Biswajyoti Sahu

Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer

High androgen receptor (AR) level in primary tumour predicts increased prostate cancer‐specific mortality. However, the mechanisms that regulate AR function in prostate cancer are poorly known. We report here a new paradigm for the forkhead protein FoxA1 action in androgen signalling. Besides pioneering the AR pathway, FoxA1 depletion elicited extensive redistribution of AR‐binding sites (ARBs) on LNCaP‐1F5 cell chromatin that was commensurate with changes in androgen‐dependent gene expression signature. We identified three distinct classes of ARBs and androgen‐responsive genes: (i) independent of FoxA1, (ii) pioneered by FoxA1 and (iii) masked by FoxA1 and functional upon FoxA1 depletion. FoxA1 depletion also reprogrammed AR binding in VCaP cells, and glucocorticoid receptor binding and glucocorticoid‐dependent signalling in LNCaP‐1F5 cells. Importantly, FoxA1 protein level in primary prostate tumour had significant association to disease outcome; high FoxA1 level was associated with poor prognosis, whereas low FoxA1 level, even in the presence of high AR expression, predicted good prognosis. The role of FoxA1 in androgen signalling and prostate cancer is distinctly different from that in oestrogen signalling and breast cancer.

Increased Expression of Androgen Receptor Sensitizes Prostate Cancer Cells to Low Levels of Androgens

Androgen receptor (AR) is known to be overexpressed in castration-resistant prostate cancer. To interrogate the functional significance of the AR level, we established two LNCaP cell sublines expressing in a stable fashion two to four times (LNCaP-ARmo) and four to six times (LNCaP-ARhi) higher level of AR than the parental cell line expressing the empty vector (LNCaP-pcDNA3.1). LNCaP-ARhi cell line grew faster than the control line in low concentrations, especially in 1 nmol/L 5alpha-dihydrotestosterone (DHT). Microarray-based transcript profiling and subsequent unsupervised hierarchical clustering showed that LNCaP-ARhi cells clustered together with VCaP cells, containing endogenous AR gene amplification and overexpression, indicating the central role of AR in the overall regulation of gene expression in prostate cancer cells. Two hundred forty genes showed >2-fold changes on DHT treatment in LNCaP-ARhi at 4 h time point, whereas only 164 and 52 showed changes in LNCaP-ARmo and LNCaP-pcDNA3.1, respectively. Many androgen-regulated genes were upregulated in LNCaP-ARhi at 10-fold lower concentration of DHT than in control cells. DHT (1 nmol/L) increased expression of several cell cycle-associated genes in LNCaP-ARhi cells. ChIP-on-chip assay revealed the presence of chromatin binding sites for AR within +/-200 kb of most of these genes. The growth of LNCaP-ARhi cells was also highly sensitive to cyclin-dependent kinase inhibitor, roscovitine, at 1nmol/L DHT. In conclusion, our results show that overexpression of AR sensitizes castration-resistant prostate cancer cells to the low levels of androgens. The activity of AR signaling pathway is regulated by the levels of both ligand and the receptor.

Androgen-regulated miR-32 targets BTG2 and is overexpressed in castration-resistant prostate cancer

The androgen receptor (AR) signaling pathway is involved in the emergence of castration-resistant prostate cancer (CRPC). Here, we identified several androgen-regulated microRNAs (miRNAs) that may contribute to the development of CRPC. Seven miRNAs, miR-21, miR-32, miR-99a, miR-99b, miR-148a, miR-221 and miR-590-5p, were found to be differentially expressed in CRPC compared with benign prostate hyperplasia (BPH) according to microarray analyses. Significant growth advantage for LNCaP cells transfected with pre-miR-32 and pre-miR-148a was found. miR-32 was demonstrated to reduce apoptosis, whereas miR-148a enhanced proliferation. Androgen regulation of miR-32 and miR-148a was confirmed by androgen stimulation of the LNCaP cells followed by expression analyses. The AR-binding sites in proximity of these miRNAs were demonstrated with chromatin immunoprecipitation (ChIP). To identify target genes for the miRNAs, mRNA microarray analyses were performed with LNCaP cells transfected with pre-miR-32 and pre-miR-148a. Expression of BTG2 and PIK3IP1 was reduced in the cells transfected with pre-miR-32 and pre-miR-148a, respectively. Also, the protein expression was reduced according to western blot analysis. BTG2 and PIK3IP1 were confirmed to be targets by 3′UTR-luciferase assays. Finally, immunostainings showed a statistically significant (P<0.0001) reduction of BTG2 protein in CRPCs compared with untreated prostate cancer (PC). The lack of BTG2 staining was also associated (P<0.01) with a short progression-free time in patients who underwent prostatectomy. In conclusion, androgen-regulated miR-32 is overexpressed in CRPC, leading to reduced expression of BTG2. Thus, miR-32 is a potential marker for aggressive disease and is a putative drug target in PC.

