Heterochromatin Research Articles

Lamins are intermediate filaments constituting the nuclear lamina which maintains the structural integrity of the nucleus and play a key role in the spatiotemporal genome organization. Mutations in lamin A/C have been associated with a plethora of diseases including dilated cardiomyopathy. In this study, we focused on lamin A mutants E161K and K97E which are widely reported in patients afflicted with dilated cardiomyopathy. We established that these mutations cause large scale disruption of the peripheral lamina and consequent heterochromatin (HC)organization, along with the formation of lamin A aggregates inside the nucleoplasm. Using coarse-grained polymer simulations, we explored the interplay between lamin, nuclear membrane, and chromatin showing that disruptions in these interactions can reproduce the experimental phenotypes. Simulations also predict altered positioning depending on HCcontent and altered lamin dynamics in E161K and K97E mutants. The predictions from the simulations were verified using 3D FISH to delineate the reorganization of chromosome territories in these mutants. Based on the disengagement of lamin A foci from HC, we subsequently investigated the dynamic properties of the mutant aggregates using advanced imaging techniques. Through this, we report that mutant lamin aggregates show internal rearrangement and external exchange at the mesoscopic-level reminiscent of liquid-liquid phase separation.

In the malaria parasite Plasmodium falciparum, the expression of many genes is regulated by heterochromatin (HC) based on the histone mark tri-methylation of histone H3 lysine 9 (H3K9me3). HC assembly involves three distinct steps: de novo nucleation, spreading and maintenance. Nucleation, which consists in formation of HC in a previously euchromatic region, determines the specific genomic locations where HC occurs. This process is not well understood in malaria parasites. Here we investigated the DNA sequence cis determinants of HC nucleation in P. falciparum, using a screening approach based on integration of fragments from different heterochromatic genes into an euchromatic locus, followed by H3K9me3 chromatin immunoprecipitation (ChIP) analysis. We found that fragments of var gene upstream regions nucleated HC efficiently, whereas fragments from the pfap2-g upstream region or from the mspdbl2 locus did not nucleate HC. Fragments from the beginning of the coding sequence (CDS) of pfap2-g nucleated HC with low efficiency, as evidenced by nucleation requiring long fragments of ~2 kb and occurring only in a fraction of the parasites. These results demonstrate that the primary DNA sequence is a main determinant of HC nucleation in P. falciparum. We also studied HC maintenance at the pfap2-g locus, which demonstrated that specific parts of the upstream region, different from the regions competent for HC nucleation, are required for maintenance. Together, our results provide initial insight into how HC is directed to specific loci and maintained in P. falciparum.

In the malaria parasite Plasmodium falciparum, the expression of many genes is regulated by heterochromatin (HC) based on the histone mark tri-methylation of histone H3 lysine 9 (H3K9me3). HC assembly involves three distinct steps: de novo nucleation, spreading and maintenance. Nucleation, which consists in formation of HC in a previously euchromatic region, determines the specific genomic locations where HC occurs. This process is not well understood in malaria parasites. Here we investigated the DNA sequence cis determinants of HC nucleation in P. falciparum, using a screening approach based on integration of fragments from different heterochromatic genes into an euchromatic locus, followed by H3K9me3 chromatin immunoprecipitation (ChIP) analysis. We found that fragments of var gene upstream regions nucleated HC efficiently, whereas fragments from the pfap2-g upstream region or from the mspdbl2 locus did not nucleate HC. Fragments from the beginning of the coding sequence (CDS) of pfap2-g nucleated HC with low efficiency, as evidenced by nucleation requiring long fragments of ~2 kb and occurring only in a fraction of the parasites. These results demonstrate that the primary DNA sequence is a main determinant of HC nucleation in P. falciparum. We also studied HC maintenance at the pfap2-g locus, which demonstrated that specific parts of the upstream region, different from the regions competent for HC nucleation, are required for maintenance. Together, our results provide initial insight into how HC is directed to specific loci and maintained in P. falciparum.

