (and downstream validation studies) identified SLC1A1, which encodes an Asp/Glu transporter named EAAT3, as an oncogenic driver in ccRCC. Results: SLC1A1 expression was elevated in pVHL-deficient cells – but in a HIF-independent manner. Inactivation of SLC1A1 (using CRISPR/Cas9) led to fitness defects in a panel of ccRCC cell lines, along with a notable depletion of nucleotide biosynthesis precursors, likely because of the importance of the Asp carbon skeleton in this pathway. Consistent with this, esterified-Asp rescued the fitness defects associated with SLC1A1 loss. Moreover, SLC1A1 upregulation (high Asp/Glu) diminished dependency on Glutaminase; whereas, SLC1A1 loss (low Asp/Glu) increased Glutaminase dependency and promoted sensitivity to Glutaminase blockade. Finally, using clinical cohorts and cell-based studies, we found that SLC1A1 can impact its own transcriptional expression, but also the expression of certain other metabolic transporters involved in Asp/Glu metabolism (e.g. SLC22A11) and nucleotide metabolism (e.g SLC28A1). We speculate, these transcriptional alterations are driven by the conversion of Glu into a-ketoglutarate, a key cofactor for KDMs. Our ongoing studies are addressing the mechanistic basis of this transcriptional regulation and exploring the feasibility of pharmacologically targeting SLC1A1 in ccRCC. Altogether, we show that ccRCCs hijack nutrient reabsorption mechanisms (e.g. SLC1A1) to drive oncogenic programs. SLC1A1 thus links epigenetic dysregulation to a HIF-independent metabolic program regulating nucleotide biosynthesis/uptake. Citation Format: Abhishek A. Chakraborty. The SLC1A1/EAAT3 dicarboxylic amino acid transporter: An actionable, HIF-independent, hub that links epigenetic dysregulation to nucleotide metabolism in kidney cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR008.

ChIP-seq profiling of H3K4me3 and H3K27me3 in an invasive insect, Bactrocera dorsalis.

Introduction: While it has been suggested that histone modifications can facilitate animal responses to rapidly changing environments, few studies have profiled whole-genome histone modification patterns in invasive species, leaving the regulatory landscape of histone modifications in invasive species unclear. Methods: Here, we screen genome-wide patterns of two important histone modifications, trimethylated Histone H3 Lysine 4 (H3K4me3) and trimethylated Histone H3 Lysine 27 (H3K27me3), in adult thorax muscles of a notorious invasive pest, the Oriental fruit fly Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), using Chromatin Immunoprecipitation with high-throughput sequencing (ChIP-seq). Results: We identified promoters featured by the occupancy of H3K4me3, H3K27me3 or bivalent histone modifications that were respectively annotated with unique genes key to muscle development and structure maintenance. In addition, we found H3K27me3 occupied the entire body of genes, where the average enrichment was almost constant. Transcriptomic analysis indicated that H3K4me3 is associated with active gene transcription, and H3K27me3 is mostly associated with transcriptional repression. Importantly, we identified genes and putative motifs modified by distinct histone modification patterns that may possibly regulate flight activity. Discussion: These findings provide the first evidence of histone modification signature in B. dorsalis, and will be useful for future studies of epigenetic signature in other invasive insect species.

Mass-Cytometry-Based Quantification of Global Histone Post-Translational Modifications at Single-Cell Resolution Across Peripheral Immune Cells in IBD.

