Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch

Abstract

Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis.

Figure 1

HIRA interacts with Akt1. (a, b) Whole-cell extracts prepared from C2C12 (a) or HEK293T (b) cells were subjected to IP using anti-p-Ser, anti-p-Thr, and control IgG antibodies, they were then probed with anti-HIRA (WC119) to detect phospho-HIRA. Two phospho-proteins, RNA polymerase II and β-catenin, were used as positive controls. (c) Sequences potentially targeted by Akt (RxRxxS/T) in vertebrate homologues were aligned. (d, e) GFP-HIRA was co-expressed with Myc-Akt1 in 293T cells. Whole-cell extracts were subjected to IP and immunoblotted using the indicated antibodies. Five percent of the input sample was loaded in parallel. *Non-specific background. (f) 293T cells were treated with MK2206 (10 μM) or LY294002 (10 μM) and the whole-cell extracts were subjected to IP using anti-HIRA(WC15) or IgG. Anti-pan-Akt, p-Akt(S473), or HIRA(WC119) antibodies were used for immunoblotting. TBP (TATA-binding protein) was used as the loading control. GFP, green fluorescent protein.

Figure 2

HIRA is phosphorylated by Akt1 at serine 650. (a) An in vitro kinase assay was performed with purified GST or GST-HIRA(640–655; WT or S650A) and 80 ng of Akt1. Reaction mixtures were subjected to SDS-PAGE followed by autoradiography to detect P³²-labeled HIRA. Akt1 shows autophosphorylation. *GST and partial degradation products. (b) Full-length HIRA WT or S650A (3 μg each) purified from insect cells was phosphorylated in the presence and absence of 80 ng of Akt1 and cold ATP. Phosphorylation of HIRA was detected using an anti-phospho-HIRA antibody (p-HIRA). (c) Verification of anti-phospho-HIRA antibody. The specificity of the purified p-HIRA antibody was tested by dot blot assay. The indicated amounts of peptides with phosphorylated Ser 650 (RPRKD(pS)RLMPV) or their unmodified counterparts (RPRKDSRLMPV) were spotted on the membrane, and immunoblotting was performed using an anti p-HIRA antibody. The antibody specifically recognized the p-peptides phosphorylated at Ser650. (d) Whole-cell extracts from 293T cells were subjected to IP using an anti-p-HIRA antibody in the presence or absence of phospho- or unmodified peptides acting as competitors. Immunoprecipitates were probed with WC119 to detect HIRA. Rabbit IgG was used as the negative control. (e) 293T cells were treated with MK2206 (10 μM) for 24 h and whole-cell extracts were used for immunoblotting with the indicated antibodies. Tubulin was used as the loading control. IP, immunoprecipitation; WT, wild type.

Figure 3

HIRA phosphorylation decreases during muscle cell differentiation. The levels of the indicated proteins were monitored in C2C12 cells grown in growth (GM) or differentiating medium for 3 days (D1–3). (a, b) C2C12 cell extracts prepared from myoblasts and myotubes were subjected to IP using an anti-p-HIRA antibody and immunoblotted with an anti-HIRA antibody. Myogenin, MHC and Ezh2 were used as differentiation markers. The p-HIRA/HIRA protein signal ratio in each sample was quantitated and compared through normalization. (c) Whole-cell extracts were analyzed by western blotting using the indicated antibodies. TBP (TATA-binding protein) served as the loading control. The p-HIRA/HIRA protein signal ratio was obtained as above from two independent experiments. (d) ChIP using an anti-p-HIRA antibody was performed to monitor the recruitment of p-HIRA on MyoD regulatory regions in C2C12 myoblasts (GM) and myotubes (D3). Chromatin solutions prepared from each were subjected to IP using an anti-p-HIRA antibody and analyzed in duplicate via quantitative real-time-PCR to measure the relative enrichment of p-HIRA at the indicated loci. The data represent the percentages of ChIP (IP/Input). Error bars represent s.d., n=2. *P<0.05. CER, core enhancer region; ChIP, chromate immunoprecipitation; DRR, distal regulatory region; PRR, proximal regulatory region; siRNA, small interfering RNA.

Figure 4

Akt1 is the major kinase of HIRA in myoblasts. (a) Knock-down of each Akt isoform using specific siRNAs. C2C12 cells were treated with control siRNA or siRNAs specifically targeting Akt1 or Akt2 for 72 h. Total RNA was isolated to determine the steady-state level of each Akt by qRT-PCR. The PCR values were normalized to β-actin and presented as values relative to the control, which was set as 1. (b, c) C2C12 cells were treated with control siRNA or siRNAs for Akt1 or Akt2 for 72 h. Whole-cell extracts were then subjected to western blotting using the indicated antibodies (b) or subjected to IP using an anti-p-HIRA antibody (c), as described in Figure 3. siRNA, small interfering RNA; qRT-PCR, quantitative reverse transcription PCR.

Figure 5

Effect of S650 mutation on protein interactions and H3/H4 histone binding. (a) Purified recombinant Flag-HIRA (WT, S650A and S650D) and GST-Asf1a were incubated in vitro and precipitated using an anti-HIRA antibody (WC15). IP pellets were probed with anti-Asf1a and anti-HIRA (WC119) antibodies. (b, c) HA-HIRA (wild type, S650A, or S650D) was expressed in 293T cells. HA-HIRA was subjected to IP using an anti-HA antibody and the endogenous proteins interacting with HIRA were detected by immunoblotting with the corresponding specific antibodies. WT, wild type.

Figure 6

Phosphorylation mimicking form of HIRA prevents the incorporation of H3.3 and the activation of muscle genes. (a) C2C12 cells stably expressing empty vectors or each type of HA-hHIRA were generated and confirmed by IP. Cell extracts were subjected to IP using an anti-HA antibody and probed with an anti-WC119 HIRA antibody that recognized both human and mouse HIRA proteins. (b) C2C12 cells were treated with siRNAs specifically targeting endogenous mouse HIRA. The mHIRA knockdown efficiency was monitored by qRT-PCR. (c) C2C12 cell lines were treated with mHIRA-targeting siRNAs, induced to differentiate for 3 days, and subjected to qRT-PCR for analysis of mRNA levels. (d) H3.3 ChIP was performed to monitor H3.3 incorporation in the MyoD regulatory regions in the indicated C2C12 cell lines. Chromatin solutions prepared from myotubes were subjected to IP using an anti-H3.3 antibody. Error bars represent s.d., n=2. IP, immunoprecipitation; mRNA, messenger RNA; qRT-PCR, quantitative reverse transcription PCR; siRNA, small interfering RNA.