Chromatin contains not only heterochromatin (HC) and euchromatins (EC) but also facultative heterochromatin (fHC), which experience the dynamic remodeling between HCs and ECs by different regulators. The regulation of fHCs involves lots of different cell functions, like genomic stability and gene transcription. Heterochromatin protein 1 (HP1) recognizes methylated H3K9 and reshapes the chromatin into the fHCs through liquid-liquid phase separation (LLPS). Among the three members of the HP1 family, HP1α can condensate by itself and HP1β forms granules with the help of TRIM28, while the HP1γ cannot phase separation alone either and the coordinator is still unclear. So, in this study, we investigated the molecular mechanism of how HP1γ interacts with TRIM66 through PxVxL motif. Based on that, we examined the key regions that controlled the TRIM66-HP1γ co-phase separation behaviors both in vitro and in vivo. Furthermore, we proved that the liquid granules of TRIM66-HP1γ and chromatin highly correlated with H3K9me3 sites, which indicated the relationship with DNA damage response. Finally, combined with our previous study, we proposed the system for how TRIM66 remodeled the chromatin into compressed fHC through the TRIM66-HP1γ-H3K9me3 axis with liquid-liquid phase separation.

The spatial organization of euchromatin (EC) and heterochromatin (HC) appears as a cell-type specific network, which seems to have an impact on gene regulation and cell fate. The spatial organization of cohesin should thus also be characteristic for a cell type since it is involved in a TAD (topologically associating domain) formation, and thus in gene regulation or DNA repair processes. Based on the previous hypotheses and results on the general importance of heterochromatin organization on genome functions in particular, the configurations of these organizational units (EC represented by H3K4me3-positive regions, HC represented by H3K9me3-positive regions, cohesins) are investigated in the cell nuclei of different cancer and non-cancerous cell types and under different anti-cancer treatments. Confocal microscopic images of the model cell systems were used and analyzed using analytical processes of quantification created in Fiji, an imaging tool box well established in different fields of science. Human fibroblasts, breast cancer and glioblastoma cells as well as murine embryonal terato-carcinoma cells were used as these cell models and compared according to the different parameters of spatial arrangements. In addition, proliferating, quiescent and from the quiescent state reactivated fibroblasts were analyzed. In some selected cases, the cells were treated with X-rays or azacitidine. Heterogeneous results were obtained by the analyses of the configurations of the three different organizational units: granulation and a loss of H3K4me3-positive regions (EC) occurred after irradiation with 4 Gy or azacitidine treatment. While fibroblasts responded to irradiation with an increase in cohesin and granulation, in breast cancer cells, it resulted in decreases in cohesin and changes in granulation. H3K9me3-positive regions (HC) in fibroblasts experienced increased granulation, whereas in breast cancer cells, the amount of such regions increased. After azacitidine treatment, murine stem cells showed losses of cohesin and granulation and an increase in the granulation of H3K9me3-positive regions. Fibroblasts that were irradiated with 2 Gy only showed irregularities in structural amounts and granulation. Quiescent fibroblasts contained less euchromatin-related H3K4me3-positive signals and cohesin levels as well as higher heterochromatin-related H3K9me3-positive signals than non-quiescent ones. In general, fibroblasts responded more intensely to X-ray irradiation than breast cancer cells. The results indicate the usefulness of model cell systems and show that, in general, characteristic differences initially existing in chromatin and cohesin organizations result in specific responses to anti-cancer treatment.

Cell senescence is emerging as a critical factor in the pathophysiology of aging bone loss. We have shown that the essential amino acid tryptophan is metabolized by IDO-1 in the periphery to generate kynurenine (KYN), and that KYN can signal though the aryl hydrocarbon receptor (AhR) transcription factor pathway to inhibit osteogenesis in bone marrow MSCs via epigenetic regulation of osteogenic genes, while also upregulating osteoclastogenic transcription factors and genes driving osteoclast activity. Further, we recently showed that KYN acting via AhR inhibits MSC autophagy while inducing senescence. Here we demonstrate that KYN metabolites downstream from KYN act via the AhR signaling pathway to inhibit autophagy and induce SASP expression and drive senescence in murine and human bone marrow MSCs. We focused on two of these metabolites, quinolinic acid (QA) and kynurenic acid (KYNA) and investigated their effects on BMSC cellular function. We demonstrated that both kynurenine pathway metabolites QA and KYNA increase biomarkers for senescence including beta-galactosidase, p21/Cdkn1 and other SASPs such as PAI-1 and TIMP-2, as well as nuclear DNA damage leading to senescent markers like H2A Ser139 phosphorylation, and the accumulation of senescence-associated hetero chromatin foci (SAHF) with H3K9-me3 labeling. Then upon treatment with the AhR inhibitor 3’4’-DMF the disruption of autophagy and induction of senescent biomarkers was blocked. Like KYN, the effects of QA and KYNA were mediated through the AhR receptor. Therefore, this presents novel therapeutic targets linked to KYN metabolite signaling via AhR to prevent senescence and bone loss.

