Inhibition of AR-mediated transcription by binding of Oct1 to a motif enriched in AR-occupied regions.

BACKGROUND: The androgen receptor (AR) plays roles in prostate development and cancer (PCa). In response to androgens, the AR binds to androgen-response elements (AREs) to modulate gene transcription. The responses of such genes are dependent on the cellular milieu and on sequences around the AREs, which attract other transcription factors. Previously, bioinformatic analysis of 62 AR-occupied regions (ARORs) in PCa cells revealed enrichment for both AREs and a TTGGCAAATA-like motif. We undertook the present study to investigate the significance of the TTGGCAAATA-like motif. METHODS: Prostate cancer cell lines, LNCaP and C4-2B, were analyzed by transient transfections of wild-type and mutant reporter constructs, electro-mobility shift assays (EMSAs), and RT-qPCR analysis of endogenous genes. RESULTS: In two of six tested ARORs, point mutations in the TTGGCAAATA-like motif resulted in inhibition of DHT-mediated enhancer activity. EMSA revealed that Oct1 bound the motif, and that the mutations that abolished DHT responsiveness in the transfection assays augmented Oct1 binding. These results suggest a role for Oct1 as a context-dependent negative coregulator of AR. Consistent with this, siRNA knockdown of Oct1 increased the DHT-mediated enhancer activity of transfected reporters as well as an endogenous AR target gene, transglutaminase 2. CONCLUSIONS: Oct1 negatively regulates DHT-mediated enhancer activity in a subset of ARORs. The enrichment of ARORs for the Oct-binding, TTGGCAAATA-like motif may reflect a mechanism that utilizes Oct1 to keep AR activity in check at some ARORs, while augmenting AR activity in other ARORs. Therefore, Oct1 may have regulatory functions in prostate development and cancer progression.

Identification of novel androgen receptor target genes in prostate cancer.

BACKGROUND: The androgen receptor (AR) plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa). However, little is known about AR target genes that mediate the receptor's roles in disease progression. RESULTS: Using Chromatin Immunoprecipitation (ChIP) Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant) and LNCaP (androgen-dependent) PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells--D-dopachrome tautomerase (DDT), Protein kinase C delta (PRKCD), Glutathione S- transferase theta 2 (GSTT2), Transient receptor potential cation channel subfamily V member 3 (TRPV3), and Pyrroline-5-carboxylate reductase 1 (PYCR1)--most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT), was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation. CONCLUSION: AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are repressed. In general, response is stronger in C4-2B compared to LNCaP cells. Some of the genes near AR-occupied regions appear to be regulated by the AR in vivo as evidenced by their expression levels in prostate cancer tumors of various stages. Several AR target genes discovered in the present study, for example PRKCD and PYCR1, may open avenues in PCa research and aid the development of new approaches for disease management.

Genomic androgen receptor-occupied regions with different functions, defined by histone acetylation, coregulators and transcriptional capacity.

BACKGROUND: The androgen receptor (AR) is a steroid-activated transcription factor that binds at specific DNA locations and plays a key role in the etiology of prostate cancer. While numerous studies have identified a clear connection between AR binding and expression of target genes for a limited number of loci, high-throughput elucidation of these sites allows for a deeper understanding of the complexities of this process. METHODOLOGY/PRINCIPAL FINDINGS: We have mapped 189 AR occupied regions (ARORs) and 1,388 histone H3 acetylation (AcH3) loci to a 3% continuous stretch of human genomic DNA using chromatin immunoprecipitation (ChIP) microarray analysis. Of 62 highly reproducible ARORs, 32 (52%) were also marked by AcH3. While the number of ARORs detected in prostate cancer cells exceeded the number of nearby DHT-responsive genes, the AcH3 mark defined a subclass of ARORs much more highly associated with such genes -- 12% of the genes flanking AcH3+ARORs were DHT-responsive, compared to only 1% of genes flanking AcH3-ARORs. Most ARORs contained enhancer activities as detected in luciferase reporter assays. Analysis of the AROR sequences, followed by site-directed ChIP, identified binding sites for AR transcriptional coregulators FoxA1, CEBPbeta, NFI and GATA2, which had diverse effects on endogenous AR target gene expression levels in siRNA knockout experiments. CONCLUSIONS/SIGNIFICANCE: We suggest that only some ARORs function under the given physiological conditions, utilizing diverse mechanisms. This diversity points to differential regulation of gene expression by the same transcription factor related to the chromatin structure.

Androgen receptor-mediated repression of novel target genes.

BACKGROUND: The androgen receptor (AR) plays a pivotal role in prostate cancer (PCa) initiation and progression. To date, studies have focused disproportionately on androgen-stimulated genes such as prostate-specific antigen (PSA), while repressed genes have gained little attention, even though they too may be involved in regulating cell growth, differentiation, and apoptosis. METHODS: ChIP Display was used to identify putative AR target genes in the ablation-resistant human PCa cell line, C4-2B. Quantitative real-time reverse transcription-PCR analysis was used to measure gene expression in cells subjected to dihydrotestosterone (DHT) timecourse and dose-response, as well as AR knock-down and bicalutamide-treatments. RESULTS: We report on three genes, KIAA1217, CHRM1, and WBSCR28, which were newly identified in a screen for AR-occupied regions in C4-2B PCa cells, and which were repressed by treatment with DHT. AR knock-down resulted in increased KIAA1217, CHRM1, and WBSCR28 mRNA, indicating that, like PSA stimulation, AR represses these three genes even in the absence of added ligand. DHT decreased KIAA1217 and CHRM1 pre-mRNA levels, suggesting AR-mediated transcriptional inhibition. Cycloheximide attenuated DHT-mediated repression of CHRM1, suggesting the requirement of new protein synthesis. Furthermore, bicalutamide treatment did not mimic, but rather antagonized DHT-mediated KIAA1217 repression. Unlike the handful of androgen-repressed genes studied thus far, AR occupancy at KIAA1217, CHRM1, and WBSCR28 was mapped outside their respective 5'-promoter regions. CONCLUSIONS: Many more genes likely share AR-mediated gene repression through distal regulatory elements. Further study of such targets and their transcriptional regulation may help explain the receptor's tumorigenicity in PCa.

Involvement of Niemann-Pick type C2 protein in hematopoiesis regulation.

Niemann-Pick type C2 (NPC2) protein has been characterized as a cholesterol-binding protein. Its loss leads to NPC2 disease, an inherited neurodegenerative disorder. When analyzing gene expression profile, we noticed high expression of both NPC2 and its receptor, mannose 6-phosphate receptor (MPR), in murine hematopoietic stem cells. NPC2 protein, in the presence of thrombopoietin (TPO), causes an increase in CFU-GEMM (colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte) and a decrease in CFU-GM (colony-forming unit-granulocyte-macrophage) colony number in colony-forming cell (CFC) assays. This effect is independent of cholesterol binding but does require the presence of MPR. With M07e cells, a TPO-dependent hematopoietic leukemia cell line, NPC2 can inhibit TPO-induced differentiation and enhance TPO-mediated anti-apoptosis effects. Strikingly, these results are not observed under the standard 20% O(2) level of the standard incubator, but rather at 7% O(2), the physiological oxygen level of bone marrow. Furthermore, NPC2 protein upregulates hypoxia inducible factor 1-alpha protein level at 7% O(2), but not at 20% O(2). Our results demonstrate that NPC2 protein plays a role in hematopoiesis at the physiologic bone marrow level of O(2).