The region 3' to Xist mediates X chromosome counting and H3 Lys-4 dimethylation within the Xist gene

A counting process senses the X chromosome/autosome ratio and ensures that X chromosome inactivation (XCI) initiates in the female (XX) but not in the male (XY) mouse embryo. Counting is regulated by the X-inactivation centre, which contains the Xist gene. Deleting 65 kb 3' to Xist in XO embryonic stem (ES) cells affects counting and results in inappropriate XCI upon differentiation. We show here that normal counting can be rescued in these deleted ES cells using cre/loxP re-insertion, and refine the location of elements controlling counting within a 20 kb bipartite domain. Furthermore, we show that the 65 kb deletion also leads to inappropriate XCI in XY differentiated ES cells, which excludes the involvement of sex-specific mechanisms in the initiation of XCI. At the chromatin level, we have found that the Xist gene corresponds to a peak of H3 Lys-4 dimethylation, which is dramatically and specifically affected by the deletion 3' to Xist. Our results raise the possibility that H3 Lys-4 dimethylation within Xist may be functionally implicated in the counting process.     

New lessons from random X-chromosome inactivation in the mouse

X-chromosome inactivation (XCI) ensures dosage compensation in mammals. Random XCI is a process where a single X chromosome is silenced in each cell of the epiblast of mouse female embryos. Operating at the level of an entire chromosome, XCI is a major paradigm for epigenetic processes. Here we review the most recent discoveries concerning the role of long noncoding RNAs, pluripotency factors, and chromosome structure in random XCI.     

The long and the short of it: RNA-directed chromatin asymmetry in mammalian X-chromosome inactivation

Mammalian X-chromosome inactivation is controlled by a multilayered silencing pathway involving both short and long non-coding RNAs, which differentially recruit the epigenetic machinery to establish chromatin asymmetries. In response to developmentally regulated small RNAs, dicer, a key effector of RNA interference, locally silences Xist on the active X-chromosome and establishes the heterochromatin conformation along the silent X-chromosome. The 1.6 kb RepA RNA initiates silencing by targeting the PRC2 polycomb complex to the inactive X-chromosome. In addition, the nuclear microenvironment is implicated in the initiation and maintenance of X-chromosome asymmetries. Here we review new findings involving these various RNA species in terms of understanding Xist gene regulation and the establishment of X-chromosome inactivation.     

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.     

Bimodal signatures of germline methylation are linked with gene expression plasticity in the coral Acropora millepora

Background

In invertebrates, genes belonging to dynamically regulated functional categories appear to be less methylated than “housekeeping” genes, suggesting that DNA methylation may modulate gene expression plasticity. To date, however, experimental evidence to support this hypothesis across different natural habitats has been lacking.

Results

Gene expression profiles were generated from 30 pairs of genetically identical fragments of coral Acropora millepora reciprocally transplanted between distinct natural habitats for 3 months. Gene expression was analyzed in the context of normalized CpG content, a well-established signature of historical germline DNA methylation. Genes with weak methylation signatures were more likely to demonstrate differential expression based on both transplant environment and population of origin than genes with strong methylation signatures. Moreover, the magnitude of expression differences due to environment and population were greater for genes with weak methylation signatures.

Conclusions

Our results support a connection between differential germline methylation and gene expression flexibility across environments and populations. Studies of phylogenetically basal invertebrates such as corals will further elucidate the fundamental functional aspects of gene body methylation in Metazoa.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1109) contains supplementary material, which is available to authorized users.     

Silencing of transgene expression in mammalian cells by DNA methylation and histone modifications in gene therapy perspective

DNA methylation and histone modifications are vital in maintaining genomic stability and modulating cellular functions in mammalian cells. These two epigenetic modifications are the most common gene regulatory systems known to spatially control gene expression. Transgene silencing by these two mechanisms is a major challenge to achieving effective gene therapy for many genetic conditions. The implications of transgene silencing caused by epigenetic modifications have been extensively studied and reported in numerous gene delivery studies. This review highlights instances of transgene silencing by DNA methylation and histone modification with specific focus on the role of these two epigenetic effects on the repression of transgene expression in mammalian cells from integrative and non-integrative based gene delivery systems in the context of gene therapy. It also discusses the prospects of achieving an effective and sustained transgene expression for future gene therapy applications.     