FoxA1 Specifies Unique Androgen and Glucocorticoid Receptor Binding Events in Prostate Cancer Cells

The forkhead protein FoxA1 has functions other than a pioneer factor, in that its depletion brings about a significant redistribution in the androgen receptor (AR) and glucocorticoid receptor (GR) cistromes. In this study, we found a novel function for FoxA1 in defining the cell-type specificity of AR- and GR-binding events in a distinct fashion, namely, for AR in LNCaP-1F5 cells and for GR in VCaP cells. We also found different, cell-type and receptor-specific compilations of cis-elements enriched adjacent to the AR- and GR-binding sites. The AR pathway is central in prostate cancer biology, but the role of GR is poorly known. We find that AR and GR cistromes and transcription programs exhibit significant overlap, and GR regulates a large number of genes considered to be AR pathway-specific. This raises questions about the role of GR in maintaining the AR pathway under androgen-deprived conditions in castration-resistant prostate cancer patients. However, in the presence of androgen, ligand-occupied GR acts as a partial antiandrogen and attenuates the AR-dependent transcription program. .

Impact of cytosine methylation on DNA binding specificities of human transcription factors.

Positives and negatives of methylated CpG When the DNA bases cytosine and guanine are next to each other, a methyl group is generally added to the pyrimidine, generating a mCpG dinucleotide. This modification alters DNA structure but can also affect function by inhibiting transcription factor (TF) binding. Yin et al. systematically analyzed the effect of CpG methylation on the binding of 542 human TFs (see the Perspective by Hughes and Lambert). In addition to inhibiting binding of some TFs, they found that mCpGs can promote binding of others, particularly TFs involved in development, such as homeodomain proteins. Science, this issue p. eaaj2239; see also p. 489 Genome-scale analysis reveals positive and negative binding of transcription factors to methylated CpG dinucleotides. INTRODUCTION Nearly all cells in the human body share the same primary genome sequence consisting of four nucleotide bases. One of the bases, cytosine, is commonly modified by methylation of its 5 position in CpG dinucleotides (mCpG). Most CpG dinucleotides in the human genome are methylated, but the level of CpG methylation varies with genetic location (promoter versus gene body), whether genes are active versus silenced, and cell type. Research has shown that the maintenance of a particular cellular state after cell division is dependent on faithful transmission of methylated CpGs, as well as inheritance of the mother cells’ repertoire of transcription factors by the daughter cells. These two mechanisms of epigenetic inheritance are linked to each other; the binding of transcription factors can be affected by cytosine methylation, and cytosine methylation can, in turn, be added or removed by proteins that associate with transcription factors. RATIONALE The genetic and epigenetic language, which imparts when and where genes are expressed, is understood at a conceptual level. However, a more detailed understanding is needed of the genomic regulatory mechanism by which methylated cytosines affect transcription factor binding. Because cytosine methylation changes DNA structure, it has the potential to affect binding of all transcription factors. However, a systematic analysis of binding of a large collection of transcription factors to all possible DNA sequences has not previously been conducted. RESULTS To globally characterize the effect of cytosine methylation on transcription factor binding, we systematically analyzed binding specificities of full-length transcription factors and extended DNA binding domains to unmethylated and CpG-methylated DNA by using methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment). We evaluated binding of 542 transcription factors and identified a large number of previously uncharacterized transcription factor recognition motifs. Binding of most major classes of transcription factors, including bHLH, bZIP, and ETS, was inhibited by mCpG. In contrast, transcription factors such as homeodomain, POU, and NFAT proteins preferred to bind methylated DNA. This class of binding was enriched in factors with central roles in embryonic and organismal development. The observed binding preferences were validated using several orthogonal methods, including bisulfite-SELEX and protein-binding microarrays. In addition, the preference of the pluripotency factor OCT4 to bind to a mCpG-containing motif was confirmed by chromatin immunoprecipitation analysis in mouse embryonic stem cells with low or high levels of CpG methylation (due to deficiency in all enzymes that methylate cytosines or contribute to their removal, respectively). Crystal structure analysis of the homeodomain proteins HOXB13, CDX1, CDX2, and LHX4 revealed three key residues that contribute to the preference of this developmentally important family of transcription factors for mCpG. The preference for binding to mCpG was due to direct hydrophobic interactions with the 5-methyl group of methylcytosine. In contrast, inhibition of binding of other transcription factors to methylated sequences was found to be caused by steric hindrance. CONCLUSION Our work constitutes a global analysis of the effect of cytosine methylation on DNA binding specificities of human transcription factors. CpG methylation can influence binding of most transcription factors to DNA—in some cases negatively and in others positively. Our finding that many developmentally important transcription factors prefer to bind to mCpG sites can inform future analyses of the role of DNA methylation on cell differentiation, chromatin reprogramming, and transcriptional regulation. Systematic analysis of the impact of CpG methylation on transcription factor binding. The bottom left panel shows the fraction of transcription factors that prefer methylated (orange) or unmethylated (teal) CpG sites, are affected in multiple ways (yellow), are not affected (green), or do not have a CpG in their motifs (gray), as determined by methylation-sensitive SELEX (top left). The structure and logos on the right highlight how HOXB13 recognizes mCpG (blue shading indicates a CpG affected by methylation). The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.

