Regulation of histone H3K4 methylation in brain development and disease

The growing list of mutations implicated in monogenic disorders of the developing brain includes at least seven genes (ARX, CUL4B, KDM5A, KDM5C, KMT2A, KMT2C, KMT2D) with loss-of-function mutations affecting proper regulation of histone H3 lysine 4 methylation, a chromatin mark which on a genome-wide scale is broadly associated with active gene expression, with its mono-, di- and trimethylated forms differentially enriched at promoter and enhancer and other regulatory sequences. In addition to these rare genetic syndromes, dysregulated H3K4 methylation could also play a role in the pathophysiology of some cases diagnosed with autism or schizophrenia, two conditions which on a genome-wide scale are associated with H3K4 methylation changes at hundreds of loci in a subject-specific manner. Importantly, the reported alterations for some of the diseased brain specimens included a widespread broadening of H3K4 methylation profiles at gene promoters, a process that could be regulated by the UpSET(KMT2E/MLL5)-histone deacetylase complex. Furthermore, preclinical studies identified maternal immune activation, parental care and monoaminergic drugs as environmental determinants for brain-specific H3K4 methylation. These novel insights into the epigenetic risk architectures of neurodevelopmental disease will be highly relevant for efforts aimed at improved prevention and treatment of autism and psychosis spectrum disorders.     

KMT2D deficiency disturbs the proliferation and cell cycle activity of dental epithelial cell line (LS8) partially via Wnt signaling

Lysine methyltransferase 2D (KMT2D), as one of the key histone methyltransferases responsible for histone 3 lysine 4 methylation (H3K4me), has been proved to be the main pathogenic gene of Kabuki syndrome disease. Kabuki patients with KMT2D mutation frequently present various dental abnormalities, including abnormal tooth number and crown morphology. However, the exact function of KMT2D in tooth development remains unclear. In this report, we systematically elucidate the expression pattern of KMT2D in early tooth development and outline the molecular mechanism of KMT2D in dental epithelial cell line. KMT2D and H3K4me mainly expressed in enamel organ and Kmt2d knockdown led to the reduction in cell proliferation activity and cell cycling activity in dental epithelial cell line (LS8). RNA-sequencing (RNA-seq) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis screened out several important pathways affected by Kmt2d knockdown including Wnt signaling. Consistently, Top/Fop assay confirmed the reduction in Wnt signaling activity in Kmt2d knockdown cells. Nuclear translocation of β-catenin was significantly reduced by Kmt2d knockdown, while lithium chloride (LiCl) partially reversed this phenomenon. Moreover, LiCl partially reversed the decrease in cell proliferation activity and G₁ arrest, and the down-regulation of Wnt-related genes in Kmt2d knockdown cells. In summary, the present study uncovered a pivotal role of histone methyltransferase KMT2D in dental epithelium proliferation and cell cycle homeostasis partially through regulating Wnt/β-catenin signaling. The findings are important for understanding the role of KMT2D and histone methylation in tooth development.     

Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

Background

A number of neurodevelopmental syndromes are caused by mutations in genes encoding proteins that normally function in epigenetic regulation. Identification of epigenetic alterations occurring in these disorders could shed light on molecular pathways relevant to neurodevelopment.

Results

Using a genome-wide approach, we identified genes with significant loss of DNA methylation in blood of males with intellectual disability and mutations in the X-linked KDM5C gene, encoding a histone H3 lysine 4 demethylase, in comparison to age/sex matched controls. Loss of DNA methylation in such individuals is consistent with known interactions between DNA methylation and H3 lysine 4 methylation. Further, loss of DNA methylation at the promoters of the three top candidate genes FBXL5, SCMH1, CACYBP was not observed in more than 900 population controls. We also found that DNA methylation at these three genes in blood correlated with dosage of KDM5C and its Y-linked homologue KDM5D. In addition, parallel sex-specific DNA methylation profiles in brain samples from control males and females were observed at FBXL5 and CACYBP.

Conclusions

We have, for the first time, identified epigenetic alterations in patient samples carrying a mutation in a gene involved in the regulation of histone modifications. These data support the concept that DNA methylation and H3 lysine 4 methylation are functionally interdependent. The data provide new insights into the molecular pathogenesis of intellectual disability. Further, our data suggest that some DNA methylation marks identified in blood can serve as biomarkers of epigenetic status in the brain.     

