Promoter‐wide analysis of Smad4 binding sites in human epithelial cells

Abstract

Smad4, the common partner Smad, is a key molecule in transforming growth factor-b (TGF-b) family signaling. Loss of Smad4 expression is found in several types of cancer, including pancreatic cancer and colon cancer, and is related to carcinogenesis. Here we identified Smad4 binding sites in the promoter regions of over 25 500 known genes by chromatin immunoprecipitation on a microarray (ChIP-chip) in HaCaT human keratinocytes. We identified 925 significant Smad4 binding sites. Approximately half of the identified sites overlapped the binding regions of Smad2 and Smad3 (Smad2 ⁄ 3, receptor-regulated Smads in TGF-b signaling), while the rest of the regions appeared dominantly occupied by Smad4 even when a different identification threshold for Smad2 ⁄ 3 binding regions was used. Distribution analysis showed that Smad4 was found in the regions relatively distant from the transcription start sites, while Smad2 ⁄ 3 binding regions were more often present near the transcription start sites. Motif analysis also revealed that activator protein 1 (AP-1) sites were especially enriched in the sites common to Smad2 ⁄ 3 and Smad4 binding regions. In contrast, GC-rich motifs were enriched in Smad4-dominant binding regions. We further determined putative target genes of Smad4 whose expression was regulated by TGF-b. Our findings revealed some general characteristics of Smad4 binding regions, and provide resources for examining the role of Smad4 in epithelial cells and cancer pathogenesis. (Cancer Sci 2009; 100: 2133-2142 M embers of the transforming growth factor-b (TGF-b) family are multifunctional proteins that regulate various biological processes. They play critical roles in cell growth, differentiation, apoptosis, motility, epithelial-to-mesenchymal transition (EMT), and extracellular matrix production. TGF-b family ligands transduce signals through heteromeric complexes of type I and type II serine ⁄ threonine kinase receptors and intracellular Smad proteins.(1,2) After ligand binding, type II receptors phosphorylate type I receptors, which then phosphorylate receptor-regulated Smads (R-Smads) at the C-terminal SSXS motif. Smad2 and Smad3 (Smad2 ⁄ 3) are TGF-b ⁄ activin ⁄ nodalspecific R-Smads, whereas Smad1, Smad5, and Smad8 (Smad1 ⁄ 5 ⁄ 8) are bone morphogenetic protein (BMP)-specific R-Smads. Phosphorylated R-Smads then form oligomers with a common partner Smad (Co-Smad), Smad4, and translocate to the nucleus where they regulate transcription of target genes. Previous reports have revealed an indispensable role of Smad4 in TGF-b-induced expression of a subset of target genes.

Transforming Growth Factor-β/SMAD Target Gene SKIL Is Negatively Regulated by the Transcriptional Cofactor Complex SNON-SMAD4

“…6C). TGF-␤ was also able to active 3TP-Lux in a SMAD4-dependent manner (data not shown) as reported previously (43,44). Intriguingly, SNON could not repress its expression when SMAD4 was absent, and as a consequence, the levels of SNON mRNA as well as the activity of SKIL promoter were increased (Fig.…”

“…SNON and SKI do not possess DNA binding ability, and they seem to be recruited to TGF-␤-responsive gene promoters through their interaction with SMAD proteins, mainly S4 (41,42). Moreover, the SKIL gene seems to be induced by TGF-␤ in either an S4-dependent or S4-independent manner (43,44).…”

Disruption of bone morphogenetic protein receptor 2 (BMPR2) in mammary tumors promotes metastases through cell autonomous and paracrine mediators

“…BMPs have been shown to be global regulators of transcriptional networks, including the repression or silencing of genes (33). We find here that BMP4 reduces the expression of CCL9 (Fig.…”

Transcriptional Control by the SMADs

“…A single SBE is not sufficient to bind an activated SMAD complex, and, in vivo, SBEs have been shown to occur as direct or inverted ( palindromic) repeats in the regulatory regions of TGF-b/NODAL/activin target genes by promoter analysis, or by whole-genome approaches, such as chromatin immunoprecipitation (ChIP)-sequencing or ChIP-on-chip analysis (Fig. 1A) (Dennler et al 1998;Wong et al 1999;Koinuma et al 2009a;Liu et al 2011;Mullen et al 2011;Morikawa et al 2013).…”