Increase In H3K4me3 Research Articles

Impacts of Hyperglycemia on Epigenetic Modifications in Human Gingival Fibroblasts and Gingiva in Diabetic Rats

Periodontal disease is considered one of the diabetic complications with high morbidity and severity. Recent studies demonstrated the involvement of the epigenome on diabetic complications. Histone modifications change chromatin architecture and gene activation. Histone modifications have been reported to alter chromatin structure and regulate gene transcription. In this study, we investigated the impacts of H3 lysine 4 trimethylation (H3K4me3) and specific histone methyltransferases of H3K4 methylation, su(var)3-9, enhancer-of-zeste, and trithorax domain 1A (SETD1A) on periodontal tissue affected by the diabetic condition. We observed the increase in H3K4me3 and SETD1A in gingival tissue of diabetic rats compared with the normal rats. Cultured human fibroblasts (hGFs) confirmed a high glucose-induced increase in H3K4me3 and SETD1A. We further demonstrated that high glucose increased the gene expression of matrix metalloproteinase (MMP) 1 and MMP13, which were canceled by sinefungin, an SETD1A inhibitor. Our investigation suggests that diabetes triggers histone modifications in the gingival tissue, resulting in gingival inflammation. Histone modifications may play crucial roles in the development of periodontal disease in diabetes.

ARID4B loss leads to activated STAT1-dependent interferon pathway in mouse embryonic stem cells and during meso/endodermal differentiation.

Proper deactivation of the pluripotency network and activation of a lineage-specific gene expression program are critical for mouse embryonic stem cell (mESC) differentiation. This is achieved by the coordinated action of transcription and chromatin factors. Our previous work identified ARID4B as a critical chromatin factor for mesoderm and endoderm differentiation. As part of a histone deacetylase complex, ARID4B plays a role in transcriptional suppression of its direct targets. Here, we investigated the mechanism of ARID4B function in mESC differentiation by focusing on genes and pathways that are upregulated in its absence. We analyzed transcriptomic results of wild-type and arid4bΔ endoderm or mesoderm differentiated cells through integrative genomics viewer and ingenuity pathway analysis. We performed real-time quantitative polymerase chain reaction for selected genes. To understand pathway activation, we performed Western blot for candidate proteins during the time-course of differentiation. We also analyzed H3K4me3, H3K27me3 and H3K27Ac ChIP-seq results to understand changes in the chromatin environment. Interferon-related genes were activated in arid4bΔ mESCs and endoderm or mesoderm directed cells. Consistent with this, higher phosphorylated STAT1 levels were found in arid4bΔ mESCs while a related phosphorylated STAT3 was unchanged. Finally, we observed a significant increase in H3K4me3 around interferon-related distal gene regulatory regions with a combination of either upregulation of H3K27Ac level or downregulation of H3K27me3 level. These results provide evidence that ARID4B is involved in the suppression of interferon-related genes in mESCs and during meso/endoderm differentiation through modulation, mainly of H3K4me3. This regulation might be important for successful mESC differentiation.

Gal-2 Increases H3K4me3 and H3K9ac in Trophoblasts and Preeclampsia

Preeclampsia (PE) is a severe pregnancy disorder with a pathophysiology not yet completely understood and without curative therapy. The histone modifications H3K4me3 and H3K9ac, as well as galectin-2 (Gal-2), are known to be decreased in PE. To gain a better understanding of the development of PE, the influence of Gal-2 on histone modification in trophoblasts and in syncytialisation was investigated. Immunohistochemical stains of 13 PE and 13 control placentas were correlated, followed by cell culture experiments. An analysis of H3K4me3 and H3K9ac was conducted, as well as cell fusion staining with E-cadherin and β-catenin—both after incubation with Gal-2. The expression of H3K4me3 and H3K9ac correlated significantly with the expression of Gal-2. Furthermore, we detected an increase in H3K4me3 and H3K9ac after the addition of Gal-2 to BeWo/HVT cells. Moreover, there was increased fusion of HVT cells after incubation with Gal-2. Gal-2 is associated with the histone modifications H3K4me3 and H3K9ac in trophoblasts. Furthermore, syncytialisation increased after incubation with Gal-2. Therefore, we postulate that Gal-2 stimulates syncytialisation, possibly mediated by H3K4me3 and H3K9ac. Since Gal-2, as well as H3K4me3 and H3K9ac, are decreased in PE, the induction of Gal-2 might be a promising therapeutic target.

