Repositioning of muscle-specific genes relative to the periphery of SC-35 domains during skeletal myogenesis

Abstract

Previous studies have shown that in a given cell type, certain active genes associate with SC-35 domains, nuclear regions rich in RNA metabolic factors and excluded from heterochromatin. This organization is not seen for all active genes; therefore, it is important to determine whether and when this locus-specific organization arises during development and differentiation of specific cell types. Here, we investigate whether gene organization relative to SC-35 domains is cell type specific by following several muscle and nonmuscle genes in human fibroblasts, committed but proliferative myoblasts, and terminally differentiated muscle. Although no change was seen for other loci, two muscle genes (Human beta-cardiac myosin heavy chain and myogenin) became localized to the periphery of an SC-35 domain in terminally differentiated muscle nuclei, but not in proliferative myoblasts or in fibroblasts. There was no apparent change in gene localization relative to either the chromosome territory or the heterochromatic compartment; thus, the gene repositioning seemed to occur specifically with respect to SC-35 domains. This gene relocation adjacent to a prominent SC-35 domain was recapitulated in mouse 3T3 cells induced into myogenesis by introduction of MyoD. Results demonstrate a cell type-specific reorganization of specific developmentally regulated loci relative to large domains of RNA metabolic factors, which may facilitate developmental regulation of genome expression.

Figure 1.

Detection of muscle-specific genes or transcripts (red) with SC-35 domains (green) in fully differentiated multinucleated myotubes (A–H) or in determined but undifferentiated myoblast cells (I, J, and K). (A and C) cMyHC RNA localizes with and frequently fills the interior regions of SC-35 domains. Areas of overlap are yellow. (C) Enlargement of a single cMyHC RNA track and SC-35 domain illustrating overlap (left-most panel). (B and D) Myogenin RNA associates with and localizes at the SC-35 domain periphery. (D) Enlargement of two domains illustrating specific and consistent association of myogenin RNA foci at the domain edge. Note the size of the myogenin RNA foci detected are approximately the size of a gene signal as seen in E and F and are considerably smaller than the domain. (E and F) Occasionally, both alleles of the transcriptionally active myogenin gene associate with the same SC-35 domain (arrows) in myotube nuclei. (F) The same myotube as in E, stained with DAPI (blue) to delineate multiple nuclei. (G and H) MyoD RNA (G) and DNA (H) do not associate with prominent SC-35 domains. Inset in G shows an enlarged view of the separation of RNA signal from the domain. (I–K) Myoblast cells were cropped to show enlargements of all three muscle-specific genes, cMyHC (I), myogenin (J), and MyoD (K), are not associated with SC-35 domains in myoblast cells before terminal differentiation.

Figure 2.

Quantitative analysis of gene organization in relation to myogenesis. The association of muscle-specific, nonmuscle, and nonexpressed sequences with SC-35 domains in differentiated myotubes, determined myoblasts, and nonmuscle fibroblast cells. Data were generated from DNA and/or RNA hybridizations. A sharp increase in association can be seen for cMyHC and myogenin in differentiated myotubes, concomitant with expression. No such change in association was seen for MyoD or for the nonmuscle-specific sequences. The data generated were gathered from examination of a minimum 50–200 signals, with the exception of albumin in myotubes where 10 signals were scored and all found to be nonassociated.

Figure 3.

Localization of muscle specific genes relative to SC-35 domains, centric heterochromatin, and chromosome territories in undifferentiated and differentiated mouse NIH3T3 (B22) (A–E and H) or human (F and G) fibroblast cells. (A–D) Colors were separated to allow distinct visualization of the mouse myogenin gene signal (red) with SC-35 domains (green) and DAPI staining of mouse centromeric heterochromatin (blue). (A and B) The mouse myogenin gene does not associate with domains in undifferentiated cells. (C and D) The mouse myogenin gene in MyoD-induced cells shows a change in association pattern such that the gene localizes to the edge of SC-35 domains, similar to that seen in human cells. The myogenin gene does not show a specific relationship to heterochromatin (blue) in either undifferentiated (A and B) or differentiated cells (C and D). The myogenin gene was occasionally associated with heterochromatin (A, right cell; upper left signal and left signal in D) despite differentiation status but was predominately unassociated. (E) Myogenin protein staining demonstrating frequency of differentiation (arrow points to one negative cell in a field of six). (F and G) Although certain muscle specific genes can change their position relative to SC-35 domains upon differentiation, the cMyHC gene localizes at the outer boundaries of its chromosome paint despite its expression status in both undifferentiated (F) and differentiated human myotubes (G). The peripheral localization was consistently apparent in two-dimensional images of whole cells. (H) Mouse NIH3T3 cells hybridized with hnRNA (red) by using a probe to Cot-1 DNA reveal regions of low hnRNA concentration in regions of DAPI-bright (blue) A-T–rich centromeres. A rim of heterochromatin is delineated by the absence of cot-1 RNA around the nuclear periphery.