Chromatin accessibility and H3K9me3 landscapes reveal long-term epigenetic effects of fetal-neonatal iron deficiency in rat hippocampus.

BACKGROUND: Iron deficiency (ID) during the fetal-neonatal period results in long-term neurodevelopmental impairments associated with pervasive hippocampal gene dysregulation. Prenatal choline supplementation partially normalizes these effects, suggesting an interaction between iron and choline in hippocampal transcriptome regulation. To understand the regulatory mechanisms, we investigated epigenetic marks of genes with altered chromatin accessibility (ATAC-seq) or poised to be repressed (H3K9me3 ChIP-seq) in iron-repleted adult rats having experienced fetal-neonatal ID exposure with or without prenatal choline supplementation. RESULTS: Fetal-neonatal ID was induced by limiting maternal iron intake from gestational day (G) 2 through postnatal day (P) 7. Half of the pregnant dams were given supplemental choline (5.0 g/kg) from G11-18. This resulted in 4 groups at P65 (Iron-sufficient [IS], Formerly Iron-deficient [FID], IS with choline [ISch], and FID with choline [FIDch]). Hippocampi were collected from P65 iron-repleted male offspring and analyzed for chromatin accessibility and H3K9me3 enrichment. 22% and 24% of differentially transcribed genes in FID- and FIDch-groups, respectively, exhibited significant differences in chromatin accessibility, whereas 1.7% and 13% exhibited significant differences in H3K9me3 enrichment. These changes mapped onto gene networks regulating synaptic plasticity, neuroinflammation, and reward circuits. Motif analysis of differentially modified genomic sites revealed significantly stronger choline effects than early-life ID and identified multiple epigenetically modified transcription factor binding sites. CONCLUSIONS: This study reveals genome-wide, stable epigenetic changes and epigenetically modifiable gene networks associated with specific chromatin marks in the hippocampus, and lays a foundation to further elucidate iron-dependent epigenetic mechanisms that underlie the long-term effects of fetal-neonatal ID, choline, and their interactions.

Normal distribution of H3K9me3 occupancy co-mediated by histone methyltransferase BcDIM5 and histone deacetylase BcHda1 maintains stable ABA synthesis in Botrytis cinerea TB-31.

Abscisic acid (ABA) is a conserved and important "sesquiterpene signaling molecule" widely distributed in different organisms with unique biological functions. ABA coordinates reciprocity and competition between microorganisms and their hosts. In addition, ABA also regulates immune and stress responses in plants and animals. Therefore, ABA has a wide range of applications in agriculture, medicine and related fields. The plant pathogenic ascomycete B. cinerea has been extensively studied as a model strain for ABA production. Nevertheless, there is a relative dearth of research regarding the regulatory mechanism governing ABA biosynthesis in B. cinerea. Here, we discovered that H3K9 methyltransferase BcDIM5 is physically associated with the H3K14 deacetylase BcHda1. Deletion of Bcdim5 and Bchda1 in the high ABA-producing B. cinerea TB-31 led to severe impairment of ABA synthesis. The combined analysis of RNA-seq and ChIP-seq has revealed that the absence of BcDIM5 and BcHda1 has resulted in significant global deficiencies in the normal distribution and level of H3K9me3 modification. In addition, we found that the cause of the decreased ABA production in the ΔBcdim5 and ΔBchda1 mutants was due to cluster gene repression caused by the emergence of hyper-H3K9me3 in the ABA gene cluster. We concluded that the ABA gene cluster is co-regulated by BcDIM5 and BcHda1, which are essential for the normal distribution of the B. cinerea TB-31 ABA gene cluster H3K9me3. This work expands our understanding of the complex regulatory network of ABA biosynthesis and provides a theoretical basis for genetic improvement of high-yielding ABA strains.

Opposite and Differently Altered Postmortem Changes in H3 and H3K9me3 Patterns in the Rat Frontal Cortex and Hippocampus.

