Identification of new regulatory sequences far upstream of the mouse monocyte chemoattractant protein-1 gene.
We systematically searched for sequences influencing the expression of the mouse monocyte chemoattractant protein-1 (MCP-1) gene (Scya2) by mapping DNase I hypersensitive sites (HS) in the chromatin of mesangial cells in a 40-kb interval around the gene. We found nine HS located between -24 kb and +12.7 kb. Three HS coincided with previously known regulatory sequences (HS-2.4, HS-1.0, and HS-0.2). We tested two of the previously unknown HS located far upstream of Scya2 (HS-19.4 and HS-16.3) in transfection experiments using luciferase reporter constructs and mouse mesangial cells as recipients. In transient transfections, both HS had a moderate effect on basal promoter activity as well as promoter activity stimulated by tumor necrosis factor-alpha. In stable transfection experiments, we found much higher activity. A DNA fragment containing HS-19.4 and HS-16.3 caused a considerable increase in the number of stably integrated luciferase copies. We determined the nucleotide sequence of the 5' flanking region to -28.6 kb. Computer-assisted sequence analysis did not yield evidence of an additional gene. These HS are located within the 5' flanking region of a gene cluster consisting of Scya2 (MCP-1), Scya7 (MCP-3), Scya11 (eotaxin), Scya12 (MCP-5), and Scya8 (MCP-2). This report represents the first comprehensive chromatin analysis of the mouse MCP-1 locus leading to the identification of a complex regulatory region located far upstream of Scya2.
Wagner
K
K
Ludwig-Maximilians-Universität, Medizinische Poliklinik, Schillerstrasse 42, D-80336 München, Germany.
Dendorfer
U
U
Chilla
S
S
Schlöndorff
D
D
Luckow
B
B
D000818:Animals; D018932:Chemokine CCL2 / Q000235:genetics*; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular; D003850:Deoxyribonuclease I / Q000378:metabolism; D005920:Glomerular Mesangium / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection
D018932:Chemokine CCL2; D002843:Chromatin; D008156:Luciferases; D003850:Deoxyribonuclease I