Identifying distinct heterochromatin regions using combinatorial epigenetic probes in live cells

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of “stressed” replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. ChIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late.

In eukaryotes, DNA is organized along with histones in nucleoprotein complexes known as chromatin, which has nucleosomes as their fundamental unit. Chromatin exists in two forms: euchromatin, corresponding to a lightly condensed structure and an easily transcribed region, and heterochromatin, a highly condensed and transcriptionally silent region. Therefore, the chromatin compaction degree interferes with regulation of gene expression, and nucleosomes act as gene silencers. In this context, using Assay for Transposase-Accessible Chromatin technique (ATAC-seq), the aims of the present study were to identify, map and characterize euchromatin regions in Longissimus dorsi muscle of Nellore cattle to understand if these regions are associated with gene expression and intramuscular fat content. Different transposase-treated nuclei concentrations were tested: 50 thousand, 75 thousand and 100 thousand, and their nucleosomal periodicity was analyzed to check the efficiency of ATAC-seq enzymatic activity. From these, 6,811, 11,121, and 11,473 euchromatin peaks were respectively found by using Model-based Analysis of ChIP-Seq (MACS2). A total of 6,212 open chromatin regions were coincident among the three nuclei concentrations, and this data was used in subsequent analyzes. Enrichment analysis performed using package RegioneR in R, considering significance of p < 0.05, revealed over-representation of peaks in 1) transcription start sites of genes expressed in skeletal muscle; 2) differentially expressed genes (GDE) associated with intramuscular fat content; 3) expression quantitative trait loci (eQTL) identified in skeletal muscle tissue. Our results indicate that skeletal muscle regulatory regions identified by ATAC-seq are involved in the control of gene expression and intramuscular fat content in cattle.

OMICS: A Journal of Integrative BiologyVol. 22, No. 9 Letter to the EditorExciting History of Tip60 and Its Companions in Carcinogenesis Across the Heterochromatin LandscapesMouhamed Idrissou, Khaldoun Rifaï, Marine Daures, Frédérique Penault-Llorca, Yves-Jean Bignon, and Dominique Bernard-GallonMouhamed IdrissouDepartment of Oncogenetics, Centre Jean Perrin, CBRV, Clermont-Ferrand, France.INSERM U 1240-IMOST, Clermont-Ferrand, France.Search for more papers by this author, Khaldoun RifaïDepartment of Oncogenetics, Centre Jean Perrin, CBRV, Clermont-Ferrand, France.INSERM U 1240-IMOST, Clermont-Ferrand, France.Search for more papers by this author, Marine DauresDepartment of Oncogenetics, Centre Jean Perrin, CBRV, Clermont-Ferrand, France.INSERM U 1240-IMOST, Clermont-Ferrand, France.Search for more papers by this author, Frédérique Penault-LlorcaDepartment of Oncogenetics, Centre Jean Perrin, CBRV, Clermont-Ferrand, France.Department of Biopathology, Centre Jean Perrin, Clermont-Ferrand, France.Search for more papers by this author, Yves-Jean BignonDepartment of Oncogenetics, Centre Jean Perrin, CBRV, Clermont-Ferrand, France.INSERM U 1240-IMOST, Clermont-Ferrand, France.Search for more papers by this author, and Dominique Bernard-GallonAddress correspondence:Dominique Bernard-Gallon, PhDDepartment of OncogeneticsCentre Jean Perrin, CBRV28 Place Henri DunantClermont-Ferrand 63001FranceE-mail Address: dominique.gallon-bernard@clermont.unicancer.frDepartment of Oncogenetics, Centre Jean Perrin, CBRV, Clermont-Ferrand, France.INSERM U 1240-IMOST, Clermont-Ferrand, France.Search for more papers by this authorPublished Online:18 Sep 2018https://doi.org/10.1089/omi.2018.0122AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byDigging Deeper into Breast Cancer Epigenetics: Insights from Chemical Inhibition of Histone Acetyltransferase TIP60 In Vitro Mouhamed Idrissou, Andre Lebert, Tiphanie Boisnier, Anna Sanchez, Fatma Zohra Houfaf Khoufaf, Frédérique Penault-Llorca, Yves-Jean Bignon, and Dominique Bernard-Gallon5 October 2020 | OMICS: A Journal of Integrative Biology, Vol. 24, No. 10 Volume 22Issue 9Sep 2018 InformationCopyright 2018, Mary Ann Liebert, Inc., publishersTo cite this article:Mouhamed Idrissou, Khaldoun Rifaï, Marine Daures, Frédérique Penault-Llorca, Yves-Jean Bignon, and Dominique Bernard-Gallon.Exciting History of Tip60 and Its Companions in Carcinogenesis Across the Heterochromatin Landscapes.OMICS: A Journal of Integrative Biology.Sep 2018.626-628.http://doi.org/10.1089/omi.2018.0122Published in Volume: 22 Issue 9: September 18, 2018Online Ahead of Print:August 14, 2018PDF download

