Mapping of a minimal BRE in Smad7 BRE-1 and identification of putative Smad binding sites. (A) Various fragments derived from the 233-bp BRE-1 were amplified by PCR and subcloned into the pGL3 vector, upstream of the E1b promoter and luciferase reporter gene. Diagrammed constructs were transiently transfected into HepG2 cells, with 0.03 μg of Smad1 and Smad4 cDNA/well, incubated overnight in the absence or presence of 5 nM BMP-7. The relative luciferase activities of cell lysates were determined. The induction (n-fold) by BMP-7 is presented. Three important regions of BRE-1 were delineated and termed box 1, box 2, and box 3. (B) Box 1 and box 2 of BRE-1 are aligned with fragments of the Dpp response elements of the tinman and Ubx genes, and BREs of the Xvent-2B, Dlx3, OPN, and Id1 promoters. Putative Smad4 binding sites (CAGC boxes) are highlighted. (C) Box 2 and box 3 of Smad7 BRE-1 are aligned to a region of the vestigial quadrant enhancer (vgQ) known to bind Mad and regions of the Ubx (72) and Smad6 promoters containing Smad1 binding sites. The MAD/Smad1 binding site sequence is highlighted.

I-BRE is a GATA- and Smad-dependent element. (A) Diagrammed clones derived from I-BRE were transiently transfected in HepG2 cells. Cells were incubated overnight in the absence (white bars) or presence (black bars) of 5 nM BMP-7. The relative luciferase activities of cell lysates were determined. Mutations created in I-BRE are indicated in bold letters under the appropriate sequences. The GATA binding sites are indicated. (B) I-BRE was ³²P-labeled and incubated with lysates of 293T cells transfected with GATA-5 and -6 cDNA. Mutations in the I-BRE probe (M1, M2, and M1/M2) correspond to mutations in panel A. +, present; −, absent. (C) Wild-type (WT) and M1/M2 mutant I-BRE (as shown in panel A) were transfected in MEF Smad4^−/− cells in the presence or absence of exogenous Smad4 cDNA. Cells were incubated overnight in the absence (white bars) or presence (black bars) of BMP-7. The relative luciferase activities of cell lysates were determined.

Exogenous expression of GATA-1, -5, and -6 rescue the I-BRE in a GATA-deficient cell line. (A) The I-BRE luciferase reporter construct was transiently transfected in Neuro-2A cells with increasing concentrations of GATA-1, -2, -3, -4, -5, or -6 cDNA. Cells were incubated overnight in the absence (white bars) or presence (black bars) of 5 nM BMP-7. The relative luciferase activities of cell lysates were determined. (B) I-BRE constructs, with wild-type (WT) sequence or GATA and HFH/HNF3 site mutations, were transiently transfected in Neuro-2A cells with GATA-5 cDNA. Cells were incubated overnight in the absence (white bars) or presence (black bars) of 5 nM BMP-7. The relative luciferase activities of cell lysates were determined. Mutations created in I-BRE are indicated in bold letters under the appropriate sequences, and the GATA binding sites are highlighted. (C) HepG2 cells were transiently transfected with the I-BRE reporter construct and siRNA against GATA-1, -5, and -6 as well as a scrambled siRNA. Cells were incubated overnight in the absence (white bars) or presence (black bars) of 5 nM BMP-7. The relative luciferase activities of cell lysates were determined at 72 h posttransfection.

BRE-1 and I-BRE respond to different concentrations of BMP-7. (A) ChIP of Smad1 bound to I-BRE and BRE-1. HepG2 cells were treated for 1 h with increasing concentrations of BMP-7. Protein-DNA complexes were cross-linked by formaldehyde treatment, cells were lysed, and DNA was sheared by sonication. Cell lysates were subjected to immunoprecipitation (IP) with an anti-Smad1 antibody (αSmad1 Ab). DNA recovered from the immunoprecipitation was quantified by real-time PCR. Diagrammed representations of PCR products are presented in Fig. 3A and 4A. PI, preimmune; −, absent. (B) The BRE-1 and I-BRE luciferase reporter constructs were transiently transfected in HepG2 and Neuro-2A cells in the presence or absence of 0.03 μg of Smad1 and Smad4 cDNA/well. Cells were incubated overnight with increasing concentrations of BMP-7. The relative luciferase activities of cell lysates were determined. (C) Smad7 expression is induced by different concentrations of BMP-7 in HepG2 and Neuro-2A cells. RNA was extracted from HepG2 (black bars) and Neuro-2A (gray bars) cells treated for 1 h with increasing concentrations of BMP-7. The RNA was reverse transcribed, and cDNA for Smad7 was quantified by real-time PCR. Levels of hypoxanthine phosphoribosyltransferase cDNA were used to normalize the results.

