Remodeling Of Chromatin Structure Research Articles

Functional Coordination of the Chromatin-Remodeling Factor AtINO80 and the Histone Chaperones NRP1/2 in Inflorescence Meristem and Root Apical Meristem.

Chromatin structure requires proper modulation in face of transcriptional reprogramming in the context of organism growth and development. Chromatin-remodeling factors and histone chaperones are considered to intrinsically possess abilities to remodel chromatin structure in single or in combination. Our previous study revealed the functional synergy between the Arabidopsis chromatin-remodeling factor INOSITOL AUXOTROPHY 80 (AtINO80) and the histone chaperone NAP1-RELATED PROTEIN 1 (NRP1) and NRP2 in somatic homologous recombination, one crucial pathway involved in repairing DNA double strand breaks. Here, we report genetic interplay between AtINO80 and NRP1/2 in regulating inflorescence meristem (IM) and root apical meristem (RAM) activities. The triple mutant atino80-5 m56-1 depleting of both AtINO80 (atino80-5) and NRP1/2 (m56-1) showed abnormal positioning pattern of floral primordia and enlargement of IM size. Higher mRNA levels of several genes involved in auxin pathway (e.g., PIN1, FIL) were found in the inflorescences of the triple mutant but barely in those of the single mutant atino80-5 or the double mutant m56-1. In particular, the depletion of AtINO80 and NRP1/2 decreased histone H3 levels within the chromatin regions of PIN1, which encodes an important auxin efflux carrier. Moreover, the triple mutant displayed a severe short-root phenotype with higher sensitivity to auxin transport inhibitor NPA. Unusual high level of cell death was also found in triple mutant root tips, accompanied by double-strand break damages revealed by γ-H2A.X loci and cortex cell enlargement. Collectively, our study provides novel insight into the functional coordination of the two epigenetic factors AtINO80 and NRP1/2 in apical meristems during plant growth and development.

Characterization of the Novel DNA Binding Activity of the BRG1 At-Hook-Bromodomain

The packaging of eukaryotic DNA with histones into chromatin acts to both compact it into the nucleus as well as provide a mechanism for regulation of the genome. Spatial and temporal remodeling of chromatin structure is critical for all DNA-templated processes and dysregulation of these pathways is associated with a number of diseases. The BRG1/BRM associated factor (BAF) complex, is an ATP-dependent remodeling complex that plays an important role in gene regulation, several subunits of which are mutated in human cancers. The Brahma related gene 1 (BRG1) subunit of BAF, which provides the ATPase activity, contains a bromodomain (BD) at its C-terminus. BDs are well-characterized readers of acetylated lysines on histones, and the BRG1-BD has been shown to bind H3K14ac. However, we have recently discovered that in addition to binding acetyl-lysine, the BRG1-BD associates with DNA, a novel function for BDs. In addition, we have demonstrated that an adjacent AT-hook contributes to a multivalent mechanism of association with DNA, increasing affinity and specificity (Morrison et al., Nature Communications, 2017). Notably, the newly identified DNA binding interface harbors several cancer-associated mutations. Here we present our continued studies on this newly recognized composite DNA binding domain. We use systematic evolution of ligands by exponential enrichment (SELEX-seq) to generate a biophysical model of sequence specificity for the domain that we are exploring structurally using NMR spectroscopy. To determine the kinetic and thermodynamic basis of association we use biolayer interferometry (BLI). In addition, we are investigating the molecular mechanism of DNA binding and how it is altered in the context of the nucleosome. Together, our results are revealing the molecular details of how this novel DNA binding domain functions to navigate chromatin, and how dysregulation of the ATBD may impact BAF function.