Overexpression of androgen receptor enhances the binding of the receptor to the chromatin in prostate cancer

Androgen receptor (AR) is overexpressed in the majority of castration-resistant prostate cancers (CRPCs). Our goal was to study the effect of AR overexpression on the chromatin binding of the receptor and to identify AR target genes that may be important in the emergence of CRPC. We have established two sublines of LNCaP prostate cancer (PC) cell line, one overexpressing AR 2–3-fold and the other 4–5-fold compared with the control cells. We used chromatin immunoprecipitation (ChIP) and deep-sequencing (seq) to identify AR-binding sites (ARBSs). We found that the number of ARBSs and the AR-binding strength were positively associated with the level of AR when cells were stimulated with low concentrations of androgens. In cells overexpressing AR, the chromatin binding of the receptor took place in 100-fold lower concentration of the ligand than in control cells. We confirmed the association of AR level and chromatin binding in two PC xenografts, one containing AR gene amplification with high AR expression, and the other with low expression. By combining the ChIP-seq and expression profiling, we identified AR target genes that are upregulated in PC. Of them, the expression of ZWINT, SKP2 (S-phase kinase-associated protein 2 (p45)) and FEN1 (flap structure-specific endonuclease 1) was demonstrated to be increased in CRPC, while the expression of SNAI2 was decreased in both PC and CRPC. FEN1 protein expression was also associated with poor prognosis in prostatectomy-treated patients. Finally, the knock-down of FEN1 with small interfering RNA inhibited the growth of LNCaP cells. Our data demonstrate that the overexpression of AR sensitizes the receptor binding to chromatin, thus, explaining how AR signaling pathway is reactivated in CRPC cells.

Androgen receptor and androgen-dependent gene expression in lung

The androgen receptor (AR) mediates the effects of male sex steroids. There are major sex differences in lung development and pathologies, including lung cancer. In this report, we show that Ar is mainly expressed in type II pneumocytes and the bronchial epithelium of murine lung and that androgen treatment increases AR protein levels in lung cells. Androgen administration altered significantly murine lung gene expression profiles; for example, by up-regulating transcripts involved in oxygen transport and down-regulating those in DNA repair and DNA recombination. Androgen exposure also affected the gene expression profile in a human lung adenocarcinoma-derived cell line, A549, by up- or down-regulating significantly some 200 transcripts, including down-regulation of genes involved in cell respiration. Dexamethasone treatment of A549 cells augmented expression of transcript sets that overlapped in part with those up-regulated by androgen in these cells. Moreover, a human lung cancer tissue array revealed that different lung cancer types are all AR-positive. Our results indicate that adult lung is an AR target tissue and suggest that AR plays a role in lung cancer biology.