WTAP-mediated m6A modification of lncRNA NORAD promotes intervertebral disc degeneration

N6-methyladenosine (m6A) is the most prevalent RNA modification at the posttranscriptional level and involved in various diseases and cellular processes. However, the underlying mechanism of m6A regulation in intervertebral disc degeneration (IVDD) remains elusive. Here, we show that methylation of the lncRNA NORAD significantly increases in senescent nucleus pulposus cells (NPCs) by m6A sequencing. Subsequent loss- and gain-of-function experiments reveal WTAP is increased in senescent NPCs due to an epigenetic increase in H3K4me3 of the promoter mediated by KDM5a, and significantly promotes NORAD m6A modification. Furthermore, YTHDF2-mediated decay of NORAD is enhanced in senescent NPCs, and then deficiency of NORAD results in less sequestraion of PUMILIO proteins, contributing to the augmented activity of PUM1/2, thus repressing the expression of target E2F3 mRNAs and promoting the cellular senescence. Here, we show interruption of NORAD m6A modification or the NORAD/PUMILIO/E2F3 axis could serve as a potential therapeutic target to inhibit the senescence of NPCs and development of IVDD.

The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by repressing pro-differentiation genes.

SUMMARYIn pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ~1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate, and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, negative elongation factor (NELF) complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency.

L-Methionine prevents β-cell damage by modulating the expression of Arx, MafA and regulation of FOXO1 in type 1 diabetic rats

L-Methionine (L-Met) is an essential sulphur-containing amino acid having a vital role in various key cellular processes. Here we investigated the effect of L-Met on streptozotocin-induced β-cell damage model of diabetes mellitus in Sprague Dawley rats. At the end of study biochemical parameters, immunoblotting, qRT-PCR and ChIP-qPCR are performed. L-Met was administered orally (250 and 500 mg/kg/day) to diabetic animals for 8 weeks improved plasma glucose and insulin levels. Pancreas immunohistochemistry showed significant increase in insulin expression, decrease in glucagon and Bax expression. Interestingly, L-Met inhibited the expression of Arx; upregulated MafA and FOXO1 which play a critical role in the maintenance of β-cell identity. Our data also showed a decrease in H3K27me3 and an increase in H3K4me3 ("bivalent domain" alteration) in diabetic rats and these recovered by L-Met. Furthermore, the chromatin-immunoprecipitation assay showed a decreased enrichment of H3K27me3 on the promoter of the FOXO1 gene in diabetic rats and L-Met prevents this decrease. Our results showed the first evidence of the involvement of H3K27me3 in regulating the expression of the FOXO1 gene and the prevention of β-cell injury by L-Met treatment. In conclusion, we report the involvement of L-Met in the modulation of α-cell identity marker (Arx), β-cell identity marker (MafA) and regulation of FOXO1 by histone methylation marks for the first time. We are of the opinion that this employed as a novel therapeutic approach for mitigating diabetes-induced β-cell death.

