Smad6 Represses Dlx3 Transcriptional Activity through Inhibition of DNA Binding

Abstract

Dlx3 (Distal-less 3) is a homeobox-containing transcription factor required for normal placental development in mice. Here we demonstrate that Dlx3 interacts with Smad6, a member of a larger family of transcriptional regulators generally thought to regulate transforming growth factor ␤/bone morphogenetic protein signaling. Immunocytochemical and immunoprecipitation studies demonstrate overlapping nuclear localization and physical interaction between Dlx3 and Smad6 in human choriocarcinoma cells and in differentiated trophoblasts from human placenta. In vitro protein interaction studies mapped the Smad6 interaction domain within Dlx3 to residues 80 -163, a region of Dlx3 that includes a portion of the homeodomain. Dlx3 and Dlx4 share homology within this region, and Dlx4 was also found to bind Smad6. Using the Esx1 gene promoter as a model for a Dlx3-responsive gene, studies demonstrate two near consensus Dlx3 binding sites within the proximal 2.3 kb of the transcription start site. Interestingly, binding of Dlx3 to one of these two sites was inhibited by interaction with Smad6. Consistent with this result, expression of an Esx1 promoter luciferase reporter was increased by overexpression of Dlx3; this effect was reversed with co-expression of Smad6. Further, small interference RNAmediated knockdown of endogenous Smad6 increased Dlx3-dependent expression of the Esx1 gene promoter. Thus, Smad6 appears to functionally interact with Dlx3, altering the ability of Dlx3 to bind target gene promoters. Smad6 appears to play a modulatory role in the regulation of Dlx3-dependent gene transcription within placental trophoblasts.

A 4 bp deletion mutation in DLX3 enhances osteoblastic differentiation and bone formation in vitro

“…Non radioisotope EMSA was performed with digoxigenin-11-dUTP (DIG)-labeled probes and gel shift kit. Briefly, double strand oligonucleotides for the wild type DLX3 homeobox domain binding element (wTT-HDBE) consensus sequence (5'-ATG ACC CCC AAT TAG TCC TGG CAG-3') and the mutant HD binding element (mGG-HDBE) consensus sequence (5'-ATG ACC CCC AAG GAG TCC TGG CAG-3') [13,21] were labeled with DIG using a terminal transferase. wTT-HDBE or mGG-HDBE consensus sequence probes (30 fmol) and nuclear protein (5 ug) were incubated in 10 ul of binding buffer containing 0.5 μg of poly(dI-dC) and 0.05 ug of L-lysine for 25 minutes at room temperature.…”

“…To test whether MT-DLX3 protein translocated into the nucleus can bind the DLX3 homeobox domain binding element (HDBE), (13,21) we performed electrophoresis mobility shift assays (EMSA) using non radioisotope labeled probe with the HDBE consensus sequence and nuclear extracts from C2C12 cells stably transfected with EV, WT-DLX3 or MT-DLX3 cDNA. As shown in figure 4 …”

Analysis of the Gene Regulatory Program Induced by the Homeobox Transcription Factor Distal-less 3 in Mouse Placenta

“…␤-Actin was used as the endogenous control for normalization of cDNA input. Relative quantification of gene expression was determined by the 2 -(⌬⌬Ct) method after first determining that the amplification efficiency of all targets and ␤-actin was identical (13). We also used commercially available ABI TaqMan gene expression assays to quantify synuclein-␣ (Snca) expression levels as well as confirm the specificity of the Roche probe sets to the hemoglobin gene family members Hba-a1, Hba-x, and Hbb-y (listed in Table 2).…”

“…Membranes were blocked using Trisbuffered saline [10 mm Tris (pH 7.6) and 150 mm NaCl] containing 0.1% Tween 20 and 5% nonfat dried milk. After 1 h of blocking, membranes were incubated with a rabbit polyclonal Dlx3 antiserum or ␤-actin as previously described (13), and protein bands representing ectopic expression were visualized using enhanced chemiluminescence (PerkinElmer, Boston, MA).…”

“…Recombinant Dlx3 was prepared by in vitro transcription and translation using a wheat germ lysate kit commercially available (Promega) for use in EMSA studies. Nuclear extracts were prepared from JEG3 choriocarcinoma cells and used in EMSA as described previously (13,14).…”