32 research outputs found
Dynamic nucleosome-depleted regions at androgen receptor enhancers in the absence of ligand in prostate cancer cells
Nucleosome positioning at transcription start sites is known to regulate gene expression by altering DNA accessibility to transcription factors; however, its role at enhancers is poorly understood. We investigated nucleosome positioning at the androgen receptor (AR) enhancers of TMPRSS2, KLK2, and KLK3/PSA in prostate cancer cells. Surprisingly, a population of enhancer modules in androgen-deprived cultures showed nucleosome-depleted regions (NDRs) in all three loci. Under androgen-deprived conditions, NDRs at the TMPRSS2 enhancer were maintained by the pioneer AR transcriptional collaborator GATA-2. Androgen treatment resulted in AR occupancy, an increased number of enhancer modules with NDRs without changes in footprint width, increased levels of histone H3 acetylation (AcH3), and dimethylation (H3K4me2) at nucleosomes flanking the NDRs. Our data suggest that, in the absence of ligand, AR enhancers exist in an equilibrium in which a percentage of modules are occupied by nucleosomes while others display NDRs. We propose that androgen treatment leads to the disruption of the equilibrium toward a nucleosome-depleted state, rather than to enhancer de novo “remodeling.” This allows the recruitment of histone modifiers, chromatin remodelers, and ultimately gene activation. The “receptive” state described here could help explain AR signaling activation under very low ligand concentrations
Absence of Inhibin Alpha and Retinoblastoma Protein Leads to Early Sertoli Cell Dysfunction
Sertoli cells, the support cells of mammalian spermatogenesis, are regulated by a number of nuclear factors and express retinoblastoma (RB) tumor suppressor protein. We hypothesized that RB is an important mediator of Sertoli cell tumorigenesis in inhibin α knockout (Inha KO) mice. In our previous mouse studies, we found that conditional knockout (cKO) of Rb in Sertoli cells caused progressive Sertoli cell dysfunction. Initially, loss of RB had no gross effect on Sertoli cell function as the mice were fertile with normal testis weights at 6 weeks of age, but by 10–14 weeks of age, mutant mice demonstrated severe Sertoli cell dysfunction and infertility. Although double knockout (dKO) of Rb and Inha did not result in exacerbation of the tumorigenic phenotype of Inha-null mice, we found that the dKO mice demonstrate an acceleration of Sertoli cell dysfunction compared to Rb cKO mice. Specifically, in contrast to Rb cKO mice, Inha/Rb dKO mice showed signs of Sertoli cell dysfunction as early as 4 weeks of age. These results demonstrate that RB is not essential for Sertoli cell tumorigenesis in Inha KO mice but that loss of Inha accelerates the infertility phenotype of Rb cKO mice
MLL2 Is Required in Oocytes for Bulk Histone 3 Lysine 4 Trimethylation and Transcriptional Silencing
Conditional knockout mouse strategies identify the histone methyltranferase MLL2 as a key player in epigenetic reprogramming of female gametes
Double knockout of <i>Inha</i> and <i>Rb</i> in Sertoli cells does not significantly affect the activin-induced wasting syndrome.
<p>Body weights (A) and liver weights (B) are not significantly different between age-matched <i>Inha<sup>−/−</sup></i> and <i>Inha/Rb</i> dKO mice (p>0.05). Control liver weights are shown for comparison. Different letters represent statistically different groups.</p
Proof of <i>Rb</i> recombination in testes of 6 week-old mice.
<p>PCR amplification of the region surrounding exon 19 was performed on DNA extracted from whole testis of <i>Inha<sup>−/−</sup></i> and <i>Inha/Rb</i> dKO mice. As shown schematically in (A), primers flanking the loxP sites amplify the recombined <i>Rb</i> conditional allele to produce a 260-bp product, which is only seen in the Cre-positive mice (B). Amplifications of a 748-bp product for the <i>Rb</i>-floxed allele and a 699-bp product for the <i>Rb</i> wild-type/null allele were used as loading controls (B).</p
Double knockout of <i>Inha</i> and <i>Rb</i> in Sertoli cells does not significantly affect disease progression.
