Chromatin Restructuring Research Articles

Expression of microRNA in male reproductive tissues and their role in male fertility.

MicroRNA (miRNA) are small non-coding RNA, approximately 22 nucleotides in length, that regulate gene expression through their ability to bind to mRNA. The role of miRNA in cellular and tissue development is well documented and their importance in male reproductive tissue development is actively being evaluated. They are present in spermatogonia, Sertoli and Leydig cells within the testis and are present in mature spermatozoa, indicating roles in normal testicular development, function and spermatogenesis. Their presence in spermatozoa has led to postulations about the roles of male miRNA during early embryonic development after fertilisation, including chromatin restructuring and possible epigenetic effects on embryo development. MiRNAs are also present in body fluids, such as blood serum, milk, ovarian follicular fluid and seminal fluid. Circulating miRNAs are stable, and aberrant expression of cellular or extracellular miRNA has been associated with multiple pathophysiological conditions, the most studied being numerous forms of cancer. Considering that miRNAs are present in spermatozoa and in seminal fluid, their stability and the relatively non-invasive procedures required to obtain these samples make miRNAs excellent candidates for use as biomarkers of male reproduction and fertility. Biomarkers, such as miRNAs, identifying fertile males would be of financial interest to the animal production industry.

Identification of Posttranslational Modifications of Endogenous Chromatin Proteins From Testicular Cells by Mass Spectrometry.

Chromatin architecture in mammalian spermatogenesis undergoes extensive structural and functional reorganization during which several testis-specific histone variants and other chromatin proteins are expressed in a stage-dependent manner. The most dramatic change in chromatin composition is observed during spermiogenesis where nucleosomal chromatin is transformed into nucleoprotamine fiber. Role of posttranslational modification (PTM) of somatic canonical histones and histone variants is well documented and effect several chromatin-templated events. PTM of testis-specific chromatin proteins is proposed to orchestrate chromatin-templated events during mammalian spermatogenesis and their identification and subsequent functional characterization is key to understand chromatin restructuring events and establishment of sperm epigenome. Here, we present protocols for the purification of endogenous testis chromatin proteins from different stages of spermatogenesis and identification of their PTM repertoire by mass spectrometry through examples of testis-specific histone variants (TH2B and HILS1), and transition proteins (TP1 and TP2).

Effect of DNA methylation on the interaction with tetrahydrocortisol–apolipoprotein A-I complex

The effect of GCNGC site methylation on the DNA binding to tetrahydrocortisol–apolipoprotein A-I complex was shown by atomic force microscopy and enzymatic analysis. Methylation of GCNGC sites alters the mechanism of interaction of tetrahydrocortisol–apolipoprotein A-I complex with DNA and leads to the aggregation of the DNA-protein complexes. It was assumed that the involvement of complexes of steroid hormones with apolipoprotein A-I in the process of chromatin restructuring is associated with the processes of DNA methylation/demethylation.

Quantitative Proteomics Uncovers Novel Factors Involved in Developmental Differentiation of Trypanosoma brucei.

Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes.

Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae

ABSTRACTCo-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) – a residue methylated by Set2 during transcription elongation – exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast.

Minor Loops in Major Folds: Enhancer-Promoter Looping, Chromatin Restructuring, and Their Association with Transcriptional Regulation and Disease.

The organization and folding of chromatin within the nucleus can determine the outcome of gene expression. Recent technological advancements have enabled us to study chromatin interactions in a genome-wide manner at high resolution. These studies have increased our understanding of the hierarchy and dynamics of chromatin domains that facilitate cognate enhancer–promoter looping, defining the transcriptional program of different cell types. In this review, we focus on vertebrate chromatin long-range interactions as they relate to transcriptional regulation. In addition, we describe how the alteration of boundaries that mark discrete regions in the genome with high interaction frequencies within them, called topological associated domains (TADs), could lead to various phenotypes, including human diseases, which we term as “TADopathies.”

