An intronic MYLK variant associated with inflammatory lung disease regulates promoter activity of the smooth muscle myosin light chain kinase isoform.

Abstract

Intronic single-nucleotide polymorphisms (SNPs) are commonly associated with complex diseases but exhibit unknown biologic functionality. Myosin light-chain kinase (MLCK), a central cytoskeletal regulator encoded by MYLK, plays a key pathophysiological role in complex diseases including acute lung injury (ALI) and asthma. We studied the potential regulatory roles of two intronic MYLK SNPs (rs936170 and rs820336) previously associated with ALI and asthma. Due to their genomic location at the junction encoding the non-muscle and smooth muscle MLCK (smMLCK) isoforms, we first identified the transcription start site (TSS) of the smMLCK isoform, and isolated a 2,954-bp DNA fragment upstream of the smMLCK TSS. Serial 5' deletion of the fragment revealed a proximal promoter region exhibiting strong promoter activity with potential inhibitory elements in the distal region. Site-directed mutageneses and luciferase reporter assays showed no effect of the distal promoter SNP rs936170 on smMLCK promoter activity. In contrast, SNP rs820336, located in an enhancer/repressor region downstream of TSS, was identified to regulate smMLCK promoter activity in an allelic-dependent manner. The A allele interrupted the binding site for Forkhead box protein N1 (FOXN1), a transcription factor governing expression of immune response genes. Silencing of FOXN1 expression (siRNA) reduced FOXN1 interaction with cis-regulatory elements in proximity to rs820336 and significantly decreased smMLCK expression. These functional insights into the involvement of intronic MYLK SNPs further strengthen the concept that MYLK contributes to inflammatory disease susceptibility and represents an attractive molecular target in complex inflammatory disorders.

• Authors: Yoo Jeong Han, Shwu-Fan Ma, Michael S Wade, Carlos Flores, Joe G N Garcia
• Journal: Journal of molecular medicine (Berlin, Germany) (J Mol Med (Berl)) Vol. 90 Issue 3 Pg. 299-308 (Mar 2012) ISSN: 1432-1440 [Electronic] Germany
• PMID: 22015949 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Calcium-Binding Proteins
• Forkhead Transcription Factors
• Protein Isoforms
• Whn protein
• MYLK protein, human
• Myosin-Light-Chain Kinase

Topics

• Acute Lung Injury (genetics, physiopathology)
• Animals
• Asthma (genetics, physiopathology)
• Calcium-Binding Proteins (genetics, metabolism)
• Cells, Cultured
• Electrophoretic Mobility Shift Assay
• Enhancer Elements, Genetic
• Forkhead Transcription Factors (genetics, metabolism)
• Gene Silencing
• Genes, Reporter
• Genetic Predisposition to Disease
• HeLa Cells
• Humans
• Introns (genetics)
• Lung Diseases (genetics)
• Muscle, Smooth (enzymology)
• Mutagenesis, Site-Directed
• Myosin-Light-Chain Kinase (genetics, metabolism)
• Polymorphism, Single Nucleotide (genetics)
• Promoter Regions, Genetic
• Protein Isoforms (genetics, metabolism)
• Rats