<p>Control mice have 100% survival until 26 weeks of age (A). During the same period, <i>Inha<sup>−/−</sup></i> (n = 25) and <i>Inha/Rb</i> dKO (n = 20) mice reach 50% survival at 14 weeks of age and are not significantly different by log-rank test (A, p>0.05). Tumor burden is also not significantly different between these two groups at 6 weeks, 10 weeks, and end of life (B, p>0.05).</p
Gene Reactivation by 5-Aza-29-Deoxycytidine–Induced Demethylation Requires SRCAP–Mediated H2A.Z Insertion to Establish Nucleosome Depleted Regions
5-Aza-29-deoxycytidine, approved by the FDA for the treatment of myelodysplastic syndrome (MDS), is incorporated into the DNA of dividing cells where it specifically inhibits DNA methylation by forming covalent complexes with the DNA methyltransferases (DNMTs). In an effort to study the correlations between DNA methylation, nucleosome remodeling, and gene reactivation, we investigate the integrated epigenetic events that worked coordinately to reprogram the methylated and closed promoters back to permissive chromatin configurations after 5-Aza-29-deoxycytidine treatment. The ChIP results indicate that H2A.Z is deposited at promoter regions by the Snf2-related CBP activator protein (SRCAP) complex following DNA demethylation. According to our genome-wide expression and DNA methylation profiles, we find that the complete re-activation of silenced genes requires the insertion of the histone variant H2A.Z, which facilitates the acquisition of regions fully depleted of nucleosome as demonstrated by NOMe–seq (Nucleosome Occupancy Methylome–sequencing) assay. In contrast, SRCAP–mediated H2A.Z deposition is not required for maintaining the active status of constitutively expressed genes. By combining Hpa II digestion with NOMe–seq assay, we show that hemimethylated DNA, which is generated following drug incorporation, remains occupied by nucleosomes. Our data highlight H2A.Z as a novel and essential factor involved in 5-Aza-29-deoxycytidine–induced gene reactivation. Furthermore, we elucidate that chromatin remodeling translates the demethylation ability of DNMT inhibitors to their downstream efficacies, suggesting future therapeuti
Retinoblastoma Protein Plays Multiple Essential Roles in the Terminal Differentiation of Sertoli Cells
Human Mass Balance and Metabolite Profiling of [C-14]-Pamiparib, a Poly (ADP-Ribose) Polymerase Inhibitor, in Patients With Advanced Cancer
Pamiparib, a selective poly (ADP‐ribose) polymerase 1/2 inhibitor, demonstrated tolerability and antitumor activity in patients with solid tumors at 60 mg orally twice daily. This phase 1 open‐label study (NCT03991494; BGB‐290‐106) investigated the absorption, metabolism, and excretion (AME) of 60 mg [(14)C]‐pamiparib in 4 patients with solid tumors. The mass balance in excreta, blood, and plasma radioactivity and plasma pamiparib concentration were determined along with metabolite profiles in plasma, urine, and feces. Unchanged pamiparib accounted for the most plasma radioactivity (67.2% ± 10.2%). Pamiparib was rapidly absorbed with a median time to maximum plasma concentration (C(max)) of 2.00 hours (range, 1.00‐3.05 hours). After reaching C(max), pamiparib declined in a biphasic manner, with a geometric mean terminal half‐life (t(1/2)) of 28.7 hours. Mean cumulative [(14)C]‐pamiparib excretion was 84.7% ± 3.5%. Pamiparib was mainly cleared through metabolism, primarily via N‐oxidation and oxidation of the pyrrolidine ring. A dehydrogenated oxidative product (M3) was the most abundant metabolite in biosamples. A mean of 2.11% and 1.11% of [(14)C]‐pamiparib was excreted as unchanged pamiparib in feces and urine, respectively, indicating near‐complete absorption and low renal clearance of parent drug. Cytochrome P450 (CYP) phenotyping demonstrated CYP2C8 and CYP3A involvement in pamiparib metabolism. These findings provide an understanding of pamiparib AME mechanisms and potential drug‐drug interaction liability