Faculty Bibliography

The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3' end. Instead, the histone mRNAs display a stem-loop structure at their 3' end. Stem-loop binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreases cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increases polyadenylation of canonical histone H3.1 mRNA possibly through downregulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic is not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones has been shown to increase sensitivity to DNA damage, as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis.

Jun dimerization protein 2 (JDP2) is a repressor of transcription factor AP-1. To investigate the transcriptional regulation of the JDP2 gene, we cloned the 5'-flanking region of the mouse JDP2 gene. Primer extension analysis revealed a new transcription start site (+1). Promoter analysis showed that the region from nt -343 to nt +177 contains basal transcriptional activity. Interestingly, the tumor suppressor p53 significantly repressed the transcriptional activity of the JDP2 promoter. Given that JDP2 inhibits expression of p53, our results suggest a negative feedback loop between JDP2 and p53, and a direct link between JDP2 and a key oncogenic pathway.

Although the androgen receptor (AR) has been implicated in the promotion of apoptosis in testicular cells (TSCs), the molecular pathway underlying AR-mediated apoptosis and its sensitivity to environmental hormones in TSCs and induced pluripotent stem cells (iPSCs) remain unclear. We generated the iPSCs from bovine TSCs via the electroporation of OCT4. The established iPSCs were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4 to maintain and stabilize the expression of stemness genes and their pluripotency. Apoptosis signaling was assessed after exposure to mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate. Here, we report that iPSCs were more resistant to MEHP-induced apoptosis than were original TSCs. MEHP also repressed the expression of AR and inactivated WNT signaling, and then led to the commitment of cells to apoptosis via the cyclin dependent kinase inhibitor p21CIP1. The loss of the frizzed receptor 7 and the gain of p21CIP were responsible for the stimulatory effect of MEHP on AR-mediated apoptosis. Our results suggest that testicular iPSCs can be used to study the signaling pathways involved in the response to environmental disruptors, and to assess the toxicity of environmental endocrine disruptors in terms of the maintenance of stemness and pluripotency.

Chromatin is subject to proofreading and repair mechanisms during the process of DNA replication, as well as repair to maintain genetic and epigenetic information and genome stability. The dynamic structure of chromatin modulates various nuclear processes, including transcription and replication, by altering the accessibility of the DNA to regulatory factors. Structural changes in chromatin are affected by the chemical modification of histone proteins and DNA, remodeling of nucleosomes, incorporation of variant histones, noncoding RNAs, and nonhistone DNA-binding proteins. Phenotypic diversity and fidelity can be balanced by controlling stochastic switching of chromatin structure and dynamics in response to the environmental disruptors and endogenous stresses. The dynamic chromatin remodeling can, therefore, serve as a sensor, through which environmental and/or metabolic agents can alter gene expression, leading to global cellular changes involving multiple interactive networks. Furthermore its recent evidence also suggests that the epigenetic changes are heritable during the development. This review will discuss the environmental sensing system for chromatin regulation and genetic and epigenetic controls from developmental perspectives.

Oxidative stress and reactive oxygen species (ROS) are associated with diseases such as cancer, cardiovascular complications, inflammation and neurodegeneration. Cellular defense systems must work constantly to control ROS levels and to prevent their accumulation. We report here that the Jun dimerization protein 2 (JDP2) has a critical role as a cofactor for transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and small Maf protein family K (MafK) in the regulation of the antioxidant-responsive element (ARE) and production of ROS. Chromatin immunoprecipitation-quantitative PCR (qPCR), electrophoresis mobility shift and ARE-driven reporter assays were carried out to examine the role of JDP2 in ROS production. JDP2 bound directly to the ARE core sequence, associated with Nrf2 and MafK (Nrf2-MafK) via basic leucine zipper domains, and increased DNA-binding activity of the Nrf2-MafK complex to the ARE and the transcription of ARE-dependent genes. In mouse embryonic fibroblasts from Jdp2-knockout (Jdp2 KO) mice, the coordinate transcriptional activation of several ARE-containing genes and the ability of Nrf2 to activate expression of target genes were impaired. Moreover, intracellular accumulation of ROS and increased thickness of the epidermis were detected in Jdp2 KO mice in response to oxidative stress-inducing reagents. These data suggest that JDP2 is required to protect against intracellular oxidation, ROS activation and DNA oxidation. qPCR demonstrated that several Nrf2 target genes such as heme oxygenase-1, glutamate-cysteine ligase catalytic and modifier subunits, the notch receptor ligand jagged 1 and NAD(P)H dehydrogenase quinone 1 are also dependent on JDP2 for full expression. Taken together, these results suggest that JDP2 is an integral component of the Nrf2-MafK complex and that it modulates antioxidant and detoxification programs by acting via the ARE.

