Ganesh N. Pandian

A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts

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.

Progress and Prospects of Pyrrole-Imidazole Polyamide–Fluorophore Conjugates as Sequence-Selective DNA Probes

Recently, the versatility of N‐methylpyrrole (Py)‐N‐methylimidazole (Im) polyamide conjugates, which have been developed from the DNA‐binding antibiotics distamycin A and netropsin, has been shown. These synthetic small molecules can permeate cells to bind with duplex DNA in a sequence‐specific manner, and hence can influence gene expression in vivo. Accordingly, several reports demonstrating the sequence specificity and biological activity of Py‐Im polyamides have accumulated. However, the benefits of Py‐Im polyamides, in particular those conjugated with fluorophores, has been overlooked. Moreover, clear directions for the employment of these attractive artificial small molecules have not yet been shown. Here, we present a detailed overview of the current and prospective applications of Py‐Im polyamide–fluorophore conjugates, including sequence‐specific recognition with fluorescence emission properties, and their potential roles in biological imaging.

Depletion of 14-3-3 Protein Exacerbates Cardiac Oxidative Stress, Inflammation and Remodeling Process via Modulation of MAPK/NF-ĸB Signaling Pathways after Streptozotocin-induced Diabetes Mellitus

Diabetic cardiomyopathy is associated with increased oxidative stress and inflammation. Mammalian 14-3-3 proteins are dimeric phosphoserine-binding proteins that participate in signal transduction and regulate several aspects of cellular biochemistry. The aim of the study presented here was to clarify the role of 14-3-3 protein in the mitogen activated protein kinase (MAPK) and nuclear factor-kB (NF-ĸB) signaling pathway after experimental diabetes by using transgenic mice with cardiac-specific expression of a dominant-negative 14-3-3 protein mutant (DN 14-3-3). Significant p-p38 MAPK activation in DN 14-3-3 mice compared to wild type mice (WT) after diabetes induction and with a corresponding up regulation of its downstream effectors, p-MAPK activated protein kinase 2 (MAPKAPK-2). Marked increases in cardiac hypertrophy, fibrosis and inflammation were observed with a corresponding up-regulation of atrial natriuretic peptide, osteopontin, connective tissue growth factor, tumor necrosis factor α, interleukin (IL)-1β, IL-6 and cellular adhesion molecules. Moreover, reactive oxygen species, left ventricular expression of NADPH oxidase subunits, p22 phox, p67 phox, and Nox4, and lipid peroxidation levels were significantly increased in diabetic DN 14-3-3mice compared to diabetic WT mice. Furthermore, myocardial NF-ĸB activation, inhibitor of kappa B-α degradation and mRNA expression of proinflammatory cytokines were significantly increased in DN 14-3-3 mice compared to WT mice after diabetes induction. In conclusion, our data suggests that depletion of 14-3-3 protein induces cardiac oxidative stress, inflammation and remodeling after experimental diabetes induction mediated through p38 MAPK, MAPKAPK-2 and NF-ĸB signaling.

Synthetic small molecules for epigenetic activation of pluripotency genes in mouse embryonic fibroblasts.

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.

Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts

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.

Synthesis and Biological Properties of Highly Sequence-Specific-Alkylating N-Methylpyrrole–N-Methylimidazole Polyamide Conjugates

Four new alkylating N-methylpyrrole-N-methylimidazole (PI) polyamide conjugates (1-4) with seven-base-pair (bp) recognition ability were synthesized. Evaluation of their DNA-alkylating activity clearly showed accurate alkylation at match site(s). The cytotoxicities of conjugates 1-4 were determined against six human cancer cell lines, and the effect of these conjugates on the expression levels of the whole human genome in A549 cells were also investigated. A few genes among the top 20 genes were commonly downregulated by each conjugate, which reflects their sequence specificity. Conversely, many of the top 10 genes were commonly upregulated, which may have been caused by alkylation damage to DNA. Moreover, the antitumor activities of the PI polyamide conjugates 2 and 3 were investigated using nude mice transplanted with DU145 or A549. The intravenous administration of each liposomal conjugate in water yielded tumor-suppressing effects specifically toward DU145 cells and not A549 cells, which was pertinent to cytotoxicity.

Development of programmable small DNA-binding molecules with epigenetic activity for induction of core pluripotency genes.

Epigenetic modifications that govern the gene expression are often overlooked with the design of artificial genetic switches. N-Methylpyrrole-N-methylimidazole (PI) hairpin polyamides are programmable small DNA binding molecules that have been studied in the context of gene regulation. Recently, we synthesized a library of compounds by conjugating PI polyamides with SAHA, a chromatin-modifier. Among these novel compounds, PI polyamide-SAHA conjugate 1 was shown to epigenetically activate pluripotency genes in mouse embryonic fibroblasts. Here, we report the synthesis of the derivatives of conjugate 1 and demonstrate that these epigenetically active molecules could be developed to improve the induction of pluripotency factors.

