Embryonic stem cells and induced pluripotent stem cells have the ability to maintain their telomere length via expression of an enzymatic complex called telomerase. Similarly, more than 85%–90% of cancer cells are found to upregulate the expression of telomerase, conferring them with the potential to proliferate indefinitely. Telomerase Reverse Transcriptase (TERT), the catalytic subunit of telomerase holoenzyme, is the rate-limiting factor in reconstituting telomerase activity in vivo. To date, the expression and function of the human Telomerase Reverse Transcriptase (hTERT) gene are known to be regulated at various molecular levels (including genetic, mRNA, protein and subcellular localization) by a number of diverse factors. Among these means of regulation, transcription modulation is the most important, as evident in its tight regulation in cancer cell survival as well as pluripotent stem cell maintenance and differentiation. Here, we discuss how hTERT gene transcription is regulated, mainly focusing on the contribution of trans-acting factors such as transcription factors and epigenetic modifiers, as well as genetic alterations in hTERT proximal promoter.

Regulatory enhancer elements in solid tumours remain poorly characterized. Here we apply micro-scale chromatin profiling to survey the distal enhancer landscape of primary gastric adenocarcinoma (GC), a leading cause of global cancer mortality. Integrating 110 epigenomic profiles from primary GCs, normal gastric tissues and cell lines, we highlight 36,973 predicted enhancers and 3,759 predicted super-enhancers respectively. Cell-line-defined super-enhancers can be subclassified by their somatic alteration status into somatic gain, loss and unaltered categories, each displaying distinct epigenetic, transcriptional and pathway enrichments. Somatic gain super-enhancers are associated with complex chromatin interaction profiles, expression patterns correlated with patient outcome and dense co-occupancy of the transcription factors CDX2 and HNF4α. Somatic super-enhancers are also enriched in genetic risk SNPs associated with cancer predisposition. Our results reveal a genome-wide reprogramming of the GC enhancer and super-enhancer landscape during tumorigenesis, contributing to dysregulated local and regional cancer gene expression.

Histone modifications and their modifying enzymes are fundamentally involved in the epigenetic regulation of adipogenesis. This study aimed to define the roles of various histone modifications and their "division of labor" in fat cell differentiation. To achieve these goals, we examined the distribution patterns of eight core histone modifications at five key adipogenic regulatory genes, Pref-1, C/EBPβ, C/EBPα, PPARγ2 and aP2, during the adipogenesis of C3H 10T1/2 mouse mesenchymal stem cells (MSCs) and 3T3-L1 preadipocytes. We found that the examined histone modifications are globally stable throughout adipogenesis but show distinct and highly dynamic distribution patterns at specific genes. For example, the Pref-1 gene has lower levels of active chromatin markers and significantly higher H3 K27 tri-methylation in MSCs compared with committed preadipocytes; the C/EBPβ gene is enriched in active chromatin markers at its 3'-UTR; the C/EBPα gene is predominantly marked by H3 K27 tri-methylation in adipogenic precursor cells, and this repressive marker decreases dramatically upon induction; the PPARγ2 and aP2 genes show increased histone acetylation on both H3 and H4 tails during adipogenesis. Further functional studies revealed that the decreased level of H3 K27 tri-methylation leads to de-repression of Pref-1 gene, while the increased level of histone acetylation activates the transcription of PPARγ2 and aP2 genes. Moreover, the active histone modification-marked 3'-UTR of C/EBPβ gene was demonstrated as a strong enhancer element by luciferase assay. Our results indicate that histone modifications are gene-specific at adipogenic regulator genes, and they play distinct roles in regulating the transcriptional network during adipogenesis.

Recent advances in the engineering of sequence-specific synthetic nucleases provide enormous opportunities for genetic manipulation of gene expression in order to study their cellular function in vivo. However, current genotyping methods to detect these programmable nuclease-induced insertion/deletion (indel) mutations in targeted human cells are not compatible for high-throughput screening of knockout clones due to inherent limitations and high cost. Here, we describe an efficient method of genotyping clonal CRISPR/Cas9-mediated mutants in a high-throughput manner involving the use of a direct lysis buffer to extract crude genomic DNA straight from cells in culture, and fluorescent PCR coupled with capillary gel electrophoresis. This technique also allows for genotyping of multiplexed gene targeting in a single clone. Overall, this time- and cost-saving technique is able to circumvent the limitations of current genotyping methods and support high-throughput screening of nuclease-induced mutants.