Regulation of human P2X1 promoter activity by beta helix-loop-helix factors in smooth muscle cells.
We isolated and characterized genomic clones of the human P2X1 receptor (hP2X1) gene in an effort to understand its tissue specific expression. The hP2X1 gene contains 12 exons spanning 20 kb, with exon sizes ranging from 59 to 143 bp. A 385 bp upstream fragment promoted hP2X1 gene expression in smooth muscle (A7R5 and primary trachealis) and fibroblast (NIH3T3) cell lines, and mutation of a consensus E box sequence (CACCTG) within this fragment (-340 to -345) did not alter basal promoter activity. However, co-transfected bHLH factors regulated activity of the 385 bp minimal P2X1 promoter in a tissue-specific manner. E12 expression inhibited and ITF2b augmented activity in A7R5 cells, but had no effect in NIH3T3 cells. ITF2a, Myo-D, and Id1 proteins had no effect on either cell line, but co-expression of ITF2a blocked E12 inhibition in A7R5 cells, while ITF2b failed to reverse the inhibition. Northern analysis of A7R5 RNA identified high levels of E12 and ITF2b transcripts, and gel shift assays using A7R5 and NIH3T3 nuclear extracts indicated the formation of a protein-DNA complex with an oligonucleotide corresponding to -330 and -348, which was abolished by base substitutions within the E box motif. Our results identify a critical E box response element in the hP2X1 promoter that binds bHLH factors and demonstrate smooth muscle specific transcriptional regulation by E proteins.
Dhulipala
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Department of Medicine, Division of Nephrology, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ, USA.
Lianos
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Kotlikoff
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D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D011401:Promoter Regions, Genetic; D018048:Receptors, Purinergic P2 / Q000235:genetics*; D058469:Receptors, Purinergic P2X; D012097:Repressor Proteins; D051820:TCF Transcription Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D000073940:Transcription Factor 4; D058125:Transcription Factor 7-Like 1 Protein; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C497453:ID1 protein, human; C497455:Idb1 protein, mouse; D051798:Inhibitor of Differentiation Protein 1; D017570:MyoD Protein; D009419:Nerve Tissue Proteins; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D012097:Repressor Proteins; D051820:TCF Transcription Factors; C544912:TCF7L1 protein, human; C544894:TCF7L2 protein, human; C090743:Tcf4 protein, mouse; C544913:Tcf7l1 protein, mouse; C544895:Tcf7l2 protein, mouse; D015534:Trans-Activators; D000073940:Transcription Factor 4; D058125:Transcription Factor 7-Like 1 Protein; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors