An Assessment of Fixed and Native Chromatin Preparation Methods to Study Histone Post-Translational Modifications at a Whole Genome Scale in Skeletal Muscle Tissue

Background

Genomic loci associated with histone marks are typically analyzed by immunoprecipitation of the chromatin followed by quantitative-PCR (ChIP-qPCR) or high throughput sequencing (ChIP-seq). Chromatin can be either cross-linked (X-ChIP) or used in the native state (N-ChIP). Cross-linking of DNA and proteins helps stabilizing their interactions before analysis. Despite X-ChIP is the most commonly used method, muscle tissue fixation is known to be relatively inefficient. Moreover, no protocol described a simple and reliable preparation of skeletal muscle chromatin of sufficient quality for subsequent high-throughput sequencing. Here we aimed to set-up and compare both chromatin preparation methods for a genome-wide analysis of H3K27me3, a broad-peak histone mark, using chicken P. major muscle tissue.

Results

Fixed and unfixed chromatin were prepared from chicken muscle tissues (Pectoralis major). Chromatin fixation, shearing by sonication or digestion and immunoprecipitation performed equivalently. High-quality Illumina reads were obtained (q30 > 93%). The bioinformatic analysis of the data was performed using epic, a tool based on SICER, and MACS2. Forty millions of reads were analyzed for both X-ChIP-seq and N-ChIP-seq experiments. Surprisingly, H3K27me3 X-ChIP-seq analysis led to the identification of only 2000 enriched regions compared to about 15,000 regions identified in the case of N-ChIP-seq. N-ChIP-seq peaks were more consistent between replicates compared to X-ChIP-seq. Higher N-ChIP-seq enrichments were confirmed by ChIP-qPCR at the PAX5 and SOX2 loci known to be enriched for H3K27me3 in myotubes and at the loci of common regions of enrichment identified in this study.

Conclusions

Our findings suggest that the preparation of muscle chromatin for ChIP-seq in cross-linked conditions can compromise the systematic analysis of broad histone marks. Therefore, native chromatin preparation should be preferred to cross-linking when a ChIP experiment has to be performed on skeletal muscle tissue, particularly when a broad source signal is considered.

     

Autologous blood transfusion augments impaired wound healing in diabetic mice by enhancing lncRNA H19 expression via the HIF-1α signaling pathway

Background

Impaired wound healing frequently occurs in diabetes mellitus (DM) and is implicated in impaired angiogenesis. Long non-coding RNA (lncRNA) H19 has been reported as being reduced in DM and played a critical role in inducing angiogenesis. Thus, we hypothesized that H19 may affect impaired wound healing in streptozotocin (STZ)-induced diabetic mice transfused with autologous blood preserved in standard preservative fluid or modified preservative fluid.

Methods

Fibroblasts in injured skin were isolated and cultured in vitro. After location of H19 in fibroblasts using fluorescence in situ hybridization (FISH), RNA-pull down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), Co immunoprecipitation (COIP) and dual luciferase reporter gene assay were used to verify the binding of H19 to HIF-1α.

Results

The modified preservative fluid preserved autologous blood increased the H19 expression in fibroblasts, and maintained better oxygen-carrying and oxygen release capacities as well as coagulation function. Furthermore, H19 promoted HIF-1α histone H3K4me3 methylation and increased HIF-1α expression by recruiting EZH2. H19 promoted fibroblast activation by activating HIF-1α signaling pathway in fibroblasts and enhanced wound healing in diabetic mice.

Conclusions

Taken together, H19 accelerated fibroblast activation by recruiting EZH2-mediated histone methylation and modulating the HIF-1α signaling pathway, whereby augmenting the process of modified preservative fluid preserved autologous blood enhancing the postoperative wound healing in diabetic mice.

     

Histone acetyltransferase CBP-related H3K23 acetylation contributes to courtship learning in <Emphasis Type="Italic">Drosophila</Emphasis>

Background

Histone modifications are critical in regulating neuronal processes. However, the impacts of individual histone modifications on learning and memory are elusive. Here, we investigated the contributions of histone H3 lysine modifications to learning and memory in Drosophila by using histone lysine-to-alanine mutants.

Results

Behavioural analysis indicated that compared to the H3WT group, mutants overexpressing H3K23A displayed impaired courtship learning. Chromatin immunoprecipitation analysis of H3K23A mutants showed that H3K23 acetylation (H3K23ac) levels were decreased on learning-related genes. Knockdown of CREB-binding protein (CBP) decreased H3K23ac levels, attenuated the expression of learning-related genes, led to a courtship learning defect and altered development of the mushroom bodies. A decline in courtship learning ability was observed in both larvae and adult treatments with ICG-001. Furthermore, treatment of Drosophila overexpressing mutated H3K23A with a CBP inhibitor did not aggravate the learning defect.

Conclusions

H3K23ac, catalysed by the acetyltransferases dCBP, contributes to Drosophila learning, likely by controlling the expression of specific genes. This is a novel epigenetic regulatory mechanism underlying neuronal behaviours.

