Fig. 1.

RFX family of proteins can bind near the start site of the COL1A1 gene. A, schematic representation of the sequence near the start site of both human COL1A1 (AF017178 accession number) and COL1A2 (AF004877 accession number) genes to show the homology between the two genes. RFX binding sites have been indicated using bold and italic bases in both genes. CpG sequences near the start sites were shown by asterisks (*). B, RFX1–3 proteins can bind to COL1A1 (−11/+10) and COL1A2 sequences in a methylation-sensitive manner as evidenced by the electrophoretic mobility shift assay. Unmethylated (U) and methylated (M) radiolabeled probes (see Table I for sequences) were incubated with the nuclear extract from rat fibroblasts cells as described under “Materials and Methods.” Protein and DNA were separated on native 4% polyacrylamide gels. The dried gel was autoradiographed at −80 °C overnight. Lanes 1 and 2 shows the methylation-sensitive binding at the COL1A2 sequence. Lanes 3 and 4 indicate no binding to either probe when the homologous sequence from COL1A1 is used as probe. Lanes 5 and 6 represent the methylation dependent binding of RFX protein at COL1A1 sequence. C, strong RFX5 complex is generated at the COL1A1 gene, similar to the COL1A2 and X-box sequence when RFX1–3 binding is removed using the methylated PBR322 sequence. Gel shift experiment was performed similarly to B in the presence a 50-fold molar excess of mPBR322. The higher mobility complex is designated as RFX5 complex.