Current understanding of histone post-translational modifications [histone modifications] across immune cell types in patients with inflammatory bowel disease [IBD] during remission and flare is limited. The present study aimed to quantify histone modifications at a single-cell resolution in IBD patients during remission and flare and how they differ compared to healthy controls. We performed a case-control study of 94 subjects [83 IBD patients and 11 healthy controls]. IBD patients had either ulcerative colitis [n = 38] or Crohn's disease [n = 45] in clinical remission or flare. We used epigenetic profiling by time-of-flight [EpiTOF] to investigate changes in histone modifications within peripheral blood mononuclear cells from IBD patients. We discovered substantial heterogeneity in histone modifications across multiple immune cell types in IBD patients. They had a higher proportion of less differentiated CD34+ haematopoietic progenitors, and a subset of CD56bright natural killer [NK] cells and γδ T cells characterized by distinct histone modifications associated with gene transcription. The subset of CD56bright NK cells had increases in several histone acetylations. An epigenetically defined subset of NK cells was associated with higher levels of C-reactive protein in peripheral blood. CD34+ monocytes from IBD patients had significantly decreased cleaved H3T22, suggesting they were epigenetically primed for macrophage differentiation. We describe the first systems-level quantification of histone modifications across immune cells from IBD patients at a single-cell resolution, revealing the increased epigenetic heterogeneity that is not possible with traditional ChIP-seq profiling. Our data open new directions in investigating the association between histone modifications and IBD pathology using other epigenomic tools.

Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modifications.

Chinese hamster ovary (CHO) cells are the most important platform for producing biotherapeutics. Random integration of a transgene into epigenetically instable regions of the genome results in silencing of the gene of interest and loss of productivity during upstream processing. Therefore, cost- and time-intensive long-term stability studies must be performed. Site-specific integration into safe harbors is a strategy to overcome these limitations of conventional cell line design. Recent publications predict safe harbors in CHO cells based on omics data sets or by learning from random integrations, but those predictions remain theory. In this study, we established a CRISPR/Cas9-mediated site-specific integration strategy based on ChIP-seq data to improve stability of recombinant CHO cells. Therefore, a ChIP experiment from the exponential and stationary growth phase of a fed-batch cultivation of CHO-K1 cells yielded 709 potentially stable integration sites. The reporter gene eGFP was integrated into three regions harboring specific modifications by CRISPR/Cas9. Targeted Cas9 nanopore sequencing showed site-specific integration in all 3 cell pools with a specificity between 23 and 73%. Subsequently, the cells with the three different integration sites were compared with the randomly integrated donor vector in terms of transcript level, productivity, gene copy numbers and stability. All site-specific integrations showed an increase in productivity and transcript levels of up to 7.4-fold. In a long-term cultivation over 70 generations, two of the site-specific integrations showed a stable productivity (>70%) independent of selection pressure.

The Retinoblastoma Tumor Suppressor Is Required for the NUP98-HOXA9-Induced Aberrant Nuclear Envelope Phenotype.

Chromosomal translocations involving the nucleoporin NUP98 gene are recurrently identified in leukemia; yet, the cellular defects accompanying NUP98 fusion proteins are poorly characterized. NUP98 fusions cause changes in nuclear and nuclear envelope (NE) organization, in particular, in the nuclear lamina and the lamina associated polypeptide 2α (LAP2α), a regulator of the tumor suppressor retinoblastoma protein (RB). We demonstrate that, for NUP98-HOXA9 (NHA9), the best-studied NUP98 fusion protein, its effect(s) on nuclear architecture largely depend(s) on RB. Morphological alterations caused by the expression of NHA9 are largely diminished in the absence of RB, both in human cells expressing the human papillomavirus 16 E7 protein and in mouse embryonic fibroblasts lacking RB. We further show that NHA9 expression associates with distinct histone modification. Moreover, the pattern of trimethylation of histone H3 lysine-27 is affected by NHA9, again in an RB-dependent manner. Our results pinpoint to an unexpected interplay between NUP98 fusion proteins and RB, which may contribute to leukemogenesis.

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available.In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells.Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.

Super-enhancer Acquisition Drives FOXC2 Expression in Middle Ear Cholesteatoma.

