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FOXA1 acts upstream of GATA2 and AR in hormonal regulation of gene expression

View Article: PubMed Central - PubMed

ABSTRACT

Hormonal regulation of gene expression by androgen receptor (AR) is tightly controlled by many transcriptional cofactors, including pioneer factors FOXA1 and GATA2, which, however, exhibit distinct expression patterns and functional roles in prostate cancer. Here, we examined how FOXA1, GATA2, and AR crosstalk and regulate hormone-dependent gene expression in prostate cancer cells. ChIP-seq analysis revealed that FOXA1 reprograms both AR and GATA2 cistrome by preferably recruiting them to FKHD-containing genomic sites. By contrast, GATA2 is unable to shift AR or FOXA1 to GATA motifs. Rather, GATA2 co-occupancy enhances AR and FOXA1 binding to nearby ARE and FKHD sites, respectively. Similarly, AR increases, but not re-programs, GATA2 and FOXA1 cistromes. Concordantly, GATA2 and AR strongly enhance the transcriptional program of each other, whereas FOXA1 regulates GATA2- and AR-mediated gene expression in a context-dependent manner due to its reprogramming effects. Taken together, our data delineated for the first time the distinct mechanisms by which GATA2 and FOXA1 regulate AR cistrome and suggest that FOXA1 acts upstream of GATA2 and AR in determining hormone-dependent gene expression in prostate cancer.

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FOXA1 acts as a pioneer factor that reprograms GATA2 cistromeA. Western blot analysis showing a slight decrease of GATA2 protein level following FOXA1 knockdown, especially in the presence of androgen.B. Venn diagram showing greatly increased GTBS following FOXA1 knockdown. Control and FOXA1-knockdown (shFOXA1) LNCaP cells were hormone-deprived and subjected to GATA2 ChIP-seq.C–D. Heatmap view (C) and average intensity plot (D) of GATA2 ChIP-seq read intensity around the three categories of GTBS identified in B.E. FKHD and GATA motif intensity around the three categories of GTBS identified in B.F–G. ChIP-PCR confirming increased GATA2 binding to target genes PSA (F) and TMPRSS2 (G) following FOXA1 knockdown. Control and FOXA1-knockdown LNCaP cells in the absence or presence of androgen were subjected to FOXA1 and GATA2 ChIP followed by qPCR analysis. Data shown are mean ± SEM in triplicate qPCR and is a representative of at least two independent experiments.
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Figure 4: FOXA1 acts as a pioneer factor that reprograms GATA2 cistromeA. Western blot analysis showing a slight decrease of GATA2 protein level following FOXA1 knockdown, especially in the presence of androgen.B. Venn diagram showing greatly increased GTBS following FOXA1 knockdown. Control and FOXA1-knockdown (shFOXA1) LNCaP cells were hormone-deprived and subjected to GATA2 ChIP-seq.C–D. Heatmap view (C) and average intensity plot (D) of GATA2 ChIP-seq read intensity around the three categories of GTBS identified in B.E. FKHD and GATA motif intensity around the three categories of GTBS identified in B.F–G. ChIP-PCR confirming increased GATA2 binding to target genes PSA (F) and TMPRSS2 (G) following FOXA1 knockdown. Control and FOXA1-knockdown LNCaP cells in the absence or presence of androgen were subjected to FOXA1 and GATA2 ChIP followed by qPCR analysis. Data shown are mean ± SEM in triplicate qPCR and is a representative of at least two independent experiments.

Mentions: The effect of FOXA1 as a pioneer factor appears unique, in that it alters chromatin accessibility to re-distribute AR, reduce AR binding to high-affinity ARE sites, and dilute AR across the genome, overall attenuating AR binding events6. We next asked whether FOXA1 might regulate other transcription factors such as GATA2 in a similar fashion. We first conducted RNA interference of FOXA1 in androgen-deprived and –replenished LNCaP cells. Western blot analysis confirmed successful FOXA1 knockdown and demonstrated a clear decrease of GATA2 protein level, mainly in the presence of androgen (Figure 4A). To determine how FOXA1 regulates GATA2 cistrome, we first performed GATA2 ChIP-seq in control and FOXA1-knockdown cells in the absence of androgen. Data analysis revealed remarkably increased number of GTBS following FOXA1 knockdown; 30,546 new GTBS were identified, while only 8,162 GTBS were lost (Figure 4B). Further, heatmap view of ChIP-seq read intensity showed greatly increased GATA2 enrichment at the gained as well as conserved GTBS (Figure 4C). Average read intensity plots demonstrated that the gained GTBS were in average much stronger than the lost ones and that the conserved GTBS were enhanced by nearly 2 fold (Figure 4D). To investigate what mediates each type of GATA2 binding events we performed motif analysis. Our data showed that the lost GTBS (category I) were strongly enriched for FKHD motif, whereas the conserved (II) and gained GTBS (III) were mediated largely by GATA2 motif (Figure 4E), supporting that FOXA1 depletion led to a shift of GTBS from FKHD- to GATA-containing regions.


