Securin overexpression correlates with the activated Rb/E2F1 pathway and histone H3 epigenetic modifications in raw areca nut-induced carcinogenesis in mice

Background Raw areca nut (RAN) consumption induces oral, esophageal and gastric cancers, which are significantly associated with the overexpression of pituitary tumor transforming gene 1/securin and chromosomal instability (CIN). An association of Securin/PTTG1 upregulation and gastric cancer in human was also demonstrated earlier. Since the molecular mechanism underlying securin upregulation remains unclear, this study intended to investigate the association of securin upregulation with the Rb-E2F1 circuit and epigenetic histone (H3) modification patterns both globally and in the promoter region of the securin gene. Methods Six groups of mice were used, and in the treated group, each mouse consumed 1 mg of RAN extract with lime per day ad libitum in the drinking water for 60 days, after which the dose was increased by 1 mg every 60 days. Histopathological evaluation of stomach tissues was performed and securin expression was analysed by immunoblotting as well as by immunohistochemistry. ChIP-qPCR assays were performed to evaluate the recruitment of different histone modifications in the core promoter region of securin gene as well as its upstream and downstream regions. Results All mice developed gastric cancer with securin overexpression after 300 days of feeding. Immunohistochemistry data revealed hyperphosphorylation of Rb and upregulation of E2F1 in the RAN-treated samples. Increased trimethylation of H3 lysine 4 and acetylation of H3 lysine 9 and 18 both globally and in the promoter region of the securin gene were observed by increasing the levels of lysine-N-methyltransferase 2A, lysine-acetyltransferase, EP-300 and PCAF after RAN treatment. ChIP-qPCR data revealed that the quantity of DNA fragments retrieved from the immunoprecipitated samples was maximum in the -83 to -192 region than further upstream and the downstream of the promoter for H3K4Me3, H3K9ac, H3K18ac and H3K9me3. Conclusions RAN-mediated pRb-inactivation induced securin upregulation, a putative E2F1 target, by inducing misregulation in chromatin remodeling in its promoter region, which led to transcriptional activation and subsequent development of chromosomal instability. Therefore, present results have led to the hypothesis that RAN-induced changes in the epigenetic landscape, securin overexpression and subsequent elevation of chromosomal instability is probably byproducts of inactivation of the pRb pathway.

H3K9 Methyltransferases Suv39h1 and Suv39h2 Control the Differentiation of Neural Progenitor Cells in the Adult Hippocampus

In the dentate gyrus of the adult hippocampus new neurons are generated from neural precursor cells through different stages including proliferation and differentiation of neural progenitor cells and maturation of newborn neurons. These stages are controlled by the expression of specific transcription factors and epigenetic mechanisms, which together orchestrate the progression of the neurogenic process. However, little is known about the involvement of histone posttranslational modifications, a crucial epigenetic mechanism in embryonic neurogenesis that regulates fate commitment and neuronal differentiation. During embryonic development, the repressive modification trimethylation of histone H3 on lysine 9 (H3K9me3) contributes to the cellular identity of different cell-types. However, the role of this modification and its H3K9 methyltransferases has not been elucidated in adult hippocampal neurogenesis. We determined that during the stages of neurogenesis in the adult mouse dentate gyrus and in cultured adult hippocampal progenitors (AHPs), there was a dynamic change in the expression and distribution of H3K9me3, being enriched at early stages of the neurogenic process. A similar pattern was observed in the hippocampus for the dimethylation of histone H3 on lysine 9 (H3K9me2), another repressive modification. Among H3K9 methyltransferases, the enzymes Suv39h1 and Suv39h2 exhibited high levels of expression at early stages of neurogenesis and their expression decreased upon differentiation. Pharmacological inhibition of these enzymes by chaetocin in AHPs reduced H3K9me3 and concomitantly decreased neuronal differentiation while increasing proliferation. Moreover, Suv39h1 and Suv39h2 knockdown in newborn cells of the adult mouse dentate gyrus by retrovirus-mediated RNA interference impaired neuronal differentiation of progenitor cells. Our results indicate that H3K9me3 and H3K9 methyltransferases Suv39h1 and Suv39h2 are critically involved in the regulation of adult hippocampal neurogenesis by controlling the differentiation of neural progenitor cells.

