Regulation of 130-kDa Smooth Muscle Myosin Light Chain Kinase Expression by an Intronic CArG Element

Chen, Meng; Zhang, Wenwu; Lu, Xiao; Hoggatt, April M.; Gunst, Susan J.; Kassab, Ghassan S.; Tune, Johnathan D.; Herring, B. Paul

doi:10.1074/jbc.m113.510362

Cited by 16 publications

(16 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This finding should be contrasted with an earlier report showing near abolishment of CNN1, another SMC-restricted gene product (34), upon CRISPR-mediated editing of a single intronic CArG box (17). Similarly, a decrease in expression of smooth muscle myosin light chain kinase with attenuated intestinal smooth muscle contractility was noted after deletion of an upstream CArG box (35). The absence of change in LMOD1 expression with the double CArG box mutation suggests that, whereas proximal promoter CArG boxes are sufficient for Lmod1 promoter activity (12), they are not necessary in their native genomic context, which would imply that other, more distal CArG boxes coordinate SRF-dependent expression of LMOD1 in both human and mouse.…”

Section: Discussioncontrasting

confidence: 54%

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice

Halim

Wilson

Oliver

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

106

108

View full text Add to dashboard Cite

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal–contractile coupling.

show abstract

Section: Discussioncontrasting

confidence: 54%

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice

Halim

Wilson

Oliver

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

106

108

View full text Add to dashboard Cite

show abstract

“…Due to the protracted time in generating genetically altered mice with this traditional approach, coupled with the complexity and redundancy of gene regulation, there have been far fewer regulatory knockouts as compared to genic knockouts. Further, genic control regions, such as enhancers, have almost always been deleted via Cre recombinase-mediated excision of 100s-1000s of nucleotides, effectively leaving a genomic cavity 21–23 . While these studies certainly provide some insight into the role of enhancers in target gene expression and animal pathophysiology, they lack resolution in narrowing down specific elements within the enhancer that mediate gene expression.…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 Genome Editing of a Single Regulatory Element Nearly Abolishes Target Gene Expression in Mice—Brief Report

Han

Slivano

Christie

et al. 2015

ATVB

View full text Add to dashboard Cite

Objective To ascertain the importance of a single regulatory element in the control of Cnn1 expression using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing. Approach and Results The CRISPR/Cas9 system was used to produce 3/18 founder mice carrying point mutations in an intronic CArG box of the smooth muscle cell (SMC)-restricted Cnn1 gene. Each founder was bred for germ line transmission of the mutant CArG box and littermate interbreeding to generate homozygous mutant (Cnn1ΔCArG/ΔCArG) mice. Quantitative RT-PCR, Western blotting, and confocal immunofluorescence microscopy showed dramatic reductions in Cnn1 mRNA and CNN1 protein expression in Cnn1ΔCArG/ΔCArG mice with no change in other SMC-restricted genes and little evidence of off-target edits elsewhere in the genome. In vivo chromatin immunoprecipitation assay revealed a sharp decrease in binding of SRF to the mutant CArG box. Loss of CNN1 expression was coincident with an increase in Ki-67 positive cells in the normal vessel wall. Conclusion CRISPR/Cas9 genome editing of a single CArG box nearly abolishes Cnn1 expression in vivo and evokes increases in SMC DNA synthesis. This facile genome editing system paves the way for a new generation of studies designed to test the importance of individual regulatory elements in living animals, including regulatory variants in conserved sequence blocks linked to human disease.

show abstract

“…Furthermore, Notch1-induced HRT2 can lead to repression of both Notch and myocardin function through destabilization of respective transcription complexes. Of note, a recent study identified a functional CArG element within intron 15 of SM-MLCK that selectively regulates 130 kDa MLCK isoform expression required for contractile activity in visceral smooth muscle (28). Whether the intronic CArG is additionally sensitive to Notch signals remains to be determined.…”