表观遗传修饰在调控寿命中的作用

表观遗传修饰是生命现象中普遍存在的一类基因调控方式,主要包括DNA甲基化、组蛋白乙酰化和组蛋白甲基化等,通常协同调控基因表达。端粒是位于真核生物染色体末端的保护性结构,在端粒以及亚端粒区域中也存在丰富的表观遗传修饰。随着研究深入,发现表观遗传修饰在调控寿命过程中扮演着重要角色,而揭示衰老的有关机制有助于我们找到延长寿命的方法,具有重大的生物学意义和临床应用前景。     

Epigenetics and mitochondrial dysfunction insights into the impact of the progression of non-alcoholic fatty liver disease

A metabolic problem occurs when regular functions of the body are disrupted due to an undesirable imbalance. Nonalcoholic fatty liver disease (NAFLD) is considered as one of the most common in this category. NAFLD is subclassified and progresses from lipid accumulation to cirrhosis before advancing to hepatocellular cancer. In spite of being a critical concern, the standard treatment is inadequate. Metformin, silymarin, and other nonspecific medications are used in the management of NAFLD. Aside from this available medicine, maintaining a healthy lifestyle has been emphasized as a means of combating this. Epigenetics, which has been attributed to NAFLD, is another essential feature of this disease that has emerged as a result of several sorts of research. The mechanisms by which DNA methylation, noncoding RNA, and histone modification promote NAFLD have been extensively researched. Another organelle, mitochondria, which play a pivotal role in biological processes, contributes to the global threat. Individuals with NAFLD have been documented to have a multitude of alterations and malfunctioning. Mitochondria are mainly concerned with the process of energy production and regulation of the signaling pathway on which the fate of a cell relies. Modulation of mitochondria leads to elevated lipid deposition in the liver. Further, changes in oxidation states result in an impaired balance between the antioxidant system and reactive oxygen species directly linked to mitochondria. Hence mitochondria have a definite role in potentiating NAFLD. In this regard, it is essential to consider the role of epigenetics as well as mitochondrial contribution while developing a medication or therapy with the desired accuracy.     

Synergistic chromatin repression of the tumor suppressor gene RARB in human prostate cancers

DNA methylation and polycomb proteins are well-known mediators of epigenetic silencing in mammalian cells. Usually described as mutually exclusive, this statement is today controversial and recent in vitro studies suggest the co-existence of both repressor systems. We addressed this issue in the study of Retinoic Acid Receptor β (RARβ), a tumor suppressor gene frequently silenced in prostate cancer. We found that the RARβ promoter is hypermethylated in all studied prostate tumors and methylation levels are positively correlated with H3K27me3 enrichments. Thus, by using bisulfite conversion and pyrosequencing of immunoprecipitated H3K27me3 chromatin, we demonstrated that DNA methylation and polycomb repression co-exist in vivo at this locus. We found this repressive association in 6/6 patient tumor samples of different Gleason score, suggesting a strong interplay of DNA methylation and EZH2 to silence RARβ during prostate tumorigenesis.     

Evidence that the methylation state of the monoamine oxidase A (MAOA) gene predicts brain activity of MAOA enzyme in healthy men

Human brain function is mediated by biochemical processes, many of which can be visualized and quantified by positron emission tomography (PET). PET brain imaging of monoamine oxidase A (MAOA)—an enzyme metabolizing neurotransmitters—revealed that MAOA levels vary widely between healthy men and this variability was not explained by the common MAOA genotype (VNTR genotype), suggesting that environmental factors, through epigenetic modifications, may mediate it. Here, we analyzed MAOA methylation in white blood cells (by bisulphite conversion of genomic DNA and subsequent sequencing of cloned DNA products) and measured brain MAOA levels (using PET and [¹¹C]clorgyline, a radiotracer with specificity for MAOA) in 34 healthy non-smoking male volunteers. We found significant interindividual differences in methylation status and methylation patterns of the core MAOA promoter. The VNTR genotype did not influence the methylation status of the gene or brain MAOA activity. In contrast, we found a robust association of the regional and CpG site-specific methylation of the core MAOA promoter with brain MAOA levels. These results suggest that the methylation status of the MAOA promoter (detected in white blood cells) can reliably predict the brain endophenotype. Therefore, the status of MAOA methylation observed in healthy males merits consideration as a variable contributing to interindividual differences in behavior.     

The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC)

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo⁵UAC) contains a unique modification, N⁶-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA₁^Val. Inactivation of yfiC gene abolishes m⁶A formation in tRNA₁^Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA₁^Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m⁶A in tRNA₁^Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.     

The last rRNA methyltransferase of E. coli revealed: The yhiR gene encodes adenine-N6 methyltransferase specific for modification of A2030 of 23S ribosomal RNA

The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR knockout gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.