Faculty Bibliography

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

Nucleosomes containing the histone variant H3.3 tend to be clustered in vivo in the neighborhood of transcriptionally active genes and over regulatory elements. It has not been clear, however, whether H3.3-containing nucleosomes possess unique properties that would affect transcription. We report here that H3.3 nucleosomes isolated from vertebrates, regardless of whether they are partnered with H2A or H2A.Z, are unusually sensitive to salt-dependent disruption, losing H2A/H2B or H2A.Z/H2B dimers. Immunoprecipitation studies of nucleosome core particles (NCPs) show that NCPs that contain both H3.3 and H2A.Z are even less stable than NCPs containing H3.3 and H2A. Intriguingly, NCPs containing H3 and H2A.Z are at least as stable as H3/H2A NCPs. These results establish an hierarchy of stabilities for native nucleosomes carrying different complements of variants, and suggest how H2A.Z could play different roles depending on its partners within the NCP. They also are consistent with the idea that H3.3 plays an active role in maintaining accessible chromatin structures in enhancer regions and transcribed regions. Consistent with this idea, promoters and enhancers at transcriptionally active genes and coding regions at highly expressed genes have nucleosomes that simultaneously carry both H3.3 and H2A.Z, and should therefore be extremely sensitive to disruption

Jun dimerization protein-2 (JDP2) is a component of the AP-1 transcription factor that represses transactivation mediated by the Jun family of proteins. Here, we examine the functional mechanisms of JDP2 and show that it can inhibit p300-mediated acetylation of core histones in vitro and in vivo. Inhibition of histone acetylation requires the N-terminal 35 residues and the DNA-binding region of JDP2. In addition, we demonstrate that JDP2 has histone-chaperone activity in vitro. These results suggest that the sequence-specific DNA-binding protein JDP2 may control transcription via direct regulation of the modification of histones and the assembly of chromatin

We have introduced the histone variant H3.3 into chicken erythroid cell lines and examined its distribution in the neighborhood of the folate receptor (FR) and beta-globin genes by using high-resolution chromatin immunoprecipitation (ChIP). Marked incorporation of tagged H3.3 into the FR gene is confined to its upstream regulatory region and is observed whether or not the gene is transcriptionally active. Incorporation is also observed over locus control regulatory elements in the absence of transcription of genes regulated by these elements, suggesting that gene activity per se is not necessarily required to replace H3 with H3.3. Other active genes display various behaviors, either incorporating H3.3 over both the coding region and upstream regulatory region or over upstream sequences only. There is, however, no straightforward correlation between sites of H3.3 incorporation and regions of enrichment in H3 acetylation and lysine-4 methylation. In the case of FR and VEGF-D, in which incorporation is confined to upstream regions, the presence of exogenous H3 results in reduced expression, whereas H3.3 stimulates expression. This finding suggests that these histone variants can be active rather than passive participants in regulation of expression

Transcription factor JDP2 served as a repressor of AP-1 and inhibited the transactivation of the c-jun gene by p300/ATF-2, by recruitment of histone deacetylase complex (HDAC3), thereby repressing the RA-induced transcription of the c-jun gene and then inhibiting the RA-mediated differentiation of F9 cells. These results suggest that HDAC3/JDP2 and p300/ATF-2 complex play a critical role in controlling the differentiation of F9 cells, in response to RA. We also found that JDP2 has the activities associated with histone binding and inhibition of histone acetyltransferase (INHAT) as well as regulation of chromatin assembly. The region that includes that includes the amino-terminal 35 amino acids adjacent to the basic region are required for histone-binding activity and the region that includes both histone-binding domain and basic region is essential for the inhibition of INHAT. Moreover, assays of nucleosome assembly in vitro demonstrated that JDP2 also has histone chaperone activity. These results revealed that JDP2 is not only a sequence specific DNA-binding protein and but also controls the transcription of AP-1 response genes by direct regulation of histone modification

Jun dimerization protein 2 (JDP2) is a novel member of AP-1 family and acts as a general repressor of a variety of transcription. JDP2 is able to bind to specific sites in target gene such as c-jun by forming homodimer or heterodimers with a Jun/ATF family member to counteract their transcriptional activity. Previously we showed that JDP2 inhibits the retinoic acid (RA) dependent transcription by recruiting a histone deacetylase 3 (HDAC3) complex to the promoter region of the target genes. We present here that JDP2 has an inhibitory activity of acetylation of all core histones mediated by histone acetyltransferase (HAT) both of p300 and PCAF in vitro. The studies of both histone-binding and HAT-inhibitory activity using a variety of recombinant JDP2(s) indicated that JDP2 might target histone itself through the histone-binding domain of JDP2, which is essential but not sufficient for the inhibition of acetylation. Therefore, our data suggested that HAT-inhibitory activity of JDP2 could in part explain the transcriptional repression of several target genes