Histone deacetylase 4 deletion broadly affects cardiac epigenetic repression and regulates transcriptional susceptibility via H3K9 methylation

Histone deacetylase 4 (HDAC4) is a member of class IIa histone deacetylases (class IIa HDACs) and is believed to possess a low intrinsic deacetylase activity. However, HDAC4 sufficiently represses distinct transcription factors (TFs) such as the myocyte enhancer factor 2 (MEF2). Transcriptional repression by HDAC4 has been suggested to be mediated by the recruitment of other chromatin-modifying enzymes, such as methyltransferases or class I histone deacetylases. However, this concept has not been investigated by an unbiased approach.Therefore, we studied the histone modifications H3K4me3, H3K9ac, H3K27ac, H3K9me2 and H3K27me3 in a genome-wide approach using HDAC4-deficient cardiomyocytes. We identified a general epigenetic shift from a ‘repressive’ to an ‘active’ status, characterized by an increase of H3K4me3, H3K9ac and H3K27ac and a decrease of H3K9me2 and H3K27me3.In HDAC4-deficient cardiomyocytes, MEF2 binding sites were considerably overrepresented in upregulated promoter regions of H3K9ac and H3K4me3. For example, we identified the promoter of Adprhl1 as a new genomic target of HDAC4 and MEF2. Overexpression of HDAC4 in cardiomyocytes was able to repress the transcription of the Adprhl1 promoter in the presence of the methyltransferase SUV39H1. On a genome-wide level, the decrease of H3K9 methylation did not change baseline expression but was associated with exercise-induced gene expression.We conclude that HDAC4, on the one hand, associates with activating histone modifications, such as H3K4me3 and H3K9ac. A functional consequence, on the other hand, requires an indirect regulation of H3K9me2. H3K9 hypomethylation in HDAC4 target genes (‘first hit’) plus a ‘second hit’ (e.g., exercise) determines the transcriptional response.

Increased expression of fragmented tRNA promoted neuronal necrosis

Neuronal necrosis induced by excessive glutamate release is well known to contribute morbidity and mortality in ischemic stroke. Over the past decades, strategies on targeting glutamate receptor did not achieve desirable clinical outcomes. Finding the downstream mechanism of the glutamate receptor activation may provide new targets to suppress the cell death. Previously, our study demonstrated that the increase of H3K4 trimethylation (H3K4me3) played a key detrimental role on neuronal necrosis; however, the mechanism of this histone modification is unclear. Through a genome-wide small RNA sequencing, we identified several tRNA-derived fragments (tRFs) and piwi-interacting RNA (piRNAs) species were enriched in glutamate-induced neuronal necrosis in rat primary neuron cultures, and this enrichment was dependent on the H3K4me3 increase. Strikingly, when we transfected several synthesized tRFs and piRNA species into neurons, the tRFs but not the piRNAs induced neuron swelling and death. The cell death morphology recapitulated neuronal necrosis induced by glutamate. For the cytotoxic effect of tRFs, our data suggested that protein synthesis was inhibited likely through induction of ribosomal stalling. By proteomic analysis of tRFs effect, the most affected pathway was enriched in the mitochondrial metabolism. Consistently, mitochondrial fragmentation was increased in neuronal necrosis, and suppression of mitochondrial fission by genetic manipulation or drug rescued neuronal necrosis. Using our previously established Drosophila model of neuronal necrosis, we found that inhibition of small RNA transcription, blocking RNA transport from nucleus to cytosol, or knocking down Ago1/2 to suppress the RNA interference effect, all rescued the fly death, suggesting transcription and processing of small RNAs contribute to neuronal necrosis. Together, these results indicate that the abnormal transcription of tRFs may play a key role downstream of the H3K4me3 increase. This provides a potential new strategy to suppress neuronal necrosis.

Abstract 2117: Immunomodulatory functions of SMYD3 in HPV-negative head and neck squamous cell carcinoma