The androgen receptor (AR) has a critical role in promoting androgen-dependent and -independent apoptosis in testicular cells. However, the molecular mechanisms that underlie the ligand-independent apoptosis, including the activity of AR in testicular stem cells, are not completely understood. In the present study, we generated induced pluripotent stem cells (iPSCs) from bovine testicular cells by electroporation of octamer-binding transcription factor 4 (OCT4). The cells were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4, which maintained and stabilized the expression of stemness genes and pluripotency. The iPSCs were used to assess the apoptosis activity following exposure to phthalate esters, including di (2-ethyhexyl) phthalates, di (n-butyl) phthalate, and butyl benzyl phthalate. Phthalate esters significantly reduced the expression of AR in iPSCs and induced a higher ratio of BAX/BCL-2, thereby favoring apoptosis. Phthalate esters also increased the expression of cyclin-dependent kinase inhibitor 1 (p21(Cip1)) in a p53-dependent manner and enhanced the transcriptional activity of p53. The forced expression of AR and knockdown of p21(Cip1) led to the rescue of the phthalate-mediated apoptosis. Overall, this study suggests that testicular iPSCs are a useful system for screening the toxicity of environmental disruptors and examining their effect on the maintenance of stemness and pluripotency, as well as for identifying the iPSC signaling pathway(s) that are deregulated by these chemicals.

Chromatin structure and gene expression are both regulated by nucleosome assembly. How environmental factors influence histone nuclear import and the nucleosome assembly pathway, leading to changes in chromatin organization and transcription, remains unknown. Acrolein (Acr) is an alpha,beta-unsaturated aldehyde, which is abundant in the environment, especially in cigarette smoke. It has recently been implicated as a potential major carcinogen of smoking-related lung cancer. Here we show that Acr forms adducts with histone proteins in vitro and in vivo and preferentially reacts with free histones rather than with nucleosomal histones. Cellular fractionation analyses reveal that Acr exposure specifically inhibits acetylations of N-terminal tails of cytosolic histones H3 and H4, modifications that are important for nuclear import and chromatin assembly. Notably, Acr exposure compromises the delivery of histone H3 into chromatin and increases chromatin accessibility. Moreover, changes in nucleosome occupancy at several genomic loci are correlated with transcriptional responses to Acr exposure. Our data provide new insights into mechanisms whereby environmental factors interact with the genome and influence genome function.

Within the genome, expressed genes marked by 'open' chromatin are often adjacent to silent, heterochromatic regions. There are also regions containing neighboring active genes with different programs of expression. In both cases, DNA sequence elements may function as insulators, either providing barriers that prevent the incursion of heterochromatic signals into open domains or acting to block inappropriate contact between the enhancer of one gene and the promoter of another. The mechanisms associated with insulation are diverse: Enhancer-blocking insulation is largely associated with the ability to stabilize the formation of loop domains within the nucleus. Barrier insulation is often associated with the ability to block propagation of silencing histone modifications. Here, we provide examples of both kinds of insulator action, derived initially from studies of the compound insulator element at the 5' end of the chicken beta-globin locus. Such elements appear to have more general regulatory roles in the genome that have been exploited to provide insulator function where necessary to demarcate separate domains within the nucleus

To understand how chromatin structure is organized by different histone variants, we have measured the genome-wide distribution of nucleosome core particles (NCPs) containing the histone variants H3.3 and H2A.Z in human cells. We find that a special class of NCPs containing both variants is enriched at 'nucleosome-free regions' of active promoters, enhancers and insulator regions. We show that preparative methods used previously in studying nucleosome structure result in the loss of these unstable double-variant NCPs. It seems likely that this instability facilitates the access of transcription factors to promoters and other regulatory sites in vivo. Other combinations of variants have different distributions, consistent with distinct roles for histone variants in the modulation of gene expression