FOXA1 acts upstream of GATA2 and AR in hormonal regulation of gene expression
FOXA1 acts as a pioneer factor that reprograms GATA2 cistromeA. Western blot analysis showing a slight decrease of GATA2 protein level following FOXA1 knockdown, especially in the presence of androgen.B. Venn diagram showing greatly increased GTBS following FOXA1 knockdown. Control and FOXA1-knockdown (shFOXA1) LNCaP cells were hormone-deprived and subjected to GATA2 ChIP-seq.C–D. Heatmap view (C) and average intensity plot (D) of GATA2 ChIP-seq read intensity around the three categories of GTBS identified in B.E. FKHD and GATA motif intensity around the three categories of GTBS identified in B.F–G. ChIP-PCR confirming increased GATA2 binding to target genes PSA (F) and TMPRSS2 (G) following FOXA1 knockdown. Control and FOXA1-knockdown LNCaP cells in the absence or presence of androgen were subjected to FOXA1 and GATA2 ChIP followed by qPCR analysis. Data shown are mean ± SEM in triplicate qPCR and is a representative of at least two independent experiments.
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Figure 4: FOXA1 acts as a pioneer factor that reprograms GATA2 cistromeA. Western blot analysis showing a slight decrease of GATA2 protein level following FOXA1 knockdown, especially in the presence of androgen.B. Venn diagram showing greatly increased GTBS following FOXA1 knockdown. Control and FOXA1-knockdown (shFOXA1) LNCaP cells were hormone-deprived and subjected to GATA2 ChIP-seq.C–D. Heatmap view (C) and average intensity plot (D) of GATA2 ChIP-seq read intensity around the three categories of GTBS identified in B.E. FKHD and GATA motif intensity around the three categories of GTBS identified in B.F–G. ChIP-PCR confirming increased GATA2 binding to target genes PSA (F) and TMPRSS2 (G) following FOXA1 knockdown. Control and FOXA1-knockdown LNCaP cells in the absence or presence of androgen were subjected to FOXA1 and GATA2 ChIP followed by qPCR analysis. Data shown are mean ± SEM in triplicate qPCR and is a representative of at least two independent experiments.
Mentions: The effect of FOXA1 as a pioneer factor appears unique, in that it alters chromatin accessibility to re-distribute AR, reduce AR binding to high-affinity ARE sites, and dilute AR across the genome, overall attenuating AR binding events6. We next asked whether FOXA1 might regulate other transcription factors such as GATA2 in a similar fashion. We first conducted RNA interference of FOXA1 in androgen-deprived and –replenished LNCaP cells. Western blot analysis confirmed successful FOXA1 knockdown and demonstrated a clear decrease of GATA2 protein level, mainly in the presence of androgen (Figure 4A). To determine how FOXA1 regulates GATA2 cistrome, we first performed GATA2 ChIP-seq in control and FOXA1-knockdown cells in the absence of androgen. Data analysis revealed remarkably increased number of GTBS following FOXA1 knockdown; 30,546 new GTBS were identified, while only 8,162 GTBS were lost (Figure 4B). Further, heatmap view of ChIP-seq read intensity showed greatly increased GATA2 enrichment at the gained as well as conserved GTBS (Figure 4C). Average read intensity plots demonstrated that the gained GTBS were in average much stronger than the lost ones and that the conserved GTBS were enhanced by nearly 2 fold (Figure 4D). To investigate what mediates each type of GATA2 binding events we performed motif analysis. Our data showed that the lost GTBS (category I) were strongly enriched for FKHD motif, whereas the conserved (II) and gained GTBS (III) were mediated largely by GATA2 motif (Figure 4E), supporting that FOXA1 depletion led to a shift of GTBS from FKHD- to GATA-containing regions.

View Article: PubMed Central - PubMed

ABSTRACT

Hormonal regulation of gene expression by androgen receptor (AR) is tightly controlled by many transcriptional cofactors, including pioneer factors FOXA1 and GATA2, which, however, exhibit distinct expression patterns and functional roles in prostate cancer. Here, we examined how FOXA1, GATA2, and AR crosstalk and regulate hormone-dependent gene expression in prostate cancer cells. ChIP-seq analysis revealed that FOXA1 reprograms both AR and GATA2 cistrome by preferably recruiting them to FKHD-containing genomic sites. By contrast, GATA2 is unable to shift AR or FOXA1 to GATA motifs. Rather, GATA2 co-occupancy enhances AR and FOXA1 binding to nearby ARE and FKHD sites, respectively. Similarly, AR increases, but not re-programs, GATA2 and FOXA1 cistromes. Concordantly, GATA2 and AR strongly enhance the transcriptional program of each other, whereas FOXA1 regulates GATA2- and AR-mediated gene expression in a context-dependent manner due to its reprogramming effects. Taken together, our data delineated for the first time the distinct mechanisms by which GATA2 and FOXA1 regulate AR cistrome and suggest that FOXA1 acts upstream of GATA2 and AR in determining hormone-dependent gene expression in prostate cancer.

No MeSH data available.


Related in: MedlinePlus