Two new chromodomain-containing proteins that associate with heterochromatin in <Emphasis Type="Italic">Sciara coprophila</Emphasis> chromosomes

We report here the molecular and cytological characterization of two proteins, ScoHET1 and ScoHET2 (for Sciara coprophila heterochromatin), which associate to constitutive heterochromatin in the dipteran S. coprophila. Both proteins, ScoHET1 of 37 kDa and ScoHET2 of 44 kDa, display two chromodomain motifs that contain the conserved residues essential for the recognition of methylated histone H3 at lysine 9. We raised antibodies to analyze the chromosomal location of ScoHET1 and ScoHET2 in somatic and germline cells. In S. coprophila polytene chromosomes, both proteins associate to the pericentromeric regions and to the heterochromatic subterminal bands of the chromosomes. In germinal nuclei, ScoHET1 and ScoHET2 proteins distribute to the heterochromatic regions of the regular chromosome complement and are abundantly present along the heterochromatic germline-limited “L” chromosomes. We investigated histone methylation modifications and found that all heterochromatic regions enriched in ScoHET1/ScoHET2 proteins exhibit high levels of di- and tri-methylated histone H3 at lysine 9. Taken together, our results support that the association of ScoHET1/ScoHET2 to heterochromatin is mediated by histone H3K9 methylation. Using 5-methylcytosine antibodies, we proved the cytological detection of DNA methylation in S. coprophila. From our observations in L germline chromosomes, heterochromatin in S. coprophila is highly enriched in DNA 5-methylcytosine residues. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High-resolution mapping of DNA methylation in human genome using oligonucleotide tiling array

DNA methylation is an epigenetic mark crucial in regulation of gene expression. Aberrant DNA methylation causes silencing of tumor suppressor genes and promotes chromosomal instability in human cancers. Most of previous studies for DNA methylation have focused on limited genomic regions, such as selected genes or promoter CpG islands (CGIs) containing recognition sites of methylation-sensitive restriction enzymes. Here, we describe a method for high-resolution analysis of DNA methylation using oligonucleotide tiling arrays. The input material is methylated DNA immunoprecipitated with anti-methylcytosine antibodies. We examined the ENCODE region (∼1% of human genome) in three human colorectal cancer cell lines and identified over 700 candidate methylated sites (CMS), where 24 of 25 CMS selected randomly were subsequently verified by bisulfite sequencing. CMS were enriched in the 5′ regulatory regions and the 3′ regions of genes. We also compared DNA methylation patterns with histone H3 and H4 acetylation patterns in the HOXA cluster region. Our analysis revealed no acetylated histones in the hypermethylated region, demonstrating reciprocal relationship between DNA methylation and histone H3 and H4 acetylation. Our method recognizes DNA methylation with little bias by genomic location and, therefore, is useful for comprehensive high-resolution analysis of DNA methylation providing new findings in the epigenomics. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Daily Variations of Homocysteine Concentration May Influence Methylation of DNA in Normal Healthy Individuals

The regulation of genetic expression is tightly controlled and well balanced in the organism by different epigenetic mechanisms such as DNA methylation and histone modifications. DNA methylation occurring after embryogenesis is seen mainly as an irreversible event. Even small changes in genomic DNA methylation might be of biological relevance, and several factors influencing DNA methylation have been identified so far, one being homocysteine. In this study, genomic DNA methylation was analyzed and homocysteine plasma levels were measured over a 24 h period in 30 healthy students (15 males and 15 females) exposed to a standard 24 h regime of daytime activity alternating with nighttime sleep. Plasma homocysteine concentrations were measured using HPLC detection. DNA was extracted from whole EDTA blood, and genomic DNA methylation was assessed by fluorescently labeled cytosine extension assay. Both homocysteine and DNA methylation showed 24 h variation. Homocysteine showed a significant daily rhythm with an evening peak and nocturnal nadir in all subjects (p<0.001). Males showed higher overall homocysteine levels compared to females (p=0.002). Genomic DNA methylation showed a significant rhythm with increased levels at night (p=0.021), which was inverse to plasma homocysteine levels.     

Establishment of paternal allele-specific DNA methylation at the imprinted mouse Gtl2 locus

The monoallelic expression of imprinted genes is controlled by epigenetic factors including DNA methylation and histone modifications. In mouse, the imprinted gene Gtl2 is associated with two differentially methylated regions: the IG-DMR, which serves as a gametic imprinting mark at which paternal allele-specific DNA methylation is inherited from sperm, and the Gtl2-DMR, which acquires DNA methylation on the paternal allele after fertilization. The timeframe during which DNA methylation is acquired at secondary DMRs during post-fertilization development and the relationship between secondary DMRs and imprinted expression have not been well established. In order to better understand the role of secondary DMRs in imprinting, we examined the methylation status of the Gtl2-DMR in pre- and post-implantation embryos. Paternal allele-specific DNA methylation of this region correlates with imprinted expression of Gtl2 during post-implantation development but is not required to implement imprinted expression during pre-implantation development, suggesting that this secondary DMR may play a role in maintaining imprinted expression. Furthermore, our developmental profile of DNA methylation patterns at the Cdkn1c- and Gtl2-DMRs illustrates that the temporal acquisition of DNA methylation at imprinted genes during post-fertilization development is not universally controlled.     