     

Proteomic profiling identifies specific histone species associated with leukemic and cancer cells

Background

Chromatin is an extraordinarily complex structure. Much of this complexity results from the presence of numerous histone post-translational modifications and histone variants. Alterations in the patterns of histone post-translational modifications are emerging as a feature of many types of cancer and have been shown to have prognostic value.

Results

We have applied a liquid chromatography/mass spectrometry-based approach to comprehensively characterize the histone proteome in primary samples from chronic lymphocytic leukemia (CLL) patients, as well as bladder and breast cancer cell culture models. When compared to non-malignant CD19+ B cells from healthy donors, the CLL histone proteome showed a distinct signature of differentially expressed species, spanning all the histones studied and including both post-translationally modified species and unmodified, non-allelic replication-dependent histone isoforms. However, the large changes in histone H3 and H4 that are characteristic of many cancer types were not observed. One of species of H2A (mass = 14,063 Da) was the most strongly associated with time to treatment in CLL patients. CLL patient samples also demonstrated histone profiles that were distinct from those of the bladder and breast cancer cells.

Conclusions

Signatures of histone profiles are complex and can distinguish between healthy individuals and CLL patients and may provide prognostic markers. In addition, histone profiles may define tissue specific malignancies.

     

Characterizing biomarkers in osteosarcoma metastasis based on an ego-network

Objectives

To characterize biomarkers that underlie osteosarcoma (OS) metastasis based on an ego-network.

Results

From the microarray data, we obtained 13,326 genes. By combining PPI data and microarray data, 10,520 shared genes were found and constructed into ego-networks. 17 significant ego-networks were identified with p < 0.05. In the pathway enrichment analysis, seven ego-networks were identified with the most significant pathway.

Conclusions

These significant ego-modules were potential biomarkers that reveal the potential mechanisms in OS metastasis, which may contribute to understanding cancer prognoses and providing new perspectives in the treatment of cancer.

     

Copper oxide nanoparticles induce anticancer activity in A549 lung cancer cells by inhibition of histone deacetylase

Objectives

Copper oxide nanoparticles (CuO NPs) promoting anticancer activity may be due to the regulation of various classes of histone deacetylases (HDACs).

Results

Green-synthesized CuO NPs significantly arrested total HDAC level and also suppressed class I, II and IV HDACs mRNA expression in A549 cells. A549 cells treated with CuO NPs downregulated oncogenes and upregulated tumor suppressor protein expression. CuO NPs positively regulated both mitochondrial and death receptor-mediated apoptosis caspase cascade pathway in A549 cells.

Conclusion

Green-synthesized CuO NPs inhibited HDAC and therefore shown apoptosis mediated anticancer activity in A549 lung cancer cell line.

     

Histone modifications induced by MDV infection at early cytolytic and latency phases

Background

Marek’s disease (MD) is a highly contagious, lymphomatous disease of chickens induced by a herpesvirus, Marek’s disease virus (MDV) that is the cause of major annual losses to the poultry industry. MD pathogenesis involves multiple stages including an early cytolytic phase and latency, and transitions between these stages are governed by several host and environmental factors. The success of vaccination strategies has led to the increased virulence of MDV and selective breeding of naturally resistant chickens is seen as a viable alternative. While multiple gene expression studies have been performed in resistant and susceptible populations, little is known about the epigenetic effects of infection.

Results

In this study, we investigated temporal chromatin signatures induced by MDV by analyzing early cytolytic and latent phases of infection in the bursa of Fabricius of MD-resistant and –susceptible birds. Major global variations in chromatin marks were observed at different stages of MD in the two lines. Differential H3K27me3 marks were associated with immune-related pathways, such as MAP kinase signaling, focal adhesion and neuroactive ligand receptor interaction, and suggested varying degrees of silencing in response to infection. Immune-related microRNAs, e.g. gga-miR-155 and gga-miR-10b, bore chromatin signatures, which suggested their contribution to MD-susceptibility. Finally, several members of the focal adhesion pathway, e.g. THBS4 and ITGA1, showed marked concordance between gene expression and chromatin marks indicating putative epigenetic regulation in response to MDV infection.

Conclusion

Our comprehensive analysis of chromatin signatures, therefore, revealed further clues about the epigenetic effects of MDV infection although further studies are necessary to elucidate the functional implications of the observed variations in histone modifications.

     

Reveal cell type-specific regulatory elements and their characterized histone code classes via a hidden Markov model

Background

With the maturity of next generation sequencing technology, a huge amount of epigenomic data have been generated by several large consortia in the last decade. These plenty resources leave us the opportunity about sufficiently utilizing those data to explore biological problems.

Results

Here we developed an integrative and comparative method, CsreHMM, which is based on a hidden Markov model, to systematically reveal cell type-specific regulatory elements (CSREs) along the whole genome, and simultaneously recognize the histone codes (mark combinations) charactering them. This method also reveals the subclasses of CSREs and explicitly label those shared by a few cell types. We applied this method to a data set of 9 cell types and 9 chromatin marks to demonstrate its effectiveness and found that the revealed CSREs relates to different kinds of functional regulatory regions significantly. Their proximal genes have consistent expression and are likely to participate in cell type-specific biological functions.