Mapping of specific BRE regulating the Smad7 gene. (A) A schematic representation of a 5.4-kbp genomic fragment cloned from a lambda phage genomic DNA library is presented. The Smad7 coding region is hatched (ORF). The TGF-β responsive element (TRE) and specific BREs (BRE-1, BRE-2, and I-BRE) are indicated. Various restriction fragments derived from the 5.4-kbp genomic clone were subcloned into the pGL3 vector, upstream of the E1b promoter and luciferase reporter gene, and are shown as black bars with the corresponding clone letter. Diagrammed constructs were transiently transfected into HepG2 cells and incubated overnight in the absence or presence of 5 nM BMP-7. For constructs A to L, 0.03 μg of Smad1 and Smad4 cDNA/well were transfected into the cells. The relative luciferase activities of cell lysates were determined. The induction (n-fold) by BMP-7 is indicated. (B) The BRE-1 fragment was transiently transfected into HepG2 cells with or without Smad1 and Smad4 cDNA. Cells were treated with BMP-7, as indicated above, and the luciferase activities of cell lysates were determined.

Smad1 binds to BRE-1 in living cells. (A) Schematic representation of PCR products amplified following ChIP assays. (B) ChIP of Smad1 bound to BRE-1. HepG2 cells were treated for 1 h with increasing concentrations of BMP-7. Protein-DNA complexes were cross-linked by formaldehyde treatment, cells were lysed, and DNA was sheared by sonication. Cell lysates were subjected to immunoprecipitation with an anti-Smad1 antibody. DNA recovered from the immunoprecipitation was amplified by PCR. PI, preimmune; −, absent. (C) Diagrammed clones derived from BRE-1 were transiently transfected in HepG2 cells with 0.03 μg of Smad1 and Smad4 cDNA/well. Cells were incubated overnight in the absence (white bars) or presence (black bars) of 5 nM BMP-7. The relative luciferase activities of cell lysates were determined. Mutations created in box 2 or box 3 are indicated in bold letters under the appropriate sequences. The putative Smad4 and Smad1 binding sites are highlighted in gray and black, respectively. WT, wild type.

I-BRE binds Smad and GATA proteins in living cells. (A) ChIP of Smad1 bound to I-BRE. HepG2 cells were treated for 1 h with increasing concentrations of BMP-7. Protein-DNA complexes were cross-linked by formaldehyde treatment, cells were lysed, and DNA was sheared by sonication. Cell lysates were subjected to immunoprecipitation (IP) with an anti-Smad1 antibody. DNA recovered from the immunoprecipitation was amplified by PCR. PCR products amplified are diagrammed. PI, preimmune. PCR of the control region is presented in Fig. 3B. (B) ChIP of GATA-6 bound to I-BRE. HepG2 cells were treated as described for panel A, and cell lysates were subjected to immunoprecipitation with the goat C-20 anti-GATA-6 (αGATA-6) antibody (Santa Cruz) or with an irrelevant goat antibody (−) (C) 293T cells were transfected with cDNA for Flag-GATA-5 and the wild-type or mutated (M1/M2 as in Fig. 4A) I-BRE reporter construct. Protein-DNA complexes were cross-linked by formaldehyde treatment, the DNA was sheared by sonication, and cell lysates were subjected to immunoprecipitation with an anti-Flag (αFlag) antibody. The DNA recovered from the immunoprecipitation was amplified by PCR with primers specific to I-BRE. (D) 293T cells were transfected with the I-BRE reporter construct and cDNA for Flag-GATA-5, Myc-Smad1, and Alk2-QD. Cells were treated as described for panel C. Lysates were subjected to immunoprecipitation with an anti-Flag antibody followed by elution with Flag peptide and immunoprecipitation of the eluate with an anti-Smad1 antibody. I-BRE recovered from the double immunoprecipitation was amplified by PCR. +, present; −, absent.

Mapping of regions in GATA-5 required for I-BRE activation. (A and B) The I-BRE was transiently transfected in Neuro-2A cells with deletion mutants of the GATA-5 cDNA. Cells were incubated overnight in the absence (white bars) or presence (black bars) of BMP-7. The relative luciferase activities of cell lysates were determined. The zinc fingers (Zn) and activation domains (AD) of GATA-5 are indicated. (C) The I-BRE was transiently transfected in Neuro-2A cells with GATA-5, GATA-2, and chimeras of the two proteins. Cells were incubated overnight in the absence (white bars) or presence (black bars) of BMP-7. The relative luciferase activities of cell lysates were determined. The zinc fingers (Zn) and BAR of GATA-5 are indicated.