Specific Chromatin Modifications Research Articles

Systematic epigenome editing captures the context-dependent instructive function of chromatin modifications

Chromatin modifications are linked with regulating patterns of gene expression, but their causal role and context-dependent impact on transcription remains unresolved. Here we develop a modular epigenome editing platform that programs nine key chromatin modifications, or combinations thereof, to precise loci in living cells. We couple this with single-cell readouts to systematically quantitate the magnitude and heterogeneity of transcriptional responses elicited by each specific chromatin modification. Among these, we show that installing histone H3 lysine 4 trimethylation (H3K4me3) at promoters can causally instruct transcription by hierarchically remodeling the chromatin landscape. We further dissect how DNA sequence motifs influence the transcriptional impact of chromatin marks, identifying switch-like and attenuative effects within distinct cis contexts. Finally, we examine the interplay of combinatorial modifications, revealing that co-targeted H3K27 trimethylation (H3K27me3) and H2AK119 monoubiquitination (H2AK119ub) maximizes silencing penetrance across single cells. Our precision-perturbation strategy unveils the causal principles of how chromatin modification(s) influence transcription and dissects how quantitative responses are calibrated by contextual interactions.

A-Z of Epigenetic Readers: Targeting Alternative Splicing and Histone Modification Variants in Cancer.

Epigenetic 'reader' proteins, which have evolved to interact with specific chromatin modifications, play pivotal roles in gene regulation. There is growing interest in the alternative splicing mechanisms that affect the functionality of such epigenetic readers in cancer etiology. The current review considers how deregulation of epigenetic processes and alternative splicing events contribute to pathophysiology. An A-Z guide of epigenetic readers is provided, delineating the antagonistic 'yin-yang' roles of full-length versus spliced isoforms, where this is known from the literature. The examples discussed underscore the key contributions of epigenetic readers in transcriptional regulation, early development, and cancer. Clinical implications are considered, offering insights into precision oncology and targeted therapies focused on epigenetic readers that have undergone alternative splicing events during disease pathogenesis. This review underscores the fundamental importance of alternative splicing events in the context of epigenetic readers while emphasizing the critical need for improved understanding of functional diversity, regulatory mechanisms, and future therapeutic potential.

Epigenetic regulation during meiosis and crossover.

Meiosis is a distinctive type of cell division that reorganizes genetic material between generations. The initial stages of meiosis consist of several crucial steps which include double strand break, homologous chromosome pairing,break repair and crossover. Crossover frequency varies depending on the position on the chromosome, higher at euchromatin region and rare at heterochromatin, centromeres, telomeres and ribosomal DNA. Crossover positioning is dependent on various factors, especially epigenetic modifications. DNA methylation, histone post-translational modifications, histone variants and non-coding RNAs are most probably playing an important role in positioning of crossovers on a chromosomal level as well as hotspot level. DNA methylation negatively regulates crossover frequency and its effect is visible in centromeres, pericentromeres and heterochromatin regions. Pericentromeric chromatin and heterochromatin mark studies have been a centre of attraction in meiosis. Crossover hotspots are associated with euchromatin regions having specific chromatin modifications such as H3K4me3, H2A.Z. and H3 acetylation. This review will provide the current understanding of the epigenetic role in plants during meiotic recombination, chromosome synapsis, double strand break and hotspots with special attention to euchromatin and heterochromatin marks. Further, the role of epigenetic modifications in regulating meiosis and crossover in other organisms is also discussed.

DNA Sequence-Dependent Properties of Nucleosome Positioning in Regions of Distinct Chromatin States in Mouse Embryonic Stem Cells.

Chromatin architecture is orchestrated, and plays crucial roles during the developmental process by regulating gene expression. In embryonic stem cells (ESCs), three types of chromatin states, including active, repressive and poised states, were previously identified and characterized with specific chromatin modification marks and different transcription activity, but it is largely unknown how nucleosomes are organized in these chromatin states. In this study, by using a DNA deformation energy model, we investigated the sequence-dependent nucleosome organization within the chromatin states in mouse ESCs. The results revealed that: (1) compared with poised genes, active genes are characterized with a higher level of nucleosome occupancy around their transcription start sites (TSS) and transcription termination sites (TTS), and both types of genes do not have a nucleosome-depleted region at their TTS, contrasting with the MNase-seq based result; (2) based on our previous DNA bending energy model, we developed an improved model capable of predicting both rotational positioning and nucleosome occupancy determined by a chemical mapping approach; (3) DNA bending-energy-based analyses demonstrated that the fragile nucleosomes positioned at both gene ends could be explained largely by enhanced rotational positioning signals encoded in DNA, but nucleosome phasing around the TSS of active genes was not determined by sequence preference; (4) the nucleosome occupancy landscape around the binding sites of some developmentally important transcription factors known to bind with different chromatin contexts, was also successfully predicted; (5) the difference of nucleosome occupancy around the TSS between CpG-rich and CpG-poor promoters was partly captured by our sequence-dependent model. Taken together, by developing an improved deformation-energy-based model, we revealed some sequence-dependent properties of the nucleosome arrangements in regions of distinct chromatin states in mouse ESCs.

The histone code of the fungal genus Aspergillus uncovered by evolutionary and proteomic analyses.

Chemical modifications of DNA and histone proteins impact the organization of chromatin within the nucleus. Changes in these modifications, catalysed by different chromatin-modifying enzymes, influence chromatin organization, which in turn is thought to impact the spatial and temporal regulation of gene expression. While combinations of different histone modifications, the histone code, have been studied in several model species, we know very little about histone modifications in the fungal genus Aspergillus, whose members are generally well studied due to their importance as models in cell and molecular biology as well as their medical and biotechnological relevance. Here, we used phylogenetic analyses in 94 Aspergilli as well as other fungi to uncover the occurrence and evolutionary trajectories of enzymes and protein complexes with roles in chromatin modifications or regulation. We found that these enzymes and complexes are highly conserved in Aspergilli, pointing towards a complex repertoire of chromatin modifications. Nevertheless, we also observed few recent gene duplications or losses, highlighting Aspergillus species to further study the roles of specific chromatin modifications. SET7 (KMT6) and other components of PRC2 (Polycomb Repressive Complex 2), which is responsible for methylation on histone H3 at lysine 27 in many eukaryotes including fungi, are absent in Aspergilli as well as in closely related Penicillium species, suggesting that these lost the capacity for this histone modification. We corroborated our computational predictions by performing untargeted MS analysis of histone post-translational modifications in Aspergillus nidulans. This systematic analysis will pave the way for future research into the complexity of the histone code and its functional implications on genome architecture and gene regulation in fungi.

Bidirectional promoters: an enigmatic genome architecture and their roles in cancers.

Bidirectional promoters are the transcription regulatory regions of genes positioned head-to-head on opposite strands. Specific sequence signals, chromatin modifications and three-dimensional structures of the transcription site facilitate the unconventional yet tightly regulated transcription proceeding in both directions from these promoters. Mutations or aberrant epigenetic changes can lead to abnormal enhanced or reduced expression from either of the bidirectionally transcribed genes resulting in tumorigenesis. Moreover, bidirectionally transcribed genes might also contribute towards the immune regulation in tumor microenvironment. In this review, we aimed to expound the characteristic features of bidirectional promoters alongside their transcriptional regulations, and ultimately, the association of these enigmatic genomic elements in different cancers.

Repurposing proscillaridin A in combination with decitabine against embryonal rhabdomyosarcoma RD cells.

Embryonal rhabdomyosarcoma (eRMS) is the most common type of rhabdomyosarcoma in children. eRMS is characterized by malignant skeletal muscle cells driven by hyperactivation of several oncogenic pathways including the MYC pathway. Targeting MYC in cancer has been extremely challenging. Recently, we have demonstrated that the heart failure drug, proscillaridin A, produced anticancer effects with specificity toward MYC expressing leukemia cells. We also reported that decitabine, a hypomethylating drug, synergizes with proscillaridin A in colon cancer cells. Here, we investigated whether proscillaridin A exhibits epigenetic and anticancer activity against eRMS RD cells, overexpressing MYC oncogene, and its combination with decitabine. We investigated the anticancer effects of proscillaridin A in eRMS RD cells in vitro. In response to drug treatment, we measured growth inhibition, cell cycle arrest, loss of clonogenicity and self-renewal capacity. We further evaluated the impact of proscillaridin A on MYC expression and its downstream transcriptomic effects by RNA sequencing. Then, we measured protein expression of epigenetic regulators and their associated chromatin post-translational modifications in response to drug treatment. Chromatin immunoprecipitation sequencing data sets were coupled with transcriptomic results to pinpoint the impact of proscillaridin A on gene pathways associated with specific chromatin modifications. Lastly, we evaluated the effect of the combination of proscillaridin A and the DNA demethylating drug decitabine on eRMS RD cell growth and clonogenic potential. Clinically relevant concentration of proscillaridin A (5nM) produced growth inhibition, cell cycle arrest and loss of clonogenicity in eRMS RD cells. Proscillaridin A produced a significant downregulation of MYC protein expression and inhibition of oncogenic transcriptional programs controlled by MYC, involved in cell replication. Interestingly, significant reduction in total histone 3 acetylation and on specific lysine residues (lysine 9, 14, 18, and 27 on histone 3) was associated with significant protein downregulation of a series of lysine acetyltransferases (KAT3A, KAT3B, KAT2A, KAT2B, and KAT5). In addition, proscillaridin A produced synergistic growth inhibition and loss of clonogenicity when combined with the approved DNA demethylating drug decitabine. Proscillaridin A produces anticancer and epigenetic effects in the low nanomolar range and its combination with decitabine warrants further investigation for the treatment of eRMS.

The Modular Circuitry of Apicomplexan Cell Division Plasticity.

The close-knit group of apicomplexan parasites displays a wide variety of cell division modes, which differ between parasites as well as between different life stages within a single parasite species. The beginning and endpoint of the asexual replication cycles is a ‘zoite’ harboring the defining apical organelles required for host cell invasion. However, the number of zoites produced per division round varies dramatically and can unfold in several different ways. This plasticity of the cell division cycle originates from a combination of hard-wired developmental programs modulated by environmental triggers. Although the environmental triggers and sensors differ between species and developmental stages, widely conserved secondary messengers mediate the signal transduction pathways. These environmental and genetic input integrate in division-mode specific chromosome organization and chromatin modifications that set the stage for each division mode. Cell cycle progression is conveyed by a smorgasbord of positively and negatively acting transcription factors, often acting in concert with epigenetic reader complexes, that can vary dramatically between species as well as division modes. A unique set of cell cycle regulators with spatially distinct localization patterns insert discrete check points which permit individual control and can uncouple general cell cycle progression from nuclear amplification. Clusters of expressed genes are grouped into four functional modules seen in all division modes: 1. mother cytoskeleton disassembly; 2. DNA replication and segregation (D&S); 3. karyokinesis; 4. zoite assembly. A plug-and-play strategy results in the variety of extant division modes. The timing of mother cytoskeleton disassembly is hard-wired at the species level for asexual division modes: it is either the first step, or it is the last step. In the former scenario zoite assembly occurs at the plasma membrane (external budding), and in the latter scenario zoites are assembled in the cytoplasm (internal budding). The number of times each other module is repeated can vary regardless of this first decision, and defines the modes of cell division: schizogony, binary fission, endodyogeny, endopolygeny.

Abstract A22: Selective inhibition of TOR complex 2 as a means to sensitize cancer cells to DNA damage agents?

Abstract The inhibition of the central growth regulatory kinase TOR, which participates in two complexes, TORC1 and TORC2, is the focus of many metabolic and cancer studies. Most studies have dealt with TORC1, the canonical target of rapamycin. However, TORC2 also plays a role in cancer development. In fission yeast, TORC2, through the activation of Gad8 (homologous to AKT in humans), is required for survival under DNA-damaging conditions. TORC2-Gad8 (AKT) is not required for Chk1-dependent DNA damage-induced cell-cycle arrest; however, perturbation of TORC2-Gad8 signaling generates an accumulation of homologous recombination (HR)-Rad52 containing repair foci. In addition, cells lacking TORC2-Gad8 become dependent on the HR pathway for their viability. Thus, we speculate that TORC2-Gad8 signaling affects HR-independent DNA repair pathways. Our most recent results suggest that TORC2-Gad8 is required for specific chromatin modifications that ensure proficient DNA damage repair. Given that the role of TORC2 in DNA damage repair is conserved in human cells, we predict that the selective inhibition of mTORC2 can improve combined therapy, by sensitizing cancer cells to DNA-damaging agents. Chemicogenetic screens that aim at isolating specific TORC2 inhibitors will be described. Citation Format: Adiel Cohen, Emese Pataki, Nuria Vital, Martin Kupiec, Ronit Weisman. Selective inhibition of TOR complex 2 as a means to sensitize cancer cells to DNA damage agents? [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A22.

Conserved and nuanced hierarchy of gene regulatory response to hypoxia.

A dynamic assembly of nuclear and cytoplasmic processes regulate gene activity. Hypoxic stress and the associated energy crisis activate a plurality of regulatory mechanisms including modulation of chromatin structure, transcriptional activation and post-transcriptional processes. Temporal control of genes is associated with specific chromatin modifications and transcription factors. Genome-scale technologies that resolve transcript subpopulations in the nucleus and cytoplasm indicate post-transcriptional processes enable cells to conserve energy, prepare for prolonged stress and accelerate recovery. Moreover, the harboring of gene transcripts associated with growth in the nucleus and macromolecular RNA-protein complexes contributes to the preferential translation of stress-responsive gene transcripts during hypoxia. We discuss evidence of evolutionary variation in integration of nuclear and cytoplasmic processes that may contribute to variations in flooding resilience.

Histone deacetylases, Mbd3/NuRD, and Tet2 hydroxylase are crucial regulators of epithelial-mesenchymal plasticity and tumor metastasis.

An epithelial-mesenchymal transition (EMT) represents a basic morphogenetic process of high cell plasticity underlying embryogenesis, wound healing, cancer metastasis and drug resistance. It involves a profound transcriptional and epigenetic reprogramming of cells. A critical role of epigenetic modifiers and their specific chromatin modifications has been demonstrated during EMT. However, it has remained elusive whether epigenetic control differs between the dynamic cell state transitions of reversible EMT and the fixed differentiation status of irreversible EMT. We have employed varying EMT models of murine breast cancer cells to identify the key players establishing epithelial-mesenchymal cell plasticity during reversible and irreversible EMT. We demonstrate that the Mbd3/NuRD complex and the activities of histone deacetylases (HDACs), and Tet2 hydroxylase play a critical role in keeping cancer cells in a highly metastatic mesenchymal state. Combinatorial interference with their functions leads to mesenchymal-epithelial transition (MET) and efficiently represses metastasis formation by invasive murine and human breast cancer cells in vivo.

TRIM66 reads unmodified H3R2K4 and H3K56ac to respond to DNA damage in embryonic stem cells

Recognition of specific chromatin modifications by distinct structural domains within “reader” proteins plays a critical role in the maintenance of genomic stability. However, the specific mechanisms involved in this process remain unclear. Here we report that the PHD-Bromo tandem domain of tripartite motif-containing 66 (TRIM66) recognizes the unmodified H3R2-H3K4 and acetylated H3K56. The aberrant deletion of Trim66 results in severe DNA damage and genomic instability in embryonic stem cells (ESCs). Moreover, we find that the recognition of histone modification by TRIM66 is critical for DNA damage repair (DDR) in ESCs. TRIM66 recruits Sirt6 to deacetylate H3K56ac, negatively regulating the level of H3K56ac and facilitating the initiation of DDR. Importantly, Trim66-deficient blastocysts also exhibit higher levels of H3K56ac and DNA damage. Collectively, the present findings indicate the vital role of TRIM66 in DDR in ESCs, establishing the relationship between histone readers and maintenance of genomic stability.

Chromatin organization regulated by EZH2-mediated H3K27me3 is required for OPN-induced migration of bone marrow-derived mesenchymal stem cells.

Osteopontin (OPN) is a chemokine-like extracellular matrix-associated protein involved in the migration of bone marrow-derived mesenchymal stem cells (BMSCs). An increasing number of studies have found that chromatin organization may affect cellular migration. However, whether OPN regulates chromatin organization is not understood, nor are the underlying molecular mechanisms. In this study, we investigated the link between chromatin organization and BMSC migration and demonstrated that OPN-mediated BMSC migration leads to elevated levels of heterochromatin marker histone H3 lysine 27 trimethylation (H3K27me3) through the methyltransferase EZH2. The expression of EZH2 reorganizes the chromatin structure of BMSCs. Pharmacological inhibition or depletion of EZH2 blocks BMSC migration. Moreover, using an atomic force microscope (AFM), we found that chromatin decondensation alters the mechanical properties of the nucleus. In addition, inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signals represses OPN-promoted chromatin condensation and cell migration. Thus, our results identify a mechanism by which ERK1/2 signalling drives specific chromatin modifications in BMSCs, which alters chromatin organization and thereby enables OPN-mediated BMSC migration.