Conclusions

These results suggest CsreHMM has the potential to help understand cell identity and the diverse mechanisms of gene regulation.

     

Dynamic changes of histone H3 lysine 9 following trimethylation in bovine oocytes and pre-implantation embryos

Objectives

We have examined dynamic changes of histone H3 lysine 9 following trimethylation (H3K9me3), the mRNA expression levels of SUV39H1 and SUV39H2 in bovine oocytes and the role in the development of in vitro fertilization (IVF) pre-implantation embryos.

Results

There were strong H3K9me3 signals in germinal vesicle (GV) oocytes but no signals in MII oocytes. H3K9me3 signals were maintained during IVF pre-implantation embryo development. SUV39H1 and SUV39H2 showed significantly higher mRNA expression levels in GV oocytes than MII oocytes (P < 0.01). SUV39H1 showed high mRNA expression level in two-cell embryos, however, SUV39H2 showed high mRNA expression level in four-cell embryos. In other development stage, SUV39H1 and SUV39H2 showed low expression levels.

Conclusion

Bovine IVF pre-implantation embryos maintain strong H3K9me3 signals and SUV39H1 and SUV39H2 are highly expressed at the early development stage of pre-implantation embryos.

     

PCI-24781 can improve in vitro and in vivo developmental capacity of pig somatic cell nuclear transfer embryos

Objective

To examine the effect of PCI-24781 (abexinostat) on the blastocyst formation rate in pig somatic cell nuclear transferred (SCNT) embryos and acetylation levels of the histone H3 lysine 9 and histone H4 lysine 12.

Results

Treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos, in comparison to the control group (25.3 vs. 10.5 %, P < 0.05). Furthermore, PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12. TUNEL assay and Hoechst 33342 staining revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos. Also, PCI-24781-treated embryos were transferred into three surrogate sows, one of whom became pregnant and two fetuses developed.

Conclusion

PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.

     

Integrated analysis of microRNA-target interactions with clinical outcomes for cancers

Background

Clinical statement alone is not enough to predict the progression of disease. Instead, the gene expression profiles have been widely used to forecast clinical outcomes. Many genes related to survival have been identified, and recently miRNA expression signatures predicting patient survival have been also investigated for several cancers. However, miRNAs and their target genes associated with clinical outcomes have remained largely unexplored.

Methods

Here, we demonstrate a survival analysis based on the regulatory relationships of miRNAs and their target genes. The patient survivals for the two major cancers, ovarian cancer and glioblastoma multiforme (GBM), are investigated through the integrated analysis of miRNA-mRNA interaction pairs.

Results

We found that there is a larger survival difference between two patient groups with an inversely correlated expression profile of miRNA and mRNA. It supports the idea that signatures of miRNAs and their targets related to cancer progression can be detected via this approach.

Conclusions

This integrated analysis can help to discover coordinated expression signatures of miRNAs and their target mRNAs that can be employed for therapeutics in human cancers.

     

re-TAMD: exploring interactions between H3 peptide and YEATS domain using enhanced sampling

Background

Analysis of preferred binding regions of a ligand on a protein is important for detecting cryptic binding pockets and improving the ligand selectivity.

Result

The enhanced sampling approach TAMD has been adapted to allow a ligand to unbind from its native binding site and explore the protein surface. This so-called re-TAMD procedure was then used to explore the interaction between the N terminal peptide of histone H3 and the YEATS domain. Depending on the length of the peptide, several regions of the protein surface were explored. The peptide conformations sampled during the re-TAMD correspond to peptide free diffusion around the protein surface.

Conclusions

The re-TAMD approach permitted to get information on the relative influence of different regions of the N terminal peptide of H3 on the interaction between H3 and YEATS.

     

Mitochondrial aconitase is a key regulator of energy production for growth and protein expression in Chinese hamster ovary cells

Introduction

Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.

Objectives

The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.

Methods

To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.

Results

Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.

Conclusion

Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.

     

Identification of cancer subtypes from single-cell RNA-seq data using a consensus clustering method

Background

Human cancers are complex ecosystems composed of cells with distinct molecular signatures. Such intratumoral heterogeneity poses a major challenge to cancer diagnosis and treatment. Recent advancements of single-cell techniques such as scRNA-seq have brought unprecedented insights into cellular heterogeneity. Subsequently, a challenging computational problem is to cluster high dimensional noisy datasets with substantially fewer cells than the number of genes.

Methods

In this paper, we introduced a consensus clustering framework conCluster, for cancer subtype identification from single-cell RNA-seq data. Using an ensemble strategy, conCluster fuses multiple basic partitions to consensus clusters.

Results

Applied to real cancer scRNA-seq datasets, conCluster can more accurately detect cancer subtypes than the widely used scRNA-seq clustering methods. Further, we conducted co-expression network analysis for the identified melanoma subtypes.

Conclusions

Our analysis demonstrates that these subtypes exhibit distinct gene co-expression networks and significant gene sets with different functional enrichment.