Gene body methylation safeguards ribosomal DNA transcription by preventing PHF6-mediated enrichment of repressive histone mark H4K20me3.

DNA methylation shows complex correlations with gene expression, and the role of promoter hypermethylation in repressing gene transcription has been well addressed. Emerging evidence indicates that gene body methylation promotes transcription; however, the underlying mechanisms remain to be further investigated. Here, using methylated DNA immunoprecipitation sequencing (MeDIP-seq), bisulfite genomic sequencing, and immunofluorescent labeling, we show that gene body methylation is indeed positively correlated with rRNA gene (rDNA) transcription. Mechanistically, gene body methylation is largely maintained by DNA methyltransferase 1 (DNMT1), deficiency or downregulation of which during myoblast differentiation or nutrient deprivation results in decreased gene body methylation levels, leading to increased gene body occupancy of plant homeodomain (PHD) finger protein 6 (PHF6). PHF6 binds to hypomethylated rDNA gene bodies where it recruits histone methyltransferase SUV4-20H2 to establish the repressive histone modification, H4K20me3, ultimately inhibiting rDNA transcription. These findings demonstrate that DNMT1-mediated gene body methylation safeguards rDNA transcription by preventing enrichment of repressive histone modifications, suggesting that gene body methylation serves to maintain gene expression in response to developmental and/or environmental stresses.

Senescence-activated enhancer landscape orchestrates the senescence-associated secretory phenotype in murine fibroblasts.

The three-dimensional configuration of the chromatin architecture is known to be crucial for alterations in the transcriptional network; however, the underlying mechanisms of epigenetic control of senescence-related gene expression by modulating the chromatin architecture remain unknown. Here, we demonstrate frequent chromosomal compartment switching during mouse embryonic fibroblasts (MEFs) replicative senescence as characterized by senescence-inactivated (SIAEs) and -activated enhancers (SAEs) in topologically associated domains (TADs). Mechanistically, SAEs are closely correlated with senescence-associated secretory phenotype (SASP) genes, which are a key transcriptional feature of an aging microenvironment that contributes to tumor progression, aging acceleration, and immunoinflammatory responses. Moreover, SAEs can positively regulate robust changes in SASP expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is capable of enhancing SAE activity, which accelerates the emergence of SAEs flanking SASPs and the secretion of downstream factors, contributing to the progression of senescence. Our results provide novel insight into the TAD-related control of SASP gene expression by revealing hierarchical roles of the chromatin architecture, transcription factors, and enhancer activity in the regulation of cellular senescence.

3' UTR lengthening as a novel mechanism in regulating cellular senescence.

Cellular senescence has been viewed as a tumor suppression mechanism and also as a contributor to individual aging. Widespread shortening of 3' untranslated regions (3' UTRs) in messenger RNAs (mRNAs) by alternative polyadenylation (APA) has recently been discovered in cancer cells. However, the role of APA in the process of cellular senescence remains elusive. Here, we found that hundreds of genes in senescent cells tended to use distal poly(A) (pA) sites, leading to a global lengthening of 3' UTRs and reduced gene expression. Genes that harbor longer 3' UTRs in senescent cells were enriched in senescence-related pathways. Rras2, a member of the Ras superfamily that participates in multiple signal transduction pathways, preferred longer 3' UTR usage and exhibited decreased expression in senescent cells. Depletion of Rras2 promoted senescence, while rescue of Rras2 reversed senescence-associated phenotypes. Mechanistically, splicing factor TRA2B bound to a core "AGAA" motif located in the alternative 3' UTR of Rras2, thereby reducing the RRAS2 protein level and causing senescence. Both proximal and distal poly(A) signals showed strong sequence conservation, highlighting the vital role of APA regulation during evolution. Our results revealed APA as a novel mechanism in regulating cellular senescence.

Changes in the position and volume of inactive X chromosomes during the G0/G1 transition.