Fil: Bolzan, Alejandro Daniel. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - La Plata. Instituto Multidisciplinario de Biologia Celular. Provincia de Buenos Aires. Gobernacion. Comision de Investigaciones Cientificas. Instituto Multidisciplinario de Biologia Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biologia Celular; Argentina

In several eukaryotic organisms, heterochromatin (HC) in the introns of genes can regulate RNA processing, including polyadenylation, but the mechanism underlying this regulation is poorly understood. By promoting distal polyadenylation, the bromo-adjacent homology (BAH) domain-containing and RNA recognition motif-containing protein ASI1 and the H3K9me2-binding protein EDM2 are required for the expression of functional full-length transcripts of intronic HC-containing genes in Arabidopsis Here we report that ASI1 and EDM2 form a protein complex in vivo via a bridge protein, ASI1-Immunoprecipitated Protein 1 (AIPP1), which is another RNA recognition motif-containing protein. The complex also may contain the Pol II CTD phosphatase CPL2, the plant homeodomain-containing protein AIPP2, and another BAH domain protein, AIPP3. As is the case with dysfunction of ASI1 and EDM2, dysfunction of AIPP1 impedes the use of distal polyadenylation sites at tested intronic HC-containing genes, such as the histone demethylase gene IBM1, resulting in a lack of functional full-length transcripts. A mutation in AIPP1 causes silencing of the 35S-SUC2 transgene and genome-wide CHG hypermethylation at gene body regions, consistent with the lack of full-length functional IBM1 transcripts in the mutant. Interestingly, compared with asi1, edm2, and aipp1 mutations, mutations in CPL2, AIPP2, and AIPP3 cause the opposite effects on the expression of intronic HC-containing genes and other genes, suggesting that CPL2, AIPP2, and AIPP3 may form a distinct subcomplex. These results advance our understanding of the interplay between heterochromatic epigenetic modifications and RNA processing in higher eukaryotes.

The heat shock response is characterized by the transcriptional activation of both hsp genes and noncoding and repeated satellite III DNA sequences located at pericentric heterochromatin. Both events are under the control of Heat Shock Factor I (HSF1). Here we show that under heat shock, HSF1 recruits major cellular acetyltransferases, GCN5, TIP60 and p300 to pericentric heterochromatin leading to a targeted hyperacetylation of pericentric chromatin. Redistribution of histone acetylation toward pericentric region in turn directs the recruitment of Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, BRD4, which are required for satellite III transcription by RNAP II. Altogether we uncover here a critical role for HSF1 in stressed cells relying on the restricted use of histone acetylation signaling over pericentric heterochromatin (HC).

