Final Report Summary - CMR (Chromatin modifiers in reprogramming)
Transcription factor-based reprogramming has enabled the generation of induced pluripotent stem cells (iPSCs). IPSCs can be used in disease modeling and derivation of rejection-free mature cell types for cell-replacement therapies. Proof-of-principle studies show that a number of genetic, complex, and malignant diseases can benefit from such cell-based therapies. Despite their immense potential, technical obstacles remain in the generation of iPSCs. This project (CMR) aimed at generating tools to overcome these obstacles by elucidating the molecular mechanisms of reprogramming. Specifically, the role of chromatin modifiers in cellular reprogramming was investigated. To identify novel chromatin-based regulators of reprogramming, and understand the molecular mechanisms of these regulators, CMR: (1) elucidated the mechanism by which inhibition of histone methyl-transferases Suv39H1 and Setd2 enhances reprogramming, and (2) identified a number of histone demethylases that act as positive or negative regulators of reprogramming. First, the role of Suv39H1 as a suppressor of reprogramming was validated using both genetic and chemical tools. Suv39H1 was shown to act during the initial phases of reprogramming. Through gene expression analysis, two pluripotency related genes, NANOG and SOX2, was shown to be regulated by this protein. Chromatin immunoprecipitation (ChIP) experiments revealed that, upon Suv39H1 inhibition, histone H3 lysine 9 methylation levels were significantly downregulated at the promoter regions of these pluripotency-associated genes.
In addition, CMR has investigated the role of histone H3 lysine 36 (H3K36) methylation during somatic cell reprogramming. Inhibition of two H3K36 methyl-transferases SetD2 and Ash1L significantly enhanced reprogramming suggesting that this chromatin mark act as a barrier in this process. Gene expression analyses revealed that loss of H3K36 methylation on its own does not directly activate the pluripotency network. In contrast, highly expressed somatic-specific genes in fibroblasts such as ZEB1 and GREM1 were significantly downregulated upon SetD2 inhibition. Analysis of available ChIP datasets revealed that these genes gain the repressive H3K27 tri-methyl mark upon acquisition of pluripotency, suggesting an antagonistic relationship between H3K36 and H3K27 tri-methyl modifications.
The final aim of CMR was the identification of histone demethylases that act as positive or negative regulators of reprogramming. To this end, a loss-of-function screen using short hairpin RNAs (shRNAs) was carried out. Knock-down of two H3K9 demethylases resulted in a significant increase in reprogramming efficiency. Conversely, inhibition of KDM2A and KDM2B, which act on H3K36 methylation, impeded reprogramming. Thus, this demethylase screen reinforced previous findings regarding the role of H3K9 and H3K36 methylation during reprograming.
The results of CMR constitutes an important contribution to our understanding of the molecular basis of reprogramming. These results enable more efficient generation of iPSCs and, therefore, are likely to have an impact on the methodologies used in the derivation of iPSCs for clinical use in the near future.
In addition, CMR has investigated the role of histone H3 lysine 36 (H3K36) methylation during somatic cell reprogramming. Inhibition of two H3K36 methyl-transferases SetD2 and Ash1L significantly enhanced reprogramming suggesting that this chromatin mark act as a barrier in this process. Gene expression analyses revealed that loss of H3K36 methylation on its own does not directly activate the pluripotency network. In contrast, highly expressed somatic-specific genes in fibroblasts such as ZEB1 and GREM1 were significantly downregulated upon SetD2 inhibition. Analysis of available ChIP datasets revealed that these genes gain the repressive H3K27 tri-methyl mark upon acquisition of pluripotency, suggesting an antagonistic relationship between H3K36 and H3K27 tri-methyl modifications.
The final aim of CMR was the identification of histone demethylases that act as positive or negative regulators of reprogramming. To this end, a loss-of-function screen using short hairpin RNAs (shRNAs) was carried out. Knock-down of two H3K9 demethylases resulted in a significant increase in reprogramming efficiency. Conversely, inhibition of KDM2A and KDM2B, which act on H3K36 methylation, impeded reprogramming. Thus, this demethylase screen reinforced previous findings regarding the role of H3K9 and H3K36 methylation during reprograming.
The results of CMR constitutes an important contribution to our understanding of the molecular basis of reprogramming. These results enable more efficient generation of iPSCs and, therefore, are likely to have an impact on the methodologies used in the derivation of iPSCs for clinical use in the near future.