Autism-associated CHD8 deficiency impairs axon development and migration of cortical neurons

BackgroundMutations in CHD8, chromodomain helicase DNA-binding protein 8, are among the most replicated and common findings in genetic studies of autism spectrum disorder (ASD). The CHD8 protein is believed to act as a transcriptional regulator by remodeling chromatin structure and recruiting histone H1 to target genes. The mechanism by which deficiency of CHD8 causes ASD has not been fully elucidated.MethodsWe examined the expression of CHD8 in human and mouse brains using both immunohistochemistry and RNA in situ hybridization. We performed in utero electroporation, neuronal culture, and biochemical analysis using RNAi to examine the functional consequences of CHD8 deficiency.ResultsWe discovered that CHD8 is expressed highly in neurons and at low levels in glia cells in both humans and mice. Specifically, CHD8 is localized predominately in the nucleus of both MAP2 and parvalbumin-positive neurons. In the developing mouse brain, expression of Chd8 peaks from E16 to E18 and then decreases significantly at P14 to adulthood. Knockdown of Chd8 results in reduced axon and dendritic growth, disruption of axon projections to the contralateral cortex, and delayed neuronal migration at E18.5 which recovers by P3 and P7.ConclusionOur findings indicate an important role for CHD8 in dendritic and axon development and neuronal migration and thus offer novel insights to further dissect the underlying molecular and circuit mechanisms of ASD caused by CHD8 deficiency.

Characterization of H3 methylation in regulating oocyte development in cyprinid fish.

Histone post-modifications are important epigenetic markers involved in multiple cellular processes via regulation of gene transcription or remodeling of chromatin structure. Oocyte development is a critical process under rigorous control to prevent the generation of aberrant gametes. However, the regulatory mechanism of oocyte early development is not well-understood due to the tiny size and poor distinguishability of the gonad in juvenile stages. Here, two cyprinid hybrid fishes, a sterile allotriploid fish and a gynogenetic hybrid fish with delayed oocyte development, provided research models to investigate the mechanisms involved. We used cytogenetic and molecular methods to confirm the pachytene arrest of oocytes in allotriploid fish and gynogenetic hybrid fish. On the basis of these developmental differences, we screened 21 different histone H3 modifications by ELISA and found that four modifications (H3K4me3, H3K9me3, H3K79me, and H3K79me3) differed significantly in the two cyprinid hybrid fishes. Changes in histone methylation at the three residues (H3K4, K9, K79) were caused by specific methyltransferases and demethylases. Our results provide new insights into the epigenetic regulation of oocyte early development in fish, a process critical for understanding of reproductive biology and with practical applications in the aquacultural breeding industry.

Genetic and epigenetic determinants establish a continuum of Hsf1 occupancy and activity across the yeast genome.

Heat shock factor 1 is the master transcriptional regulator of molecular chaperones and binds to the same cis-acting heat shock element (HSE) across the eukaryotic lineage. In budding yeast, Hsf1 drives the transcription of ∼20 genes essential to maintain proteostasis under basal conditions, yet its specific targets and extent of inducible binding during heat shock remain unclear. Here we combine Hsf1 chromatin immunoprecipitation sequencing (seq), nascent RNA-seq, and Hsf1 nuclear depletion to quantify Hsf1 binding and transcription across the yeast genome. We find that Hsf1 binds 74 loci during acute heat shock, and these are linked to 46 genes with strong Hsf1-dependent expression. Notably, Hsf1’s induced DNA binding leads to a disproportionate (∼7.5-fold) increase in nascent transcription. Promoters with high basal Hsf1 occupancy have nucleosome-depleted regions due to the presence of “pioneer factors.” These accessible sites are likely critical for Hsf1 occupancy as the activator is incapable of binding HSEs within a stably positioned, reconstituted nucleosome. In response to heat shock, however, Hsf1 accesses nucleosomal sites and promotes chromatin disassembly in concert with the Remodels Structure of Chromatin (RSC) complex. Our data suggest that the interplay between nucleosome positioning, HSE strength, and active Hsf1 levels allows cells to precisely tune expression of the proteostasis network.

Controversial Impact of Sirtuins in Chronic Non-Transmissible Diseases and Rehabilitation Medicine.

