Gene regulatory elements, networks, and mechanisms

National Institute Of Environmental Health Sciences

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Abstract

Understanding how an environmental stimulant affects cellular physiology to give rise to a specific phenotypic outcome is a fundamental step toward understanding development and pathogenesis. During development and in response to environmental insults, various signaling cascades culminate in the activation of key chromatin remodeling enzymes and transcription factors, which collectively modulate the chromatin architecture to establish and/or maintain gene expression programs controlling cell identity. Our laboratory uses integrative interdisciplinary approaches merging systems biology, functional genomics, and biochemistry to map, reconstruct, and characterize developmentally- and environmentally-responsive gene networks that control fundamental biological processes ranging from transcription and signal transduction to cellular response to changes in the environment. Specifically, we seek to understand how transcription regulators and epigenetic modifications regulate gene expression programs controling cell fate decisions during cellular development and differentiation. Bivalent chromatin is characterized by the simultaneous presence of H3K4me3 and H3K27me3, histone modifications generally associated with transcriptionally active and repressed chromatin, respectively. Prevalent in embryonic stem cells (ESCs), bivalency is postulated to poise/prime lineage-controlling developmental genes for rapid activation during embryogenesis while maintaining a transcriptionally repressed state in the absence of activation cues; however, this hypothesis remains to be directly tested. Most gene promoters DNA-hypermethylated in adult human cancers are bivalently marked in ESCs, and it was speculated that bivalency predisposes them for aberrant de novo DNA methylation and irreversible silencing in cancer, but evidence supporting this model was largely lacking. We showed that bivalent chromatin does not poise genes for rapid activation but protects promoters from de novo DNA methylation. Bivalent genes in ESCs that are frequent targets of aberrant hypermethylation in cancer are particularly strongly associated with loss of H3K4me3/bivalency in cancer. Altogether, our findings suggested that bivalency protects reversibly repressed genes from irreversible silencing and that loss of bivalency may make them more susceptible to aberrant DNA methylation in diseases such as cancer and that Bivalency may thus represent a distinct regulatory mechanism for maintaining epigenetic plasticity. In our ongoing studies, we seek to determine whether deletion of Mll2 (chiefly responsible for H3K4me3 at bivalent chromatin) in mouse ESCs abrogates protection against de novo DNA methylation during pre- to post-implantation epiblast differentiation Previously, we predicted an essential role for nuclear export receptor protein Crm1, in regulation of ESC identity. Crm1, also known as Exportin1/Xpo1, is best known for its role in nuclear export of proteins containing short, hydrophobic nuclear export signal (NES) motifs. Through a combination of genome-wide, biochemical, and genetic studies over the last eight years on Crm1s function in mouse ESCs, we have established a non-canonical role for Crm1 on chromatin in promoting PRC2-mediated repressed chromatin state. Briefly, we found that (A) Crm1 preferentially binds to key developmental in ESCs (B) Crm1s NES-binding cleft is necessary for Crm1 binding to chromatin (C) Crm1 colocalizes with Erk2 on chromatin, and Crm1 binding to chromatin is MAPK/ERK signaling-dependent (D) Crm1 is recruited to chromatin in an Erk2- and NES-dependent manner (E) Crm1 colocalizes and interacts with the repressive Polycomb complex PRC2, specifically at PRC2 nucleation sites but not PRC2 spreading sites (F) Crm1 promotes PRC2-mediated H3K27me3 repressive domain formation, and (G) Crm1 C-terminal tail is necessary for chromatin binding, PRC2 interaction, H3K27me3 domain formation, and ESC differentiation. The manuscript containing these findings is currently under preparation for submission. In our ongoing studies, we seek to determine mechanisms that recruit Crm1 to chromatin, and the function of chromatin-bound Crm1 during early embyronic development. Collectively, our studies provide a foundation for defining the mechanism and scope of developmentally- and environmentally- responsive gene networks.