Distinct histone modifications regulate gene expression in certain diseases, but little is known about histone epigenetics in middle ear cholesteatoma. It is known that histone acetylation destabilizes the nucleosome and chromatin structure and induces gene activation. The association of histone acetylation with chronic inflammatory diseases has been indicated in recent studies. In this study, we examined the localization of variously modified histone H3 acetylation at lysine 9, 14, 18, 23, and 27 in paraffin-embedded sections of human middle ear cholesteatoma (cholesteatoma) tissues and the temporal bones of an animal model of cholesteatoma immunohistochemically. As a result, we found that there was a significant increase of the expression levels of H3K27ac both in human cholesteatoma tissues and the animal model. In genetics, super-enhancers are clusters of enhancers that drive the transcription of genes involved in cell identity. Super-enhancers were originally defined using the H3K27ac signal, and then we used H3K27ac chromatin immunoprecipitation followed by sequencing to map the active cis-regulatory landscape in human cholesteatoma. Based on the results, we identified increased H3K27ac signals as super-enhancers of the FOXC2 loci, as well as increased protein of FOXC2 in cholesteatoma. Recent studies have indicated that menin-MLL inhibitor could suppress tumor growth through the control of histone H3 modification. In this study, we demonstrated that the expression of FOXC2 was inhibited by menin-MLL inhibitor in vivo. These findings indicate that FOXC2 expression under histone modifications promoted the pathogenesis of cholesteatoma and suggest that it may be a therapeutic target of cholesteatoma.

Integrative genomic analysis of early neurogenesis reveals a temporal genetic program for differentiation and specification of preplate and Cajal-Retzius neurons.

Neurogenesis in the developing neocortex begins with the generation of the preplate, which consists of early-born neurons including Cajal-Retzius (CR) cells and subplate neurons. Here, utilizing the Ebf2-EGFP transgenic mouse in which EGFP initially labels the preplate neurons then persists in CR cells, we reveal the dynamic transcriptome profiles of early neurogenesis and CR cell differentiation. Genome-wide RNA-seq and ChIP-seq analyses at multiple early neurogenic stages have revealed the temporal gene expression dynamics of early neurogenesis and distinct histone modification patterns in early differentiating neurons. We have identified a new set of coding genes and lncRNAs involved in early neuronal differentiation and validated with functional assays in vitro and in vivo. In addition, at E15.5 when Ebf2-EGFP+ cells are mostly CR neurons, single-cell sequencing analysis of purified Ebf2-EGFP+ cells uncovers molecular heterogeneities in CR neurons, but without apparent clustering of cells with distinct regional origins. Along a pseudotemporal trajectory these cells are classified into three different developing states, revealing genetic cascades from early generic neuronal differentiation to late fate specification during the establishment of CR neuron identity and function. Our findings shed light on the molecular mechanisms governing the early differentiation steps during cortical development, especially CR neuron differentiation.

Unique Epigenetic Programming Distinguishes Regenerative Spermatogonial Stem Cells in the Developing Mouse Testis.

SummarySpermatogonial stem cells (SSCs) both self-renew and give rise to progenitors that initiate spermatogenic differentiation in the mammalian testis. Questions remain regarding the extent to which the SSC and progenitor states are functionally distinct. Here we provide the first multiparametric integrative analysis of mammalian germ cell epigenomes comparable with that done for >100 somatic cell types by the ENCODE Project. Differentially expressed genes distinguishing SSC- and progenitor-enriched spermatogonia showed distinct histone modification patterns, particularly for H3K27ac and H3K27me3. Motif analysis predicted transcription factors that may regulate spermatogonial subtype-specific fate, and immunohistochemistry and gene-specific chromatin immunoprecipitation analyses confirmed subtype-specific differences in target gene binding of a subset of these factors. Taken together, these results show that SSCs and progenitors display distinct epigenetic profiling consistent with these spermatogonial subtypes being differentially programmed to either self-renew and maintain regenerative capacity as SSCs or lose regenerative capacity and initiate lineage commitment as progenitors.

