技术资料

The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs,but Sox4,Sox5,Sox6,Sox8,Sox9,Sox11,Sox12,Sox13,and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover,the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming,albeit at low efficiency. By molecular dissection of the C-terminus of Sox17,we found that the β-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK,we analyzed ChIP-sequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2. View Publication

With defined culture protocol,human embryonic stem cells (hESCs) are able to generate cardiomyocytes in vitro,therefore providing a great model for human heart development,and holding great potential for cardiac disease therapies. In this study,we successfully generated a highly pure population of human cardiomyocytes (hCMs) (backslashtextgreater95% cTnT+) from hESC line,which enabled us to identify and characterize an hCM-specific signature,at both the gene expression and DNA methylation levels. Gene functional association network and gene-disease network analyses of these hCM-enriched genes provide new insights into the mechanisms of hCM transcriptional regulation,and stand as an informative and rich resource for investigating cardiac gene functions and disease mechanisms. Moreover,we show that cardiac-structural genes and cardiac-transcription factors have distinct epigenetic mechanisms to regulate their gene expression,providing a better understanding of how the epigenetic machinery coordinates to regulate gene expression in different cell types. View Publication

Once set,the inactive status of the X chromosome in female somatic cells is preserved throughout subsequent cell divisions. The inactive status of the X chromosome is characterized by many features,including late replication. In contrast to induced pluripotent stem cells (iPSCs) in mice,the X chromosome in human female iPSCs usually remains inactive after reprogramming of somatic cells to the pluripotent state,although recent studies point to the possibility of reactivation of the X chromosome. Here,we demonstrated that,during reprogramming,the inactive X chromosome switches from late to synchronous replication,with restoration of the transcription of previously silenced genes. This process is accompanied by accumulation of a new epigenetic mark or intermediate of the DNA demethylation pathway,5-hydroxymethylcytosine (5hmC),on the activated X chromosome. Our results indicate that the active status of the X chromosome is better confirmed by early replication and the reappearance of 5hmC,rather than by appearance of histone marks of active chromatin,removal of histone marks of inactive chromatin,or an absence of XIST coating. View Publication

Understanding the root causes of autoimmune diseases is hampered by the inability to access relevant human tissues and identify the time of disease onset. To examine the interaction of immune cells and their cellular targets in type 1 diabetes,we differentiated human induced pluripotent stem cells into pancreatic endocrine cells,including $\beta$ cells. Here,we describe an in vitro platform that models features of human type 1 diabetes using stress-induced patient-derived endocrine cells and autologous immune cells. We demonstrate a cell-type-specific response by autologous immune cells against induced pluripotent stem cell-derived $\beta$ cells,along with a reduced effect on $\alpha$ cells. This approach represents a path to developing disease models that use patient-derived cells to predict the outcome of an autoimmune response. View Publication