Transcription factor-based reprogramming of differentiated cells to induced pluripotent stem cells (iPSCs) has revolutionized the field of stem cell biology and greatly increased the opportunities for disease modeling and cell-based regenerative therapies. While iPSCs offer tremendous potential, a number of technical challenges remain in the generation of iPSCs that hamper their use in clinical settings. A comprehensive understanding of the molecular mechanisms of cellular reprogramming is essential for devising methods to overcome these challenges. Our research is aimed at understanding the molecular mechanisms of reprogramming and identifying genes that are important for iPSC generation. Specifically, we utilize forward genetics tools (i.e. shRNA and CRISPR-Cas9 technology) and small molecule inhibitors to identify the role chromatin modifying enzymes play during reprogramming.
Our long term research goal is to understand the mechanisms that maintain cell identity and determine whether perturbations of these pathways can result in efficient and faithful reprogramming to pluripotency.
Bromodomain inhibition of the coactivators CBP/EP300 facilitate cellular reprogramming
Here we identify acetyl-lysine competitive inhibitors targeting the bromodomains of coactivators CBP and EP300 as potent enhancers of reprogramming. These inhibitors accelerate reprogramming, are critical during its early stages and, when combined with DOT1L inhibition, enable efficient derivation of human iPSCs with OCT4 and SOX2. In contrast, catalytic inhibition of CBP/EP300 prevents iPSC formation, suggesting distinct functions for different co-activator domains in reprogramming. CBP/EP300 bromodomain inhibition decreases somatic-specific gene expression, histone H3 lysine 27 acetylation (H3K27Ac) and chromatin accessibility at target promoters and enhancers. The master mesenchymal transcription factor PRRX1 is one such functionally important target of CBP/EP300 bromodomain inhibition. Collectively, these results show that CBP/EP300 bromodomains sustain cell type specific gene expression and maintain cell identity. Click on the graphical abstract below to read the paper.
Understanding the Role of Chromatin Modifiers in Somatic Cell Reprogramming
We have employed a loss-of-function approach to interrogate the role of genes in DNA and histone methylation pathways and identified Dot1L, the only known H3K79 methyl-transferase, as a potent suppressor of cellular reprogramming. Inhibition of Dot1L either by RNA interference or a small molecule inhibitor accelerated reprogramming, significantly increased the yield of iPSCs, and substituted for two of the exogenous transcription factors used in reprogramming. To determine the role of additional chromatin modifiers in reprogramming we are currently pursuing a combination of candidate-based and high-throughput molecular approaches.