Tissue-specific and steroid-dependent interaction of transcription factors with the oestrogen-inducible apoVLDL II promoter in vivo.

Abstract

Using in vivo dimethylsulphate footprinting, we have analysed protein–DNA interactions within the promoter region of the oestrogen‐inducible gene encoding chicken apo very low density lipoprotein II (apoVLDL II). Most of the guanosine–protein contacts found, are located within the 230‐bp DNA 5′ flanking the gene and can be grouped into separate protein‐binding sites. Two of these sites resemble the oestrogen‐responsive element (ERE) which is the target site for the oestrogen receptor. A third site has some features in common with the chicken ovalbumin upstream promoter element binding the COUP transcription factor. All protein contacts identified are present in the apoVLDL‐II‐expressing liver exclusively, and are not found in the hormone‐naive liver, in erythrocytes or the oviduct tubular gland. Our results demonstrate the binding in vivo of a protein, presumably the oestrogen receptor, to the ERE and suggest that the hormone activates transcription by establishing a transcription complex comprising several factors at the apoVLDL II promoter.

Aberrant Silencing of the CpG Island-Containing Human O6-Methylguanine DNA Methyltransferase Gene Is Associated with the Loss of Nucleosome-Like Positioning

“…SF767, U138, CLA, and A1235 cultured cells, as well as human T cells, were analyzed for in vivo DNAprotein interactions at nt 1 to 1190 of the MGMT CpG island by ligand-mediated PCR (LMPCR) under previously described conditions (6). LMPCR-suitable DNA devoid of DNA-protein interactions was generated as previously described (25,38,42). The sets of nested primers used in the analysis of the transcribed strand of the MGMT CpG island were as follows: nt 60 to 282, 5Ј-GCTCCCTCT GAAGGCTCCAG-3Ј (LMP19), 5Ј-GAGTGTCCTCTGCTCCCTC CGAAG-3Ј (LMP20), and 5Ј-GAGTGTCCTCTGCTCCCTCCGAAGGCTC-3Ј (LMP21); nt 282 to 487, 5Ј-GTGAGGTACTGGGAGTTAGGAC-3Ј (LMP22), 5Ј-CAAC ATAGCTTCTCTGGTGGACACAA-3Ј (LMP23), and 5Ј-CAACATAGCTTCT CTGGTGGACACAATTC-3Ј (LMP24); nt 480 to 801, 5Ј-TGCCCCCACGGCC CCCTGACA-3Ј (LMP13), 5Ј-TCTCTGCTGGTCTGGGGGTCCCTGA-3Ј (LMP14), and 5Ј-TCTCTGCTGGTCTGGGGGTCCCTGACTAG-3Ј (LMP15); nt 703 to 864, 5Ј-CGGCCCATTTGGCAAACTAAG-3Ј (LMP1), 5Ј-AGGCAC AGACCTCAGGCGGAAGCT-3Ј (LMP2), and 5Ј-AGGCACAGACCTCAGG CGGAAGCTGGGA-3Ј (LMP3); nt 864 to 1075, 5Ј-GATGCGCAGACTGCCT CAG-3Ј (LMP4), 5Ј-TGGGCATGCGCCGACCCGGTC-3Ј (LMP5), and 5Ј-TG GGCATGCGCCGACCCGGTCGGG-3Ј (LMP6); and nt 1076 to 1190, 5Ј-CCC GGATATGCTGGGACAGC-3Ј (LMP7), 5Ј-CCGCGCCCCTGAACGCTTTG CGTC-3Ј (LMP8), and 5Ј-CCGCGCCCCTGAACGCTTTGCGTCCCGA-3Ј (LMP9).…”

In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1.

“…The reaction was quenched by adding a 10-fold volume of ice-cold PBS followed by centrifugation. After washing with ice-cold PBS, nuclei were prepared (Wijnholds et al 1988}, and DNA was purified [300 ~g/ml proteinase K, 20 mM Tris-HC1 (pH 8), 20 mM NaC1, 20 mM EDTA, 1% SDS for 3 hr at 37°C]. After phenol/chloroform extraction, the DNA was precipitated in ethanol and redissolved in 10 mM Tris-HC1 (pH 7.8}, 1 mM EDTA.…”

Reactivation of apolipoprotein II gene transcription by cycloheximide reveals two steps in the deactivation of estrogen receptor-mediated transcription.

“…Analysis of the mechanisms of transcriptional regulation of these genes as well as the study of mRNA turnover in avian liver cells has been hampered by the absence of a suitable homologous cell line. Studies of gene activation and estrogen-dependent alterations in chromatin structure and DNA methylation patterns have been restricted to animal studies or tissue homogenates (2,3,14,15,38,42,67,68 …”