A large body of evidence reports about the positive effects of physical activity in pathophysiological conditions associated with aging. Physical exercise, alone or in combination with other medical therapies, unquestionably causes reduction of symptoms in chronic non-transmissible diseases often leading to significant amelioration or complete healing. The molecular basis of this exciting outcome—however, remain largely obscure. Epigenetics, exploring at the interface between environmental signals and the remodeling of chromatin structure, promises to shed light on this intriguing matter possibly contributing to the identification of novel therapeutic targets. In this review, we shall focalize on the role of sirtuins (Sirts) a class III histone deacetylases (HDACs), which function has been frequently associated, often with a controversial role, to the pathogenesis of aging-associated pathophysiological conditions, including cancer, cardiovascular, muscular, neurodegenerative, bones and respiratory diseases. Numerous studies, in fact, demonstrate that Sirt-dependent pathways are activated upon physical and cognitive exercises linking mitochondrial function, DNA structure remodeling and gene expression regulation to designed medical therapies leading to tangible beneficial outcomes. However, in similar conditions, other studies assign to sirtuins a negative pathophysiological role. In spite of this controversial effect, it is doubtless that studying sirtuins in chronic diseases might lead to an unprecedented improvement of life quality in the elderly.

Brg1 subunit of chromatin remodelling complex is present during Chara vulgaris (Charophyceae) spermatogenesis

ABSTRACTDuring spermatogenesis, cells developed as a result of numerous mitotic and meiotic divisions transform into mature spermatozoids. In spermatids, remodelling of chromatin structure takes place which is connected with nuclear protein exchange, DNA double strand breaks and epigenetic modifications. Chromatin remodelling complexes, which have mostly been studied in animals, also participate in this process. The Brg1 protein, a functional homologue of the yeast Swi2/Snf2 catalytic subunit of the SWI/SNF complex, is engaged in regulation of cell proliferation and highly expressed in round spermatids in mammals. Immunocytochemical studies with the anti-Brg1 antibody revealed positive reactions in nuclei of the green alga Chara vulgaris at the 64-cell proliferative stage and in spermatid nuclei at the I/II–VII spermiogenesis stages. The most intensive reaction was observed at the early spermiogenesis stages (I/II–III), while at the initial stages of a proliferative phase (4-, 8- and 16-cell) the reaction was not observed, and at 32-cell and VII stages the immunosignals were very weak. Ultrastructural studies with the immunogold technique confirmed the results of the immunocytochemical studies. The highest numbers of gold grains were observed at stages I/II and III of spermiogenesis, and together they constituted above 48% of the total number of gold grains. A much lower, but still substantial, amount of these grains was observed at the 64-cell stage and IV stage (>15% and 17%), respectively. Percentage analysis revealed the lowest number of gold particles at stage VII (3.72%). The significant presence of Brg1 protein at early spermiogenesis stages is correlated with acetylation of the H4K12 histone. It may also be hypothesized that in C. vulgaris the Brg1 subunit participates in processes important for proper chromatin condensation and facilitates maintenance of the correct shape of the spermatid nucleus. On the basis of earlier and current studies it seems that chromatin remodelling in spermatids of this model alga proceeds according to the model presented for mammals.

β-adrenergic Receptor Stimulation Revealed a Novel Regulatory Pathway via Suppressing Histone Deacetylase 3 to Induce Uncoupling Protein 1 Expression in Mice Beige Adipocyte.

Browning of adipose tissue has been prescribed as a potential way to treat obesity, marked by the upregulation of uncoupling protein 1 (Ucp1). Several reports have suggested that histone deacetylase (HDAC) might regulate Ucp1 by remodelling chromatin structure, although the mechanism remains unclear. Herein, we investigate the effect of β-adrenergic receptor (β-AR) activation on the chromatin state of beige adipocyte. β-AR-stimulated Ucp1 expression via cold (in vivo) and isoproterenol (in vitro) resulted in acetylation of histone activation mark H3K27. H3K27 acetylation was also seen within Ucp1 promoter upon isoproterenol addition, favouring open chromatin for Ucp1 transcriptional activation. This result was found to be associated with the downregulation of class I HDAC mRNA, particularly Hdac3 and Hdac8. Further investigation showed that although HDAC8 activity decreased, Ucp1 expression was not altered when HDAC8 was activated or inhibited. In contrast, HDAC3 mRNA and protein levels were simultaneously downregulated upon isoproterenol addition, resulting in reduced recruitment of HDAC3 to the Ucp1 enhancer region, causing an increased H3K27 acetylation for Ucp1 upregulation. The importance of HDAC3 inhibition was confirmed through the enhanced Ucp1 expression when the cells were treated with HDAC3 inhibitor. This study highlights the novel mechanism of HDAC3-regulated Ucp1 expression during β-AR stimulation.