Section: Discussionmentioning

confidence: 99%

“…Plasmid Constructs-Expression plasmids for intracellular Notch1 (pcDNA3-ICN1), flag-tagged myocardin (pFLAGmyocardin) and MLCK-luciferase reporter constructs (p6476 and p389) were described previously (14,26,28). Expression plasmids for wild-type HRT2 (pHRT2-V5) and HRT2 mutants (HRT2:BHO, HRT2:BHOϩ, and HRT2[B-]) were described previously (26,29).…”

Section: Methodsmentioning

confidence: 99%

Autoregulatory Control of Smooth Muscle Myosin Light Chain Kinase Promoter by Notch Signaling

Basu

Proweller

2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

Smooth muscle myosin light chain kinase (SM-MLCK) is the key enzyme responsible for phosphorylation of regulatory myosin light chain (MLC 20 ), resulting in actin-myosin cross-bridging and force generation in vascular smooth muscle required for physiological vasoreactivity and blood pressure control. In this study, we investigated the combinatorial role of myocardin/serum response factor (SRF) and Notch signaling in the transcriptional regulation of MLCK gene expression. Promoter reporter analyses in rat A10 smooth muscle cells revealed a bimodal pattern of MLCK promoter activity and gene expression upon stimulation with constitutively active Notch1 in presence of myocardin or by Jagged1 ligand stimulation. An initial Notch1-induced increase in MLCK transcription was followed by loss in promoter sensitivity, which could be restored with further Notch1 dose escalation. Real-time PCR analyses revealed that endogenous levels of Hairy Related Transcription (HRT) factor 2 (HRT2) peaked concurrently with inhibitory concentrations of Notch1. Forced expression of HRT2 demonstrated simultaneous repression of both myocardin-and Notch1-induced MLCK promoter activity. HRT2-mediated repression was further confirmed by HRT2 truncations and siHRT2 treatments that rescued MLCK promoter activity and gene expression. Chromatin immunoprecipitation studies revealed both Jagged1 ligand-and Notch1-enhanced myocardin/SRF complex formation at the promoter CArG element. In contrast, heightened levels of HRT2 concomitantly disrupted myocardin/SRF and Notch transcription complex formation at respective CArG and CSL binding elements. Taken together, SM-MLCK promoter activity appears highly sensitive to the relative levels of Notch1 signaling, HRT2, and myocardin. These findings identify a novel Notch-dependent HRT2 autoregulatory circuit coordinating transcriptional regulation of SM-MLCK.The contractile activity of vascular smooth muscle cells (VSMCs) 2 serves to regulate physiological vascular resistance and blood pressure and dysfunctional vasoconstriction or relaxation underlies peripheral hypertensive disease, the most common diagnosis and leading risk factor of cardiovascular mortality (1). Mature, differentiated VSMCs express a number of tissue-specific contractile genes such as smooth muscle ␣-actin (SM ␣-actin), SM22␣, calponin, h-caldesmon, smoothelin-B, smooth muscle-myosin heavy chain (SM-MHC), and regulatory genes including myosin light chain kinase (MLCK) critical for effective constrictor function (2). These genes are known to be transcriptionally regulated in part by serum response factor (SRF) and its co-activator, myocardin (3-5). SRF belongs to the MADS box family of transcription factors, which have the characteristic feature of mediating homodimerization and DNA binding and recruiting a variety of transcriptional cofactors that influence DNA binding affinity, transcriptional activity, and target gene specificity (6, 7). Myocardin is a member of the SAP domain family of nuclear proteins responsible for activation of many...

show abstract

Regulation of 130-kDa Smooth Muscle Myosin Light Chain Kinase Expression by an Intronic CArG Element

Cited by 16 publications

References 52 publications

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice

CRISPR-Cas9 Genome Editing of a Single Regulatory Element Nearly Abolishes Target Gene Expression in Mice—Brief Report

Autoregulatory Control of Smooth Muscle Myosin Light Chain Kinase Promoter by Notch Signaling

Contact Info

Product

Resources

About