Identification of an histone H3 acetylated/K4-methylated-bound intragenic enhancer regulatory for urokinase receptor expression.
The transcriptionally regulated urokinase-type plasminogen activator receptor (u-PAR) contributes to cancer progression. Although previous studies have identified multiple 5' regulatory elements, these cis motifs cannot fully account for u-PAR expression prompting a search for hitherto uncharacterized regulatory elements. DNase I hypersensitivity and chromatin immunoprecipitation assays using u-PAR-expressing colon cancer cells indicated a hypersensitive region (+665/+2068) in intron 1 enriched with acetylated histone 3 (H3) and H3 methylated at lysine 4, markers of regulatory regions. The +665/+2068 region increased transcription from a u-PAR-promoter in an orientation- and distance-independent manner fulfilling the criteria of an enhancer. Optimal stimulation of the u-PAR promoter by phorbol ester required this enhancer. Systematic truncations combined with DNase I footprinting revealed two protected regions (+1060/+1099 and +1123/+1134) with deletion of the latter practically abolishing enhancer activity. The +1123/+1134 region harbored non-consensus activator protein-1 and Ets1 binding sites bound with c-Jun (and/or the related JunD/JunB) and c-Fos (and/or the related FosB/Fra-1/Fra-2) as revealed with chromatin immunoprecipitation. Further, nuclear extract from resected colon cancers showed elevated protein binding to a +1123/+1134-spanning probe coordinate with elevated u-PAR protein. Thus, we have defined a novel intragenic enhancer in the u-PAR gene required for constitutive and inducible expression.
Wang
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Department of Cancer Biology, MD Anderson Cancer Center, Houston, TX 77030, USA.
Yan
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Asangani
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Allgayer
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H
Boyd
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D000107:Acetylation; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D003110:Colonic Neoplasms / Q000235:genetics; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000737:chemistry; D004742:Enhancer Elements, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006657:Histones / Q000378:metabolism; D006801:Humans; D007438:Introns; D011401:Promoter Regions, Genetic; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D014157:Transcription Factors / Q000378:metabolism
D002843:Chromatin; D006657:Histones; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D014157:Transcription Factors; D003850:Deoxyribonuclease I