Histone deacetylation directs DNA methylation in survivin gene silencing

DNA methylation and histone acetylation are major epigenetic modifications in gene silencing. In our previous research, we found that the methylated oligonucleotide (SurKex) complementary to a region of promoter of survivin could induce DNA methylation in a site-specific manner leading to survivin silencing. Here, we further studied the role of histone acetylation in survivin silencing and the relationship between histone acetylation and DNA methylation.First we observed the levels of histone H4 and H4K16 acetylation that were decreased after SurKex treatment by using the chromatin immunoprecipitation (ChIP) assay. Next, we investigated the roles of histone acetylation and DNA methylation in survivin silencing after blockade of histone deacetylation with Trichostatin A (TSA). We assessed survivin mRNA expression by RT-PCR, measured survivin promoter methylation by bisulfite sequencing and examined the level of histone acetylation by the ChIP assay. The results showed that histone deacetylation blocked by TSA reversed the effects of SurKex on inhibiting the expression of survivin mRNA, inducing a site-specific methylation on survivin promoter and decreasing the level of histone acetylation. Finally, we examined the role of histone acetylation in the expression of DNA methyltransferase 1 (DNMT1) mRNA. The results showed that histone deacetylation blocked by TSA reversed the increasing effect of histone deacetylation on the expression of survivin mRNA. This study suggests that histone deacetylation guides SurKex-induced DNA methylation in survivin silencing possibly through increasing the expression of DNMT1 mRNA.     

A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme

The lysine methyltransferase (KMT) SETMAR is implicated in the response to and repair of DNA damage, but its molecular function is not clear. SETMAR has been associated with dimethylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates, we utilized a strategy on the basis of quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 of the mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. Furthermore, we show that SETMAR methylates snRNP70 Lys-130 in cells. Because snRNP70 is a key early regulator of 5′ splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates.     

Epigenetic profiling of heterochromatic satellite DNA

Sugar beet (Beta vulgaris) chromosomes consist of large heterochromatic blocks in pericentromeric, centromeric, and intercalary regions comprised of two different highly abundant DNA satellite families. To investigate DNA methylation at single base resolution at heterochromatic regions, we applied a method for strand-specific bisulfite sequencing of more than 1,000 satellite monomers followed by statistical analyses. As a result, we uncovered diversity in the distribution of different methylation patterns in both satellite families. Heavily methylated CG and CHG (H=A, T, or C) sites occur more frequently in intercalary heterochromatin, while CHH sites, with the exception of CAA, are only sparsely methylated, in both intercalary and pericentromeric/centromeric heterochromatin. We show that the difference in DNA methylation intensity is correlated to unequal distribution of heterochromatic histone H3 methylation marks. While clusters of H3K9me2 were absent from pericentromeric heterochromatin and restricted only to intercalary heterochromatic regions, H3K9me1 and H3K27me1 were observed in all types of heterochromatin. By sequencing of a small RNA library consisting of 6.76 million small RNAs, we identified small interfering RNAs (siRNAs) of 24 nucleotides in size which originated from both strands of the satellite DNAs. We hypothesize an involvement of these siRNAs in the regulation of DNA and histone methylation for maintaining heterochromatin.     

Cofactors‐loaded quaternary structure of lysine‐specific demethylase 5C (KDM5C) protein: Computational model

The KDM5C gene (also known as JARID1C and SMCX) is located on the X chromosome and encodes a ubiquitously expressed 1560‐aa protein, which plays an important role in lysine methylation (specifically reverses tri‐ and di‐methylation of Lys4 of histone H3). Currently, 13 missense mutations in KDM5C have been linked to X‐linked mental retardation. However, the molecular mechanism of disease is currently unknown due to the experimental difficulties in expressing such large protein and the lack of experimental 3D structure. In this work, we utilize homology modeling, docking, and experimental data to predict 3D structures of KDM5C domains and their mutual arrangement. The resulting quaternary structure includes KDM5C JmjN, ARID, PHD1, JmjC, ZF domains, substrate histone peptide, enzymatic cofactors, and DNA. The predicted quaternary structure was investigated with molecular dynamic simulation for its stability, and further analysis was carried out to identify features measured experimentally. The predicted structure of KDM5C was used to investigate the effects of disease‐causing mutations and it was shown that the mutations alter domain stability and inter‐domain interactions. The structural model reported in this work could prompt experimental investigations of KDM5C domain‐domain interaction and exploration of undiscovered functionalities. Proteins 2016; 84:1797–1809. © 2016 Wiley Periodicals, Inc.