A Fusarium graminearum strain-comparative proteomic approach identifies regulatory changes triggered by agmatine

Plant pathogens face different environmental clues depending on the stage of the infection cycle they are in. Fusarium graminearum infects small grain cereals producing trichothecenes type B (TB) that act as virulence factor in the interaction with the plant and have important food safety implications. This study addresses at the proteomic level the effect of an environmental stimulus (such as the presence of a polyamine like agmatine) possibly encountered by the fungus when it is already within the plant. Because biological diversity affects the proteome significantly, a multistrain (n=3) comparative approach was used to identify consistent effects caused on the fungus by the nitrogen source (agmatine or glutamic acid). Proteomics analyses were performed by the use of 2D-DIGE. Results showed that agmatine augmented TB production but not equally in all strains. The polyamine reshaped drastically the proteome of the fungus activating specific pathways linked to the translational control within the cell. Chromatin restructuring, ribosomal regulations, protein and mRNA processing enzymes were modulated by the agmatine stimulus as well as metabolic, structural and virulence-related proteins, suggesting the need to reshape specifically the fungal cell for TB production, a key step for the pathogen spread within the spike. Biological significanceInduction of toxin synthesis by plant compounds plays a crucial role in toxin contamination of food and feed, in particular trichothecenes type B produced mainly by F. graminearum on wheat. This work describes the level of diversity of 3 strains facing 2 toxin inducing plant derived compounds. This knowledge is of use for the research community on toxigenic Fusarium strains in cereals for understanding the role of fungal diversity in toxin inducibility. This work also suggests that environmental clues that can be found within the plant during infection (like different nitrogen compounds) are crucial stimuli for reshaping the proteome profile and consequently the specialization profiling of the fungus, ultimately leading to very different toxin contamination levels in the plant.

RDNA genetic imbalance and nucleolar chromatin restructuring is induced by distant hybridization between Raphanus sativus and Brassica alboglabra.

The expression of rDNA in hybrids inherited from only one progenitor refers to nucleolar dominance. The molecular basis for choosing which genes to silence remains unclear. We report genetic imbalance induced by distant hybridization correlates with formation of rDNA genes (NORs) in the hybrids between Raphanus sativus L. and Brassica alboglabra Bailey. Moreover, increased CCGG methylation of rDNA in F1 hybrids is concomitant with Raphanus-derived rDNA gene silencing and rDNA transcriptional inactivity revealed by nucleolar configuration restriction. Newly formed rDNA gene locus occurred through chromosomal in F1 hybrids via chromosomal imbalance. NORs are gained de novo, lost, and/or transposed in the new genome. Inhibition of methyltransferases leads to changes in nucleolar architecture, implicating a key role of methylation in control of nucleolar dominance and vital nucleolar configuration transition. Our findings suggest that gene imbalance and methylation-related chromatin restructuring is important for rDNA gene silencing that may be crucial for synthesis of specific proteins.

Minor grove binding ligands disrupt PARP-1 activation pathways

PARP-1 is a nuclear enzyme regulating transcription, chromatin restructuring, and DNA repair. PARP-1 is activated by interaction with NAD+, DNA, and core histones. Each route of PARP-1 activation leads to somewhat different outcomes. PARP-1 interactions with core histones control PARP-1 functions during transcriptional activation in euchromatin. DNA-dependent regulation of PARP-1 determines its localization in heterochromatin and PARP-1-dependent silencing. Here we address the biological significance of DNA-dependent PARP-1 regulation in vitro and in vivo. We report that minor grove binding ligands (MGBLs) specifically target PARP-1 interaction with DNA, and, hence, the DNA-dependent pathway of PARP-1 activation. By obstructing its interaction with DNA molecules, MGBLs block PARP-1 activity in vitro and in vivo, as we demonstrate using Drosophila, as well as human cancer-derived cells. We also demonstrate synergistic inhibition of PARP-1, combining MGBLs with conventional NAD+-dependent inhibitors in human cancer cells. These results suggest that combining different classes of PARP-1 inhibitors can precisely modulate PARP-1 activity in living cells, thus holding promise for new avenues of cancer treatment.

Krüppel-associated Box (KRAB)-associated Co-repressor (KAP-1) Ser-473 Phosphorylation Regulates Heterochromatin Protein 1β (HP1-β) Mobilization and DNA Repair in Heterochromatin

The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-β chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-β, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-β from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-β exchange from heterochromatin, promoting DNA repair.