A Synthetic Small Molecule for Targeted Transcriptional Activation of Germ Cell Genes in a Human Somatic Cell

In nature, coordinated genetic and/or epigenetic mechanisms govern the global transcriptional reprogramming that dictates cell fate. In contrast to genetic modifications, which cause irreversible changes of the cellular phenotype, epigenetic modifications can be reversed. The plasticity of epigenetic modifications facilitates the development of novel drugs, including small molecules that are capable of restoring incurable dysfunctions associated with faulty transcriptional machinery. Chromatin-modifying enzymes act both as facilitators and barriers by switching the transcriptional networks “on” and “off” to modulate the chromatin topology that governs the cellular phenotype. In particular, chromatin remodeling mediated by histone acetylation could induce global changes in the transcriptional status of a cell. Accordingly, small molecules that could inhibit histone deacetylase (HDAC), a chromatin-modifying enzyme, were shown to induce global changes in the acetylation profile, but in a non-selective manner. The development of small molecules that trigger targeted transcriptional activation could ensure better efficacy and a reduction of long-term side effects. However, precise control of gene expression is difficult, as epigenetic modifications are not insulated events. Therefore, epigenetically active small molecules with a DNA recognition domain are required. N-methylpyrrole (P) and Nmethylimidazole (I) polyamides are able to recognize each of the four Watson–Crick base pairs by binding to the minor groove of the DNA. As a novel chemical approach to dictate cell fate through targeted transcriptional activation, we have developed a small molecule that has access to both the genetic and epigenetic environment. This small molecule, namely SAHA-PIP, contains the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and hairpin pyrrole–imidazole polyamides (PIPs), which are capable of sequence-specific DNA recognition. In mouse fibroblasts, we first demonstrated the remarkable ability of SAHA-PIPs to induce differential activation of pluripotent stem-cell-associated genes and fine-tuned these molecules for enhanced efficacy. 8] Likewise, SAHA-PIP-mediated targeted transcriptional activation may trigger unusual activation of the silent gene network(s) in somatic cells. Gametogenesis is one of those silent biological processes in somatic cells. Meiosis is a highly specialized cell-division process in multicellular eukaryotes that is specific to germ cells. Aberrations in the orderly meiotic process are a prominent cause of human infertility. In mammalian spermatogenesis, the meiotic phase involves a series of intricate processes, such as chromosome remodeling and genetic recombination. Male mammalian germ cells express distinct populations of Piwi-interacting RNAs to govern the silencing of transposable elements in the germline at the pre-pachytene and pachytene stages of meiosis. MOV10L1, a germ-cell-specific putative RNA helicase, functions upstream of Piwi proteins to maintain post-meiotic genome integrity. A recent study revealed that the epigenetic disruption of the PIWI pathway could be associated with spermatogenic disorders in infertile male human patients. As SAHA-PIPs selectively activate a set of pluripotency genes in mouse fibroblasts, they may have a similar effect in human dermal fibroblasts (HDFs) to modulate the typically conserved gene network that is associated with pluripotency and/or gametogenesis. Herein, we report the remarkable ability of the SAHA-PIP K, which was developed as part of a SAHA-PIP library (A to F), to trigger targeted transcriptional activation of germ-cell-specific and PIWI-pathway genes in HDFs. Global changes in gene transcription were analyzed using a SurePrint G3 Human GEv 2 8 60 K Microarray (Agilent Technologies) after treating a library of synthetic SAHA-PIPs (A–F) with HDFs based on reported standardization studies. 8] Gene ontology analysis of initial microarray data [*] L. Han, S. Junetha, C. Anandhakumar, J. Taniguchi, A. Saha, T. Bando, Prof. H. Sugiyama Department of Chemistry, Graduate School of Science Kyoto University Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan) E-mail: hs@kuchem.kyoto-u.ac.jp L. Han Shanghai Key Laboratory of Chemical Biology, State Key Laboratory of Bioreactor Engineering, School of Pharmacy East China University of Science and Technology Meilong Road 130, Shanghai, 200237 (China) G. N. Pandian, S. Sato, Prof. H. Sugiyama Institute for Integrated Cell-Material Sciences (WPI-iCeMS) Kyoto University Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501 (Japan)

Programmable genetic switches to control transcriptional machinery of pluripotency

Transcriptional activators play a central role in the regulation of gene expression and have the ability to manipulate the specification of cell fate. Pluripotency is a transient state where a cell has the potential to develop into more than one type of mature cell. The induction of pluripotency in differentiated cells requires extensive chromatin reorganization regulated by core transcriptional machinery. Several small molecules have been shown to enhance the efficiency of somatic cell reprogramming into pluripotent stem cells. However, entirely chemical‐based reprogramming remains elusive. Recently, we reported that selective DNA‐binding hairpin pyrrole‐imidazole polyamides conjugated with histone deacetylase inhibitor could mimic natural transcription factors and epigenetically activate certain pluripotency‐associated genes. Here, we review the need to develop selective chromatin‐modifying transcriptional activators for somatic genome reprogramming.

Chemically induced pluripotent stem cells (CiPSCs): a transgene-free approach

Induced pluripotent stem cells (iPSCs) could be generated by a single gene Oct4 and chemical compounds, in which exogenous expression of Oct4 was indispensable for reprogramming. Recent advances in chemical-mediated cellular reprogramming suggest that small molecules alone (i.e. without Yamanaka factors) can successfully establish iPSCs from mouse somatic cells.