Dynamic Activity of Histone H3-Specific Chaperone Complexes in Oncogenesis

Canonical histone H3.1 and variant H3.3 deposit at different sites of the chromatin via distinct histone chaperones. Histone H3.1 relies on chaperone CAF-1 to mediate replication-dependent nucleosome assembly during S-phase, while H3.3 variant is regulated and incorporated into the chromatin in a replication-independent manner through HIRA and DAXX/ATRX. Current literature suggests that dysregulated expression of histone chaperones may be implicated in tumor progression. Notably, ectopic expression of CAF-1 can promote a switch between canonical H3.1 and H3 variants in the chromatin, impair the chromatic state, lead to chromosome instability, and impact gene transcription, potentially contributing to carcinogenesis. This review focuses on the chaperone proteins of H3.1 and H3.3, including structure, regulation, as well as their oncogenic and tumor suppressive functions in tumorigenesis.

Chromatin alterations during the epididymal maturation of mouse sperm refine the paternally inherited epigenome

Background Paternal lifestyle choices and male exposure history have a critical influence on the health and fitness of the next generation. Accordingly, defining the processes of germline programming is essential to resolving how the epigenetic memory of paternal experiences transmits to their offspring. Established dogma holds that all facets of chromatin organization and histone posttranslational modification are complete before sperm exits the testes. However, recent clinical and animal studies suggest that patterns of DNA methylation change during epididymal maturation. In this study, we used complementary proteomic and deep-sequencing approaches to test the hypothesis that sperm posttranslational histone modifications change during epididymal transit. Results Using proteomic analysis to contrast immature spermatozoa and mature sperm isolated from the mouse epididymis, we find progressive changes in multiple histone posttranslational modifications, including H3K4me1, H3K27ac, H3K79me2, H3K64ac, H3K122ac, H4K16ac, H3K9me2, and H4K20me3. Interestingly, some of these changes only occurred on histone variant H3.3, and most involve chromatin modifications associated with gene enhancer activity. In contrast, the bivalent chromatin modifications, H3K4me3, and H3K27me3 remained constant. Using chromatin immunoprecipitation coupled with deep sequencing, we find that changes in histone h3, lysine 27 acetylation (H3K27ac) involve sharpening broad diffuse regions into narrow peaks centered on the promoter regions of genes driving embryonic development. Significantly, many of these regions overlap with broad domains of H3K4me3 in oocytes and ATAC-seq signatures of open chromatin identified in MII oocytes and sperm. In contrast, histone h3, lysine 9 dimethylation (H3K9me2) becomes enriched within the promoters of genes driving meiosis and in the distal enhancer regions of tissue-specific genes sequestered at the nuclear lamina. Maturing sperm contain the histone deacetylase enzymes HDAC1 and HDAC3, suggesting the NuRD complex may drive some of these changes. Finally, using Western blotting, we detected changes in chromatin modifications between caput and caudal sperm isolated from rams (Ovis aries), inferring changes in histone modifications are a shared feature of mammalian epididymal maturation. Conclusions These data extend our understanding of germline programming and reveal that, in addition to trafficking noncoding RNAs, changes in histone posttranslational modifications are a core feature of epididymal maturation.

Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization

Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. The organization of the interface between the kinetochore and the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, remains incompletely understood. A 16-subunit complex, the constitutive centromere associated network (CCAN), bridges CENP-A to the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has also important implications for the mechanism of specific recognition of the CENP-A nucleosome. A supported model depicts the interaction as fuzzy and identifies the disordered CCAN subunit CENP-C as only determinant of specificity. A more speculative model identifies both CENP-C and CENP-N as specificity determinants, but implies CENP-A may be in a hemisome rather than in a classical octamer.