Alzheimer’s Disease Biomarkers and Epigenetic Intermediates Following Exposure to Pb In Vitro

Late onset Alzheimer's disease (LOAD) is typical of the majority of Alzheimer's disease (AD) cases (~90%), and has no clear genetic association. Previous studies from our lab suggest that an epigenetic component could be involved. Developmental exposure of primates and rodents to lead (Pb) predetermined the expression of AD-related genes, such as the amyloid-β precursor protein (AβPP), later in life. In addition to AβPP, the preponderance of genes that were reprogrammed was rich in CpG dinucleotides implicating DNA methylation and chromatin restructuring in their regulation. To examine the involvement of epigenetic intermediates in Pb-induced alterations in gene expression, differentiated SH-SY5Y cells were exposed to a series of Pb concentrations (5-100 μM) for 48 h and were analyzed for the protein expression of AβPP, β-site amyloid precursor protein cleaving enzyme 1 (BACE1), specificity protein 1 and 3 (Sp1, Sp3) and epigenetic intermediates like DNA methyltransferase 1, 3a (Dnmt1, Dnmt3a) and methyl CpG binding protein 2 (MeCP2) involved in DNA methylation six days after the exposure had ceased. Western blot analysis indicated a significant latent elevation in AD biomarkers as well as the transcription factors Sp1 and Sp3, accompanied by a significant reduction in the protein levels of DNA methylating enzymes. RT-PCR analysis of Dnmt1, Dnmt3a and MeCP2 indicated a significant down-regulation of the mRNA levels. These data suggest that Pb interferes with DNA methylating capacity in these cells, thus altering the expression of AD-related genes.

Pregnancy status and choline intake alter DNA integrity, epigenetic marks and gene expression

This study sought to systemically elucidate the effects of pregnancy and maternal choline intake on genomic markers. Healthy third trimester pregnant (n=26, wk 27 gestation) and non‐pregnant (n=21) women were randomized to controlled choline intakes of 480 or 930 mg/day for 12‐wk. Leukocyte samples were acquired at wk 0 and 12. Pregnant women had higher DNA damage compared to non‐pregnant women (P < 0.001), suggesting insufficient defense against oxidative stress. Genome‐wide transcript profiling revealed the up‐regulation of genes related to immune response, consistent with an activated host defense system towards the end of pregnancy. Pregnant women responded to the higher choline intake with increased (P < 0.05) histone 3 lysine 4 di‐methylation (H3K4me2), a marker of transcription activation. In sum, pregnancy altered markers of genomic stability and modulated the expression of immune‐related genes in leukocytes. The epigenetic modification of histones among pregnant women consuming varied choline intakes may have downstream effects on chromatin restructuring and gene transcription.Grant Funding Source: Funded in part by USDA, ENC and the Beef Checkoff through NCBA

ChemInform Abstract: Chemical Biology of Histone Acetyltransferase Natural Compounds Modulators

AbstractReview: 130 refs.

Interplay of Dynamic Transcription and Chromatin Remodeling: Lessons from Yeast

Regulation of transcription involves dynamic rearrangements of chromatin structure. The budding yeast Saccharomyces cerevisiae has a variety of highly conserved factors necessary for these reconstructions. Chromatin remodelers, histone modifiers and histone chaperones directly associate to promoters and open reading frames of exposed genes and facilitate activation and repression of transcription. We compare two distinct patterns of induced transcription: Sustained transcribed genes switch to an activated state where they remain as long as the induction signal is present. In contrast, single pulsed transcribed genes show a quick and strong induction pulse resulting in high transcript levels followed by adaptation and repression to basal levels. We discuss intensively studied promoters and coding regions from both groups for their co-factor requirements during transcription. Interplay between chromatin restructuring factors and dynamic transcription is highly variable and locus dependent.

Chemical biology of Histone acetyltransferase natural compounds modulators

Histone acetyltransferases (HATs) are a class of epigenetic enzymes crucial for chromatin restructuring and transcriptional regulation in eukaryotic cells, thus being a promising target for therapeutic development. Nonetheless, differently from histone deacetylases (HDACs) inhibitors, there is still paucity of small-molecule modulators of HAT activity. After a decline during past decade, natural products and their derivatives could be once again a valuable tool in the lead discovery process and meet such need of Novel Chemical Entities (NCEs). In this review, we will provide a comprehensive summary on the discovery of small-molecule HAT modulators from naturally occurring molecular scaffolds.

