Cellular reprogramming involves profound alterations in genome-wide gene expression that is precisely controlled by a hypothetical epigenetic code. Small molecules have been shown to artificially induce epigenetic modifications in a sequence independent manner. Recently, we showed that specific DNA binding hairpin pyrrole-imidazole polyamides (PIPs) could be conjugated with chromatin modifying histone deacetylase inhibitors like SAHA to epigenetically activate certain pluripotent genes in mouse fibroblasts. In our steadfast progress to improve the efficiency of SAHA-PIPs, we identified a novel compound termed, δ that could dramatically induce the endogenous expression of Oct-3/4 and Nanog. Genome-wide gene analysis suggests that in just 24 h and at nM concentration, δ induced multiple pluripotency-associated genes including Rex1 and Cdh1 by more than ten-fold. δ treated MEFs also rapidly overcame the rate-limiting step of epithelial transition in cellular reprogramming by switching “” the complex transcriptional gene network.

Considering the essential role of chromatin remodeling in gene regulation, their directed modulation is of increasing importance. To achieve gene activation by epigenetic modification, we synthesized a series of pyrrole-imidazole polyamide conjugates (PIPs) that can bind to predetermined DNA sequences, and attached them with suberoylanilide hydroxamic acid (SAHA), a potent histone deacetylase inhibitor. As histone modification is associated with pluripotency, these new types of conjugates, termed SAHA-PIPs, were screened for their effect on the expression of induced pluripotent stem cell (iPSC) factors. We found certain SAHA-PIPs that could differentially up-regulate the endogenous expression of Oct-3/4, Nanog, Sox2, Klf4 and c-Myc. SAHA and other SAHA-PIPs did not show such induction; this implies a role for PIPs and their sequence specificity in this differential gene activation. Chromatin immunoprecipitation analysis suggested that SAHA-PIP-mediated gene induction proceeds by histone H3 Lys9 and Lys14 acetylation and Lys4 trimethylation, which are epigenetic features associated with transcriptionally active chromatin.

The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation.

Germ cell gene switch: The dynamic epigenome coordinates the timely genome‐wide transcriptional activation that governs interindividual diversity, for example, in germ cells, which differ from somatic cells through their ability to undergo meiosis. Now, an epigenetically active synthetic small molecule can trigger unusual activation of the typically conserved PIWI gene that regulates the meiotic process in a human somatic cell.

A nontransgenic approach to reprogram mouse somatic cells into induced pluripotent stem cells using only small molecules got achieved to propose a potential clinical-friendly cellular reprogramming strategy. Consequently, the screening and identification of small molecules capable of inducing pluripotency genes in human cells are increasingly a focus of research. Because cellular reprogramming is multifactorial in nature, there is a need for versatile small molecules capable of modulating the complicated gene networks associated with pluripotency. We have developed a targeting small molecule called SAHA-PIP comprising the histone deacetylase inhibitor SAHA and the sequence-specific DNA binding pyrrole-imidazole polyamides for modulating distinct gene networks. Here, we report the identification of a SAHA-PIP termed Ì that could trigger genome-wide epigenetic reprogramming and turn ON the typically conserved core pluripotency gene network. Through independent lines of evidence, we report for the first time a synthetic small molecule inducer that target and activate the OCT-3/4 regulated pluripotency genes in human dermal fibroblasts.