Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus.
Rac2 is a Rho family GTPase expressed specifically in hematopoietic cells. The 4.5 kb proximal Rac2 gene promoter exhibits strong but promiscuous activity following either transient or stable transfection into tissue culture cells, indicating that additional cis-elements are required to silence Rac2 expression in non-hematopoietic cells. A bacterial artificial chromosome (BAC) containing the human Rac2 gene, including as little as 1.6 kb of upstream and 8 kb of downstream sequence, exhibits hematopoietic-restricted expression in transgenic mice. The Rac2 genomic locus exhibits distinct patterns of DNA methylation in expressing versus non-expressing cells. Cells that lack Rac2 expression exhibit increased cytosine methylation in the sequences flanking the gene, whereas cells that express Rac2 exhibit increased cytosine methylation within the body of the Rac2 gene. Treatment of non-expressing cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine reduces cytosine methylation of the Rac2 gene locus and is sufficient to induce Rac2 gene expression. Conversely, treatment with the histone deacetylase (HDAC) inhibitor trichostatin A fails to induce Rac2 gene expression. These findings define a genomic fragment sufficient to direct hematopoietic-specific expression of Rac2, and reveal the importance of cytosine methylation in the repression of Rac2 expression in non-hematopoietic cells.
Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Cancer Research Building, 1044 W. Walnut St., Room 327, Indianapolis, IN 46202, USA.
Paula D
PD
Ladd
Jill Sergesketter
JS
Butler
David G
DG
Skalnik
D044503:5-Methylcytosine / Q000378:metabolism; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D018387:Codon, Initiator / Q000235:genetics; D003596:Cytosine / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019175:DNA Methylation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000235:genetics*; D006413:Hematopoietic System / Q000166:cytology / Q000378:metabolism*; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D020014:K562 Cells; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D024363:Transcription Initiation Site; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000235:genetics*; D000097766:RAC2 GTP-Binding Protein
D044503:5-Methylcytosine; D001374:Azacitidine; D018387:Codon, Initiator; D003596:Cytosine; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases; D006877:Hydroxamic Acids; D008156:Luciferases; D011993:Recombinant Fusion Proteins; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein; C012589:trichostatin A