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) causes approximately 10,000 deaths annually in the United States, with human-papilloma-virus (HPV)-negative patients having a particularly poor prognosis. Pembrolizumab, a programmed-death-1 (PD-1) antibody, was recently approved as a first-line therapy for patients with relapsed disease, however response rates are as low as 19%, underscoring the need for an improved understanding of immunotherapy resistance mechanisms. We have previously shown that HPV-negative HNSCC tumors with higher mRNA levels of SET and MYND Domain Containing 3 (SMYD3), a chromatin modifier known to trimethylated H3K4 (H3K4me3), have significantly lower mRNA levels of CD8 and CD8+ T-cell attracting chemokines, and that SMYD3 depletion in HNSCC cells in vitro leads to upregulation of CD8+ T-cell attracting chemokines. The goal of this work was to elucidate epigenetic mechanisms of immune evasion mediated by SMYD3 in HPV-negative HNSCC. Methods: siRNA- mediated SMYD3 knockdown was performed for 3 days to investigate immuno-modulatory epigenetic mechanisms mediated by SMYD3. RT-qPCR for immune-related genes, Western blotting for histone marks, ChIP-sequencing for H3K4me3, ChIP-qPCRs to determine genomic interactions of SMYD3 and ATAC-sequencing were performed. SMYD3 and CD8 immunohistochemical staining (IHC) of normal squamous, dysplastic epithelium and HPV-negative HNSCC tumors was also performed. Results: siRNA mediated knockdown of SMYD3 resulted in enrichment of inflammatory pathways and upregulation of type 1 interferon response and antigen presentation machinery genes in HNSCC cell lines. Immunoblotting for histone marks showed no reduction in H3K4me3 following SMYD3 depletion for 3 days. ChIP-sequencing for H3K4me3 showed differential distribution pattern with an increase of H3K4me3 peaks on immune-related genes, and a decrease of H3K4me3 on UHRF1, a reader of the repressive histone mark H3K9me3. RNA-seq, RT-PCR and ChIP-qPCR showed that SMYD3 may regulate the transcription of UHRF1 by binding to its promoter. SMYD3 depletion also led to decreased protein levels of UHRF1. ChIP for UHRF1 and H3K9me3 showed that UHRF1 binds directly on the promoters of CXCL9 and CXCL10 which are enriched for H3K9me3. IHC on Normal Squamous Epithelium and Squamous Cell Carcinoma tissues of increasing cancer stages showed increase in SMYD3 levels with the higher stage of cancer, and an inverse correlation with CD8+ T cell infiltration within the tumors. Conclusion: This is the first study reporting that the chromatin modifier SMYD3 may attenuate type I IFN responses in HPV-negative HNSCC by regulating the expression of a major epigenetic regulator, UHRF1, which may silence the expression of immune-related genes through H3K9me3. Citation Format: Nupur Nigam, Benjamin Jonah Bernard, Kyunghee Burkitt, Sohyoung Kim, Yvette Robbins, Richard L. Bennett, Jonathan D. Licht, Vassiliki Saloura. Immunomodulatory functions of SMYD3 in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2117.

Genome wide analysis of heparan sulfate assembly

It is well known that the arrangement of the sulfated sugar residues in heparan sulfate specify the location of binding sites for heparan sulfate-binding proteins, and that binding regulates important biological processes including cell proliferation and development. Most of the enzymes involved in heparan sulfate biosynthesis have been cloned, purified and studied biochemically, but there is much less information regarding the mechanisms that control the spatial and temporal expression of heparan sulfate in cells and tissues. To study this question, a lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human melanoma cell line. High-throughput screening assays based on binding and uptake of guanidino-neomycin, a small molecule ligand that binds to heparan sulfate, was developed. The top hit was the epigenetic factor KDM2B, a histone demethylase. RNAseq revealed that inactivation of KDM2B altered the expression of several sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the binding of human fibroblast growth factor 1 (FGF1), FGF7, FGF10, S100 calcium-binding protein A12 and antithrombin, whereas binding of FGF2, interleukin-8 and neuropilin-1 was not affected. ChiPseq analysis showed an increase in H3K4me3 extending from the promoter in SULF1 into the gene body, suggesting that KDM2B normally represses SULF1 expression. Network analysis showed that inactivation of KDM2B also led to marked alterations in the expression of many extracellular matrix genes, suggesting that KDM2B serves as a master regulator of the extracellular matrix.

