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DOT-ty about Reprogramming - Chromatin-modifying enzymes as modulators of reprogramming

From Nature 
By Stuart P. Atkinson

Cellular reprogramming through the forced expression of specific transcription factors in somatic cells to produce induced pluripotent stem cells (iPSCs) has been long understood to involve a genome wide change in the chromatin environment from a largely repressive environment, non-conducive to wide-spread gene expression to a largely permissive environment that permits the expression of genes required for pluripotency and any potential lineage choice during differentiation. However, the chromatin modifying factors that are important to the reprogramming process are largely unknown, even though several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming (Hawkins et al and Mikkelsen et al). Now, using short hairpin RNAs (shRNAs) to target genes important to DNA and histone methylation pathways, researchers from the laboratory of George Q. Daley have identified the histone H3 lysine 79 methyltransferase DOT1L as an important chromatin modifying factor in the reprogramming process (Onder et al).

Fibroblasts differentiated from the H1 hESC line were infected with shRNAs and reprogrammed with the OSKM reprogramming factors and iPSCs formation was scored through Tra-1-60 staining. Downregulation of 7 genes caused a reduction in reprogramming efficiency; the repressive histone methyltransferases EHMT1 and SETDB1 and Polycomb repressive complex components BMI1, RING1, EZH2, EED and SUZ12. This suggests that a major part of the reprogramming process is the specific and efficient downregulation of the somatic cell genome during reprogramming. Interestingly, suppression of three gene enhanced reprogramming; YY1, a context-dependent transcriptional activator or repressor (Shi et al), SUV39H1, histone H3K9 methyltransferase implicated in heterochromatin formation (Schotta et al) and DOT1L a histone H3 lysine 79 methyltransferase that had not previously been studied in the context of reprogramming (Jones et al). This enhancement could be reversed upon the expression of an shRNA resistant wild-type DOT1L but not a catalytically inactive one suggesting that the catalytic activity of DOT1L is important. Suppression of DOT1L by shRNA, and also by a small molecule inhibitor (EPZ004777 (Daigle et al)), increased the reprogramming activity between threefold and six fold, and iPSCs generated displayed all of the hallmarks of pluripotency. Similar results were also observed for murine reprogramming, and so DOT1L’s function during reprogramming was further investigated.

Inhibition of DOT1L did not affect retroviral transgene expression, cellular proliferation, apoptosis or changes in the cell cycle. Inhibitor studies in human fibroblasts also identified the first two weeks of programming as being the crucial window, with significantly greater numbers of Tra-1-60-positive cell clusters on day 10 and day 14 indicating that the emergence of iPSCs is accelerated upon DOT1L inhibition. DOT1L inhibition could also allow for two factor reprogramming by functionally replacing the KLF4 and MYC transgenes, but could not replace OCT4 or SOX2 in any combination. Additionally, these two factor iPSCs exhibited all the hallmarks of pluripotency. The molecular mechanisms behind higher efficiency reprogramming under conditions of DOT1L inhibition were analysed through global gene expression analysis. At day 6 of reprogramming, shRNA cells had relatively few changes as compared to control OSKM cells (45 genes), while inhibitor treated cells, perhaps due to more complete inhibition exhibited more gene expression changes (580 changes) and finally shDOT1L OSM cells showed 94 gene changes. Common genes upregulated included the core pluripotency network factors NANOG and LIN28 which may account for the enhancement in reprogramming. This result was further validated in shDOT1L fibroblasts upon OSM or OS transduction, and found that other core pluripotency factors (ZFP42 and DNMT3B) were not upregulated, suggesting a more direct upregulation of NANOG and LIN28 rather than an upregulation in the entire pluripotency network, while suppression of NANOG or LIN28 abrogated OCT4 and SOX2 mediated shDOT1L fibroblast reprogramming. Further, DOT1L inhibition upregulated NANOG expression during OSL and LIN28 during OSN mediated reprogramming and significantly increased the efficiency of OSN and OSL reprogramming.

Changes to chromatin upon DOT1L inhibition were obviously expected and ChIP-Seq analysis was undertaken for di-methylated lysine 79 of histone H3 (DiMeK79H3), a permissive modification promoted by DOT1L, and tri-methylation of lysine 27 H3 (TriMeK27H3), a repressive modification. DOT1L inhibition in OSKM-reprogramming downregulated DiMeK79H3 at 348 genes, with an enrichment for genes associated with the induction of a mesenchymal state (SNAI2, TGFB2 and TGFBR1). The vast majority of the identified genes lacked DiMeK79H3 in the pluripotent state (272 out of the 348 devoid of H3K79me2 in ESCs), suggesting they were destined for transcriptional silencing during reprogramming, overall suggesting that DOT1L inhibition reduces DiMeK79 at EMT-associated genes, transcriptionally silencing them, and thereby promoting reprogramming. Gene expression of genes with reduced MeK79H3 also decreased and was further associated by an increase in MeK27H3, while this modification was depleted at SOX2 and ECAD, reflecting their activation and remained unchanged at promoters of other lineage master regulator genes (OLIG2, MYOD1, NKX2-1 and GATA4), indicating a level of specificity. Overexpression of the mesenchymal regulators (TWIST1, SNAI1 and ZEB1) or addition of TGFB2 counteracted the effect of DOT1L inhibition in reprogramming and also the upregulation of NANOG and LIN28 suggesting that the DOT1L inhibition-mediated upregulation of these genes is indirect.

Overall, DOT1L inhibition aids the early to middle stage of reprogramming of mouse and human cells to accelerate and increase the efficiency of the reprogramming process possibly through the downregulation of a mesenchymal gene expression program. Further, iPSCs generated following DOT1L inhibition display all of the hallmarks of pluripotency, while suppression of DOT1L allowed generation of two factor iPSCs (NANOG AND LIN28 only) implicating NANOG and LIN28 in the enhancement of reprogramming. These findings also uncover some general findings; that factors generating repressive chromatin domains are barriers to reprogramming with the potential to be targeted during reprogramming and that such a targeting strategy has the potential to replace exogenous reprogramming factors in iPSC production generating safer iPSC cells for clinical use.



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