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DNA binding sites target nuclear NFATc1 to heterochromatin regions in adult skeletal muscle fibers.

ABSTRACT: We have previously demonstrated that Ca²+/calcineurin-dependent dephosphorylation of the transcription factor nuclear factor of activated T cells subtype 1 (NFATc1) during repetitive skeletal muscle activity causes NFAT nuclear translocation and concentration in subnuclear NFAT foci. We now show that NFAT nuclear foci colocalize with heterochromatin regions of intense staining by DAPI or TO-PRO-3 that are present in the nucleus prior to NFATc1 nuclear entry. Nuclear NFATc1 also colocalizes with the heterochromatin markers trimethyl-histone H3 (Lys9) and heterochromatin protein 1?. Mutation of the NFATc1 DNA binding sites prevents entry and localization of NFATc1 in heterochromatin regions. However, fluorescence in situ hybridization shows that the NFAT-regulated genes for slow and fast myosin heavy chains are not localized within the heterochromatin regions. Fluorescence recovery after photobleaching shows that within a given nucleus, NFATc1 redistributes relatively rapidly (t(¹/?) < 1 min) between NFAT foci. Nuclear export of an NFATc1 mutant not concentrated in NFAT foci is accelerated following nuclear entry during fiber activity, indicating buffering of free nuclear NFATc1 by NFATc1 within the NFAT foci. Taken together, our results suggest that NFAT foci serve as nuclear storage sites for NFATc1, allowing it to rapidly mobilize to other nuclear regions as required.

SUBMITTER: Shen T

PROVIDER: S-EPMC3062432 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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DNA binding sites target nuclear NFATc1 to heterochromatin regions in adult skeletal muscle fibers.

Shen Tiansheng T Liu Yewei Y Contreras Minerva M Hernández-Ochoa Erick O EO Randall William R WR Schneider Martin F MF

Histochemistry and cell biology 20100924 4

We have previously demonstrated that Ca²+/calcineurin-dependent dephosphorylation of the transcription factor nuclear factor of activated T cells subtype 1 (NFATc1) during repetitive skeletal muscle activity causes NFAT nuclear translocation and concentration in subnuclear NFAT foci. We now show that NFAT nuclear foci colocalize with heterochromatin regions of intense staining by DAPI or TO-PRO-3 that are present in the nucleus prior to NFATc1 nuclear entry. Nuclear NFATc1 also colocalizes with ...[more]

PMID: 20865272

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Project description:In skeletal muscle, excitation-contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca(2+) channel (Ca(V)1.1) to be communicated to the type 1 ryanodine-sensitive Ca(2+) release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein-protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca(V)1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing ?(1S) subunit of Ca(V)1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary ?(1a) subunit of Ca(V)1.1 is a conduit for this intermolecular communication. However, a direct role for ?(1a) has been difficult to test because ?(1a) serves two other functions that are prerequisite for conformational coupling between Ca(V)1.1 and RYR1. Specifically, ?(1a) promotes efficient membrane expression of Ca(V)1.1 and facilitates the tetradic ultrastructural arrangement of Ca(V)1.1 channels within plasma membrane-SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established ? subunit-interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca(2+) transients without greatly affecting Ca(V)1.1 targeting, intramembrane gating charge movement, or releasable SR Ca(2+) store content. In contrast, a ?(1a)-binding-deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca(2+) release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca(V)1.1 from RYR1-mediated SR Ca(2+) release via its ability to interact with ?(1a). Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the ?(1a) subunit in skeletal-type EC coupling.

Project description:Nuclear Ca(2+) is important for the regulation of several nuclear processes such as gene expression. Localized Ca(2+) signals (LCSs) in skeletal muscle fibers of mice have been mainly studied as Ca(2+) release events from the sarcoplasmic reticulum. Their location with regard to cell nuclei has not been investigated. Our study is based on the hypothesis that LCSs associated with nuclei are present in skeletal muscle fibers of adult mice. Therefore, we carried out experiments addressing this question and we found novel Ca(2+) signals associated with nuclei of skeletal muscle fibers (with possibly attached satellite cells). We measured localized nuclear and perinuclear Ca(2+) signals (NLCSs and PLCSs) alongside cytosolic localized Ca(2+) signals (CLCSs) during a hypertonic treatment. We also observed NLCSs under isotonic conditions. The NLCSs and PLCSs are Ca(2+) signals in the range of micrometer [FWHM (full width at half maximum): 2.75 ± 0.27 μm (NLCSs) and 2.55 ± 0.17 μm (PLCSs), S.E.M.]. Additionally, global nuclear Ca(2+) signals (NGCSs) were observed. To investigate which type of Ca(2+) channels contribute to the Ca(2+) signals associated with nuclei in skeletal muscle fibers, we performed measurements with the RyR blocker dantrolene, the DHPR blocker nifedipine or the IP3R blocker Xestospongin C. We observed Ca(2+) signals associated with nuclei in the presence of each blocker. Nifedipine and dantrolene had an inhibitory effect on the fraction of fibers with PLCSs. The situation for the fraction of fibers with NLCSs is more complex indicating that RyR is less important for the generation of NLCSs compared to the generation of PLCSs. The fraction of fibers with NLCSs and PLCSs is not reduced in the presence of Xestospongin C. The localized perinuclear and intranuclear Ca(2+) signals may be a powerful tool for the cell to regulate adaptive processes as gene expression. The intranuclear Ca(2+) signals may be particularly interesting in this respect.

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DNA binding sites target nuclear NFATc1 to heterochromatin regions in adult skeletal muscle fibers.

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DNA binding sites target nuclear NFATc1 to heterochromatin regions in adult skeletal muscle fibers.

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