Sssfh1, a Gene Encoding a Putative Component of the RSC Chromatin Remodeling Complex, Is Involved in Hyphal Growth, Reactive Oxygen Species Accumulation, and Pathogenicity in Sclerotinia sclerotiorum.

SFH1 (for Snf5 homolog) protein, comprised in the RSC (Remodels Structure of Chromatin) chromatin remodeling complex, functions as a transcription factor (TF) to specifically regulate gene transcription and chromatin remodeling. As one of the well-conserved TFs in eukaryotic organisms, little is known about the roles of SFH1 protein in the filamentous fungi. In Sclerotinia sclerotiorum, one of the notorious plant fungal pathogens, there are nine proteins predicted to contain GATA-box domain according to GATA family TF classification, among which Sssfh1 (SS1G_01151) encodes a protein including a GATA-box domain and a SNF5 domain. Here, we characterized the roles of Sssfh1 in the developmental process and fungal pathogenicity by using RNA interference (RNAi)-based gene silencing in S. sclerotiorum. RNA-silenced strains with significantly reduced Sssfh1 RNA levels exhibited slower hyphal growth and decreased reactive oxygen species (ROS) accumulation in hyphae compared to the wild-type (WT) strain. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays demonstrated that SsSFH1 interacts with SsMSG5, a MAPK phosphatase in S. sclerotiorum. Furthermore, Sssfh1-silenced strains exhibited enhanced tolerance to NaCl and H2O2. Results of infection assays on soybean and common bean (Phaseolus vulgaris) leaves indicated that Sssfh1 is required for full virulence of S. sclerotiorum during infection in the susceptible host plants. Collectively, our results suggest that the TF SsSFH1 is involved in growth, ROS accumulation and virulence in S. sclerotiorum.

Abstract 115: Chromatin Microenvironments With Distinct Functionality During Cardiac Stress

Loss of the chromatin structural protein CTCF induces heart failure. Chromatin capture experiments (Hi-C), which detect genomic interactions, revealed global remodeling of chromatin structure after cardiac-specific depletion of CTCF or pressure overload (TAC). In the present study, we investigated epigenomic features (histone marks and DNA methylation) contributing to distinct behaviors of chromatin microenvironments. CTCF-KO or TAC induced gene expression changes mediated by a shift in compartmentalization between active and inactive chromatin (~4% of the genome). At the transcriptome level, genes significantly dysregulated in disease were involved in cardiac function ( e.g. , Mybpc3, Lmna , Hand2 ), chromatin structure ( e.g. , Hmgb2 , Rad21 ), and histone modification ( e.g. , Hdac4 , Mapk14, Foxo1 ). Our Hi-C data allowed us to correlate changes in gene expression with localization within the nucleus, showing efficient segregation of differentially expressed genes into specific neighborhoods with similar expression behavior. Analyses of histone marks in these neighborhoods reveal enrichment of activating and inhibiting marks, H3K9ac and H3K9me3, respectively, in the specialized chromatin environments associated with gene expression or silencing. DNA methylation studies showed that transcriptionally active neighborhoods tended to have unmethylated CpGs, while repressive environments were methylated. Our Hi-C data also showed how active environments positively influence the expression of the genes that physically interact with them. For example, 351 unique genes with interacting partner genes had increased H3K9ac after TAC, and their 3D interacting partners included 200 upregulated genes and 104 downregulated genes, demonstrating similar transcriptional behavior in areas with similar epigenomic features and anatomical distribution in the nucleus. Heart failure involves conserved structural reprogramming of these chromatin microenvironments.

Proteasomal degradation of the histone acetyl transferase p300 contributes to beta-cell injury in a diabetes environment

In type 2 diabetes, amyloid oligomers, chronic hyperglycemia, lipotoxicity, and pro-inflammatory cytokines are detrimental to beta-cells, causing apoptosis and impaired insulin secretion. The histone acetyl transferase p300, involved in remodeling of chromatin structure by epigenetic mechanisms, is a key ubiquitous activator of the transcriptional machinery. In this study, we report that loss of p300 acetyl transferase activity and expression leads to beta-cell apoptosis, and most importantly, that stress situations known to be associated with diabetes alter p300 levels and functional integrity. We found that proteasomal degradation is the mechanism subserving p300 loss in beta-cells exposed to hyperglycemia or pro-inflammatory cytokines. We also report that melatonin, a hormone produced in the pineal gland and known to play key roles in beta-cell health, preserves p300 levels altered by these toxic conditions. Collectively, these data imply an important role for p300 in the pathophysiology of diabetes.