In female mammals, each cell silences one X chromosome by converting it into transcriptionally inert heterochromatin. The inactivation is concomitant with epigenetic changes including methylation of specific histone residues and incorporation of macroH2A. Such epigenetic changes may exert influence on the positioning of the inactive X chromosome (Xi) within the nucleus beyond the level of chromatin structure. However, the dynamic positioning of the inactive X chromosome during cell cycle remains unclear. Here, we show that H3K27me3 is a cell-cycle-independent marker for the inactivated X chromosomes in WI38 cells. By utilizing this marker, three types of Xi locations in the nuclei are classified, which are envelope position (associated with envelope), mid-position (between the envelope and nucleolus), and nucleolus position (associated with the nucleolus). Moreover, serial-section analysis revealed that the inactive X chromosomes in the mid-position appear to be sparser and less condensed than those associated with the nuclear envelope or nucleolus. During the transition from G0 to G1 phase, the inactive X chromosomes tend to move from the envelope position to the nucleolus position in WI38 cells. Our results imply a role of chromosome positioning in maintaining the organization of the inactive X chromosomes in different cell phases.

Genome and epigenome analysis of monozygotic twins discordant for congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) is the leading non-infectious cause of death in infants. Monozygotic (MZ) twins share nearly all of their genetic variants before and after birth. Nevertheless, MZ twins are sometimes discordant for common complex diseases. The goal of this study is to identify genomic and epigenomic differences between a pair of twins discordant for a form of congenital heart disease, double outlet right ventricle (DORV). RESULTS: A monoamniotic monozygotic (MZ) twin pair discordant for DORV were subjected to genome-wide sequencing and methylation analysis. We identified few genomic differences but 1566 differentially methylated regions (DMRs) between the MZ twins. Twenty percent (312/1566) of the DMRs are located within 2 kb upstream of transcription start sites (TSS), containing 121 binding sites of transcription factors. Particularly, ZIC3 and NR2F2 are found to have hypermethylated promoters in both the diseased twin and additional patients suffering from DORV. CONCLUSIONS: The results showed a high correlation between hypermethylated promoters at ZIC3 and NR2F2 and down-regulated gene expression levels of these two genes in patients with DORV compared to normal controls, providing new insight into the potential mechanism of this rare form of CHD.

NuRD blocks reprogramming of mouse somatic cells into pluripotent stem cells.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of a defined set of transcription factors requires epigenetic changes in pluripotency genes. Nuclear reprogramming is an inefficient process and the molecular mechanisms that reset the epigenetic state during iPSC generation are largely unknown. Here, we show that downregulation of the nucleosome remodeling and deacetylation (NuRD) complex is required for efficient reprogramming. Overexpression of Mbd3, a subunit of NuRD, inhibits induction of iPSCs by establishing heterochromatic features and silencing embryonic stem cell-specific marker genes, including Oct4 and Nanog. Depletion of Mbd3, on the other hand, improves reprogramming efficiency and facilitates the formation of pluripotent stem cells that are capable of generating viable chimeric mice, even in the absence of c-Myc or Sox2. The results establish Mbd3/NuRD as an important epigenetic regulator that restricts the expression of key pluripotency genes, suggesting that drug-induced downregulation of Mbd3/NuRD may be a powerful means to improve the efficiency and fidelity of reprogramming.

Long Non-coding RNA ZFPM2-AS1: A Novel Biomarker in the Pathogenesis of Human Cancers.

Due to their biological activities in regulating dosage compensation, epigenetics, and cell differentiation, long non-coding RNAs (lncRNA) have been recognized as important regulators of the beginning and development of human malignancies. LncRNA dysregulation has a significant impact on a range of cellular functions, including proliferation, migration, invasion, and anti-apoptosis activity. Recently, aberrant expression of the long non-coding RNA zinc finger protein multitype 2 antisense RNA 1 (ZFPM2-AS1) was observed in a range of solid tumors and correlated significantly with tumor size, histological differentiation, lymph node metastasis, malignant tumor (TNM) stage, short survival, and prognosis. Additional mechanical analysis indicated that ZFPM2-AS1 was involved in several cellular activities, including proliferation, migration, invasion, cell cycle progression, and apoptosis, through microRNAs (miRNAs), signaling pathways, and other biological components or proteins. This review summarizes the current status of research on ZFPM2-AS1 in various human malignancies and discusses its mechanism of action and clinical significance in tumor development and progression.