A Dual-Mode Fluorescence Strategy for Screening HAT Modulators

Histone acetyltransferases (HATs) are an important class of epigenetic enzymes involved in chromatin restructuring and transcriptional regulation. We describe in this paper a novel approach for the identification and characterization of HAT inhibitors using both fluorescence resonance energy transfer (FRET) and fluorescence polarization. Expressed protein ligation (EPL) was used to label HATs PCAF and p300 with Dabcyl (Dab) as FRET acceptors. Methoxycoumarin (Mca) is conjugated to HAT substrate analogues to function as fluorescent donors, namely, H3CoA20Mca for interacting with PCAF and LysCoAMca for p300. When a ligand-protein interaction occurs, the fluorescent intensity of the donor fluorophore decreases due to FRET quenching by the Dab acceptor. Meanwhile, the formation of ligand-protein complexes causes reduction of the molecular mobility of the donor fluorophore, resulting in increased fluorescence anisotropy. Thus, dual modes of fluorescence measurement, FRET and anisotropy, are integrated in the same assay system. In particular, we demonstrated that both FRET and anisotropy measurements can be used to effectively detect and characterize HAT inhibitors. The developed strategy should be useful in the search of new anticancer drugs that target the substrate interfaces of the HAT targets, as well as find values in mechanistic study of HATs.

Chromosomal Protein HMGN1 Enhances the Heat Shock-induced Remodeling of Hsp70 Chromatin

The nucleosome-binding protein HMGN1 affects the structure and function of chromatin; however, its role in regulating specific gene expression in living cells is not fully understood. Here we use embryonic fibroblasts from Hmgn1(+/+) and Hmgn1(-/-) mice to examine the effect of HMGN1 on the heat shock-induced transcriptional activation of Hsp70, a well characterized gene known to undergo a rapid chromatin re-structuring during transcriptional activation. We find that loss of HMGN1 decreases the levels of Hsp70 transcripts at the early stages of heat shock. HMGN1 enhances the rate of heat shockinduced changes in the Hsp70 chromatin but does not affect the chromatin structure before induction, an indication that it does not predispose the gene to rapid activation. Heat shock elevates the levels of H3K14 acetylation in the Hsp70 chromatin of wild type cells more efficiently than in the chromatin of Hmgn1(-/-) cells, whereas treatment with histone deacetylase inhibitors abrogates the effects of HMGN1 on the heat shock response. We suggest that HMGN1 enhances the rate of heat shock-induced H3K14 acetylation in the Hsp70 promoter, thereby enhancing the rate of chromatin remodeling and the subsequent transcription during the early rounds of Hsp70 activation when the gene is still associated with histones in a nucleosomal conformation.

Epigenetics in Male Germ Cells

Epigenetics in Male Germ Cells

Role of BRCT motif containing proteins in Chk1 activation

Chk1, along with Chk2, regulates processes such as DNA replication, cell cycle control, chromatin restructuring and apoptosis. DNA damage/replication stress activates Chk1 by phosphorylation from the PI3/PI4 family of kinases. Activation of Chk1 is thought to be mediated by proteins containing the BRCA1 C-terminal domain (BRCT). We previously identified a potential complex of four Chk1-associated proteins by immunoprecipitation, western blotting and mass spectrometry, one of which is BRCA1. Germline mutations in BRCA1 are responsible for many cases of hereditary breast cancer, and cells deficient in BRCA1 sustain spontaneous aberrations in chromosome structure. Such findings indicate that BRCA1 is essential for suppressing genome instability.

γH2AX signalling during sperm chromatin remodelling in the mouse zygote

In the mouse, the paternal post-meiotic chromatin is assumed to be devoid of DNA repair after nuclear elongation and protamine-induced compaction. Hence, DNA lesions induced thereafter will have to be restored upon gamete fusion in the zygote. Misrepair of such lesions often results in chromosome type aberrations at the first cleavage division, suggesting that the repair event takes place prior to S-phase. During this stage of the zygotic cell cycle, the paternal chromatin transits from a protamine- to a nucleosome-based state. We addressed the question whether the canonical signalling pathway to DNA double strand breaks (DSBs), the phosphorylated form of histone H2AX (γH2AX) is active during chromatin restructuring of the male genetic complement in the zygote. Here, we describe the detailed characterization of γH2AX signalling in the early stages of zygotic development up to the appearance of the pronuclei. We have found the γH2AX signalling pathway to be already active during sperm chromatin remodelling after gamete fusion in a dose dependent manner, reflecting the amount of DSBs present in the sperm nucleus after in vivo male irradiation. Using DNA damaging compounds to induce lesions in the early zygote, differences in DSB sensitivity and γH2AX processing between paternal and maternal chromatin were found, suggesting differences in DNA repair capacity between the parental chromatin sets.