264 research outputs found
Nucleic Acids Res
Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after gamma-irradiation (gamma-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and gammaH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-alpha and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure
Comparative Analyses of SUV420H1 Isoforms and SUV420H2 Reveal Differences in Their Cellular Localization and Effects on Myogenic Differentiation
Methylation of histone H4 on lysine 20 plays critical roles in chromatin structure and function via mono- (H4K20me1), di- (H4K20me2), and trimethyl (H4K20me3) derivatives. In previous analyses of histone methylation dynamics in mid-gestation mouse embryos, we documented marked changes in H4K20 methylation during cell differentiation. These changes were particularly robust during myogenesis, both in vivo and in cell culture, where we observed a transition from H4K20me1 to H4K20me3. To assess the significance of this change, we used a gain-of-function strategy involving the lysine methyltransferases SUV420H1 and SUV420H2, which catalyze H4K20me2 and H4K20me3. At the same time, we characterized a second isoform of SUV420H1 (designated SUV420H1_i2) and compared the activity of all three SUV420H proteins with regard to localization and H4K20 methylation.Immunofluorescence revealed that exogenous SUV420H1_i2 was distributed throughout the cell, while a substantial portion of SUV420H1_i1 and SUV420H2 displayed the expected association with constitutive heterochromatin. Moreover, SUV420H1_i2 distribution was unaffected by co-expression of heterochromatin protein-1α, which increased the targeting of SUV420H1_i1 and SUV420H2 to regions of pericentromeric heterochromatin. Consistent with their distributions, SUV420H1_i2 caused an increase in H4K20me3 levels throughout the nucleus, whereas SUV420H1_i1 and SUV420H2 facilitated an increase in pericentric H4K20me3. Striking differences continued when the SUV420H proteins were tested in the C2C12 myogenic model system. Specifically, although SUV420H1_i2 induced precocious appearance of the differentiation marker Myogenin in the presence of mitogens, only SUV420H2 maintained a Myogenin-enriched population over the course of differentiation. Paradoxically, SUV420H1_i1 could not be expressed in C2C12 cells, which suggests it is under post-transcriptional or post-translational control.These data indicate that SUV420H proteins differ substantially in their localization and activity. Importantly, SUV420H2 can induce a transition from H4K20me1 to H4K20me3 in regions of constitutive heterochromatin that is sufficient to enhance myogenic differentiation, suggesting it can act an as epigenetic ‘switch’ in this process
The Role of p300 Histone Acetyltransferase in UV-Induced Histone Modifications and MMP-1 Gene Transcription
Matrix metalloproteinase (MMP)-1 promotes ultraviolet (UV)-triggered long-term detrimental effects such as cancer formation and premature skin aging. Although histone modifications may play a crucial role in the transcriptional regulation of MMP-1, the relationship between UV-induced histone modification and MMP-1 expression is not completely understood. Here, we identify regulators of histone acetylation that may link UV-mediated DNA damage and MMP-1 induction by UV in cultured human dermal fibroblasts (HDFs) in vitro. UV irradiation of HDFs induced MMP-1 expression and increased the level of phosphorylation of H2AX (γ-H2AX), p53 and the acetylation of histone H3 (acetyl-H3). Total histone deacetylase (HDAC) enzymatic activity was decreased by UV irradiation, while histone acetyltransferase (HAT) activity was increased. Suppression of p300 histone acetyltransferase (p300HAT) activity by the p300HAT inhibitor anacardic acid (AA) or by down-regulation of p300 by siRNA prevented UV-induced MMP-1 expression and inhibited UV-enhanced γ-H2AX, p53 level, and acetyl-H3. Using chromatin immunoprecipitation assays, we observed that γ-H2AX, p53, acetyl-H3, p300 and c-Jun were consistently recruited by UV to a distinct region (−2067/−1768) adjacent to the p300 binding site (−1858/−1845) in the MMP-1 promoter. In addition, these recruitments of γ-H2AX, p53, acetyl-H3, p300 and c-Jun to the p300-2 site were significantly abrogated by post-treatment with AA. Furthermore, overexpression of p300 increased the basal and UV-induced MMP-1 promoter activity. Our results suggest that p300HAT plays a critical role in the transcriptional regulation of MMP-1 by UV
Cellular senescence and chromatin organisation
Despite the potential importance of senescence in tumour suppression, its effector mechanism is poorly understood. Recent studies suggest that alterations in the chromatin environment might add an additional layer of stability to the phenotype. In this review, recent discoveries on the interplay between senescence and chromatin biology are overviewed
Acetonic Extract of Buxus sempervirens Induces Cell Cycle Arrest, Apoptosis and Autophagy in Breast Cancer Cells
Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC50 ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC50 of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC50 did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer
NF-Y Dependent Epigenetic Modifications Discriminate between Proliferating and Postmitotic Tissue
