130-kDa smooth muscle myosin light chain kinase is transcribed from a CArG-dependent, internal promoter within the mouse mylk gene

Feng Yin, April M. Hoggatt, Jiliang Zhou, B. Paul Herring

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

The 130-kDa smooth muscle myosin light chain kinase (smMLCK) is a Ca 2+/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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume290
Issue number6
DOIs
StatePublished - Jun 1 2006
Externally publishedYes

Fingerprint

Smooth Muscle Myosins
Myosin-Light-Chain Kinase
Genes
Serum Response Factor
Assays
Muscle
antineoplaston A10
Reporter Genes
Introns
Smooth Muscle Myocytes
Binding Sites
Cells
Chromatin Immunoprecipitation
Electrophoretic Mobility Shift Assay
Fibroblasts
Muscle Contraction
Luciferases
Small Interfering RNA
Chromatin
Smooth Muscle

Keywords

  • GATA
  • Gene expression
  • Myocardin
  • Serum response factor
  • Telokin
  • Transcriptional regulation

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

130-kDa smooth muscle myosin light chain kinase is transcribed from a CArG-dependent, internal promoter within the mouse mylk gene. / Yin, Feng; Hoggatt, April M.; Zhou, Jiliang; Herring, B. Paul.

In: American Journal of Physiology - Cell Physiology, Vol. 290, No. 6, 01.06.2006.

Research output: Contribution to journalArticle

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abstract = "The 130-kDa smooth muscle myosin light chain kinase (smMLCK) is a Ca 2+/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.",
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