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. 2009 Dec;23(12):2038-47.
doi: 10.1210/me.2009-0092. Epub 2009 Oct 21.

Upstream stimulatory factor 2, a novel FoxA1-interacting protein, is involved in prostate-specific gene expression

Qian Sun  1 , Xiuping Yu, David J Degraff, Robert J Matusik
Affiliations

Upstream stimulatory factor 2, a novel FoxA1-interacting protein, is involved in prostate-specific gene expression

Qian Sun et al. Mol Endocrinol. 2009 Dec.

Abstract

The forkhead protein A1 (FoxA1) is critical for the androgenic regulation of prostate-specific promoters. Prostate tissue rescued from FoxA1 knockout mice exhibits abnormal prostate development, typified by the absence of expression of differentiation markers and inability to engage in secretion. Chromatin immunoprecipitation and coimmunoprecipitation studies revealed that FoxA1 is one of the earliest transcription factors that binds to prostate-specific promoters, and that a direct protein-protein interaction occurs between FoxA1 and androgen receptor. Interestingly, evidence of the interaction of FoxA1 with other transcription factors is lacking. The upstream stimulatory factor 2 (USF2), an E-box-binding transcription factor of the basic-helix-loop-helix-leucine-zipper family, binds to a consensus DNA sequence similar to FoxA1. Our in vitro and in vivo studies demonstrate the binding of USF2 to prostate-specific gene promoters including the probasin promoter, spermine-binding protein promoter, and prostate-specific antigen core enhancer. Furthermore, we show a direct physical interaction between FoxA1 and USF2 through the use of immunoprecipitation and glutathione-S-transferase pull-down assays. This interaction is mediated via the forkhead DNA-binding domain of FoxA1 and the DNA-binding domain of USF2. In summary, these data indicate that USF2 is one of the components of the FoxA1/androgen receptor transcriptional protein complex that contributes to the expression of androgen-regulated and prostate-specific genes.

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Figures

Figure 1
Figure 1
Identification of USF2-binding site in PSA core enhancer. A, PSA1 EMSA probe. The probe PSA1 used in our EMSA experiments encompasses sequence located at −4122/−4109 bp of the PSA gene core enhancer, inclusive of AREIII and a FoxA1-binding site, which also contains the USF2-binding site. B, EMSA. LNCaP nuclear extracts were incubated with radiolabeled PSA1 probe. Strong complexes formed in lanes 2–5. The very top complex was shifted by USF2 antibody (lane 4), but not USF1 or GATA3 antibodies (lanes 3 and 5). N.E., Nuclear extract.
Figure 2
Figure 2
USF2 occupies PB promoter, SBP promoter, and PSA enhancer in vivo. A, Schematic diagram of the 5′-upstream regions of PB, SBP, and PSA genes. The diagram shows the amplified regions including distal regions (negative controls) and the USF2-binding regions. B, C, and D, ChIP. After an overnight IP with indicated antibodies, formaldehyde cross-linking was reversed, and DNA fragments were extracted, followed by real-time PCR amplification (see Materials and Methods).
Figure 3
Figure 3
IP. A, LNCaP cell lysates were immunoprecipitated with FoxA1 antibody (lane 3). Water (lane 1) and goat serum (lane 2) were used as negative controls. LNCaP cell lysate (Input) served as a positive control. Western blotting was performed using USF2 antibody. B, LNCaP cell lysates were immunoprecipitated with USF2 antibody (lanes 3, 4, and 5), water (lane 1), and rabbit IgG (lane 2, negative control). LNCaP cell lysate (Input) served as a positive control. EB was added to remove the influence of DNA presence on observed protein-protein interactions (0 μg/ml EB in lane 3, 50 μg/ml EB in lane 4, 100 μg/ml EB in lane 5). Western blot was performed using FoxA1 antibody. IB, Immunoblotting.
Figure 4
Figure 4
A and B, Schematic diagrams showing a series of FoxA1 and USF2 subdomains used in vitro GST pull-down assays. The FoxA1 FH domain and USF2 transcription activation domain are highlighted in black. Two conserved FoxA1 C-terminal domains designated as region 2 and 3 are in gray boxes (see Materials and Methods). C, Western blot using anti-V5 antibody shows eight in vitro synthesized V5-labled FoxA1 subdomains and a V5-labeled LacZ protein. D, Eight V5-labeled FoxA1 subdomains as well as a LacZ protein were synthesized in vitro and incubated with the GST-bound USF2 to identify the USF2-interacting region in FoxA1. E, Four purified truncated GST-USF2 fusion proteins were bound to glutathione agarose beads, followed by incubation with V5-labeled FoxA1 protein synthesized in vitro. Western blot was performed to identify the FoxA1-interacting domain in USF2. CT, C-terminal; FH, forkhead; IB, immunoblotting; NT, N-terminal.
Figure 5
Figure 5
A, USF2 knockdown in LNCaP cells. Western blotting shows an approximately 50% decrease in USF2 expression after 48 h knockdown, and an approximately 95% decrease after 72 h knockdown. B, Steady-state levels of PSA mRNA were determined after USF2 knockdown in LNCaP cells by quantitative real-time PCR. Total RNA was extracted from USF2 knockdown LNCaP cells and control LNCaP cells treated in the presence and absence of DHT.
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