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. 2001 Aug 15;29(16):3439-47.
doi: 10.1093/nar/29.16.3439.

Differential localization of HDAC4 orchestrates muscle differentiation

E A Miska  1 , E Langley, D Wolf, C Karlsson, J Pines, T Kouzarides
Affiliations

Differential localization of HDAC4 orchestrates muscle differentiation

E A Miska et al. Nucleic Acids Res. .

Abstract

The class II histone deacetylases HDAC4 and HDAC5 interact specifically with the myogenic MEF2 transcription factor and repress its activity. Here we show that HDAC4 is cytoplasmic during myoblast differentiation, but relocates to the nucleus once fusion has occurred. Inappropriate nuclear entry of HDAC4 following overexpression suppresses the myogenic programme as well as MEF2-dependent transcription. Activation of the Ca(2+)/calmodulin signalling pathway via constitutively active CaMKIV prevents nuclear entry of HDAC4 and HDAC4-mediated inhibition of differentiation. Consistent with a role of phosphorylation in HDAC4 cytoplasmic localisation, HDAC4 binds to 14-3-3 proteins in a phosphorylation-dependent manner. Together these data establish a role for HDAC4 in muscle differentiation. Recently, HDAC5 has also been implicated in muscle differentiation. However, despite the functional similarities of HDAC4 and HDAC5, their intracellular localisations are opposed, suggesting a distinct role for these enzymes during muscle differentiation.

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Figures

Figure 1
Figure 1
HDAC4 relocalises from the cytoplasm to the nucleus after myoblast fusion. (A) C2C12 myoblasts were microinjected with pcHDAC4-GFP. Four hours after injection living cells were visualised using fluorescence microscopy. To visualise HDAC4–GFP in myotubes, myoblasts were transferred to differentiation medium, incubated for 2 days and microinjected with pcHDAC4-GFP. Living myotubes were visualised using fluorescence microscopy. (B) Cytoplasmic and nuclear extracts were prepared from one 15 cm dish each of either 40–50% confluent myoblasts or myotubes after 5 days differentiation. Extracts were mixed with HDAC4-specific antibody (HD4B) and immunoprecipitated using protein A/G beads. Immunoprecipitates were separated using SDS–PAGE and western blotting was performed using a second HDAC4-specific antibody (HD4A). (C) C2C12 myoblasts were microinjected with pcHDAC4-GFP and living cells were visualised using fluorescence microscopy. Leptomycin B (LMB) was added to the medium at 20 nM and HDAC4–GFP fluorescence was monitored for 20 min.
Figure 2
Figure 2
HDAC4 inhibits C2C12 differentiation and MEF2 expression. (A) C2C12 myoblasts were transfected either with pcHDAC4-Flag, pBJ5-HDAC5-Flag, pcHDAC1-Flag or pcGAL4 and pEGFP as a transfection marker (100 ng each plasmid). After transfection cells were transferred to differentiation medium for 4 days before fixing. GFP staining was visualised using confocal microscopy. (B) C2C12 myoblasts were transfected with 1 µg 3×MCK-MEF2-luc and 200 ng either pcHDAC4-Flag or pcHDAC1-Flag. After transfection cells were transferred to differentiation medium for 2 days. Cell extracts were prepared and analysed for luciferase activity.
Figure 3
Figure 3
Both the MEF2-binding and catalytic domains of HDAC4 are required for inhibition of differentiation. (A) Schematic representation of the HDAC4 deletion constructs used. (B) C2C12 myoblasts were transfected either with pcHDAC4-Myc/His, pcHDAC4(118–611)-Myc/His, pcHDAC4(118–313)-Myc/His, pcHDAC4(612–1084)-Myc/His, pcHDAC4-D840N-Myc/His or pcGAL4 and pEGFP as a tranfection marker (100 ng each plasmid). After 4 days in differentiation medium, GFP staining was visualised using confocal microscopy.
Figure 4
Figure 4
Activated CaMKIV and MKK6b overcome HDAC4-induced inhibition of MEF2-dependent transcription and C2C12 differentiation. (A) C2C12 myoblasts were transfected with 1 µg 3×MCK-MEF2-luc, 100 ng pcHDAC4-Myc/His, 500 ng pRSV-CaMKIV* or 500 ng pMKK6b(E) as specified for each lane. After transfection, cells were incubated in differentiation medium. After 24 h cell extracts were prepared and analysed for luciferase activity. (B) C2C12 myoblasts were transfected either with pcGAL4 or pcHDAC4-Myc/His (100 ng each), either in the presence or absence of pRSV-CaMKIV* or pMKK6b(E) (200 ng each). 100 ng pEGFP was co-transfected as a transfection marker (100 ng each plasmid). After transfection cells were transferred to differentiation medium for 4 days before fixing. GFP staining was visualised using confocal microscopy.
Figure 5
Figure 5
Active CaMKIV, but not MKK6b, regulates HDAC4 localisation. (A) C2C12 myoblasts were transfected with 100 ng pcHDAC4-GFP. After transfection cells were transferred to differentiation medium for 24 h before fixing. GFP staining was visualised using confocal microscopy. HDAC4-GFP localised either to the cytoplasm or the nucleus. (B) C2C12 myoblasts were transfected with 100 ng pcHDAC4-GFP either in the presence or absence of pRSV-CaMKIV* or pMKK6b(E) (500 ng each). After transfection cells were transferred to differentiation medium for 24 h before fixing. Prior to fixing staurosporine (20 µM) or SB203580 (10 µM) was added to the culture medium for 1 h, as indicated. GFP staining was visualised using confocal microscopy. Percentages of cells displaying cytoplasmic staining are given (n > 200). (C) C2C12 myoblasts were transfected with 100 ng pcHDAC4-GFP either in the presence or absence of pRSV-CaMKIV* or pMKK6b(E) (200 ng each). After transfection cells were transferred to differentiation medium for 4 days before fixing. GFP staining was visualised using confocal microscopy.
Figure 6
Figure 6
HDAC4 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. 293 cells were transfected with 5 µg pcHDAC4-Flag. Thirty-six hours after transfection cells were lysed either with or without prior treatment with staurosporine (20 µM) for 60 min. (A) Cell lysates were incubated with 1 µg recombinant purified GST–14-3-3 fusion protein pre-bound to glutathione beads. Bound material was subjected to SDS–PAGE and western analysis using anti-Flag antibody. (B) Input of GST–14-3-3 fusion proteins separated using SDS–PAGE and visualised using Coomassie staining. (C) Input of cell lysates subjected to SDS–PAGE and western analysis using anti-Flag antibody.
Figure 7
Figure 7
A model for the role of HDAC4 during C2C12 differentiation. In myoblasts HDAC4 is held in the cytoplasm, where it associates with 14-3-3 proteins in a phosphorylation-dependent manner. Cytoplasmic retention of HDAC4, which is stimulated by CaMKIV activity, allows MEF2-dependent transcriptional activation via CBP/p300 histone acetyltransferases. After myotube fusion HDAC4 relocates to the nucleus, where it associates with MEF2 to repress transcription.
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