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. 2009 Jan 15;69(2):709-17.
doi: 10.1158/0008-5472.CAN-08-3415.

Genome-wide promoter analysis of the SOX4 transcriptional network in prostate cancer cells

Christopher D Scharer  1 , Colleen D McCabe, Mohamed Ali-Seyed, Michael F Berger, Martha L Bulyk, Carlos S Moreno
Affiliations

Genome-wide promoter analysis of the SOX4 transcriptional network in prostate cancer cells

Christopher D Scharer et al. Cancer Res. .

Abstract

SOX4 is a critical developmental transcription factor in vertebrates and is required for precise differentiation and proliferation in multiple tissues. In addition, SOX4 is overexpressed in many human malignancies, but the exact role of SOX4 in cancer progression is not well understood. Here, we have identified the direct transcriptional _targets of SOX4 using a combination of genome-wide localization chromatin immunoprecipitation-chip analysis and transient overexpression followed by expression profiling in a prostate cancer model cell line. We have also used protein-binding microarrays to derive a novel SOX4-specific position-weight matrix and determined that SOX4 binding sites are enriched in SOX4-bound promoter regions. Direct transcriptional _targets of SOX4 include several key cellular regulators, such as EGFR, HSP70, Tenascin C, Frizzled-5, Patched-1, and Delta-like 1. We also show that SOX4 _targets 23 transcription factors, such as MLL, FOXA1, ZNF281, and NKX3-1. In addition, SOX4 directly regulates expression of three components of the RNA-induced silencing complex, namely Dicer, Argonaute 1, and RNA Helicase A. These data provide new insights into how SOX4 affects developmental signaling pathways and how these changes may influence cancer progression via regulation of gene networks involved in microRNA processing, transcriptional regulation, the TGFbeta, Wnt, Hedgehog, and Notch pathways, growth factor signaling, and tumor metastasis.

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Figures

Figure 1
Figure 1
(A) Affymetrix U133A GeneChip microarray analysis of SOX4 overexpression and knockdown in LNCaP prostate cancer cells. Overexpression of SOX4 leads to increased EGFR expression while siRNA knockdown of SOX4 results in decreased EGFR expression. (B) Schematic showing the location of the SOX4 binding site in the first intron of the EGFR (Top) and ERBB2 (Bottom) genes. Arrows denote location of the SOX4 binding site. (C) ChIP assay of FLAG-SOX4 bound to the introns of EGFR, ERBB2 and TLE1 PSMA is shown as a negative control. SOX4 bound DNA is specifically amplified in the FLAG IP lane from FLAG-SOX4 expressing cells (lane 3) and not control cells (lane 5) or with a non-specific antibody (lanes 2 and 4). (D) Luciferase reporter assays with SOX4 binding sites showing activation in the presence of SOX4 compared to empty vector. * indicates p-value less than 0.01 by students T-test and error bars indicate 1 SD (n = 3 independent biological replicates performed on separate days).
Figure 2
Figure 2
(A) Graph showing enrichment in the three HA-SOX4 lanes over the average of the two YFP replicates for the SOX4 _target gene FMO4 Y-axis is the signal intensity across the genomic coordinates on the X-axis. (B) qPCR ChIP analysis of 10 randomly selected genes verified in both the RWPE-1 and LNCaP cell lines. Graph shows fold enrichment of the HA-SOX4 IP over the YFP negative control IP. Numbers above the bars represent the mean log2 of fold enrichment of ChIP-chip signal for the probes contained in the peak relative to YFP. Error bars indicate 1 SD (n = 3 technical replicates). (C) and (D) Genes that were verified by conventional ChIP assay. HA-SOX4 and YFP cells were subjected to conventional ChIP followed by PCR in both the LNCaP (C) and RWPE-1 (D) prostate cell lines. Six genes verified in the LNCaP cell lines and five in the RWPE-1 cell lines.
Figure 3
Figure 3
(A) Heat map (top) illustrating Illumina expression data of the 1,766 significant genes as determined by SAM analysis. Red indicates overexpressed and green denotes underexpressed genes. Venn diagram (bottom) depicts the overlap between 3,470 ChIP-chip SOX4 direct _target genes, the Illumina expression data set of 1,766 genes, and the Affymetrix expression dataset of 465 genes. (B) qPCR expression analysis of SOX4 direct _target genes after SOX4 overexpression in LNCaP cells. All ten genes were upregulated over a vector control transfection, similar to values determined by the Illumina array with a p-value less than 0.005 by students T-test. Error bars indicate 1 SD (n = 3 independent biological replicates performed on separate days). (C)DICER protein expression is upregulated by SOX4 HA-SOX4 or vector control were transfected into LNCaP cells and immunoblots were probed for DICER, SOX4, and PP2Ac as a loading control. (D) PBM-derived 8mer PWM for SOX4 displayed both graphically and numerically for each base position derived from incubation of recombinant GST-SOX4-DBD with a universal ‘all 8-mer’ double-stranded DNA protein-binding microarray. With stringent criteria (core similarity ≥ 0.85, matrix similarity ≥ 0.75) we find 60% of the peaks in the 282 high-confidence promoters contain SOX4 binding sites.
Figure 4
Figure 4
IPA analysis of direct _target genes graphically illustrating the cellular location of the SOX4 transcriptional _target genes. SOX4 regulates a host of nuclear and membrane localized proteins as well as multiple components of the RISC complex. Red indicates _target genes upregulated by SOX4, green denotes downregulated genes and white represents genes for which no expression change was detected.
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