Person:

Shi, Yujiang

The genome-wide distribution patterns of the ‘6th base’ 5-hydroxymethylcytosine (5hmC) in many tissues and cells have recently been revealed by hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by high throughput sequencing or tiling arrays. However, it has been challenging to directly compare different data sets and samples using data generated by this method. Here, we report a new comparative hMeDIP-seq method, which involves barcoding different input DNA samples at the start and then performing hMeDIP-seq for multiple samples in one hMeDIP reaction. This approach extends the barcode technology from simply multiplexing the DNA deep sequencing outcome and provides significant advantages for quantitative control of all experimental steps, from unbiased hMeDIP to deep sequencing data analysis. Using this improved method, we profiled and compared the DNA hydroxymethylomes of mouse ES cells (ESCs) and mouse ESC-derived neural progenitor cells (NPCs). We identified differentially hydroxymethylated regions (DHMRs) between ESCs and NPCs and uncovered an intricate relationship between the alteration of DNA hydroxymethylation and changes in gene expression during neural lineage commitment of ESCs. Presumably, the DHMRs between ESCs and NPCs uncovered by this approach may provide new insight into the function of 5hmC in gene regulation and neural differentiation. Thus, this newly developed comparative hMeDIP-seq method provides a cost-effective and user-friendly strategy for direct genome-wide comparison of DNA hydroxymethylation across multiple samples, lending significant biological, physiological and clinical implications.

Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs’ role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes.

Summary Naked mole rat (NMR) is a valuable model for aging and cancer research due to its exceptional longevity and cancer resistance. We observed that the reprogramming efficiency of NMR fibroblasts in response to OSKM was drastically lower than that of mouse fibroblasts. Expression of SV40 LargeT antigen (LT) dramatically improved reprogramming of NMR fibroblasts. Inactivation of Rb alone, but not p53, was sufficient to improve reprogramming efficiency, suggesting that NMR chromatin may be refractory to reprogramming. Analysis of the global histone landscape revealed that NMR had higher levels of repressive H3K27 methylation marks and lower levels of activating H3K27 acetylation marks than mouse. ATAC-seq revealed that in NMR, promoters of reprogramming genes were more closed than mouse promoters, while expression of LT led to massive opening of the NMR promoters. These results suggest that NMR displays a more stable epigenome that resists de-differentiation, contributing to the cancer resistance and longevity of this species.