Ubiquitination of Histone H2B by Proteasome Subunit RPT6 Controls Histone Methylation Chromatin Dynamics During Memory Formation

BackgroundPosttranslational histone modifications play a critical role in the regulation of gene transcription underlying synaptic plasticity and memory formation. One such epigenetic change is histone ubiquitination, a process that is mediated by the ubiquitin–proteasome system in a manner similar to that by which proteins are normally targeted for degradation. However, histone ubiquitination mechanisms are poorly understood in the brain and in learning. In this article, we describe a new role for the ubiquitin–proteasome system in histone crosstalk, showing that learning-induced monoubiquitination of histone H2B (H2Bubi) is required for increases in the transcriptionally active H3 lysine 4 trimethylation (H3K4me3) mark at learning-related genes in the hippocampus. MethodsUsing a series of molecular, biochemical, electrophysiological, and behavioral experiments, we interrogated the effects of short interfering RNA–mediated knockdown and CRISPR (clustered regularly interspaced short palindromic repeats)-mediated upregulation of ubiquitin ligases, deubiquitinating enzymes and histone methyltransferases in the rat dorsal hippocampus during memory consolidation. ResultsWe show that H2Bubi recruits H3K4me3 through a process that is dependent on the 19S proteasome subunit RPT6 and that a loss of H2Bubi in the hippocampus prevents learning-induced increases in H3K4me3, gene transcription, synaptic plasticity, and memory formation. Furthermore, we show that CRISPR–dCas9-mediated increases in H2Bubi promote H3K4me3 and memory formation under weak training conditions and that promoting histone methylation does not rescue memory impairments resulting from loss of H2Bubi. ConclusionsThese results suggest that H2B ubiquitination regulates histone crosstalk in learning by way of nonproteolytic proteasome function, demonstrating a novel mechanism by which histone modifications are coordinated in response to learning.

The BTB Transcription Factors ZBTB11 and ZFP131 Maintain Pluripotency by Repressing Pro-Differentiation Genes

In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ~1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, NELF complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency.

Modification of the Histone Landscape with JAK Inhibition in Myeloproliferative Neoplasms.

Simple SummaryThe introduction of JAK inhibitors has provided objective benefits to many patients with myeloproliferative neoplasms. However, the overall efficacy of these drugs is limited and improved therapeutic options are required. Epigenetic dysregulation is increasingly observed to play a role in these disorders. This study evaluates the changing landscape of histone modifications in myeloproliferative neoplasm (MPN) cell line models and patient samples from the MAJIC clinical trial following administration of the JAK inhibitor ruxolitinib, demonstrating that ruxolitinib has an epigenetic effect. This changing histone modification landscape may offer a target for further therapy to augment treatment.Dysregulation of epigenetic processes is increasingly understood to play a role in the pathogenesis of myeloproliferative neoplasms (MPNs). Ruxolitinib, a JAK/STAT inhibitor, has proved a useful addition to the therapeutic arsenal for these disorders, but has limited disease modifying activity. We determined the effect of JAK inhibition on the histone landscape of MPN cells in cell line models of MPNs and validated using samples from the MAJIC randomised clinical trial of ruxolitinib in polycythaemia vera and essential thrombocythaemia. We demonstrated an epigenetic modifying effect of ruxolitinib using a histone modification assay. The majority of 21 histone H3 modifications were upregulated, with H3K27me3 and H3K36me2 significant in the combined cell line results. Chromatin immunoprecipitation and sequencing (CHIP-seq) for three marks of interest, H3K4me1, H3K4me3 and H3K27ac, was consistent with the histone modification assay showing a significant increase in H3K4me3 and H3K27ac peaks at promoter regions, both marks of active transcription. In contrast, RNA sequencing demonstrates a coordinated reduction in gene expression in a number of cell pathways including PI3K-AKT signalling, transcriptional misregulation in cancer and JAK-STAT signalling in spite of these histone changes. This highlights the complex mechanisms of transcriptional control within the cells which was reflected in analysis of the histone landscape in patient samples following ruxolitinib treatment.

