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Integrated analysis identifies a class of androgen-responsive genes regulated by short combinatorial long-range mechanism facilitated by CTCF.

Taslim C, Chen Z, Huang K, Huang TH, Wang Q, Lin S - Nucleic Acids Res. (2012)

Bottom Line: In this study, we carried out an integrated analysis combining several types of high-throughput data, including genome-wide distribution data of H3K4 di-methylation (H3K4me2), CCCTC binding factor (CTCF), AR and FoxA1 cistrome data as well as androgen-regulated gene expression data.We found that a subset of androgen-responsive genes was significantly enriched near AR/H3K4me2 overlapping regions and FoxA1 binding sites within the same CTCF block.Our results suggest a relatively short combinatorial long-range regulation mechanism facilitated by CTCF blocking.

View Article: PubMed Central - PubMed

Affiliation: Department of Statistics, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Recently, much attention has been given to elucidate how long-range gene regulation comes into play and how histone modifications and distal transcription factor binding contribute toward this mechanism. Androgen receptor (AR), a key regulator of prostate cancer, has been shown to regulate its target genes via distal enhancers, leading to the hypothesis of global long-range gene regulation. However, despite numerous flows of newly generated data, the precise mechanism with respect to AR-mediated long-range gene regulation is still largely unknown. In this study, we carried out an integrated analysis combining several types of high-throughput data, including genome-wide distribution data of H3K4 di-methylation (H3K4me2), CCCTC binding factor (CTCF), AR and FoxA1 cistrome data as well as androgen-regulated gene expression data. We found that a subset of androgen-responsive genes was significantly enriched near AR/H3K4me2 overlapping regions and FoxA1 binding sites within the same CTCF block. Importantly, genes in this class were enriched in cancer-related pathways and were downregulated in clinical metastatic versus localized prostate cancer. Our results suggest a relatively short combinatorial long-range regulation mechanism facilitated by CTCF blocking. Under such a mechanism, H3K4me2, AR and FoxA1 within the same CTCF block combinatorially regulate a subset of distally located androgen-responsive genes involved in prostate carcinogenesis.

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Related in: MedlinePlus

Genes with AR in the same block exhibit higher levels of gene expression fold change. (A) Illustrations of two types of blocks that are being compared in (B). AR-ARG block is defined as block with both AR binding site (purple box) and androgen-responsive gene (purple arrow). Nearby non-AR block is defined as block adjacent to AR-ARG blocks without AR binding site. (B) Expression fold-change of all genes in AR-ARG blocks (blocks with androgen-responsive gene and AR binding site) are significantly higher than genes in nearby non-AR blocks. (C) Heatmap showing the log2 fold-change of expression level of all genes in nearby non-AR blocks (black bar), genes in upAR-ARG blocks (blocks with upregulated androgen-responsive gene and AR, red bar) and genes in downAR-ARG blocks (blocks with downregulated androgen-responsive genes and AR, green bar). Color represents log2 fold-change of expression level of genes after 4 h DHT versus 0 hr DHT (basal level). (D) Expression level of androgen-responsive genes in AR blocks are significantly higher than those in blocks without AR (non-AR blocks). (E) Silencing of CTCF decreases CTCF protein expression. LNCaP cells were transfected with siControl or siCTCF, and treated with vehicle or DHT for 4 h. Western blots were performed using antibodies indicated. (F) Silencing of CTCF decreases CTCF binding at the CTCF blocks with AR (regions 1–4) and without AR (regions 5–6). LNCaP cells were transfected with siControl or siCTCF, and stimulated with vehicle or DHT. ChIP assays were performed using antibodies against CTCF. (G) Silencing of CTCF significantly decreases expression fold changes of responsive genes in blocks with AR. siControl or siCTCF transfected LNCaP cells were stimulated with vehicle or DHT for 4 h. Total RNA was isolated and amplified with gene-specific primers.
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gks139-F2: Genes with AR in the same block exhibit higher levels of gene expression fold change. (A) Illustrations of two types of blocks that are being compared in (B). AR-ARG block is defined as block with both AR binding site (purple box) and androgen-responsive gene (purple arrow). Nearby non-AR block is defined as block adjacent to AR-ARG blocks without AR binding site. (B) Expression fold-change of all genes in AR-ARG blocks (blocks with androgen-responsive gene and AR binding site) are significantly higher than genes in nearby non-AR blocks. (C) Heatmap showing the log2 fold-change of expression level of all genes in nearby non-AR blocks (black bar), genes in upAR-ARG blocks (blocks with upregulated androgen-responsive gene and AR, red bar) and genes in downAR-ARG blocks (blocks with downregulated androgen-responsive genes and AR, green bar). Color represents log2 fold-change of expression level of genes after 4 h DHT versus 0 hr DHT (basal level). (D) Expression level of androgen-responsive genes in AR blocks are significantly higher than those in blocks without AR (non-AR blocks). (E) Silencing of CTCF decreases CTCF protein expression. LNCaP cells were transfected with siControl or siCTCF, and treated with vehicle or DHT for 4 h. Western blots were performed using antibodies indicated. (F) Silencing of CTCF decreases CTCF binding at the CTCF blocks with AR (regions 1–4) and without AR (regions 5–6). LNCaP cells were transfected with siControl or siCTCF, and stimulated with vehicle or DHT. ChIP assays were performed using antibodies against CTCF. (G) Silencing of CTCF significantly decreases expression fold changes of responsive genes in blocks with AR. siControl or siCTCF transfected LNCaP cells were stimulated with vehicle or DHT for 4 h. Total RNA was isolated and amplified with gene-specific primers.

