130-kDa smooth muscle myosin light chain kinase is transcribed from a CArG-dependent, internal promoter within the mouse mylk gene.
The 130-kDa smooth muscle myosin light chain kinase (smMLCK) is a Ca2+/CaM-regulated enzyme that plays a pivotal role in the initiation of smooth muscle contraction and regulation of cellular migration and division. Despite the critical importance of smMLCK in these processes, little is known about the mechanisms regulating its expression. In this study, we have identified the proximal promoter of smMLCK within an intron of the mouse mylk gene. The mylk gene encodes at least two isoforms of MLCK (130 and 220 kDa) and telokin. Luciferase reporter gene assays demonstrated that a 282-bp fragment (-167 to +115) of the smMLCK promoter was sufficient for maximum activity in A10 smooth muscle cells and 10T1/2 fibroblasts. Deletion of the 16 bp between -167 and -151, which included a CArG box, resulted in a nearly complete loss of promoter activity. Gel mobility shift assays and chromatin immunoprecipitation assays demonstrated that serum response factor (SRF) binds to this CArG box both in vitro and in vivo. SRF knockdown by short hairpin RNA decreased endogenous smMLCK expression in A10 cells. Although the SRF coactivator myocardin induced smMLCK expression in 10T1/2 cells, myocardin activated the promoter only two- to fourfold in reporter gene assays. Addition of either intron 1 or 6 kb of the 5' upstream sequence did not lead to any further activation of the promoter by myocardin. The proximal smMLCK promoter also contains a consensus GATA-binding site that bound GATA-6. GATA-6 binding to this site decreased endogenous smMLCK expression, inhibited promoter activity in smooth muscle cells, and blocked the ability of myocardin to induce smMLCK expression. Altogether, these data suggest that SRF and SRF-associated factors play a key role in regulating the expression of smMLCK.
Yin
Feng
F
Dept. of Cellular and Integrative Physiology, Indiana Univ. School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5120, USA.
Hoggatt
April M
AM
Zhou
Jiliang
J
Herring
B Paul
BP
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D050993:GATA6 Transcription Factor / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D026362:Serum Response Factor / Q000235:genetics / Q000378:metabolism; D024745:Smooth Muscle Myosins / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D000099251:Myocardin
D004268:DNA-Binding Proteins; D050993:GATA6 Transcription Factor; D009219:Myosin-Light-Chain Kinase; D009687:Nuclear Proteins; D026362:Serum Response Factor; D024745:Smooth Muscle Myosins; D015534:Trans-Activators; D000099251:Myocardin