Loss of Tet1 associated 5-hydroxymethylcytosine is concomitant with aberrant promoter hypermethylation in liver cancer

Aberrant hypermethylation of CpG islands (CGI) in human tumors occurs predominantly at repressed genes in the host tissue, but the preceding events driving this phenomenon are poorly understood. In this study, we temporally tracked epigenetic and transcriptomic perturbations which occur in a mouse model of liver carcinogenesis. Hypermethylated CGI events in the model were predicted by enrichment of the DNA modification 5-hydroxymethylcytosine (5hmC) and the histone H3 modification H3K27me3 at silenced promoters in the host tissue. During cancer progression, CGI underwent hypo-hydroxymethylation prior to hypermethylation, whilst retaining H3K27me3. In livers from mice deficient in Tet1, a tumor suppressor involved in cytosine demethylation, we observed a similar loss of promoter core 5hmC, suggesting that reduced Tet1 activity at CGI may contribute to epigenetic dysregulation observed during hepatocarcinogenesis. Consistent with this possibility, mouse liver tumors exhibited reduced Tet1 protein levels. Similar to humans, DNA methylation changes at CGI in mice did not appear to be direct drivers of hepatocellular carcinoma progression, rather, dynamic changes in H3K27me3 promoter deposition correlated strongly with tumor-specific activation and repression of transcription. Overall, our results suggest that loss of promoter-associated 5hmC in liver tumors licenses reprogramming of DNA methylation at silent CGI during progression

Neuronal MeCP2 is expressed at near histone-octamer levels and globally alters the chromatin state

MeCP2 is a nuclear protein with an affinity for methylated DNA that can recruit histone deacetylases. Deficiency or excess of MeCP2 causes severe neurological problems, suggesting that the number of molecules per cell must be precisely regulated. We quantified MeCP2 in neuronal nuclei and found that it is nearly as abundant as the histone octamer. Despite this high abundance, MeCP2 associates preferentially with methylated regions and high-throughput sequencing showed that its genome-wide binding tracks methyl-CpG density. MeCP2 deficiency results in global changes in neuronal chromatin structure, including elevated histone acetylation and a doubling of histone H1. Neither change is detectable in glia, where MeCP2 occurs at lower levels. The mutant brain also shows elevated transcription of repetitive elements. Our data argue that MeCP2 may not act as a gene-specific transcriptional repressor in neurons, but might instead dampen transcriptional noise genome-wide in a DNA methylation-dependent manner

Tissue-specific gene repositioning by muscle nuclear membrane proteins enhances repression of critical developmental genes during myogenesis

Whether gene repositioning to the nuclear periphery during differentiation adds another layer of regulation to gene expression remains controversial. Here, we resolve this by manipulating gene positions through targeting the nuclear envelope transmembrane proteins (NETs) that direct their normal repositioning during myogenesis. Combining transcriptomics with high-resolution DamID mapping of nuclear envelope-genome contacts, we show that three muscle-specific NETs, NET39, Tmem38A, and WFS1, direct specific myogenic genes to the nuclear periphery to facilitate their repression. Retargeting a NET39 fragment to nucleoli correspondingly repositioned a target gene, indicating a direct tethering mechanism. Being able to manipulate gene position independently of other changes in differentiation revealed that repositioning contributes ⅓ to ⅔ of a gene’s normal repression in myogenesis. Together, these NETs affect 37% of all genes changing expression during myogenesis, and their combined knockdown almost completely blocks myotube formation. This unequivocally demonstrates that NET-directed gene repositioning is critical for developmental gene regulation

Cell type–specific DNA methylation at intragenic CpG islands in the immune system

Human and mouse genomes contain a similar number of CpG islands (CGIs), which are discrete CpG-rich DNA sequences associated with transcription start sites. In both species, ∼50% of all CGIs are remote from annotated promoters but, nevertheless, often have promoter-like features. To determine the role of CGI methylation in cell differentiation, we analyzed DNA methylation at a comprehensive CGI set in cells of the mouse hematopoietic lineage. Using a method that potentially detects ∼33% of genomic CpGs in the methylated state, we found that large differences in gene expression were accompanied by surprisingly few DNA methylation changes. There were, however, many DNA methylation differences between hematopoietic cells and a distantly related tissue, brain. Altered DNA methylation in the immune system occurred predominantly at CGIs within gene bodies, which have the properties of cell type–restricted promoters, but infrequently at annotated gene promoters or CGI flanking sequences (CGI “shores”). Unexpectedly, elevated intragenic CGI methylation correlated with silencing of the associated gene. Differentially methylated intragenic CGIs tended to lack H3K4me3 and associate with a transcriptionally repressive environment regardless of methylation state. Our results indicate that DNA methylation changes play a relatively minor role in the late stages of differentiation and suggest that intragenic CGIs represent regulatory sites of differential gene expression during the early stages of lineage specification