Differential enrichment of H3K9me3 at annotated satellite DNA repeats in human cell lines and during fetal development in mouse

Background Trimethylation of histone H3 on lysine 9 (H3K9me3) at satellite DNA sequences has been primarily studied at (peri)centromeric regions, where its level shows differences associated with various processes such as development and malignant transformation. However, the dynamics of H3K9me3 at distal satellite DNA repeats has not been thoroughly investigated. Results We exploit the sets of publicly available data derived from chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-Seq), produced by the The Encyclopedia of DNA Elements (ENCODE) project, to analyze H3K9me3 at assembled satellite DNA repeats in genomes of human cell lines and during mouse fetal development. We show that annotated satellite elements are generally enriched for H3K9me3, but its level in cancer cell lines is on average lower than in normal cell lines. We find 407 satellite DNA instances with differential H3K9me3 enrichment between cancer and normal cells including a large 115-kb cluster of GSATII elements on chromosome 12. Differentially enriched regions are not limited to satellite DNA instances, but instead encompass a wider region of flanking sequences. We found no correlation between the levels of H3K9me3 and noncoding RNA at corresponding satellite DNA loci. The analysis of data derived from multiple tissues identified 864 instances of satellite DNA sequences in the mouse reference genome that are differentially enriched between fetal developmental stages. Conclusions Our study reveals significant differences in H3K9me3 level at a subset of satellite repeats between biological states and as such contributes to understanding of the role of satellite DNA repeats in epigenetic regulation during development and carcinogenesis.

KDM4A regulates myogenesis by demethylating H3K9me3 of myogenic regulatory factors

Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.

Histone demethylase KDM4A overexpression improved the efficiency of corrected human tripronuclear zygote development

Human zygotes are difficult to obtain for research because of limited resources and ethical debates. Corrected human tripronuclear (ch3PN) zygotes obtained by removal of the extra pronucleus from abnormally fertilized tripronuclear (3PN) zygotes are considered an alternative resource for basic scientific research. In the present study, 8-cell and blastocyst formation efficiency were significantly lower in both 3PN and ch3PN embryos than in normal fertilized (2PN) embryos, while histone H3 lysine 9 trimethylation (H3K9me3) levels were much higher. It was speculated that the aberrant H3K9me3 level detected in ch3PN embryos may be related to low developmental competence. Microinjection of 1000 ng/µl lysine-specific demethylase 4 A (KDM4A) mRNA effectively reduced the H3K9me3 level and significantly increased the developmental competence of ch3PN embryos. The quality of ch3PN zygotes improved as the grading criteria, cell number and pluripotent expression significantly increased in response to KDM4A mRNA injection. Developmental genes related to zygotic genome activation (ZGA) were also upregulated. These results indicate that KDM4A activates the transcription of the ZGA program by enhancing the expression of related genes, promoting epigenetic modifications and regulating the developmental potential of ch3PN embryos. The present study will facilitate future studies of ch3PN embryos and could provide additional options for infertile couples.

USP9X-mediated KDM4C deubiquitination promotes lung cancer radioresistance by epigenetically inducing TGF-β2 transcription

Radioresistance is regarded as the main barrier to effective radiotherapy in lung cancer. However, the underlying mechanisms of radioresistance remain elusive. Here, we show that lysine-specific demethylase 4C (KDM4C) is overexpressed and correlated with poor prognosis in lung cancer patients. We provide evidence that genetical or pharmacological inhibition of KDM4C impairs tumorigenesis and radioresistance in lung cancer in vitro and in vivo. Moreover, we uncover that KDM4C upregulates TGF-β2 expression by directly reducing H3K9me3 level at the TGF-β2 promoter and then activates Smad/ATM/Chk2 signaling to confer radioresistance in lung cancer. Using tandem affinity purification technology, we further identify deubiquitinase USP9X as a critical binding partner that deubiquitinates and stabilizes KDM4C. More importantly, depletion of USP9X impairs TGF-β2/Smad signaling and radioresistance by destabilizing KDM4C in lung cancer cells. Thus, our findings demonstrate that USP9X-mediated KDM4C deubiquitination activates TGF-β2/Smad signaling to promote radioresistance, suggesting that targeting KDM4C may be a promising radiosensitization strategy in the treatment of lung cancer.