Sequential changes in histone modifications shape transcriptional responses underlying microglia polarization by glioma.

Microglia, resident myeloid cells of the central nervous system (CNS), act as immune sentinels that contribute to maintenance of physiological homeostasis and respond to any perturbation in CNS. Microglia could be polarized by various stimuli to perform dedicated functions and instigate inflammatory or pro-regenerative responses. Microglia and peripheral macrophages accumulate in glioblastomas (GBMs), malignant brain tumors, but instead of initiating antitumor responses, these cells are polarized to the pro-invasive and immunosuppressive phenotype which persists for a long time and contributes to a "cold" immune microenvironment of GBMs. Molecular mechanisms underlying this long-lasting "microglia memory" are unknown. We hypothesized that this state may rely on epigenetic silencing of inflammation-related genes. In this study, we show that cultured microglia pre-exposed to glioma-conditioned medium (GCM) acquire a "transcriptional memory" and display reduced expression of inflammatory genes after re-stimulation with lipopolysaccharide. Unstimulated microglia have unmethylated DNA and active histone marks at selected gene promoters indicating chromatin accessibility. Adding GCM increases expression and enzymatic activity of histone deacetylases (Hdac), leading to erasure of histone acetylation at tested genes. Later inflammatory genes acquire repressive histone marks (H3K27 trimethylation), which correlates with silencing of their expression. GCM induced genes acquire active histone marks. Hdac inhibitors block GCM-induced changes of histone modifications and restore microglia ability to initiate effective inflammatory responses. Altogether, we show a scenario of distinct histone modifications underlying polarization of microglia by glioma. We demonstrate contribution of epigenetic mechanisms to glioma-induced "transcriptional memory" in microglia resulting in the tumor-supportive phenotype.

The Arabidopsis epigenetic regulator ICU11 as an accessory protein of Polycomb Repressive Complex 2

Molecular mechanisms enabling the switching and maintenance of epigenetic states are not fully understood. Distinct histone modifications are often associated with ON/OFF epigenetic states, but how these states are stably maintained through DNA replication, yet in certain situations switch from one to another remains unclear. Here, we address this problem through identification of Arabidopsis INCURVATA11 (ICU11) as a Polycomb Repressive Complex 2 accessory protein. ICU11 robustly immunoprecipitated in vivo with PRC2 core components and the accessory proteins, EMBRYONIC FLOWER 1 (EMF1), LIKE HETEROCHROMATIN PROTEIN1 (LHP1), and TELOMERE_REPEAT_BINDING FACTORS (TRBs). ICU11 encodes a 2-oxoglutarate-dependent dioxygenase, an activity associated with histone demethylation in other organisms, and mutant plants show defects in multiple aspects of the Arabidopsis epigenome. To investigate its primary molecular function we identified the Arabidopsis FLOWERING LOCUS C (FLC) as a direct target and found icu11 disrupted the cold-induced, Polycomb-mediated silencing underlying vernalization. icu11 prevented reduction in H3K36me3 levels normally seen during the early cold phase, supporting a role for ICU11 in H3K36me3 demethylation. This was coincident with an attenuation of H3K27me3 at the internal nucleation site in FLC, and reduction in H3K27me3 levels across the body of the gene after plants were returned to the warm. Thus, ICU11 is required for the cold-induced epigenetic switching between the mutually exclusive chromatin states at FLC, from the active H3K36me3 state to the silenced H3K27me3 state. These data support the importance of physical coupling of histone modification activities to promote epigenetic switching between opposing chromatin states.

Quantifying epigenetic stability with minimum action paths.