Temporal and spatial epigenetic modifications in the brain occur during ontogenetic development, pathophysiological disorders, and aging. When epigenetic marks, such as histone methylations, in brain autopsies or biopsy samples are studied, it is critical to understand their postmortem/surgical stability. For this study, the frontal cortex and hippocampus of adult rats were removed immediately (controls) or after a postmortem delay of 15, 30, 60, 90, 120, or 150 min. The patterns of unmodified H3 and its trimethylated form H3K9me3 were analyzed in frozen samples for Western blot analysis and in formalin-fixed tissues embedded in paraffin for confocal microscopy. We found that both the unmodified H3 and H3K9me3 showed time-dependent but opposite changes and were altered differently in the frontal cortex and hippocampus with respect to postmortem delay. In the frontal cortex, the H3K9me3 marks increased approximately 450% with a slow parallel 20% decrease in the unmodified H3 histones after 150 min. In the hippocampus, the change was opposite, since H3K9me3 marks decreased steadily by approximately 65% after 150 min with a concomitant rapid increase of 20-25% in H3 histones at the same time. Confocal microscopy located H3K9me3 marks in the heterochromatic regions of the nuclei of all major cell types in the control brains: oligodendrocytes, astrocytes, neurons, and microglia. Therefore, epigenetic marks could be affected differently by postmortem delay in different parts of the brain.

DNA satellite and chromatin organization at mouse centromeres and pericentromeres.

BACKGROUND: Centromeres are essential for faithful chromosome segregation during mitosis and meiosis. However, the organization of satellite DNA and chromatin at mouse centromeres and pericentromeres is poorly understood due to the challenges of assembling repetitive genomic regions. RESULTS: Using recently available PacBio long-read sequencing data from the C57BL/6 strain, we find that contrary to the previous reports of their homogeneous nature, both centromeric minor satellites and pericentromeric major satellites exhibit a high degree of variation in sequence and organization within and between arrays. While most arrays are continuous, a significant fraction is interspersed with non-satellite sequences, including transposable elements. Using chromatin immunoprecipitation sequencing (ChIP-seq), we find that the occupancy of CENP-A and H3K9me3 chromatin at centromeric and pericentric regions, respectively, is associated with increased sequence enrichment and homogeneity at these regions. The transposable elements at centromeric regions are not part of functional centromeres as they lack significant CENP-A enrichment. Furthermore, both CENP-A and H3K9me3 nucleosomes occupy minor and major satellites spanning centromeric-pericentric junctions and a low yet significant amount of CENP-A spreads locally at centromere junctions on both pericentric and telocentric sides. Finally, while H3K9me3 nucleosomes display a well-phased organization on major satellite arrays, CENP-A nucleosomes on minor satellite arrays are poorly phased. Interestingly, the homogeneous class of major satellites also phase CENP-A and H3K27me3 nucleosomes, indicating that the nucleosome phasing is an inherent property of homogeneous major satellites. CONCLUSIONS: Our findings reveal that mouse centromeres and pericentromeres display a high diversity in satellite sequence, organization, and chromatin structure.

The Significance of Modified Histone H3 in Epithelial Dysplasia and Oral Cancer.

BACKGROUND: Oral carcinogenesis is complex and influenced by both genetic and epigenetic changes. Altered histone modification is the epigenetic event that plays a role in cancer development and progression. Distinct modification patterns of histones have been shown to affect patient prognosis in selected cancers. This study aimed to evaluate the profiles of histone H3 modification in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC) in association with the clinical-pathologic characteristics. METHODS: One hundred patients were divided into 4 groups: low-grade OED, high-grade OED, OSCC, and normal oral mucosa (NOM). The levels of 3 types of histone modification-the H3K18ac, H3K9me3, and H3K9ac-were analysed immunohistochemically. Their expression profiles were compared and correlated with prognostically relevant clinical and pathologic features. RESULTS: The H3K18ac and H3K9me3 were upregulated in OSCC, compared with OED and NOM. In contrast, the H3K9ac was downregulated in low-grade OED but increased in high-grade OED and OSCC. The hyperacetylations of H3K18 and H3K9 significantly correlated with advanced cancer depth of invasion and high T stage, respectively. CONCLUSIONS: Histone H3 acetylation and methylation at lysine residues are differentially involved in the multistep oral carcinogenesis and impact aggressive cancer phenotypes. The effect of H3K9ac appears early in OED development, whilst the increased H3K18ac and H3K9me3 may be vital in the emergence of OSCC.

ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline.

H3K9 trimethylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that in male meiosis, ATF7IP2 amasses on autosomal and X-pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X-pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global up-regulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.

Mode of SUV420H2 heterochromatin localization through multiple HP1 binding motifs in the heterochromatic targeting module.