Dynamics of Eukaryotic Histone Exchange with Single Molecule Resolution

Remodelling chromatin structure is important for regulating gene expression, DNA replication and repair and other fundamental nuclear processes. The basic chromatin unit is the nucleosome. SWR1 is a multi-subunit complex, whose chromatin remodelling activity is associated with regulation of gene expression in heterochromatin regions of chromosomes in plants and mammals, and with the cellular response to DNA damage. In yeast, the simplest eukaryotic organism, the SWR1 complex is responsible for the ATP-dependent nucleosome remodelling by exchanging its canonical H2A histone with Htz1 variant (also known as H2A.Z in mammalian cells). In spite of a large number of genetic, biochemical and structural studies on SWR1, its detailed histone exchange mechanism remains largely unknown. To investigate the mechanism of histone exchange by SWR1, we have developed a single-molecule FRET assay, which monitors the interaction between individual nucleosomes and yeast SWR1 complexes in real time. The data show distinct dynamic behaviours in the presence or absence of ATP, or in the presence of non-hydrolysable ATP analogue. We hypothesize that the observed dynamics are important for the removal of canonical H2A histones or deposition of the histone variant Htz1. We anticipate that our data will help elucidate the molecular mechanism of histone exchange by SWR1.

Characterization of the Novel DNA Binding Activity of the BRG1 At-Hook-Bromodomain and Effect of Cancer Mutations

The packaging of eukaryotic DNA with histones into chromatin acts to both compact it into the nucleus as well as provide a mechanism for regulation of the genome. Spatial and temporal remodeling of chromatin structure is critical for all DNA-templated processes and dysregulation of these pathways is associated with a number of diseases. The human switching/sucrose non-fermenting (SWI/SNF) complex, is an ATP-dependent remodeling complex that plays an important role in gene regulation, several subunits of which are mutated in human cancers. The Brahma related gene 1 (BRG1) subunit of hSWI/SNF, which provides the ATPase activity, contains a bromodomain (BD) at its C-terminus. BDs are well-characterized readers of acetylated lysines on histones, and the BRG1-BD has been shown to bind H3K14ac. However, we have recently discovered that in addition to binding acetyl-lysine, the BRG1-BD associates with DNA, a novel function for BDs. In addition, we have demonstrated that an adjacent AT-hook contributes to a multivalent mechanism of association with DNA, increasing affinity and specificity (Morrison et al., Nature Communications, 2017). Notably, the newly identified DNA binding interface harbors several cancer-associated mutations. Here we present our continued studies on this newly recognized composite DNA binding domain. We use systematic evolution of ligands by exponential enrichment (SELEX-seq) to generate a biophysical model of sequence specificity for the domain that we are exploring structurally using NMR spectroscopy and X-ray crystallography. To determine the kinetic and thermodynamic basis of association we use biolayer interferometry (BLI). In addition, we are investigating how DNA binding is altered in the context of the nucleosome, and the effect of known cancer mutations on the DNA binding activity. Together, our results are revealing the molecular details of how this novel DNA binding domain functions.

Targeting histone deacetylase 4/ubiquitin-conjugating enzyme 9 impairs DNA repair for radiosensitization of hepatocellular carcinoma cells in mice.