Metastasis-related methyltransferase 1 (Merm1) represses the methyltransferase activity of Dnmt3a and facilitates RNA polymerase I transcriptional elongation.

Stimulatory regulators for DNA methyltransferase activity, such as Dnmt3L and some Dnmt3b isoforms, affect DNA methylation patterns, thereby maintaining gene body methylation and maternal methylation imprinting, as well as the methylation landscape of pluripotent cells. Here we show that metastasis-related methyltransferase 1 (Merm1), a protein deleted in individuals with Williams-Beuren syndrome, acts as a repressive regulator of Dnmt3a. Merm1 interacts with Dnmt3a and represses its methyltransferase activity with the requirement of the binding motif for S-adenosyl-L-methionine. Functional analysis of gene regulation revealed that Merm1 is capable of maintaining hypomethylated rRNA gene bodies and co-localizes with RNA polymerase I in the nucleolus. Dnmt3a recruits Merm1, and in return, Merm1 ensures the binding of Dnmt3a to hypomethylated gene bodies. Such interplay between Dnmt3a and Merm1 facilitates transcriptional elongation by RNA polymerase I. Our findings reveal a repressive factor for Dnmt3a and uncover a molecular mechanism underlying transcriptional elongation of rRNA genes.

TGF-ß signaling alters H4K20me3 status via miR-29 and contributes to cellular senescence and cardiac aging.

Cellular senescence is a well-orchestrated programmed process involved in age-related pathologies, tumor suppression and embryonic development. TGF-ß/Smad is one of the predominant pathways that regulate damage-induced and developmentally programmed senescence. Here we show that canonical TGF-ß signaling promotes senescence via miR-29-induced loss of H4K20me3. Mechanistically, oxidative stress triggers TGF-ß signaling. Activated TGF-ß signaling gives rise to acute accumulation of miR-29a and miR-29c, both of which directly suppress their novel target, Suv4-20h, thus reducing H4K20me3 abundance in a Smad-dependent manner, which compromises DNA damage repair and genome maintenance. Loss of H4K20me3 mediated by the senescent TGF-ß/miR-29 pathway contributes to cardiac aging in vivo. Disruption of TGF-ß signaling restores H4K20me3 and improves cardiac function in aged mice. Our study highlights the sequential mechanisms underlying the regulation of senescence, from senescence-inducing triggers to activation of responsive signaling followed by specific epigenetic alterations, shedding light on potential therapeutic interventions in cardiac aging.

The chromatin remodeling factor CSB recruits histone acetyltransferase PCAF to rRNA gene promoters in active state for transcription initiation.

The promoters of poised rRNA genes (rDNA) are marked by both euchromatic and heterochromatic histone modifications and are associated with two transcription factors, UBF and SL1 that nucleate transcription complex formation. Active rRNA genes contain only euchromatic histone modifications and are loaded with all components of transcriptional initiation complex including RNA polymerase I. Coupled with histone acetylation and RNA polymerase I targeting, poised promoters can be converted to active ones by ATP-dependent chromatin remodeling factor CSB for initiation of rDNA transcription. However, it is not clear how dynamic histone modifications induce the assembly of polymerase I transcription initiation complex to active promoters during such conversion. Here we show that a complex consisting of CSB, RNA polymerase I and histone acetyltransferase PCAF is present at the rDNA promoters in active state. CSB is required for the association of PCAF with rDNA, which induces acetylation of histone H4 and histone H3K9. Overexpression of CSB promotes the association of PCAF with rDNA. Knockdown of PCAF leads to decreased levels of H4ac and H3K9ac at rDNA promoters, prevents the association of RNA polymerase I and inhibits pre-rRNA synthesis. The results demonstrate that CSB recruits PCAF to rDNA, which allows histone acetylation that is required for the assembly of polymerase I transcription initiation complex during the transition from poised to active state of rRNA genes, suggesting that CSB and PCAF play cooperative roles to establish the active state of rRNA genes by histone acetylation.