Chromatin can adopt multiple stable, heritable states with distinct histone modifications and varying levels of gene expression. Insight on the stability and maintenance of such epigenetic states can be gained by mathematical modeling of stochastic reaction networks for histone modifications. Analytical results for the kinetic networks are particularly valuable. Compared to computationally demanding numerical simulations, they often are more convenient at evaluating the robustness of conclusions with respect to model parameters. In this communication, we developed a second-quantization-based approach that can be used to analyze discrete stochastic models with a fixed, finite number of particles using a representation of the SU(2) algebra. We applied the approach to a kinetic model of chromatin states that captures the feedback between nucleosomes and the enzymes conferring histone modifications. Using a path-integral expression for the transition probability, we computed the epigenetic landscape that helps to identify the emergence of bistability and the most probable path connecting the two steady states. We anticipate the generalizability of the approach will make it useful for studying more complicated models that couple epigenetic modifications with transcription factors and chromatin structure.

Unique Epigenetic Programming Distinguishes Regenerative Spermatogonial Stem Cells in The Developing Mouse Testis

Spermatogonial stem cells (SSCs) both self-renew and give rise to progenitor spermatogonia that initiate spermatogenic differentiation in the mammalian testis. Questions remain regarding the extent to which the SSC and progenitor states are functionally distinct. Here we provide the first multiparametric integrative analysis of mammalian germ cell epigenomes comparable to that done for >100 somatic cell types by the ENCODE Project. Differentially expressed genes distinguishing SSC- and progenitor-enriched spermatogonia showed distinct histone modification patterns, particularly related to enrichment of H3K27ac and H3K27me3. Motif-enrichment analysis predicted transcription factors that may regulate spermatogonial subtype-specific fate, and immunohistochemistry and gene-specific chromatin immunoprecipitation analyses confirmed subtype-specific differences in target gene binding of a subset of these factors. Taken together, these results suggest that SSCs and progenitors represent distinct spermatogonial subtypes differentially programmed to either self-renew and maintain regenerative capacity as SSCs, or lose regenerative capacity and initiate lineage commitment as progenitors.

PIRCh-seq: functional classification of non-coding RNAs associated with distinct histone modifications

We develop PIRCh-seq, a method which enables a comprehensive survey of chromatin-associated RNAs in a histone modification-specific manner. We identify hundreds of chromatin-associated RNAs in several cell types with substantially less contamination by nascent transcripts. Non-coding RNAs are found enriched on chromatin and are classified into functional groups based on the patterns of their association with specific histone modifications. We find single-stranded RNA bases are more chromatin-associated, and we discover hundreds of allele-specific RNA-chromatin interactions. These results provide a unique resource to globally study the functions of chromatin-associated lncRNAs and elucidate the basic mechanisms of chromatin-RNA interactions.

Integrative Analysis from the Epigenome to Translatome Uncovers Patterns of Dominant Nuclear Regulation during Transient Stress.

Gene regulation is a dynamic process involving changes ranging from the remodeling of chromatin to preferential translation. To understand integrated nuclear and cytoplasmic gene regulatory dynamics, we performed a survey spanning the epigenome to translatome of Arabidopsis (Arabidopsis thaliana) seedlings in response to hypoxia and reoxygenation. This included chromatin assays (examining histones, accessibility, RNA polymerase II [RNAPII], and transcription factor binding) and three RNA assays (nuclear, polyadenylated, and ribosome-associated). Dynamic patterns of nuclear regulation distinguished stress-induced and growth-associated mRNAs. The rapid upregulation of hypoxia-responsive gene transcripts and their preferential translation were generally accompanied by increased chromatin accessibility, RNAPII engagement, and reduced Histone 2A.Z association. Hypoxia promoted a progressive upregulation of heat stress transcripts, as evidenced by RNAPII binding and increased nuclear RNA, with polyadenylated RNA levels only elevated after prolonged stress or reoxygenation. Promoters of rapidly versus progressively upregulated genes were enriched for cis-elements of ethylene-responsive and heat shock factor transcription factors, respectively. Genes associated with growth, including many encoding cytosolic ribosomal proteins, underwent distinct histone modifications, yet retained RNAPII engagement and accumulated nuclear transcripts during the stress. Upon reaeration, progressively upregulated and growth-associated gene transcripts were rapidly mobilized to ribosomes. Thus, multilevel nuclear regulation of nucleosomes, transcript synthesis, accumulation, and translation tailor transient stress responses.plantcell;31/11/2573/FX1F1fx1.

