Developmental changes in the methylation status of regulatory elements in the murine alpha 1(I) collagen gene

Abstract

Regulatory elements contributing to the tissue-specific regulation of the murine alpha 1(I) collagen (Colla1) gene have previously been identified in its promoter region and first intron. Because several lines of evidence indicate that DNA methylation may be involved in the tissue-specific regulation of Colla1 gene expression, we have analyzed the methylation status of the 5' region of the gene by restriction analysis and a methylation-dependent PCR assay. All sites tested were unmethylated in sperm DNA. The region surrounding the start site of transcription was partially or completely methylated in undifferentiated embryonal cell lines, suggesting that it may be marked by de novo methylation during early embryonic development. In differentiated cells and adult tissues, the Colla1 promoter was completely demethylated in collagen-producing and some nonproducing cells, and partially methylated in other nonproducing cells. The first intron was unmethylated in collagen-producing as well as nonproducing cells. Only sites in the first exon showed an inverse correlation with transcriptional activity, i.e., they were unmethylated in cells that express the gene, but predominantly methylated in cells that do not. Our results indicate that the methylation status of a small area (less than 1 kb) downstream of the Colla1 promoter, but not of the promoter itself or the first intron, may be critical for transcriptional activity of the promoter, presumably through an indirect mechanism.

Figure 1

Col1a1 gene expression in murine cell lines and tissues. RNA was isolated from the indicated cell lines and tissues and analyzed by RNase protection assay as described (Chan et al., 1991) using a probe from the Col1a1 first exon that protects 112 nucleotides of Col1a1 mRNA. Ten μg total RNA from the indicated cell lines and 40 μg total RNA from the indicated tissues were used. The autoradiogram on the left was exposed overnight, and the one on the right for four days.

Figure 2

A. Summary of regulatory elements and potential factor-binding sites in the 5′ region of the murine Col1a1 gene. The number of nucleotides relative to the transcription start site (+1) is shown above the map. Filled boxes represent exons, and open boxes show the regions where positive (+) or negative (−) regulatory elements have been located. Filled ovals above the map indicate the location of potential factor-binding sites identified by DNasel footprints and gel shift experiments. Arrows point to previously mapped DNase hypersensitive sites (for details, see the text). B. Location of restriction sites for Hpa II, Hha I, and Tha I (↓) in the 5′ region of the murine Col1a1 gene and summary of their methylation status in different cell types. ○, unmethylated; ●, methylated; ◒, partially methylated. C. Nucleotide sequence of the Col1a1 promoter and first exon (Harbers et al., 1984). The TATAA box, the footprints 1–4 referred to in the text, and restriction sites for the methylation-sensitive enzymes Hpa II, Hha I, and Tha I are underlined. The start site of transcription is +1, and the translation start codon ATG is underlined. The / between nucleotides 193 and 194 shows the beginning of the first intron.

Figure 3

A. Methylation status of the regulatory elements in the Col1a1 promoter. Genomic DNA from the indicated cell lines and tissues was analyzed by restriction digestion and Southern blot hybridization. Digestion with Pvu II creates a 2.5 kb fragment when hybridized to the indicated probe. Further digestion with Hpa II or Hha I yields the fragments shown in the diagram, depending on the methylation status of the sites. B. Methylation status of closely adjacent Hpa II sites in the Col1a1 promoter analyzed by methylation-dependent PCR assay. Genomic DNA from 3T3 and WEHI-3B cells was digested with Hpa II and amplified by PCR as described in Materials and Methods. In uncut DNA, fragments of 117 bp and 145 bp, respectively, were amplified when primer pairs flanking the Hpa II sites were used. Amplification was completely abolished by prior digestion of the DNA with Hpa II, indicating that both Hpa II sites were unmethylated in both cell types.

Figure 3

A. Methylation status of the regulatory elements in the Col1a1 promoter. Genomic DNA from the indicated cell lines and tissues was analyzed by restriction digestion and Southern blot hybridization. Digestion with Pvu II creates a 2.5 kb fragment when hybridized to the indicated probe. Further digestion with Hpa II or Hha I yields the fragments shown in the diagram, depending on the methylation status of the sites. B. Methylation status of closely adjacent Hpa II sites in the Col1a1 promoter analyzed by methylation-dependent PCR assay. Genomic DNA from 3T3 and WEHI-3B cells was digested with Hpa II and amplified by PCR as described in Materials and Methods. In uncut DNA, fragments of 117 bp and 145 bp, respectively, were amplified when primer pairs flanking the Hpa II sites were used. Amplification was completely abolished by prior digestion of the DNA with Hpa II, indicating that both Hpa II sites were unmethylated in both cell types.

Figure 4

Methylation status of Hpa II and Hha I sites in the first exon and intron of the murine Col1a1 gene. Genomic DNA from the indicated cell lines and tissues was digested with Bgl II (A), which creates a 2.6 kb fragment, Pvu II (B), which creates a 2.5 kb fragment, or Pst I and Bgl II (C and D), which create a 0.8 kb fragment when hybridized with the indicated probes. Further digestion with Hpa II and Hha I (A, B, and C) or Tha I (D) yields the fragments shown in the diagrams, depending on the methylation status of the restriction sites.

Figure 4

Methylation status of Hpa II and Hha I sites in the first exon and intron of the murine Col1a1 gene. Genomic DNA from the indicated cell lines and tissues was digested with Bgl II (A), which creates a 2.6 kb fragment, Pvu II (B), which creates a 2.5 kb fragment, or Pst I and Bgl II (C and D), which create a 0.8 kb fragment when hybridized with the indicated probes. Further digestion with Hpa II and Hha I (A, B, and C) or Tha I (D) yields the fragments shown in the diagrams, depending on the methylation status of the restriction sites.