Inhibition of colorectal tumor formation using a new miR‐210 therapeutic inhibitor that targets the Lnc RNA Xist and Nme1

In this report we investigate the role of microRNA‐210 (miR‐210) in the etiology of colorectal cancer (CRC) using both cell‐based and xenograft mouse models. We show that miR‐210 expression is higher in the colon cancer cell line SW620 versus normal colon epithelium and has been associated with decreased cancer survival rates. Stable expression of our new miR‐210 inhibitor changes the morphology, proliferation and gene expression of SW620 cells and tumor growth. RNAseq identified potential targets of miR‐210 and qPCR has verified several of these targets. We find that inhibition of miR‐210 can act as a tumor suppressor and reduce tumor formation of SW620 colon cancer cells in xenograft mouse models. Direct comparison with capecitabine treatments demonstrates the effectiveness of the miR‐210 inhibitor treatment. The direct administration of the miR‐210 inhibitor (MiRIS) to existing tumors can inhibit tumor growth in both NSG and Foxnu/j mouse models. The inhibition of miR‐210 can cause alterations in hypoxia responses and upregulation of several tumor suppressor genes, including Nme1. We demonstrate that the long non‐coding transcript XIST is regulated by miR‐210 and XIST expression is decreased in colon tumors. We show that XIST expression correlates with the expression of the tumor suppressor gene Nme1. Furthermore, the inhibition of miR‐210 effects epigenetic marks of the NME1 loci, with increases in H3K4me3 and H3K27ac near the transcription start site (TSS). We show that our new miR‐210 inhibitor can be used as an effective colorectal cancer therapeutic tool.

Arabidopsis SDG8 Potentiates the Sustainable Transcriptional Induction of the Pathogenesis-Related Genes PR1 and PR2 During Plant Defense Response.

Post-translational covalent modifications of histones play important roles in modulating chromatin structure and are involved in the control of multiple developmental processes in plants. Here we provide insight into the contribution of the histone lysine methyltransferase SET DOMAIN GROUP 8 (SDG8), implicated in histone H3 lysine 36 trimethylation (H3K36me3), in connection with RNA polymerase II (RNAPII) to enhance Arabidopsis immunity. We showed that even if the sdg8-1 loss-of-function mutant, defective in H3K36 methylation, displayed a higher sensitivity to different strains of the bacterial pathogen Pseudomonas syringae, effector-triggered immunity (ETI) still operated, but less efficiently than in the wild-type (WT) plants. In sdg8-1, the level of the plant defense hormone salicylic acid (SA) was abnormally high under resting conditions and was accumulated similarly to WT at the early stage of pathogen infection but quickly dropped down at later stages. Concomitantly, the transcription of several defense-related genes along the SA signaling pathway was inefficiently induced in the mutant. Remarkably, albeit the defense genes PATHOGENESIS-RELATED1 (PR1) and PR2 have retained responsiveness to exogenous SA, their inductions fade more rapidly in sdg8-1 than in WT. At chromatin, while global levels of histone methylations were found to be stable, local increases of H3K4 and H3K36 methylations as well as RNAPII loading were observed at some defense genes following SA-treatments in WT. In sdg8-1, the H3K36me3 increase was largely attenuated and also the increases of H3K4me3 and RNAPII were frequently compromised. Lastly, we demonstrated that SDG8 could physically interact with the RNAPII C-terminal Domain, providing a possible link between RNAPII loading and H3K36me3 deposition. Collectively, our results indicate that SDG8, through its histone methyltransferase activity and its physical coupling with RNAPII, participates in the strong transcriptional induction of some defense-related genes, in particular PR1 and PR2, to potentiate sustainable immunity during plant defense response to bacterial pathogen.

Genome-Wide Identification of Histone Modifications Involved in Placental Development in Pigs.