Several strategies to improve the efficacy of radiation therapy against hepatocellular carcinoma (HCC) have been investigated. One approach is to develop radiosensitizing compounds. Because histone deacetylase 4 (HDAC4) is highly expressed in liver cancer and known to regulate oncogenesis through chromatin structure remodeling and controlling protein access to DNA, we postulated that HDAC4 inhibition might enhance radiation's effect on HCC cells. HCC cell lines (Huh7 and PLC5) and an ectopic xenograft were pretreated with HDAC inhibitor or short hairpin RNA to knock down expression of HDAC4 and then irradiated (2.5-10.0 Gy). We evaluated cell survival by a clonogenic assay; apoptosis by Annexin V immunofluorescence; γH2AX, Rad51, and HDAC4 by immunofluorescence staining; HDAC4, Rad51, and ubiquitin-conjugating enzyme 9 (Ubc9) in HCC cell nuclei by cell fractionation and confocal microscopy; physical interaction between HDAC4/Rad51/Ubc9 by immunoprecipitation; and the downstream targets of HDAC4 knockdown by immunoblotting. Both HDAC4 knockdown and HDAC inhibitor enhanced radiation-induced cell death and reduced homologous recombination repair of DNA double-strand breaks and protein kinase B activation, leading to increased apoptosis. HDAC4 knockdown with or without an HDAC inhibitor significantly delayed tumor growth in a radiation-treated xenograft model. Radiation stimulated nuclear translocation of Rad51 in an HDAC4-dependent manner and the binding of Ubc9 directly to HDAC4, which led to Ubc9 acetylation. Moreover, these effects were accompanied by HDAC4/Ubc9/Rad51 complex dissociation through inhibiting nuclear translocation. Conclusion: HDAC4 signaling blockade enhances radiation-induced lethality in HCC cells and xenografts. These findings raise the possibility that HDAC4/Ubc9/Rad51 complex in DNA repair may be a target for radiosensitization of HCC. (Hepatology 2018;67:586-599).

Identifying Cell Cycle Modulators That Selectively Target ARID1A Deficiency Using High-Throughput Image-Based Screening

ARID1A, a component of the chromatin remodeling complex SWI/SNF, is an evolutionarily conserved complex that uses the energy of adenosine triphosphate hydrolysis to remodel chromatin structure and functions as a master regulator of gene transcription. Recent genomic studies have revealed that ARID1A is one of the most frequently mutated genes in human cancers. However, therapeutic approaches that selectively target ARID1A-mutant tumors are not yet clinically available. Our previous study showed that ARID1A facilitates chromatin response and cell cycle checkpoint activation after DNA damage. Therefore, an ARID1A deficiency may result in therapeutic vulnerabilities in cell cycle modulators. The goals of our study were to develop a novel screening approach, based on fluorescent ubiquitination-based cell cycle indicators (FUCCI), and to identify chemical agents that can selectively modulate the cell cycle transition in ARID1A-deficient cancer cells. Using this high-throughput assay, we screened 2643 compounds and identified six potential chemical modulators that can selectively modulate the cell cycle in ARID1A-deficient cells; these agents may be useful for developing new therapeutics for ARID1A-mutant tumors. In summary, our study demonstrates that FUCCI cell-based high-content screening is a powerful and effective approach for identifying cell cycle modulators and can be applied to multigenotypic screening for targeted cancer therapeutics.

Establishing nucleosome architecture and stability at promoters: Roles of pioneer transcription factors and the RSC chromatin remodeler.

Improvements in deep sequencing, together with methods to rapidly deplete essential transcription factors (TFs) and chromatin remodelers, have recently led to a more detailed picture of promoter nucleosome architecture in yeast and its relationship to transcriptional regulation. These studies revealed that ∼40% of all budding yeast protein-coding genes possess a unique promoter structure, where we propose that an unusually unstable nucleosome forms immediately upstream of the transcription start site (TSS). This "fragile" nucleosome (FN) promoter architecture relies on the combined action of the essential RSC (Remodels Structure of Chromatin) nucleosome remodeler and pioneer transcription factors (PTFs). FNs are associated with genes whose expression is high, coupled to cell growth, and characterized by low cell-to-cell variability (noise), suggesting that they may promote these features. Recent studies in metazoans suggest that the presence of dynamic nucleosomes upstream of the TSS at highly expressed genes may be conserved throughout evolution.

Genomic effects of glucocorticoids.

Glucocorticoids and their receptor (GR) have been an important area of research because of their pleiotropic physiological functions and extensive use in the clinic. In addition, the association between GR and glucocorticoids, which is highly specific, leads to rapid nuclear translocation where GR associates with chromatin to regulate gene transcription. This simplified model system has been instrumental for studying the complexity of transcription regulation processes occurring at chromatin. In this review we discuss our current understanding of GR action that has been enhanced by recent developments in genome wide measurements of chromatin accessibility, histone marks, chromatin remodeling and 3D chromatin structure in various cell types responding to glucocorticoids.