Chromatin Immunoprecipitation in Early Mouse Embryos.

Epigenetic regulation is achieved at many levels by different factors such as tissue-specific transcription factors, members of the basal transcriptional apparatus, chromatin-binding proteins, and noncoding RNAs. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method that allows elucidating gene regulation at the molecular level by assessing if chromatin modifications or proteins are present at a specific locus. Initially, the majority of ChIP experiments were performed on cultured cell lines and more recently this technique has been adapted to a variety of tissues in different model organisms. Using ChIP on mouse embryos, it is possible to document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development and to get biological meaning from observations made on tissue culture analyses. We describe here a ChIP protocol on freshly isolated mouse embryonic somites for in vivo analysis of muscle specific transcription factor binding on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.

Chromatin-associated regulation of sorbitol synthesis in flower buds of peach.

PpeS6PDH gene is postulated to mediate sorbitol synthesis in flower buds of peach concomitantly with specific chromatin modifications. Perennial plants have evolved an adaptive mechanism involving protection of meristems within specialized structures named buds in order to survive low temperatures and water deprivation during winter. A seasonal period of dormancy further improves tolerance of buds to environmental stresses through specific mechanisms poorly known at the molecular level. We have shown that peach PpeS6PDH gene is down-regulated in flower buds after dormancy release, concomitantly with changes in the methylation level at specific lysine residues of histone H3 (H3K27 and H3K4) in the chromatin around the translation start site of the gene. PpeS6PDH encodes a NADPH-dependent sorbitol-6-phosphate dehydrogenase, the key enzyme for biosynthesis of sorbitol. Consistently, sorbitol accumulates in dormant buds showing higher PpeS6PDH expression. Moreover, PpeS6PDH gene expression is affected by cold and water deficit stress. Particularly, its expression is up-regulated by low temperature in buds and leaves, whereas desiccation treatment induces PpeS6PDH in buds and represses the gene in leaves. These data reveal the concurrent participation of chromatin modification mechanisms, transcriptional regulation of PpeS6PDH and sorbitol accumulation in flower buds of peach. In addition to its role as a major translocatable photosynthate in Rosaceae species, sorbitol is a widespread compatible solute and cryoprotectant, which suggests its participation in tolerance to environmental stresses in flower buds of peach.

Transcriptional and epigenetic analyses of the DMD locus reveal novel cis‑acting DNA elements that govern muscle dystrophin expression

The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2Mb and accounts for approximately 0,1% of the entire human genome. Mutations in this gene cause Duchenne and Becker Muscular Dystrophy, X-linked Dilated Cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16h, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques.Our results unveil a surprisingly powerful processivity of the RNA polymerase II along the entire 2.2Mb of the DMD locus with just one site of pausing around intron 52. We also discovered epigenetic marks highlighting the existence of four novel cis‑DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA.Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies.

DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease.

Single-molecule kinetic analysis of HP1-chromatin binding reveals a dynamic network of histone modification and DNA interactions.

Chromatin recruitment of effector proteins involved in gene regulation depends on multivalent interaction with histone post-translational modifications (PTMs) and structural features of the chromatin fiber. Due to the complex interactions involved, it is currently not understood how effectors dynamically sample the chromatin landscape. Here, we dissect the dynamic chromatin interactions of a family of multivalent effectors, heterochromatin protein 1 (HP1) proteins, using single-molecule fluorescence imaging and computational modeling. We show that the three human HP1 isoforms are recruited and retained on chromatin by a dynamic exchange between histone PTM and DNA bound states. These interactions depend on local chromatin structure, the HP1 isoforms as well as on PTMs on HP1 itself. Of the HP1 isoforms, HP1α exhibits the longest residence times and fastest binding rates due to DNA interactions in addition to PTM binding. HP1α phosphorylation further increases chromatin retention through strengthening of multivalency while reducing DNA binding. As DNA binding in combination with specific PTM recognition is found in many chromatin effectors, we propose a general dynamic capture mechanism for effector recruitment. Multiple weak protein and DNA interactions result in a multivalent interaction network that targets effectors to a specific chromatin modification state, where their activity is required.