Mentions: CTCF has been touted as an enhancer insulator in the literature. For example, Chan and Song (21) provided some evidence that CTCF can block distal action of ER. We set out to find whether CTCF has a similar role in relation to AR by trying to obtain answers to the following questions: Does AR potentially regulate genes in the same block? Can AR also regulate genes in nearby blocks? To this end, we compared the log fold change of the expression levels of all genes in ‘AR-ARG blocks’ to the log fold change of the expression of all genes in ‘nearby-no-AR blocks’ (i.e. blocks nearest to AR-ARG blocks but not containing AR themselves). The log fold change is computed from the expression levels of genes after 4-h of DHT stimulation versus vehicle control. To exclude genes that are too far to be regulated by AR, we filter out genes that are more than 100 kb away from any AR binding site. We have 1064 total genes in 296 ‘AR-ARG blocks’ and 71 total genes in 58 ‘nearby-no-AR blocks’. Figure 2A provides an illustration of the genes in the two types of blocks being compared (i.e. all genes in ‘AR-ARG blocks’ versus all genes in ‘nearby-no-AR blocks’). Figure 2B shows that the change of expression levels of all genes in ‘AR-ARG blocks’ is significantly higher than genes located in ‘nearby-no-AR blocks’ (Mann–Whitney test, P-value = 1.42 × 10−5). Figure 2C shows the individual gene expression fold change in ‘upAR-ARG blocks’ (red bar, ‘AR blocks with up-regulated gene’), ‘downAR-ARG blocks’ (green bar, ‘AR blocks with down-regulated gene’) and in ‘nearby-no-AR blocks’ (black bar, ‘blocks without AR nearest to AR-ARG blocks’). Although there are some responsive genes, in general genes in ‘nearby-no-AR blocks’ do not show differential expression (with black lines spread out through the entire range), whereas genes in ‘upAR-ARG blocks’ and in ‘downAR-ARG blocks’ mostly show high and low expression level, respectively. This indicates that distal AR may regulate genes within the same ‘CTCF block’. Consistent with this notion, we have experimentally demonstrated that 4 distal AR binding sites located at −12 kb, −14 kb, −20 kb and −73 kb away from transcription start site (TSS) of TMPRSS2 gene form chromatin loops with the TMPRSS2 promoter within the same CTCF block (10).Figure 2.