Discovery of small molecule antagonists of human Retinoblastoma Binding Protein 4 (RBBP4)

RBBP4 is a nuclear WD40 motif-containing protein widely implicated in various cancers and a putative drug target. It interacts with multiple proteins within diverse complexes such as nucleosome remodeling and deacetylase (NuRD) complex and polycomb repressive complex 2 (PRC2), as well as histone H3 and H4 through two distinct binding sites. B-cell lymphoma/leukemia 11A (BCL11A), friend of GATA-1 (FOG-1), plant homeodomain finger protein 6 (PHF6) and histone H3 bind to the top of the donut-shaped seven-bladed β-propeller fold of RBBP4, while suppressor of zeste 12 (SUZ12), metastasis associated protein 1 (MTA1) and histone H4 bind to a pocket on the side of the WD40 repeats of this protein. Here, we report the discovery of the first small molecule antagonists of the RBBP4 top pocket, competing with interacting peptides from proteins such as BCL11A and histone H3. We also determined the first crystal structure of RBBP4 in complex with a small molecule (OICR17251), paving the path for structure-guided design and optimization towards more potent antagonists.

Sodium valproate is effective against Botrytis cinerea infection of tomato by enhancing histone H3 acetylation-directed gene transcription and triggering tomato fruits immune response

Botrytis cinerea causes grey mold resulting in enormous financial loss. Fungicide resistance of B. cinerea has become a serious issue in food safety and agricultural environmental protection. Sodium valproate (SV) has been used in clinical trials, thus it is excellent candidate for fungicide development considering its safety. However, the antifungal activity remains unclear. SV was effective against B. cinerea by enhancing acetylation of histone H3, including H3K9ac, H3K14ac, and H3K56ac. A transcriptomics analysis revealed that the expression of 1,557 genes changed significantly in response to SV. A pathway enrichment analysis identified 16 significant GO terms, in which molecular functions were mainly involved. In addition, the expression levels of 13 genes involved in B. cinerea virulence and 5 genes involved in tomato immune response were altered by the SV treatment. These results indicate that SV inhibits B. cinerea by enhancing acetylation of histone H3 and modifying gene transcription. Thus, SV is an effective, safe potential antifungal agent for control of both pre- and post-harvest losses caused by B. cinerea.

Modulation of IL-6 Expression by KLF4-Mediated Transactivation and PCAF-Mediated Acetylation in Sublytic C5b-9-Induced Rat Glomerular Mesangial Cells

Interleukin-6 (IL-6) overproduction has been considered to contribute to inflammatory damage of glomerular mesangial cells (GMCs) in human mesangial proliferative glomerulonephritis (MsPGN) and its rat model called Thy-1 nephritis (Thy-1N). However, the regulatory mechanisms of IL-6 expression in GMCs upon sublytic C5b-9 timulation remain poorly understood. We found that Krüppel-like factor 4 (KLF4) bound to the IL-6 promoter (−618 to −126 nt) and activated IL-6 gene transcription. Furthermore, lysine residue 224 of KLF4 was acetylated by p300/CBP-associated factor (PCAF), which was important for KLF4-mediated transactivation. Moreover, lysine residue 5 on histone H2B and lysine residue 9 on histone H3 at the IL-6 promoter were also acetylated by PCAF, which resulted in an increase in IL-6 transcription. Besides, NF-κB activation promoted IL-6 expression by elevating the expression of PCAF. Overall, these findings suggest that sublytic C5b-9-induced the expression of IL-6 involves KLF4-mediated transactivation, PCAF-mediated acetylation of KLF4 and histones, and NF-κB activation in GMCs.

Histone H3 deacetylation promotes host cell viability for efficient infection by Listeria monocytogenes

For many intracellular bacterial pathogens manipulating host cell survival is essential for maintaining their replicative niche, and is a common strategy used to promote infection. The bacterial pathogen Listeria monocytogenes is well known to hijack host machinery for its own benefit, such as targeting the host histone H3 for modification by SIRT2. However, by what means this modification benefits infection, as well as the molecular players involved, were unknown. Here we show that SIRT2 activity supports Listeria intracellular survival by maintaining genome integrity and host cell viability. This protective effect is dependent on H3K18 deacetylation, which safeguards the host genome by counteracting infection-induced DNA damage. Mechanistically, infection causes SIRT2 to interact with the nucleic acid binding protein TDP-43 and localise to genomic R-loops, where H3K18 deacetylation occurs. This work highlights novel functions of TDP-43 and R-loops during bacterial infection and identifies the mechanism through which L. monocytogenes co-opts SIRT2 to allow efficient infection.