Distinct Regulation of the Expression of Satellite DNAs in the Beetle Tribolium castaneum

In the flour beetle, Tribolium castaneum (peri)centromeric heterochromatin is mainly composed of a major satellite DNA TCAST1 interspersed with minor satellites. With the exception of heterochromatin, clustered satellite repeats are found dispersed within euchromatin. In order to uncover a possible satellite DNA function within the beetle genome, we analysed the expression of the major TCAST1 and a minor TCAST2 satellite during the development and upon heat stress. The results reveal that TCAST1 transcription was strongly induced at specific embryonic stages and upon heat stress, while TCAST2 transcription is stable during both processes. TCAST1 transcripts are processed preferentially into piRNAs during embryogenesis and into siRNAs during later development, contrary to TCAST2 transcripts, which are processed exclusively into piRNAs. In addition, increased TCAST1 expression upon heat stress is accompanied by the enrichment of the silent histone mark H3K9me3 on the major satellite, while the H3K9me3 level at TCAST2 remains unchanged. The transcription of the two satellites is proposed to be affected by the chromatin state: heterochromatin and euchromatin, which are assumed to be the prevalent sources of TCAST1 and TCAST2 transcripts, respectively. In addition, distinct regulation of the expression might be related to diverse roles that major and minor satellite RNAs play during the development and stress response.

Heat Stress Affects H3K9me3 Level at Human Alpha Satellite DNA Repeats

Satellite DNAs are tandemly repeated sequences preferentially assembled into large arrays within constitutive heterochromatin and their transcription is often activated by stress conditions, particularly by heat stress. Bioinformatic analyses of sequenced genomes however reveal single repeats or short arrays of satellite DNAs dispersed in the vicinity of genes within euchromatin. Here, we analyze transcription of a major human alpha satellite DNA upon heat stress and follow the dynamics of “silent” H3K9me3 and “active” H3K4me2/3 histone marks at dispersed euchromatic and tandemly arranged heterochromatic alpha repeats. The results show H3K9me3 enrichment at alpha repeats upon heat stress, which correlates with the dynamics of alpha satellite DNA transcription activation, while no change in H3K4me2/3 level is detected. Spreading of H3K9me3 up to 1–2 kb from the insertion sites of the euchromatic alpha repeats is detected, revealing the alpha repeats as modulators of local chromatin structure. In addition, expression of genes containing alpha repeats within introns as well as of genes closest to the intergenic alpha repeats is downregulated upon heat stress. Further studies are necessary to reveal the possible contribution of H3K9me3 enriched alpha repeats, in particular those located within introns, to the silencing of their associated genes.

16 Improvement of porcine cloned embryo developmental competence via KDM4A overexpression and H3K9me3 methyltransferase inhibitor treatment