Integrated analysis identifies a class of androgen-responsive genes regulated by short combinatorial long-range mechanism facilitated by CTCF.

Taslim C, Chen Z, Huang K, Huang TH, Wang Q, Lin S - Nucleic Acids Res. (2012)

Genes with AR in the same block exhibit higher levels of gene expression fold change. (A) Illustrations of two types of blocks that are being compared in (B). AR-ARG block is defined as block with both AR binding site (purple box) and androgen-responsive gene (purple arrow). Nearby non-AR block is defined as block adjacent to AR-ARG blocks without AR binding site. (B) Expression fold-change of all genes in AR-ARG blocks (blocks with androgen-responsive gene and AR binding site) are significantly higher than genes in nearby non-AR blocks. (C) Heatmap showing the log2 fold-change of expression level of all genes in nearby non-AR blocks (black bar), genes in upAR-ARG blocks (blocks with upregulated androgen-responsive gene and AR, red bar) and genes in downAR-ARG blocks (blocks with downregulated androgen-responsive genes and AR, green bar). Color represents log2 fold-change of expression level of genes after 4 h DHT versus 0 hr DHT (basal level). (D) Expression level of androgen-responsive genes in AR blocks are significantly higher than those in blocks without AR (non-AR blocks). (E) Silencing of CTCF decreases CTCF protein expression. LNCaP cells were transfected with siControl or siCTCF, and treated with vehicle or DHT for 4 h. Western blots were performed using antibodies indicated. (F) Silencing of CTCF decreases CTCF binding at the CTCF blocks with AR (regions 1–4) and without AR (regions 5–6). LNCaP cells were transfected with siControl or siCTCF, and stimulated with vehicle or DHT. ChIP assays were performed using antibodies against CTCF. (G) Silencing of CTCF significantly decreases expression fold changes of responsive genes in blocks with AR. siControl or siCTCF transfected LNCaP cells were stimulated with vehicle or DHT for 4 h. Total RNA was isolated and amplified with gene-specific primers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3367180&req=5

