|Targeting Small Molecules Hits the Spot for Reprogramming|
“A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts”
Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is known to involve the genome-wide remodelling of chromatin and it has been suggested that small molecules which can affect the chromatin landscape could be used to reactivate the endogenous pluripotency network in place of transcription factor transduction (Onder et al andHirai et al). Researchers from the laboratory of Hiroshi Sugiyama at Kyoto University, Japan have previously shown that the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) can be conjugated with selective DNA-binding hairpin pyrrole-imidazole polyamides (PIPs) (Ohtsuki et al), allowing the upregulation of pluripotency-associated factors through the targeted increase in permissive histone modifications at the promoter regions of Oct4 and Nanog in mouse embryonic fibroblasts (MEFs) (Pandian et al, ChemBioChem, 2011). However, the induction values observed were very low (Pandian et al, ChemBioChem, 2011 and Pandian et al, Bioorg Med Chem, 2012) and so the laboratory sought to synthesise new SAHA-PIPs with improved recognition ability (Pandian et al, Sci Reps, 2012).
Initially, 16 new SAHA-PIP conjugates were designed to improve the recognition of GC-rich sequences present at promoter regions which represent sites for DNA binding factors. One of these new conjugates (d) could induce endogenous Oct4 and Nanog expression in MEFs by up to 50-fold within 24 hours and, in some cases, this was comparable to levels observed in mouse embryonic stem cells (mESCs). Other factors tested included Sox2, which was induced 8-fold, while Myc and Klf4 were not induced. Neither SAHA treatment alone nor treatment with a non-functional d allowed for the induction of any tested pluripotency associated factors, suggesting that the specific targeting of SAHA through PIP allowed for the selective gene expression through directed histone deacetylase inhibition. The effective concentration for d was observed to be at 100nM although, encouragingly, no cytotoxic effects were observed in MEFs even at 1 mM.
Sridharan et al) (36% of elevated genes in MEFs), including Rex1 and Dppa4, which were induced by 70-fold and 7-fold respectively, although the levels in mESCs were 595-fold and 241-fold, respectively. Further analysis of genome wide transcriptional changes in response to d uncovered a pattern of gene upregulation indicative of a mesenchymal to epithelial transition (MET), an important rate-limiting step during the reprogramming of the somatic genome. This included the rapid induction of Cdh1 and Cldn7 and the downregulation of epithelial to mesenchymal transition (EMT) markers (Fgf5, Snai1, Snai2 and Zeb2); overall suggesting that d treatment can initiate cellular reprogramming (Samavarchi-Tehrani et al).
Analysis of histone acetylation levels after treatment with d found increased levels of acetylation of histone H3 at the promoter regions of Oct4, Nanog, Dppa4, Rex1 and Cdh1 whereas treatment with SAHA alone, or with the non-functional d, had no notable effect on acetylation at these sites. Interestingly, acetylation levels of histone H3 in d-treated MEFs were comparable and occasionally surpassed the levels observed in mESCs. Overall this suggests that gene induction is caused by the elevation of histone acetylation at these gene promoters thereby allowing access to DNA binding factors which can mediate gene expression.
Multiple studies have previously shown that the generation of iPSCs can be improved using small molecules which affect chromatin modifications (Huangfu et al, Mali et al and Shi et al). This study now shows that through the use of a targeting strategy - the conjugation of PIP moieties, this activity can be localised to areas with transcription factor binding sites, allowing for the induction of endogenous expression of reprogramming-associated gene expression and genes associated with MET, a significant barrier to somatic cell reprogramming (Li et al). Hopefully, future work will study the additional efficiency that d could allow alongside common reprogramming protocols and, importantly, its implementation in human somatic cell reprogramming.
Hirai, H. et al.
Huangfu, D. et al.
Li, R. et al.
Mali, P. et al.
Ohtsuki, A. et al.
Onder, T. T. et al.
Pandian, G. N. et al.
Pandian, G. N. et al.
Pandian, GN. et al.
Samavarchi-Tehrani, P. et al.
Shi, Y. et al.
Sridharan, R. et al.
Article originally appeared in Science Reports.
STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.