Fil: Chiarini, Franco Ezequiel. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto Multidisciplinario de Biologia Vegetal. Universidad Nacional de Cordoba. Facultad de Ciencias Exactas Fisicas y Naturales. Instituto Multidisciplinario de Biologia Vegetal; Argentina

Diferenciación cromosómica en tres especies de Leptostemonum (Solanum, Solanaceae) endémicos de islas oceánicas. Los cromosomas de Solanum nelsonii, S. sandwicense (endémicosde las islas Hawaii) y de S. vespertilio (de las islas Canarias) fueron estudiados por medio de tinción clásica, bandeo CMA/DAPI y FISH con sondas para los genes de ADNr de 18-5.8-26S y 5S. El objetivofue comprobar cambios cromosómicos (número cromosómico, cariotipo, patrones de heterocromatina y loci de ADNr) durante la evolución de estos taxones, en comparación con sus parientes continentales. Una aparente estasis cromosómica fue confirmada para las tres especies, en lo que respecta a número cromosómico y cariotipos. Sin embargo, también se encontró evidencia de cambios de secuencias crípticos, acumulativos. La especiación en estas especies no estaría asociada a grandes rearreglos cromosómicos o poliploidía, sino más bien a divergencia genética.

Abstract Cellular senescence is a highly ‘stable’ state of cell cycle arrest induced by various pathophysiological stimuli, including: telomere dysfunction; abnormal mitotic stress; DNA damage, and; some types of cytokines. Senescent cells typically exhibit distinct morphological changes, including: enlargement of both the cytoplasm and nucleus; prominent nucleoli; and cytoplasmic vacuoles. In addition, a number of biochemical and molecular markers of senescence have been described, which are summarized in our recent review articles. While it is widely accepted that both of the p53 and p16/RB tumor suppressor pathways are involved in senescence, the precise mechanisms underling the phenotype are still elusive. The senescence phenotype can be heterogeneous and, dependent on the stress or cell type, the ‘quality’ of the phenotype varies. Importantly, several ‘effector mechanisms’ that can modulate the senescence phenotype have been identified, including: senescence associated heterochromatic foci (SAHFs) and epigenetic gene regulation; the DNA damage response (DDR); senescence associated secretory phenotype (SASP); and the autophagy/mTOR pathway. Senescence markers are often associated with senescence effectors, and combinations of these markers and effectors have been useful in extending the concept of the senescence quality, not only within cultured cell models but also to in vivo systems. We, and others, have previously shown that normal human diploid fibroblasts (HDFs) often exhibit dramatic heterochromatin (HC) alterations during senescence (i.e. SAHFs) in a p16/RB dependent manner and have proposed that SAHF formation is associated with the stability of the phenotype. SAHFs were originally described in oncogene-induced senescence (OIS) and, to a lesser extent, in replicative and DNA damage-induced senescent HDFs. Thus the intensity to which SAHFs are provoked appears to be context dependent. In addition, non-histone chromosomal architectural proteins, HMGA1 and HMGA2, which have been implicated in cancer, are essential structural components of SAHFs. Depletion of HMGA1 in particular disrupts SAHFs almost completely, rendering senescence arrest unstable. We have recently shown that SAHFs are distinct non-overlapping multi-layer structures, in which H3K9me3 (a constitutive HC mark) is enriched in the ‘core’ of SAHFs, surrounded by a layer of H3K27me3 (a facultative HC mark), which separates the core from the transcriptionally active H3K4/36me3 regions. The data also suggest that this multi-layered structure of HC is achieved through a ‘spatial reorganization’ of pre-existing HC without the massive spreading of HC. It was recently reported that Lamin B1, which forms a fibrillar network at nuclear lamina, is down-regulated during senescence. Constitutive HC is often associated with the nuclear envelope in mammals, and our data suggest that the depletion of Lamin B1 disrupts the HC anchoring to nuclear lamina and facilitates its spatial repositioning during senescence. In addition, a series of studies from the Adams laboratory have shown that, as an early event in senescence, translocation of the HIRA histone chaperone complex to PML bodies is required for SAHF formation. Thus SAHF formation appears to involve multiple events and ‘modular processes’. However, how individual gene regulation is associated with SAHFs remains to be elucidated. The p53 transcription factor is a frequently mutated tumor suppressor. Levels of p53 are typically regulated through its stability, thus, upon stress stimuli, p53 can be up-regulated very quickly. This acutely induced p53 has been used as a major model system for studying genome-wide p53 targets. However, the long-term activation of p53 might be more important in chronic phenotypes such as senescence. We have investigated genome-wide p53 DNA binding profiles in our HDF model, where we can acutely (acute DDR, acDDR) or chronically (OIS) activate p53. Our data suggest that acute- and chronic-p53 profiles are highly distinctive, with the latter being preferentially associated with CpG islands. In addition, our preliminary data suggest that during OIS p53 can also bind uniquely to a region called the epidermal differentiation complex (a tissue specific gene complex, which is heterochromatic in fibroblasts) where the genes are stably silenced. This locus is enriched for both H3K9me3 and Lamin B1, and is primarily located at the nuclear periphery in normal HDFs. During OIS, this locus appears to be internalized and spread in the inter-SAHF space. In addition, some genes within the locus are specifically expressed in OIS cells in a p53-dependent manner, reinforcing the critical role for the spatial reorganization of the genome in gene regulation. Currently we are investigating a functional role for such ‘ectopic’ gene regulation by p53 during senescence. Citation Format: Masashi Narita. Chromatin structure change and aberrant gene expression during senescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY10-01. doi:10.1158/1538-7445.AM2015-SY10-01

