Transcription occurs in pulses in muscle fibers - PubMed (original) (raw)

Transcription occurs in pulses in muscle fibers

S Newlands et al. Genes Dev. 1998.

Abstract

We report a novel mechanism of gene regulation in skeletal muscle fibers. Within an individual myofiber nucleus, not all muscle loci are transcriptionally active at a given time and loci are regulated independently. This phenomenon is particularly remarkable because the nuclei within a myofiber share a common cytoplasm. Both endogenous muscle-specific and housekeeping genes and transgenes are regulated in this manner. Therefore, despite the uniform protein composition of the contractile apparatus along the length of the fiber, the loci that encode this structure are not transcribed continuously. The total number of active loci for a particular gene is dynamic, changing during fetal development, regeneration, and in the adult, and potentially reflects the growth status of the fiber. The data reveal that transcription in particular stages of muscle fiber maturation occurs in pulses and is defined by a stochastic mechanism.

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Figures

Figure 1

Figure 1

Myonuclei in regenerating muscle fibers are not transcriptionally equivalent. Transcripts of endogenous genes in adult mouse regenerating muscle were detected using in situ hybridization. The brown/black staining around the periphery of the nucleus indicates localization of the mRNA. (a) α-SA transcript was detected in extensor digitorum longus (EDL) muscle at day 35 after regeneration surgery. (b) TnIs transcript was detected in day 21 regenerating soleus muscle. The solid black arrows indicate myonuclei expressing the gene; the open arrows show a myonucleus in the same fiber that is not expressing the gene. Scale bar, 25 μm.

Figure 2

Figure 2

Transgene expression does not occur in all myofiber nuclei. Sections of regenerating fibers at day 35 postsurgery from transgenic mice showing nuclei expressing (solid arrows) or not expressing (open arrows) the transgene. The nuclei have been counterstained with nuclear fast red. (a) Soleus from a TnIsUSE–95X1nucZ mouse assayed for β-galactosidase activity. The nucleus stains blue if it contains β-galactosidase activity. (b) EDL from a −2000HSA–CAT mouse assayed for CAT transcripts by in situ hybridization. Purple staining in and around the nucleus indicates localization of transcripts. (c) EDL from a HMG–CoA–lacZ homozygous female assayed for β-galactosidase activity. Scale bar, 50 μm.

Figure 3

Figure 3

Confocal scanning microscopy of myofiber nuclei. (a) Four 0.5-μm optical sections from the middle of a complete scan through a regenerating myofiber of a TnIsUSE–95X1nucZ soleus. β-Galactosidase protein was detected using a rhodamine-labeled antibody. Arrows indicate two nonexpressing nuclei. (b) Same subset of myonuclei visualized with propidium iodide. Arrows indicate the same nuclei as in a. The intensity of the PI signal was equivalent in the presence or absence of β-galactosidase indicating that the lack of β-galactosidase was not caused by a lack of nuclear material. Scale bar, 10 μm.

Figure 4

Figure 4

Isolated whole fibers also show heterogeneous gene activity. FDB muscles from TnIsUSE–95X1nucZ adult mice were treated enzymatically to dissociate the myofibers and remove the satellite cells. The isolated fibers were assayed for β-galactosidase-activity. (a) A fiber from a nonnmanipulated FDB muscle. The solid arrow indicates an expressing nucleus; open arrows indicates nonexpressing nuclei. (b) A regenerating fiber from FDB muscle 21 days after surgery. This shows a domain of transgene expression (solid arrow) flanked by domains of transgene inactivity (open arrows). Scale bar, 50 μm.

Figure 5

Figure 5

The proportion of expressing myonuclei reflects developmental age in mouse muscles. (a) Isolated fibers from six FDB muscles of three adult TnIsUSE–95X1nucZ mice were scored individually to determine the percentage of β-galactosidase expressing nuclei per fiber. Fibers (98) were scored with an average of 34 nuclei per fiber. Sections of hindlimbs from four dpc 16.5 TnIsUSE–95X1nucZ fetuses were scored for the number of β-galactosidase expressing versus nonexpressing myonuclei per fiber in Quad (b) and TA (c) muscles. Fibers (303) from Quad and 170 fibers from TA were counted with an average of nine nuclei per fiber (minimum of six). The percent values obtained in each sample were grouped into fifth percentile intervals. For each sample, the number of fibers was plotted against the percent of expressing nuclei per fiber.

Figure 6

Figure 6

The proportion of expressing nuclei in myofibers changes during regeneration. Regenerating EDL muscles of −2000HSA–CAT transgenics collected at days 14, 21, and 35 after surgery. In situ hybridization (purple staining in and around the nucleus) to α-SA (a) and −2000HSA–CAT mRNA (b) shows a decrease in the proportion of expressing nuclei in a myofiber over the time course of regeneration. Scale bar, 50 μm.

Figure 7

Figure 7

The distribution and abundance of the spliceosome protein, SC-35, is similar in β-galactosidase-expressing and nonexpressing myonuclei. β-Galactosidase and SC-35 were detected with antibodies in regenerating soleus from a TnIsUSE–95X1nucZ mouse. (a,b) Confocal optical scans of myofiber nuclei shown as combined algorithmic images. (a) Nucleus containing β-galactosidase (red) and SC-35 (yellow). (b) β-Galactosidase-negative nucleus (as determined by enzymatic assay—data not shown) containing SC-35 (green). The DNA was visualized with PI (red). (c,d) Black-and-white image of SC-35 distribution in a and b, respectively. A comparison of c and d shows that the distribution and abundance of SC-35 does not appear to differ in the presence or absence of β-galactosidase protein. Scale bar, 5 μm.

Figure 8

Figure 8

The distribution of active loci appears to be independent. The distribution of an endogenous transcript (α-SA or TnIs) was codetected with the expression of β-galactosidase in TnIsUSE–95X1nucZ regenerating soleus muscles. All combinations of β-galactosidase activity and endogenous gene expression were found in the fibers. (a) A regenerating fiber at 21 days with β-galactosidase activity (blue nuclei) and α-SA transcripts as detected by in situ hybridization (brown staining around the periphery of nuclei). The large arrowhead indicates a nucleus with β-galactosidase activity only; the small arrowhead indicates a nucleus expressing both α-SA transcripts and β-galactosidase. (b) A regenerating fiber at day 35 assayed for α-SA transcripts and β-galactosidase activity. Nucleus expressing α-SA only (arrow), and both α-SA and β-galactosidase (small arrowhead). (c) A regenerating fiber at day 35 assayed for TnIs transcripts and β-galactosidase activity. Nucleus expressing neither β-galactosidase nor TnIs (open arrow), TnIs only (solid arrow), and both TnIs and β-galactosidase (arrowhead). Scale bar, 50 μm.

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