Methyl-CpG-binding domain 9 (MBD9) is required for H2A.Z incorporation into chromatin at a subset of H2A.Z-enriched regions in the Arabidopsis genome

The SWR1 chromatin remodeling complex, which deposits the histone variant H2A.Z into nucleosomes, has been well characterized in yeast and animals, but its composition in plants has remained uncertain. We used the conserved SWR1 subunit ACTIN RELATED PROTEIN 6 (ARP6) as bait in tandem affinity purification experiments to isolate associated proteins from Arabidopsis thaliana. We identified all 11 subunits found in yeast SWR1 and the homologous mammalian SRCAP complexes, demonstrating that this complex is conserved in plants. We also identified several additional proteins not previously associated with SWR1, including Methyl-CpG-BINDING DOMAIN 9 (MBD9) and three members of the Alfin1-like protein family, all of which have been shown to bind modified histone tails. Since mbd9 mutant plants were phenotypically similar to arp6 mutants, we explored a potential role for MBD9 in H2A.Z deposition. We found that MBD9 is required for proper H2A.Z incorporation at thousands of discrete sites, which represent a subset of the genomic regions normally enriched with H2A.Z. We also discovered that MBD9 preferentially interacts with acetylated histone H4 peptides, as well as those carrying mono- or dimethylated H3 lysine 4, or dimethylated H3 arginine 2 or 8. Considering that MBD9-dependent H2A.Z sites show a distinct histone modification profile, we propose that MBD9 recognizes particular nucleosome modifications via its PHD- and Bromo-domains and thereby guides SWR1 to these sites for H2A.Z deposition. Our data establish the SWR1 complex as being conserved across eukaryotes and suggest that MBD9 may be involved in targeting the complex to specific genomic sites through nucleosomal interactions. The finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex, along with the synergistic phenotype of arp6;mbd9 double mutants, suggests that MBD9 also has important roles beyond H2A.Z deposition.

Predicting enhancers in mammalian genomes using supervised hidden Markov models

BackgroundEukaryotic gene regulation is a complex process comprising the dynamic interaction of enhancers and promoters in order to activate gene expression. In recent years, research in regulatory genomics has contributed to a better understanding of the characteristics of promoter elements and for most sequenced model organism genomes there exist comprehensive and reliable promoter annotations. For enhancers, however, a reliable description of their characteristics and location has so far proven to be elusive. With the development of high-throughput methods such as ChIP-seq, large amounts of data about epigenetic conditions have become available, and many existing methods use the information on chromatin accessibility or histone modifications to train classifiers in order to segment the genome into functional groups such as enhancers and promoters. However, these methods often do not consider prior biological knowledge about enhancers such as their diverse lengths or molecular structure.ResultsWe developed enhancer HMM (eHMM), a supervised hidden Markov model designed to learn the molecular structure of promoters and enhancers. Both consist of a central stretch of accessible DNA flanked by nucleosomes with distinct histone modification patterns. We evaluated the performance of eHMM within and across cell types and developmental stages and found that eHMM successfully predicts enhancers with high precision and recall comparable to state-of-the-art methods, and consistently outperforms those in terms of accuracy and resolution.ConclusionseHMM predicts active enhancers based on data from chromatin accessibility assays and a minimal set of histone modification ChIP-seq experiments. In comparison to other ’black box’ methods its parameters are easy to interpret. eHMM can be used as a stand-alone tool for enhancer prediction without the need for additional training or a tuning of parameters. The high spatial precision of enhancer predictions gives valuable targets for potential knockout experiments or downstream analyses such as motif search.