To study the clinicopathological and immunohistochemical features, histogenesis and prognosis of pleuropulmonary blastoma (PPB) in children. PPB specimens from 16 pediatric cases with an age ranging from 1 year and 7 months to 5 years and 3 months (mean age of 3 years) were retrieved and analyzed by routine histological, immunohistochemical and electron methods. Among 16 patients, there were 2 type I, 7 type II and 7 type III PPB cases. Type I PPB as multilocular cystic structure, consisted of thin fibrous wall lining the respiratory epithelium, subepithelial primitive blastema or immature mesenchymal cells, with or without rhabdomyoblastic differentiation or cartilage; Type II PPB as cystic-solid tumor, comparing with type I, consisted of intracystic components with appearance of anaplastic tumor cells. Type III PPB consisted of completely solid mass, the same as the solid region of type II, had mixed pattern including blastema, undifferentiated spindle-cell proliferations and sarcomas. In addition, anaplastic tumor cells and intra-and extra- cytoplasmic eosinophilic globules were also commonly present. Epithelial components in PPB were benign. Immunohistochemical study showed primitive mesenchymal differentiation of tumors. All cases were positive for vimentin, desmin, myogenin and SMA in tumors with skeletal muscle differentiation, S-100 was positive in tumors with cartilage differentiation. All tumors were negative for synaptophysin, CD99, and CD117. Benign epithelial components were positive for AE1/AE3 and EMA. In 12 cases, electron microscopy revealed few organelles in the primitive mesenchymal cells and rich heterochromatin in mesenchymal cells, the latter also demonstrating cytoplasmic myofilament dysplasia. Nine cases had clinical follow-up ranging from 5 to 48 months, of which 4 patients died. PPB is a rare lung neoplasm of children under the age of 6 years, with distinct pathological morphology. PPB may arise from lung or pleura mesenchymal cells and has a poor clinical outcome.

Mounting evidences suggest that the ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein) which encode 2 subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres,and multiple endocrine neoplasia type 1 (MEN-1) genes are significantly mutated in most patients with pancreatic neuroendocrine tumors (pNETs),as are genes encoding key molecules of the mammalian target of rapamycin (mTOR) signaling pathway.These mutated genes promote deregulation of epigenetic processes such as chromatin remodeling,histone modification and activation of alternative lengthening of telomeres,and thus combined alteration of these genes may contribute to drive tumorigenesis and metastasis of pNETs which are characterized by complex patterns of phenotypes.These findings may have great significance in the diagnosis and treatment of pNETs and predicting the prognosis,as well as providing clinical implications for targeted cancer therapy. Key words: Pancreas ; Neuroendocrine neoplasms ; Genetic mutation; Epigenetics

