Association of lamin A/C with muscle gene-specific promoters in myoblasts

Abstract

The A-type and B-type lamins form a filamentous meshwork underneath the inner nuclear membrane called the nuclear lamina, which is an important component of nuclear architecture in metazoan cells. The lamina interacts with large, mostly repressive chromatin domains at the nuclear periphery. In addition, genome-lamina interactions also involve dynamic association of lamin A/C with gene promoters in adipocytes. Mutations in the human lamin A gene cause a spectrum of hereditary diseases called the laminopathies which affect muscle, cardiac and adipose tissues. Since most mutations in lamin A/C affect skeletal muscle, we investigated lamin-chromatin interactions at promoters of muscle specific genes in both muscle and non-muscle cell lines by ChIP-qPCR. We observed that lamin A/C was specifically associated with promoter regions of muscle genes in myoblasts but not in fibroblasts. Lamin A/C dissociated from the promoter regions of the differentiation specific MyoD, myogenin and muscle creatine kinase genes when myoblasts were induced to differentiate. In the promoter regions of the myogenin and MyoD genes, the binding of lamin A/C in myoblasts inversely correlated with the active histone mark, H3K4me3. Lamin A/C binding on muscle genes was reduced and differentiation potential was enhanced on treatment of myoblasts with a histone deacetylase inhibitor. These findings suggest a role for lamina-chromatin interactions in muscle differentiation and have important implications for the pathological mechanisms of striated muscle associated laminopathies.

Keywords: Genome–lamina interactions; Laminopathies; Lamins; Muscle differentiation; Myogenin; Nuclear lamina.

Fig. 1

Lamin A/C occupancy on promoters of MyoD1, Pax7 and Myf5. ChIP-qPCR analysis with lamin A/C antibody on promoters of myogenic genes in myoblasts (open bars), myotubes differentiated for 48 h (solid bars) and NIH3T3 fibroblasts (grey bars). Fold enrichment over IgG±S.D is represented for (A) MyoD1 promoter, (B) Pax7 promoter and (C) Myf5 promoter. (D) Percentage of input values for unspecific IgG and lamin A/C (LA) obtained at representative promoter regions (MyoD −0.64 kb and Pax7 −3.1 kb) are shown to indicate specificity of lamin A/C ChIP. (p<0.05*, <0.01**).

Fig. 2

Lamin A/C occupancy on promoters of Myog and Mck. ChIP-qPCR analysis with lamin A/C antibody on promoters of differentiation-specific genes in myoblasts (open bars), myotubes differentiated for 48 h (solid bars) and NIH3T3 fibroblasts (grey bars). Fold enrichment over IgG±S.D is represented for promoter regions of (A) Myog and (B) Mck (p<0.01**).

Fig. 3

Lamin A/C occupancy on promoters of non-muscle genes. ChIP-qPCR analysis with lamin A/C antibody on promoters of non-muscle genes in myoblasts (open bars), myotubes differentiated for 48 h (solid bars) and NIH3T3 fibroblasts (grey bars). Fold enrichment over IgG±S.D is represented. (p<0.05*, <0.01**).

Fig. 4

Binding of histone activation mark H3K4me3 on muscle genes. ChIP-qPCR analysis with antibody to active histone mark H3K4me3 on promoter regions of (A) MyoD1 and (B) Myog in myoblasts (open bars) and myotubes (solid bars). Fold enrichment over IgG±S.D is represented. (p<0.05*, <0.01**).

Fig. 5

Lamin A/C occupancy in myoblasts treated with sodium butyrate. ChIP-qPCR analysis with lamin A/C antibody on promoter regions of muscle genes in untreated myoblasts (open bars) and myoblasts treated with 5 mM sodium butyrate in GM for 24 h (solid bars). Fold enrichment over IgG±S.D is represented for promoters of (A) Myog, (B) Mck, (C) MyoD1, (D) Pax7 and (E) Myf5. (F) Quantification of myogenin expression by immunofluorescence analysis in normal myoblasts (MB), normal myoblasts differentiated to myotubes in DM (MT), myoblasts treated with 5 mM sodium butyrate (tr MB) and then differentiated in DM in the absence of sodium butyrate (tr MB in DM). (p<0.05*).