Parp1 Deficiency Confers Defects in Chromatin Surveillance and Remodeling During Reprogramming by Nuclear Transfer.

Accumulating evidence suggests that cloned mice production by the injection of a somatic cell nucleus into an enucleated oocyte is inefficient. DNA damage and chromatin remodeling failures that occur during embryogenesis following nuclear transfer (NT) might explain the poor development of cloned embryos. To avoid these problems, it is important to elucidate somatic chromatin remodeling after NT. Because polyADP-ribosylation, which is catalyzed mainly by poly(ADP-ribose) polymerase 1 (Parp1), is a major post-translational modification that facilitates DNA repair and chromatin remodeling, we examined the effects of Parp1 deficiency in developing NT embryos. Parp1 was located within the pseudo-pronuclei (PPN) of NT eggs. We observed that NT eggs, after activation by Sr2+, formed PPN with significantly more efficiency in Parp1-null embryos than in wild-type NT embryos. However, most the Parp1-null embryos stopped developing by the four-cell stage. Immunostaining for γH2AX foci, a marker of DNA double strand breaks, showed longer retention in the PPN of Parp1-/- donor NT embryos than in wild-type NT embryos, suggesting that, in the absence of Parp1, DNA breaks are slowly repaired and consequently, entry into the S phase might be delayed. Furthermore, decreases in histone H3 acetylation, H3 monomethylation at lysine 4, and H3 trimethylation at lysine 27 after the Sr2+ activation step were observed in the PPN of Parp1-/- donor embryos. Taken together, our data suggest that Parp1 is involved in the plastic remodeling of chromatin structure after NT by supporting DNA repair and specific histone code modifications.

Effects of ageing and senescence on pancreatic β-cell function.

Ageing is generally associated with deterioration of organ function and regenerative potential. In the case of pancreatic β-cells, an age-related decline in proliferative potential is well documented, and was proposed to contribute to the increased prevalence of type 2 diabetes in the elderly. The effects of ageing on β-cell function, namely glucose-stimulated insulin secretion (GSIS), have not been studied as extensively. Recent work revealed that, surprisingly, β-cells of mature mice and humans secrete more insulin than young β-cells in response to high glucose concentrations, potentially serving to counteract age-related peripheral insulin resistance. This functional change appears to be orchestrated by p16(Ink4A) -driven cellular senescence and downstream remodelling of chromatin structure and DNA methylation, enhancing the expression of genes controlling β-cell function. We propose that activation of the cellular senescence program drives life-long functional maturation of β-cells, due to β-cell hypertrophy, enhanced glucose uptake and more efficient mitochondrial metabolism, in parallel to locking these cells in a non-replicative state. We speculate that the beneficial aspects of this process can be harnessed to enhance GSIS. Other age-related mechanisms, which are currently poorly understood, act to increase basal insulin secretion levels also in low glucose conditions. This leads to an overall reduction in the amplitude of insulin secretion between low and high glucose at old age, which may contribute to a deterioration in metabolic control.

DNA Methylation and Chromatin Regulation during Fleshy Fruit Development and Ripening.

Fruit ripening is a developmental process that results in the leaf-like carpel organ of the flower becoming a mature ovary primed for dispersal of the seeds. Ripening in fleshy fruits involves a profound metabolic phase change that is under strict hormonal and genetic control. This work reviews recent developments in our understanding of the epigenetic regulation of fruit ripening. We start by describing the current state of the art about processes involved in histone post-translational modifications and the remodeling of chromatin structure and their impact on fruit development and ripening. However, the focus of the review is the consequences of changes in DNA methylation levels on the expression of ripening-related genes. This includes those changes that result in heritable phenotypic variation in the absence of DNA sequence alterations, and the mechanisms for their initiation and maintenance. The majority of the studies described in the literature involve work on tomato, but evidence is emerging that ripening in other fruit species may also be under epigenetic control. We discuss how epigenetic differences may provide new targets for breeding and crop improvement.