Sulforaphane (SFN) supplementation is well tolerated and exhibits cancer‐selective cytotoxicity in preclinical models. SFN decreases global DNA‐methyltransferase (DNMT) and histone deacetylase (HDAC) activity in prostate cancer (PCa) cells, two classes of chromatin modifying enzymes (CME) involved in heterochromatin (HC) establishment and stability. HC marks co‐occur due to coordinated CME activity; therefore, we hypothesized that deficiencies in DNMT and HDAC activity would lead to alterations in histone methyltransferase (HMT) activity. SFN (15 µM) led to a global decrease in histone H3 lysine 9 trimethylation (H3K9me3) in PC3 PCa cells. Investigation into the underlying mechanism suggests SFN influences the H3K9 HMT SUV39H1. SFN leads to SUV39H1 ubiquitination and acetylation, posttranslational marks that are associated with SUV39H1 instability and reduced enzymatic activity. SUV39H1 modification coincided with decreased HC stability. Inhibition of SUV39H1 activity by small molecule inhibitor is cytotoxic to PCa cells, suggesting SUV39H1 inactivation contributes to the cancer‐suppressive activity associated with SFN. Our results suggest that destabilization of HC plays an active role in mediating cytotoxicity in response to SFN and supports further development of SFN as a therapeutic option and SUV39H1 as a therapeutic target for PCa.Grant Funding Source: Supported by NIH P01 CA090890

Background The retina ischemia reperfusion injury (RIRI) can lead to apoptosis,which is associated with many genes.It is very significant to explore the protection of drugs on RIRI-induced apoptosis.Objective This study was to explore the relationship between the expression of Nogo-A and the cell apoptosis as well as the therapeutic effect of NEP1-40 in RIRI retina.Methods The device of raising intraocular pressure (IOP)was used to elevate the IOP for 60 minutes and then restore the IOP to normal for the establishment of RIRI models.Seventy-eight SD rats were randomized to the normal group,RIRI group and NEP1-40 group.PBS of 5 ml/(kg · d)was injected intraperitoneally in the rats of the RIRI group,and NEP1-40 of 8 mg/(L · kg) was used in the same way in the NEP1-40 group.The rats were sacrificed in overdose anesthesia at 6 hours,12 hours and 1 day,2,3,7 days after RIRI to prepare the retinal specimens.The ultrastructure of rat retinas was examined under the transmission electron microscope.Cell apoptosis was assessed by the TUNEL method,and the apoptotic index (AI) was calculated.The expressions of Nogo-A protein and mRNA in rat retinas were detected by immunohistochemistry and semiquantitative reverse transcription PCR (RT-PCR).Results The ultrastructure of retinal cells were normal in the normal group.Retinal cell organelle dissolution,mitochondria swelling,cavitation,chromatin edge heterochromatin and apoptotic body were seen in the rats of the RIRI group from 12 hours through 7 days after injection.However,only the slight loose of outer membranous disk,inner and outer nuclear layer and retinal ganglion cells,the nucleus gap broadening,shortness of some mitochondrial cristae in the NEP1-40 group.TUNEL-positive cells appeared 6 hours after RIRI and reached peak in 1 day,and gradually declined after that in the RIRI group.A similar pattern was seen in the rats of the NEP1-40 group with a more mild manifestation.Significant differences were seen in the AI values among the different groups at various time points (Fgroup =100.850,P =0.000 ; Ftime =34.309,P =0.000),and the AI values were significantly higher in the RIRI group and NEP1-40 group compared with normal group;while the AI values in the NEP1-40 group was lower than those of the RIRI group (all at P＜0.05).The expressions of Nogo-A protein and mRNA showed a coincident pattern with apoptosis procedure,with a gradually elevated level from 6 hours through 7 days after RIRI and a peak in 2 days,and those in the NEP1-40 group were weaker in comparison with the RIRI group in various time points.Significant differences were detected in the expression of Nogo-A protein and the expression of Nogo-A mRNA among different groups and various time points(protein:Fgroup =164.139,P =0.000;Ftime =21.772,P =0.000.mRNA:Fgroup =93.889,P =0.000 ; Ftime =6.349,P =0.000).Conclusions Nogo-A probably plays an important role in RIRI.NEP1-40 can protect retinal cells from apoptosis following RIRI through down-regulating the expression of Nogo-A. Key words: NEP1-40; Retina; Ischemia reperfusion injury; Nogo-A; Apoptosis