Constitutive heterochromatin is transcriptionally repressed and densely packed chromatin, typically harboring histone H3 Lys9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1). SUV420H2, a histone H4 Lys20 methyltransferase, is recruited to heterochromatin by binding to HP1 through its Heterochromatic Targeting Module (HTM). Here, we have identified three HP1 binding motifs within the HTM. Both the full-length HTM and its N-terminal region (HTM-N), which contains the first and second motifs, stabilized HP1 on heterochromatin. The intervening region between the first and second HP1 binding motifs in HTM-N was also crucial for HP1 binding. In contrast, the C-terminal region of HTM (HTM-C), containing the third motif, destabilized HP1 on chromatin. An HTM V374D mutant, featuring a Val374 to Asp substitution in the second HP1 binding motif, localizes to heterochromatin without affecting HP1 stability. These data suggest that the second HP1 binding motif in the SUV420H2 HTM is critical for locking HP1 on H3K9me3-enriched heterochromatin. HTM V374D, tagged with a fluorescent protein, can serve as a live-cell probe to visualize HP1-bound heterochromatin.

Molecular functional characterization of the setdb1 and its potential target gene sox5 illuminate the histone modification-mediated orchestration of gonadal development in Chinese tongue sole (Cynoglossus semilaevis).

SET (SuVar3-9, Enhancer of Zeste, Trithorax) domain bifurcated histone lysine methyltransferase 1, setdb1, is the predominant histone lysine methyltransferase catalyzing H3K9me3. Prior studies have illustrated that setdb1 and H3K9me3 critically regulate sex differentiation and gametogenesis. However, the molecular details by which setdb1 is involved in these processes in fish have been poorly reported. Here, we cloned and characterized the setdb1 ORF (open reading frame) sequence from Chinese tongue sole (Cynoglossus semilaevis). The setdb1 ORF sequence was 3,669 bp, encoding a 1,222-amino-acid protein. Phylogenetic analysis showed that setdb1 was structurally conserved. qRT-PCR revealed that setdb1 had a high expression level in the testes at 12 mpf (months post fertilization). Single-cell RNA-seq data at 24 mpf indicated that setdb1 was generally expressed in spermatogenic cells at each stage except for sperm and was centrally expressed in oogonia. H3K9me3 modification was observed in gonads with the immunofluorescence technique. Furthermore, the overexpression experiment suggested that sox5 was a candidate target of setdb1. sox5 was abundantly expressed in male and pseudomale gonads at 24 mpf. Single-cell RNA-seq data showed that sox5 was mainly expressed in spermatogonia and its expression gradually declined with differentiation. Taken together, our findings imply that setdb1 regulates sox5 transcription in gonads, which provides molecular clues into histone modification-mediated orchestration of sex differentiation and gametogenesis.

YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia†.

Genomic integrity is critical for sexual reproduction, ensuring correct transmission of parental genetic information to the descendant. To preserve genomic integrity, germ cells have evolved multiple DNA repair mechanisms, together termed as DNA damage response. The RNA N6-methyladenosine is the most abundant mRNA modification in eukaryotic cells, which plays important roles in DNA damage response, and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) is a well-acknowledged N6-methyladenosine reader protein regulating the mRNA decay and stress response. Despite this, the correlation between YTHDF2 and DNA damage response in germ cells, if any, remains enigmatic. Here, by employing a Ythdf2-conditional knockout mouse model as well as a Ythdf2-null GC-1 mouse spermatogonial cell line, we explored the role and the underlying mechanism for YTHDF2 in spermatogonial DNA damage response. We identified that, despite no evident testicular morphological abnormalities under the normal circumstance, conditional mutation of Ythdf2 in adult male mice sensitized germ cells, including spermatogonia, to etoposide-induced DNA damage. Consistently, Ythdf2-KO GC-1 cells displayed increased sensitivity and apoptosis in response to DNA damage, accompanied by the decreased SET domain bifurcated 1 (SETDB1, a histone methyltransferase) and H3K9me3 levels. The Setdb1 knockdown in GC-1 cells generated a similar phenotype, but its overexpression in Ythdf2-null GC-1 cells alleviated the sensitivity and apoptosis in response to DNA damage. Taken together, these results demonstrate that the N6-methyladenosine reader YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia, which provides novel insights into the mechanisms underlying spermatogonial genome integrity maintenance and therefore contributes to safe reproduction.