Aberrant epigenetic reprogramming is a major reason for the developmental failure of somatic cell nuclear transfer (SCNT) embryos. Histone H3 lysine 9 trimethylation (H3K9me3), a histone marker for transcriptional repression, is considered a key barrier to the development of cloned embryos. In the present study, we found that H3K9me3 was much higher in SCNT embryos than in IVF embryos at the 4-cell and 2-cell stages; H3K9me3 demethylase KDM4A mRNA was injected into cloned embryos 5h after activation. The intensity of H3K9me3 modification decreased obviously after microinjection of KDM4A mRNA, and the developmental efficiency of porcine cloned embryos was enhanced significantly compared with control (32.2±4.2 vs. 21.0±3.5; P<0.05). Moreover, we evaluated the effect of chaetocin, an inhibitor of histone methyltransferase suv39h1/2, on SCNT embryo development. The results showed that 10 nM chaetocin not only suppressed the H3K9me3 modification in porcine embryonic fibroblast but also downregulated the expression of SUV39H1, SUV39H2, and KDM4D. However, treatment of cloned embryos with 10 nM chaetocin efficiently decreased the H3K9me3 level. Importantly, chaetocin treatment at the 4-cell stage for 6h significantly increased the blastocyst rate (57.8±4.3 vs. 43.5±1.8; P<0.05) compared with the control group. Furthermore, because a recent study showed that a high level of H3K9me3 appears in the reprogramming resistance region of nuclear transfer embryos during zygogene activation (ZGA), we examined the expression levels of ZGA-related genes. The qPCR results showed that the expression of ZGA-related genes increased significantly in SCNT embryos with chaetocin treatment compared with the control. These results suggested that chaetocin treatment can improve the efficiency of SCNT reprogramming during ZGA. In summary, our results suggested that H3K9me3 acts as an epigenetic barrier in porcine SCNT reprogramming and that a suv39h1/2 inhibitor can effectively reduce the H3K9me3 level in the early reprogramming phase and further improve the invitro developmental competence. Due to the existence of a variety of abnormal epigenetic mechanisms during somatic cell reprogramming, the combined use of small-molecule inhibitors is required in future studies. Data analyses were performed using SPSS software. Significance was set at P<0.05 unless otherwise specified. The results are expressed as the mean±standard deviation. This work was supported by the National Natural Science Foundation of China (grant number 31601942); the Postdoctoral Science Foundation of Heilongjiang Province (grant number LBH-Z17010); and the Fund for the National Key Research and Development program of China-Stem Cell and Translational Research (2016YFA0100200).

379 TREATMENT OF 293T CELLS WITH XENOPUS EGG EXTRACT INDUCES NOT-STRESS-RELATED EPIGENETIC REMODELING OF RIBOSOMAL GENES

Undefined mechanisms involved in reprogramming of somatic cells by Xenopus leavis egg extract have, to date, prevented clinical applications for cell replacement therapy. The aim of this study was to evaluate the immediate response of somatic cells to exposure to Xenopus leavis egg extract using ribosomal genes (rDNA) as a sensitive marker for stress and/or chromatin remodeling. Human epithelial 293T cells in standard culture (control), starved in glucose-free medium (stress-control), or treated for 1 h with egg extract were fixed at 6 and 24 h after treatment and analyzed for pre-rRNA synthetic activity by quantitative RT-PCR; level of H3K9me3 by immunofluorescence; and for occupancy of rDNA promoter by markers of normal activity (UBF), stress silencing (SIRT1, SUV39H1), and remodeling (SNF2H) using chromatin immunoprecipitation. Relative levels of pre-rRNA decreased in all treated groups to 20 to 35% of control levels. Chromatin immunoprecipitation did not reveal any significant effect of starvation or extract treatment on UBF and SIRT1 binding to the rDNA promoter, presumably because of the pleiotropic property of these proteins. However, starvation specifically led to enhanced SUV39H1 binding 6 h after treatment, indicating early cell response to stress. No differences were found in SUV39H1 binding between extract-treated cells and control. In contrast, binding of SNF2H at 6 h increased in extract-treated cells but decreased in starved cells. The relative level of H3K9me3 increased first after 24 h of starvation equally in all cells, suggesting later involvement of this histone modification in rDNA silencing. Extract treatment, however, led to a decrease in H3K9me3 level at 6 h, and after 24 h, 2 main cell populations were observed: one (85.4% of cells) that retained decreased H3K9me3 and one (12.4% of cells) with significantly enhanced levels. Moreover, H3K9me3 foci in the last group were associated with the periphery of presumptive nucleoli. Thus, extract treatment apparently does not initiate a stress-induced silencing of the rDNA genes; rather, it activates SNF2H-dependent chromatin remodeling, resulting in a long-term decrease in pre-rRNA synthetic activity. The research was supported by Carlsberg Foundation 2008-01-0105.