Tissue‐specific pioneer factors associate with androgen receptor cistromes and transcription programs

Androgen receptor (AR) binds male sex steroids and mediates physiological androgen actions in target tissues. ChIP‐seq analyses of AR‐binding events in murine prostate, kidney and epididymis show that in vivo AR cistromes and their respective androgen‐dependent transcription programs are highly tissue specific mediating distinct biological pathways. This high order of tissue specificity is achieved by the use of exclusive collaborating factors in the three androgen‐responsive tissues. We find two novel collaborating factors for AR signaling in vivo—Hnf4α (hepatocyte nuclear factor 4α) in mouse kidney and AP‐2α (activating enhancer binding protein 2α) in mouse epididymis—that define tissue‐specific AR recruitment. In mouse prostate, FoxA1 serves for the same purpose. FoxA1, Hnf4α and AP‐2α motifs are over‐represented within unique AR‐binding loci, and the cistromes of these factors show substantial overlap with AR‐binding events distinct to each tissue type. These licensing or pioneering factors are constitutively bound to chromatin and guide AR to specific genomic loci upon hormone exposure. Collectively, liganded receptor and its DNA‐response elements are required but not sufficient for establishment of tissue‐specific transcription programs.

Determinants of Receptor- and Tissue-Specific Actions in Androgen Signaling

The physiological androgens testosterone and 5α-dihydrotestosterone regulate the development and maintenance of primary and secondary male sexual characteristics through binding to the androgen receptor (AR), a ligand-dependent transcription factor. In addition, a number of nonreproductive tissues of both genders are subject to androgen regulation. AR is also a central target in the treatment of prostate cancer. A large number of studies over the last decade have characterized many regulatory aspects of the AR pathway, such as androgen-dependent transcription programs, AR cistromes, and coregulatory proteins, mostly in cultured cells of prostate cancer origin. Moreover, recent work has revealed the presence of pioneer/licensing factors and chromatin modifications that are important to guide receptor recruitment onto appropriate chromatin loci in cell lines and in tissues under physiological conditions. Despite these advances, current knowledge related to the mechanisms responsible for receptor- and tissue-specific actions of androgens is still relatively limited. Here, we review topics that pertain to these specificity issues at different levels, both in cultured cells and tissues in vivo, with a particular emphasis on the nature of the steroid, the response element sequence, the AR cistromes, pioneer/licensing factors, and coregulatory proteins. We conclude that liganded AR and its DNA-response elements are required but are not sufficient for establishment of tissue-specific transcription programs in vivo, and that AR-selective actions over other steroid receptors rely on relaxed rather than increased stringency of cis-elements on chromatin.

Androgen receptor uses relaxed response element stringency for selective chromatin binding and transcriptional regulation in vivo

The DNA-binding domains (DBDs) of class I steroid receptors—androgen, glucocorticoid, progesterone and mineralocorticoid receptors—recognize a similar cis-element, an inverted repeat of 5′-AGAACA-3′ with a 3-nt spacer. However, these receptors regulate transcription programs that are largely receptor-specific. To address the role of the DBD in and of itself in ensuring specificity of androgen receptor (AR) binding to chromatin in vivo, we used SPARKI knock-in mice whose AR DBD has the second zinc finger replaced by that of the glucocorticoid receptor. Comparison of AR-binding events in epididymides and prostates of wild-type (wt) and SPARKI mice revealed that AR achieves selective chromatin binding through a less stringent sequence requirement for the 3′-hexamer. In particular, a T at position 12 in the second hexamer is dispensable for wt AR but mandatory for SPARKI AR binding, and only a G at position 11 is highly conserved among wt AR-preferred response elements. Genome-wide AR-binding events agree with the respective transcriptome profiles, in that attenuated AR binding in SPARKI mouse epididymis correlates with blunted androgen response in vivo. Collectively, AR-selective actions in vivo rely on relaxed rather than increased stringency of cis-elements on chromatin. These elements are, in turn, poorly recognized by other class I steroid receptors.