Insights into the Role of Histone Methylation in Brain Aging and Potential Therapeutic Interventions.

Epigenetic mechanisms play a primary role in the cellular damage associated with brain aging. Histone posttranslational modifications represent intrinsic molecular alterations essential for proper physiological functioning, while divergent expression and activity have been detected in several aspects of brain aging. Aberrant histone methylation has been involved in neural stem cell (NSC) quiescence, microglial deficits, inflammatory processes, memory impairment, cognitive decline, neurodegenerative diseases, and schizophrenia. Herein, we provide an overview of recent studies on epigenetic regulation of brain tissue aging, mainly focusing on the role of histone methylation in different cellular and functional aspects of the aging process. Emerging targeting strategies of histone methylation are further explored, including neuroprotective drugs, natural compounds, and lifestyle modifications with therapeutic potential towards the aging process of the brain.

DNA methylation-dependent epigenetic regulation of Verticillium dahliae virulence in plants.

As a conserved epigenetic mark, DNA cytosine methylation, at the 5' position (5-mC), plays important roles in multiple biological processes, including plant immunity. However, the involvement of DNA methylation in the determinants of virulence of phytopathogenic fungi remains elusive. In this study, we profiled the DNA methylation patterns of the phytopathogenic fungus Verticillium dahliae, one of the major causal pathogens of Verticillium wilt disease that causes great losses in many crops, and explored its contribution in fungal pathogenicity. We reveal that DNA methylation modification is present in V. dahliae and is required for its full virulence in host plants. The major enzymes responsible for the establishment of DNA methylation in V. dahliae were identified. We provided evidence that DNA methyltransferase-mediated establishment of DNA methylation pattern positively regulates fungal virulence, mainly through repressing a conserved protein kinase VdRim15-mediated Ca2+ signaling and ROS production, which is essential for the penetration activity of V. dahliae. In addition, we further demonstrated that histone H3 lysine 9 trimethylation (H3K9me3), another heterochromatin marker that is closely associated with 5-mC in eukaryotes, also participates in the regulation of V. dahliae pathogenicity, through a similar mechanism. More importantly, DNA methyltransferase genes VdRid, VdDnmt5, as well as H3K9me3 methyltransferase genes, were greatly induced during the early infection phase, implying that a dynamic regulation of 5-mC and H3K9me3 homeostasis is required for an efficient infection. Collectively, our findings uncover an epigenetic mechanism in the regulation of phytopathogenic fungal virulence. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00117-5.

PAK1-dependent mechanotransduction enables myofibroblast nuclear adaptation and chromatin organization during fibrosis.

Myofibroblasts are responsible for scarring during fibrosis. The scar propagates mechanical signals inducing a radical transformation in myofibroblast cell state and increasing profibrotic phenotype. Here, we show mechanical stress from progressive scarring induces nuclear softening and de-repression of heterochromatin. The parallel loss of H3K9Me3 enables a permissive state for distinct chromatin accessibility and profibrotic gene regulation. Integrating chromatin accessibility profiles with RNA expression provides insight into the transcription network underlying the switch in profibrotic myofibroblast states, emphasizing mechanoadaptive regulation of PAK1 as key drivers. Through genetic manipulation in liver and lung fibrosis, loss of PAK1-dependent signaling impairs the mechanoadaptive response in vitro and dramatically improves fibrosis in vivo. Moreover, we provide human validation for mechanisms underpinning PAK1-mediated mechanotransduction in liver and lung fibrosis. Collectively, these observations provide insight into the nuclear mechanics driving the profibrotic chromatin landscape in fibrosis, highlighting actomyosin-dependent mechanisms as potential therapeutic targets in fibrosis.

Optogenetics for sensors: On-demand fluorescent labeling of histone epigenetics.