Cell Fate Maintenance and Reprogramming During the Oocyte-to-Embryo Transition.

This chapter reviews our current understanding of the mechanisms that regulate reprogramming during the oocyte-to-embryo transition (OET). There are two major events reshaping the transcriptome during OET. One is the clearance of maternal transcripts in the early embryo, extensively reviewed by others. The other event, which is the focus of this chapter, is the embryonic (or zygotic) genome activation (EGA). The mechanisms controlling EGA can be broadly divided into transcriptional and posttranscriptional. The former includes the regulation of the basal transcription machinery, the regulation by specific transcription factors and chromatin modifications. The latter is performed mostly via specific RNA-binding proteins (RBPs). Different animal models have been used to decipher the regulation of EGA. These models are often biased for the specific type of regulation, which is why we discuss the models ranging from invertebrates to mammals. Whether these biases stem from incomplete understanding of EGA in these models, or reflect evolutionarily distinct solutions to EGA regulation, is a key unresolved problem in developmental biology. As the mechanisms controlling developmental reprogramming can, and in some cases have been shown to, function in differentiated cells subjected to induced reprogramming, our understanding of EGA regulation may have implications for the efficiency of induced reprogramming and, thus, for regenerative medicine.

194 EFFECT OF SMALL MOLECULE INHIBITORS ON Oct-4, Nanog, AND Sox-2 EXPRESSION IN BOVINE FETAL FIBROBLAST CELLS

Successful reprogramming of somatic cells towards pluripotency requires the epigenetic marks characteristic of the differentiated cell type first be erased in order to inactivate the somatic cell program and activate the embryonic program. However, the majority of cells that undergo reprogramming become trapped in a partially reprogrammed state that is characterised by the down-regulation of somatic cell marker genes, incomplete reactivation of pluripotency genes, maintenance of viral expression, and the inability to form chimeras. Several small molecules, which act on specific signaling pathways or chromatin modifications, have been shown to improve both the kinetics and efficiency of reprogramming. These chemical modifiers aid in overcoming the roadblocks encountered during the reprogramming process by inducing the necessary epigenetic modifications needed to silence the somatic cell genome and reactivate the embryonic stem cell genome. Chemical treatment of cells before reprogramming can remodel the epigenetic landscape to be more like that of embryonic stem cell by removing the repressive epigenetic marks and relaxing chromatin structure to allow the reprogramming factors easier access to target genes. In the present study, we assessed the effect of pretreatment with small molecules on the expression of Oct-4, Nanog, and Sox-2 in bovine fetal fibroblast cells. Chemical treatment consisted of 3 small molecules: PD0325901, a mitogen-activated protein kinase inhibitor; CHIR99021, a glycogen synthase kinase-3 inhibitor; and NuP0178 (NuPotential, Baton Rouge, LA, USA), a G9a histone methyltransferase inhibitor. Cells were seeded at a density of 0.7 × 106 and expanded before being divided evenly into 2 groups. Control cells were cultured in complete culture medium (DMEM with 10% fetal bovine serum), whereas treatment cells were cultured in complete culture medium containing 0.5 mM PD0325901, 3 mM CHIR99021, and 1.8 mM NuP0178. Messenger RNA was isolated from cell cultures using Dynabeads® mRNA DIRECT™ Kit (Dynal Inc., Lake Success, NY, USA) on Day 7 and 14, and the resulting RNA products were transcribed into cDNA using Bio-Rad iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantitative RT–PCR was performed to measure transcript levels of Oct-4, Nanog, Sox-2, and poly adenylate polymerase in treated and untreated cells at Days 7 and 14. Transcript levels were quantified by relative quantification using the ΔΔCt method and expressed as ratios of target genes (Oct-4, Nanog, Sox-2) to the reference gene (PAP) and normalized against a calibrator consisting of untreated bovine fetal fibroblast cells. No difference in expression levels between untreated and treated cells was detected at either Day 7 or 14. Currently, we are utilising chromatin immunoprecipitation to examine chromatin and DNA methylation patterns around the promoters of Oct-4, Nanog, and Sox-2 to further elucidate the effects of treatment with this combination of small molecule inhibitors.