Development of placental folds is a critical event affecting placental function in pigs because it can increase surface area for improvement in capillary density as gestation advances. However, the molecular mechanisms of the event are not well defined. Histone modifications have important roles in gene regulation. To investigate their effects on regulation of genes controlling porcine placental development, RNA-seq and ChIP-seq of porcine placental tissues from gestational days 50 (establishment stage of placental folds) and 95 (expanding stage of placental folds) were carried out in this study. The differentially expressed genes were identified and of which the down- and up-regulated genes are related to endoplasmic reticulum (ER) stress and angiogenesis, respectively. In addition, we mapped the genome-wide profiles of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac), which are associated with transcriptional activation. A number of differential modification regions between the 2 gestational stages were identified and majority of them are those with increased signals of H3K4me3 (14,576 out of 16,931). Furthermore, we observed that the increase of H3K4me3 is significantly correlated with the elevated expression levels of the neighboring genes, and notably, these genes were enriched in pathways related to blood vessel formation and microvascular permeability. Taken together, the findings suggest important roles of histone modifications on placental remolding in response to developmental changes.

Waves of chromatin modifications in mouse dendritic cells in response to LPS stimulation

BackgroundThe importance of transcription factors (TFs) and epigenetic modifications in the control of gene expression is widely accepted. However, causal relationships between changes in TF binding, histone modifications, and gene expression during the response to extracellular stimuli are not well understood. Here, we analyze the ordering of these events on a genome-wide scale in dendritic cells in response to lipopolysaccharide (LPS) stimulation.ResultsUsing a ChIP-seq time series dataset, we find that the LPS-induced accumulation of different histone modifications follows clearly distinct patterns. Increases in H3K4me3 appear to coincide with transcriptional activation. In contrast, H3K9K14ac accumulates early after stimulation, and H3K36me3 at later time points. Integrative analysis with TF binding data reveals potential links between TF activation and dynamics in histone modifications. Especially, LPS-induced increases in H3K9K14ac and H3K4me3 are associated with binding by STAT1/2 and were severely impaired in Stat1−/− cells.ConclusionsWhile the timing of short-term changes of some histone modifications coincides with changes in transcriptional activity, this is not the case for others. In the latter case, dynamics in modifications more likely reflect strict regulation by stimulus-induced TFs and their interactions with chromatin modifiers.

Cycles of gene expression and genome response during mammalian tissue regeneration

BackgroundCompensatory liver hyperplasia—or regeneration—induced by two-thirds partial hepatectomy (PH) permits the study of synchronized activation of mammalian gene expression, particularly in relation to cell proliferation. Here, we measured genomic transcriptional responses and mRNA accumulation changes after PH and sham surgeries.ResultsDuring the first 10–20 h, the PH- and sham-surgery responses were very similar, including parallel early activation of cell-division-cycle genes. After 20 h, however, whereas post-PH livers continued with a robust and coordinate cell-division-cycle gene-expression response before returning to the resting state by 1 week, sham-surgery livers returned directly to a resting gene-expression state. Localization of RNA polymerase II (Pol II), and trimethylated histone H3 lysine 4 (H3K4me3) and 36 (H3K36me3) on genes dormant in the resting liver and activated during the PH response revealed a general de novo promoter Pol II recruitment and H3K4me3 increase during the early 10–20 h phase followed by Pol II elongation and H3K36me3 accumulation in gene bodies during the later proliferation phase. H3K36me3, generally appearing at the first internal exon, was preceded 5′ by H3K36me2; 3′ of the first internal exon, in about half of genes H3K36me3 predominated and in the other half H3K36me2 and H3K36me3 co-existed. Further, we observed some unusual gene profiles with abundant Pol II but little evident H3K4me3 or H3K36me3 modification, indicating that these modifications are neither universal nor essential partners to Pol II transcription.ConclusionsPH and sham surgical procedures on mice reveal striking early post-operatory gene expression similarities followed by synchronized mRNA accumulation and epigenetic histone mark changes specific to PH.

