In vivo footprinting with limiting amounts of embryo tissues: a role for C/EBP beta in early hepatic development.
Regulatory factors important for the developmental control of genes have been identified by genetic studies or by examining the ontological expression profiles of proteins that were originally characterized in adult tissues; direct biochemical studies of transcription factors within small amounts of embryo tissues have been limited. We have found that the ligation-mediated PCR (LM-PCR) technique can detect specific dimethylsulfate modifications in genomic DNA from as few as several thousand cells, making it technically feasible to identify protein-DNA interactions in pools of nascent embryo tissues. Herein we show that LM-PCR can reveal methylation protections on the albumin gene enhancer in embryonic mouse hepatocytes, indicating occupancy of a C/EBP factor binding site. Comparison of the in vivo protection pattern with that obtained from the in vitro analysis of different C/EBP isoforms suggests that in embryonic hepatocytes, C/EBP beta is bound to the albumin gene enhancer. Detailed protocols are provided so that the approach can be used to study other genes in developing embryos.
Bossard
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P
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
McPherson
C E
CE
Zaret
K S
KS
D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004622:Embryo, Mammalian; D004742:Enhancer Elements, Genetic; D008099:Liver / Q000196:embryology* / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012709:Serum Albumin / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012709:Serum Albumin; D014157:Transcription Factors; D004247:DNA