CRISPR-Cas9-Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin-Brief Report

Abstract

Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research.

Keywords: allele; epitope; mice; muscle, smooth; myocardin.

Figure 1.. In vivo MYOCD protein expression and binding activity.

(A) Western blot of MYOCD^3xFLAG in wildtype (+/+) versus heterozygous and homozygous FLAG(FL)-tagged MYOCD in indicated male mouse tissues. HEK-293 cells untransfected (−) or transfected (+) with 3xFLAG-tagged v1 isoform of MYOCD served as a control. (B) Parallel Western blots of MYOCD^3xFLAG or MYOCD^3xHA tagged tissues. (C) Tissue profile of MYOCD^3xFLAG expression in indicated female tissues. Similar results were seen in several independent experiments over the course of two years. (D) In vivo ChIP-qPCR in homozygous Myocd^3xFLAG aortae for the indicated target genes using FLAG or IgG control. Data are expressed as fold enrichment of FLAG sequence over IgG control. All results were confirmed in at least one independent experiment. Data passed test for normality and paired t-test showed significantly increased enrichment of PCR products with the anti-FLAG antibody, p < 0.01.

Figure 2.. In vitro MYOCD protein expression.

(A) Immunofluorescence microscopy of MYOCD^3xFLAG in low-passaged mouse aortic SMC (MASMC). Note abundant nuclear staining in Myocd^3xFLAG MASMC versus wild-type controls. (B) Quantitative RT-PCR following siRNA knockdown of Myocd mRNA in cultured MASMC. (C) Immunofluor-escence microscopy of MYOCD^3xFLAG in MYH11 positive MASMC treated with si scrambled control (siCtrl, top) or siMyocd (bottom). Scale bar here and in panel A is 10 μm. (D) Quantitative analysis (by two independent, blinded investigators) of number of MYOCD-FLAG positive cells co-staining for MYH11. See also Figure VC in the online-only Data Supplement.