gks139-F2: Genes with AR in the same block exhibit higher levels of gene expression fold change. (A) Illustrations of two types of blocks that are being compared in (B). AR-ARG block is defined as block with both AR binding site (purple box) and androgen-responsive gene (purple arrow). Nearby non-AR block is defined as block adjacent to AR-ARG blocks without AR binding site. (B) Expression fold-change of all genes in AR-ARG blocks (blocks with androgen-responsive gene and AR binding site) are significantly higher than genes in nearby non-AR blocks. (C) Heatmap showing the log2 fold-change of expression level of all genes in nearby non-AR blocks (black bar), genes in upAR-ARG blocks (blocks with upregulated androgen-responsive gene and AR, red bar) and genes in downAR-ARG blocks (blocks with downregulated androgen-responsive genes and AR, green bar). Color represents log2 fold-change of expression level of genes after 4 h DHT versus 0 hr DHT (basal level). (D) Expression level of androgen-responsive genes in AR blocks are significantly higher than those in blocks without AR (non-AR blocks). (E) Silencing of CTCF decreases CTCF protein expression. LNCaP cells were transfected with siControl or siCTCF, and treated with vehicle or DHT for 4 h. Western blots were performed using antibodies indicated. (F) Silencing of CTCF decreases CTCF binding at the CTCF blocks with AR (regions 1–4) and without AR (regions 5–6). LNCaP cells were transfected with siControl or siCTCF, and stimulated with vehicle or DHT. ChIP assays were performed using antibodies against CTCF. (G) Silencing of CTCF significantly decreases expression fold changes of responsive genes in blocks with AR. siControl or siCTCF transfected LNCaP cells were stimulated with vehicle or DHT for 4 h. Total RNA was isolated and amplified with gene-specific primers.
Mentions: CTCF has been touted as an enhancer insulator in the literature. For example, Chan and Song (21) provided some evidence that CTCF can block distal action of ER. We set out to find whether CTCF has a similar role in relation to AR by trying to obtain answers to the following questions: Does AR potentially regulate genes in the same block? Can AR also regulate genes in nearby blocks? To this end, we compared the log fold change of the expression levels of all genes in ‘AR-ARG blocks’ to the log fold change of the expression of all genes in ‘nearby-no-AR blocks’ (i.e. blocks nearest to AR-ARG blocks but not containing AR themselves). The log fold change is computed from the expression levels of genes after 4-h of DHT stimulation versus vehicle control. To exclude genes that are too far to be regulated by AR, we filter out genes that are more than 100 kb away from any AR binding site. We have 1064 total genes in 296 ‘AR-ARG blocks’ and 71 total genes in 58 ‘nearby-no-AR blocks’. Figure 2A provides an illustration of the genes in the two types of blocks being compared (i.e. all genes in ‘AR-ARG blocks’ versus all genes in ‘nearby-no-AR blocks’). Figure 2B shows that the change of expression levels of all genes in ‘AR-ARG blocks’ is significantly higher than genes located in ‘nearby-no-AR blocks’ (Mann–Whitney test, P-value = 1.42 × 10−5). Figure 2C shows the individual gene expression fold change in ‘upAR-ARG blocks’ (red bar, ‘AR blocks with up-regulated gene’), ‘downAR-ARG blocks’ (green bar, ‘AR blocks with down-regulated gene’) and in ‘nearby-no-AR blocks’ (black bar, ‘blocks without AR nearest to AR-ARG blocks’). Although there are some responsive genes, in general genes in ‘nearby-no-AR blocks’ do not show differential expression (with black lines spread out through the entire range), whereas genes in ‘upAR-ARG blocks’ and in ‘downAR-ARG blocks’ mostly show high and low expression level, respectively. This indicates that distal AR may regulate genes within the same ‘CTCF block’. Consistent with this notion, we have experimentally demonstrated that 4 distal AR binding sites located at −12 kb, −14 kb, −20 kb and −73 kb away from transcription start site (TSS) of TMPRSS2 gene form chromatin loops with the TMPRSS2 promoter within the same CTCF block (10).Figure 2.

Bottom Line: In this study, we carried out an integrated analysis combining several types of high-throughput data, including genome-wide distribution data of H3K4 di-methylation (H3K4me2), CCCTC binding factor (CTCF), AR and FoxA1 cistrome data as well as androgen-regulated gene expression data.We found that a subset of androgen-responsive genes was significantly enriched near AR/H3K4me2 overlapping regions and FoxA1 binding sites within the same CTCF block.Our results suggest a relatively short combinatorial long-range regulation mechanism facilitated by CTCF blocking.

View Article: PubMed Central - PubMed

Affiliation: Department of Statistics, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Recently, much attention has been given to elucidate how long-range gene regulation comes into play and how histone modifications and distal transcription factor binding contribute toward this mechanism. Androgen receptor (AR), a key regulator of prostate cancer, has been shown to regulate its target genes via distal enhancers, leading to the hypothesis of global long-range gene regulation. However, despite numerous flows of newly generated data, the precise mechanism with respect to AR-mediated long-range gene regulation is still largely unknown. In this study, we carried out an integrated analysis combining several types of high-throughput data, including genome-wide distribution data of H3K4 di-methylation (H3K4me2), CCCTC binding factor (CTCF), AR and FoxA1 cistrome data as well as androgen-regulated gene expression data. We found that a subset of androgen-responsive genes was significantly enriched near AR/H3K4me2 overlapping regions and FoxA1 binding sites within the same CTCF block. Importantly, genes in this class were enriched in cancer-related pathways and were downregulated in clinical metastatic versus localized prostate cancer. Our results suggest a relatively short combinatorial long-range regulation mechanism facilitated by CTCF blocking. Under such a mechanism, H3K4me2, AR and FoxA1 within the same CTCF block combinatorially regulate a subset of distally located androgen-responsive genes involved in prostate carcinogenesis.

Show MeSH
Related in: MedlinePlus