Post-translational modifications of histones to a large extent determine the functional state of chromatin loci. Dynamic visualization of histone modifications with genetically encoded fluorescent sensors makes it possible to monitor changes in the epigenetic state of a single living cell. At the same time, the sensors can potentially compete with endogenous factors recognizing these modifications. Thus, prolonged binding of the sensors to chromatin can affect normal epigenetic regulation. Here, we report an optogenetic sensor for live-cell visualization of histone H3 methylated at lysine-9 (H3K9me3) named MPP8-LAMS (MPP8-based light-activated modification sensor). MPP8-LAMS consists of several fusion protein parts (from N- to C-terminus): i) nuclear export signal (NES), ii) far-red fluorescent protein Katushka, iii) H3K9me3-binding reader domain of the human M phase phosphoprotein 8 (MPP8), iv) the light-responsive AsLOV2 domain, which exposes a nuclear localization signal (NLS) upon blue light stimulation. In the dark, due to the NES, MPP8-LAMS is localized in the cytosol. Under blue light illumination, MPP8-LAMS underwent an efficient translocation from cytosol to nucleus, enabling visualization of H3K9me3-enriched loci. Such an on-demand visualization minimizes potential impact on cell physiology as most of the time the sensor is separated from its target. In general, the present work extends the application of optogenetics to the area of advanced use of genetically encoded sensors.

Characterization of H3K9me3 and DNA methylation co-marked CpG-rich regions during mouse development.

BACKGROUND: H3K9me3 and DNA methylation co-marked CpG-rich regions (CHMs) are functionally important in mouse pre-implantation embryos, but their characteristics in other biological processes are still largely unknown. RESULTS: In this study, we performed a comprehensive analysis to characterize CHMs during 6 mouse developmental processes, identifying over 2,600 CHMs exhibiting stable co-mark of H3K9me3 and DNA methylation patterns at CpG-rich regions. We revealed the distinctive features of CHMs, including elevated H3K9me3 signals and a significant presence in euchromatin and the potential role in silencing younger long terminal repeats (LTRs), especially in some ERVK subfamilies. The results highlight the distinct nature of universal CHMs compared to CpG-rich nonCHMs in terms of location, LTR enrichment, and DNA sequence features, enhancing our understanding of CpG-rich regions' regulatory roles. CONCLUSIONS: This study characterizes the features of CHMs in multiple developmental processes and broadens our understanding of the regulatory roles of CpG-rich regions.

A complex interplay between H2A.Z and HP1 isoforms regulates pericentric heterochromatin.

Pericentric heterochromatin (PCH) plays an essential role in the maintenance of genome integrity and alterations in PCH have been linked to cancer and aging. HP1 α, ß, and γ, are hallmarks of constitutive heterochromatin that are thought to promote PCH structure through binding to heterochromatin-specific histone modifications and interaction with a wide range of factors. Among the less understood components of PCH is the histone H2A variant H2A.Z, whose role in the organization and maintenance of PCH is poorly defined. Here we show that there is a complex interplay between H2A.Z and HP1 isoforms in PCH. While the loss of HP1α results in the accumulation of H2A.Z.1 in PCH, which is associated with a significant decrease in its mobile fraction, H2A.Z.1 binds preferentially to HP1ß in these regions. Of note, H2A.Z.1 downregulation results in increased heterochromatinization and instability of PCH, reflected by accumulation of the major epigenetic hallmarks of heterochromatin in these regions and increased frequency of chromosome aberrations related to centromeric/pericentromeric defects. Our studies support a role for H2A.Z in genome stability and unveil a key role of H2A.Z in the regulation of heterochromatin-specific epigenetic modifications through a complex interplay with the HP1 isoforms.

ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline.

H3K9 tri-methylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that, in male meiosis, ATF7IP2 amasses on autosomal and X pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global upregulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.

CELF2 Sustains a Proliferating/OLIG2+ Glioblastoma Cell Phenotype via the Epigenetic Repression of SOX3.

Glioblastomas (GBs) are incurable brain tumors. The persistence of aggressive stem-like tumor cells after cytotoxic treatments compromises therapeutic efficacy, leading to GBM recurrence. Forcing the GBM cells to irreversibly abandon their aggressive stem-like phenotype may offer an alternative to conventional cytotoxic treatments. Here, we show that the RNA binding protein CELF2 is strongly expressed in mitotic and OLIG2-positive GBM cells, while it is downregulated in differentiated and non-mitotic cells by miR-199a-3p, exemplifying GBM intra-tumor heterogeneity. Using patient-derived cells and human GBM samples, we demonstrate that CELF2 plays a key role in maintaining the proliferative/OLIG2 cell phenotype with clonal and tumorigenic properties. Indeed, we show that CELF2 deficiency in patient-derived GSCs drastically reduced tumor growth in the brains of nude mice. We further show that CELF2 promotes TRIM28 and G9a expression, which drive a H3K9me3 epigenetic profile responsible for the silencing of the SOX3 gene. Thus, CELF2, which is positively correlated with OLIG2 and Ki67 expression in human GBM samples, is inversely correlated with SOX3 and miR-199a-3p. Accordingly, the invalidation of SOX3 in CELF2-deficient patient-derived cells rescued proliferation and OLIG2 expression. Finally, patients expressing SOX3 above the median level of expression tend to have a longer life expectancy. CELF2 is therefore a crucial target for the malignant potential of GBM and warrants attention when developing novel anticancer strategies.