An Arabidopsis E3 ligase HUB2 increases histone H2B monoubiquitination and enhances drought tolerance in transgenic cotton

SummaryThe HUB2 gene encoding histone H2B monoubiquitination E3 ligase is involved in seed dormancy, flowering timing, defence response and salt stress regulation in Arabidopsis thaliana. In this study, we used the cauliflower mosaic virus (CaMV) 35S promoter to drive AtHUB2 overexpression in cotton and found that it can significantly improve the agricultural traits of transgenic cotton plants under drought stress conditions, including increasing the fruit branch number, boll number, and boll‐setting rate and decreasing the boll abscission rate. In addition, survival and soluble sugar, proline and leaf relative water contents were increased in transgenic cotton plants after drought stress treatment. In contrast, RNAi knockdown of GhHUB2 genes reduced the drought resistance of transgenic cotton plants. AtHUB2 overexpression increased the global H2B monoubiquitination (H2Bub1) level through a direct interaction with GhH2B1 and up‐regulated the expression of drought‐related genes in transgenic cotton plants. Furthermore, we found a significant increase in H3K4me3 at the DREB locus in transgenic cotton, although no change in H3K4me3 was identified at the global level. These results demonstrated that AtHUB2 overexpression changed H2Bub1 and H3K4me3 levels at the GhDREB chromatin locus, leading the GhDREB gene to respond quickly to drought stress to improve transgenic cotton drought resistance, but had no influence on transgenic cotton development under normal growth conditions. Our findings also provide a useful route for breeding drought‐resistant transgenic plants.

Sporadic DUX4 expression in FSHD myocytes is associated with incomplete repression by the PRC2 complex and gain of H3K9 acetylation on the contracted D4Z4 allele

BackgroundFacioscapulohumeral muscular dystrophy 1 (FSHD1) has an autosomal dominant pattern of inheritance and primarily affects skeletal muscle. The genetic cause of FSHD1 is contraction of the D4Z4 macrosatellite array on chromosome 4 alleles associated with a permissive haplotype causing infrequent sporadic expression of the DUX4 gene. Epigenetically, the contracted D4Z4 array has decreased cytosine methylation and an open chromatin structure. Despite these genetic and epigenetic changes, the majority of FSHD myoblasts are able to repress DUX4 transcription. In this study we hypothesized that histone modifications distinguish DUX4 expressing and non-expressing cells from the same individuals.ResultsFSHD myocytes containing the permissive 4qA haplotype with a long terminal D4Z4 unit were sorted into DUX4 expressing and non-expressing groups. We found similar CpG hypomethylation between the groups of FSHD-affected cells suggesting that CpG hypomethylation is not sufficient to trigger DUX4 expression. A survey of histone modifications present at the D4Z4 region during cell lineage commitment revealed that this region is bivalent in FSHD iPS cells with both H3K4me3 activating and H3K27me3 repressive marks present, making D4Z4 poised for DUX4 activation in pluripotent cells. After lineage commitment, the D4Z4 region becomes univalent with H3K27me3 in FSHD and non-FSHD control myoblasts and a concomitant increase in H3K4me3 in a small fraction of cells. Chromatin immunoprecipitation (ChIP) for histone modifications, chromatin modifier proteins and chromatin structural proteins on sorted FSHD myocytes revealed that activating H3K9Ac modifications were ~ fourfold higher in DUX4 expressing FSHD myocytes, while the repressive H3K27me3 modification was ~ fourfold higher at the permissive allele in DUX4 non-expressing FSHD myocytes from the same cultures. Similarly, we identified EZH2, a member of the polycomb repressive complex involved in H3K27 methylation, to be present more frequently on the permissive allele in DUX4 non-expressing FSHD myocytes.ConclusionsThese results implicate PRC2 as the complex primarily responsible for DUX4 repression in the setting of FSHD and H3K9 acetylation along with reciprocal loss of H3K27me3 as key epigenetic events that result in DUX4 expression. Future studies focused on events that trigger H3K9Ac or augment PRC2 complex activity in a small fraction of nuclei may expose additional drug targets worthy of study.