The regulation of gene transcription requires posttranslational modifications of histones that, in concert with chromatin remodeling factors, shape the structure of chromatin. It is currently under intense investigation how this structure is modulated, in particular in the context of proliferation and differentiation. Compelling evidence suggests that the transcription factor NF-Y acts as a master regulator of cell cycle progression, activating the transcription of many cell cycle regulatory genes. However, the underlying molecular mechanisms are not yet completely understood. Here we show that NF-Y exerts its effect on transcription through the modulation of the histone “code”. NF-Y colocalizes with nascent RNA, while RNA polymerase II is I phosphorylated on serine 2 of the YSPTSPS repeats within its carboxyterminal domain and histones are carrying modifications that represent activation signals of gene expression (H3K9ac and PAN-H4ac). Comparing postmitotic muscle tissue from normal mice and proliferating muscles from mdx mice, we demonstrate by chromatin immunoprecipitation (ChIP) that NF-Y DNA binding activity correlates with the accumulation of acetylated histones H3 and H4 on promoters of key cell cycle regulatory genes, and with their active transcription. Accordingly, p300 is recruited onto the chromatin of NF-Y target genes in a NF-Y-dependent manner, as demonstrated by Re-ChIP. Conversely, the loss of NF-Y binding correlates with a decrease of acetylated histones, the recruitment of HDAC1, and a repressed heterochromatic state with enrichment of histones carrying modifications known to mediate silencing of gene expression (H3K9me3, H3K27me2 and H4K20me3). As a consequence, NF-Y target genes are downregulated in this context. In conclusion, our data indicate a role of NF-Y in modulating the structure and transcriptional competence of chromatin in vivo and support a model in which NF-Y-dependent histone “code” changes contribute to the proper discrimination between proliferating and postmitotic cells in vivo and in vitro
Histone acetyltransferase inhibitor II induces apoptosis in glioma cell lines via the p53 signaling pathway
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field
Specific Loss of Histone H3 Lysine 9 Trimethylation and HP1γ/Cohesin Binding at D4Z4 Repeats Is Associated with Facioscapulohumeral Dystrophy (FSHD)
Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy in which no mutation of pathogenic gene(s) has been identified. Instead, the disease is, in most cases, genetically linked to a contraction in the number of 3.3 kb D4Z4 repeats on chromosome 4q. How contraction of the 4qter D4Z4 repeats causes muscular dystrophy is not understood. In addition, a smaller group of FSHD cases are not associated with D4Z4 repeat contraction (termed “phenotypic” FSHD), and their etiology remains undefined. We carried out chromatin immunoprecipitation analysis using D4Z4–specific PCR primers to examine the D4Z4 chromatin structure in normal and patient cells as well as in small interfering RNA (siRNA)–treated cells. We found that SUV39H1–mediated H3K9 trimethylation at D4Z4 seen in normal cells is lost in FSHD. Furthermore, the loss of this histone modification occurs not only at the contracted 4q D4Z4 allele, but also at the genetically intact D4Z4 alleles on both chromosomes 4q and 10q, providing the first evidence that the genetic change (contraction) of one 4qD4Z4 allele spreads its effect to other genomic regions. Importantly, this epigenetic change was also observed in the phenotypic FSHD cases with no D4Z4 contraction, but not in other types of muscular dystrophies tested. We found that HP1γ and cohesin are co-recruited to D4Z4 in an H3K9me3–dependent and cell type–specific manner, which is disrupted in FSHD. The results indicate that cohesin plays an active role in HP1 recruitment and is involved in cell type–specific D4Z4 chromatin regulation. Taken together, we identified the loss of both histone H3K9 trimethylation and HP1γ/cohesin binding at D4Z4 to be a faithful marker for the FSHD phenotype. Based on these results, we propose a new model in which the epigenetic change initiated at 4q D4Z4 spreads its effect to other genomic regions, which compromises muscle-specific gene regulation leading to FSHD pathogenesis
Chromatin Remodeling Pathways in Smooth Muscle Cell Differentiation, and Evidence for an Integral Role for p300
Phenotypic alteration of vascular smooth muscle cells (SMC) in response to injury or inflammation is an essential component of vascular disease. Evidence suggests that this process is dependent on epigenetic regulatory processes. P300, a histone acetyltransferase (HAT), activates crucial muscle-specific promoters in terminal (non-SMC) myocyte differentiation, and may be essential to SMC modulation as well.We performed a subanalysis examining transcriptional time-course microarray data obtained using the A404 model of SMC differentiation. Numerous chromatin remodeling genes (up to 62% of such genes on our array platform) showed significant regulation during differentiation. Members of several chromatin-remodeling families demonstrated involvement, including factors instrumental in histone modification, chromatin assembly-disassembly and DNA silencing, suggesting complex, multi-level systemic epigenetic regulation. Further, trichostatin A, a histone deacetylase inhibitor, accelerated expression of SMC differentiation markers in this model. Ontology analysis indicated a high degree of p300 involvement in SMC differentiation, with 60.7% of the known p300 interactome showing significant expression changes. Knockdown of p300 expression accelerated SMC differentiation in A404 cells and human SMCs, while inhibition of p300 HAT activity blunted SMC differentiation. The results suggest a central but complex role for p300 in SMC phenotypic modulation.Our results support the hypothesis that chromatin remodeling is important for SMC phenotypic switching, and detail wide-ranging involvement of several epigenetic modification families. Additionally, the transcriptional coactivator p300 may be partially degraded during SMC differentiation, leaving an activated subpopulation with increased HAT activity and SMC differentiation-gene specificity
- …