Trimethylation profile of histones H3 lysine 4 and 9 in late preantral and early antral caprine follicles grown in vivo versus in vitro in the presence of anethole.

This study assessed the histones methylation profile (H3K4me3 and H3K9me3) in late preantral (PA) and early antral (EA) caprine follicles grown in vivo and in vitro, and the anethole effect during in vitro culture of PA follicles. Uncultured in vivo-grown follicles (PA, n = 64; EA, n = 73) were used as controls to assess the methylation profile and genes' expression related to apoptosis cascade (BAX, proapoptotic; BCL2, antiapoptotic), steroidogenesis (CYP17, CYP19A1), and demethylation (KDM1AX1, KDM1AX2, KDM3A). The isolated PA follicles (n = 174) were cultured in vitro for 6 days in α-MEM+ in either absence (control) or presence of anethole. After culture, EA follicles were evaluated for methylation, mRNA abundance, and morphometry. Follicle diameter increased after culture, regardless of treatment. The methylation profile and the mRNA abundance were similar between in vivo-grown PA and EA follicles. Anethole treatment led to higher H3K4me3 fluorescence intensity in EA follicles. The mRNA abundances of BAX, CYP17, and CYP19A1 were higher, and BCL2 and KDM3A were lower in in vitro-grown EA follicles than in vivo-grown follicles. In conclusion, in vitro follicle culture affected H3K4me3 fluorescence intensity, mRNA abundance of apoptotic genes, and steroidogenic and demethylase enzymes compared with in vivo-grown follicles.

Cooperation between IRTKS and deubiquitinase OTUD4 enhances the SETDB1-mediated H3K9 trimethylation that promotes tumor metastasis via suppressing E-cadherin expression.

Elevated expression and genetic aberration of IRTKS, also named as BAIAP2L1, have been observed in many tumors, especially in tumor progression. however, the molecular and cellular mechanisms involved in the IRTKS-enhanced tumor progression are obscure. Here we show that higher IRTKS level specifically increases histone H3 lysine 9 trimethylation (H3K9me3) by promoting accumulation of the histone methyltransferase SETDB1. Furthermore, we reveal that IRTKS recruits the deubiquitinase OTUD4 to remove Lys48-linked polyubiquitination at K182/K1050 sites of SETDB1, thus blocking SETDB1 degradation via the ubiquitin-proteasome pathway. Interestingly, the enhanced IRTKS-OTUD4-SETDB1-H3K9me3 axis leads to a general decrease in chromatin accessibility, which inhibits transcription of CDH1 encoding E-cadherin, a key molecule essential for maintaining epithelial cell phenotype, and therefore results in epithelial-mesenchymal transition (EMT) and malignant cell metastasis. Clinically, the elevated IRTKS levels in tumor specimens correlate with SETDB1 levels, but negatively associate with survival time. Our data reveal a novel mechanism for the IRTKS-enhanced tumor progression, where IRTKS cooperates with OTUD4 to enhance SETDB1-mediated H3K9 trimethylation that promotes tumor metastasis via suppressing E-cadherin expression. This study also provides a potential approach to reduce the activity and stability of the known therapeutic target SETDB1 possibly through regulating IRTKS or deubiquitinase OTUD4.

H3K9 and H4K20 methyltransferases are directly involved in the heterochromatinization of the paternal chromosomes in male Planococcus citri embryos.

Using a new method for bulk preparation of early stage embryos, we have investigated the role played by putative Planococcus citri H3K9 and H4K20 histone methyl transferases (HMTases) in regulating heterochromatinization of the imprinted paternal chromosomal set in male embryos. We found that H3K9 and H420 HMTases are required for heterochromatinization of the paternal chromosomes. We present evidence that both HMTases maintain the paternal "imprint" during the cleavage divisions when both parental chromosome sets are euchromatic. A testable model that accommodates our findings is proposed.