Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 10 (1), 77-84

Pax7 Activates Myogenic Genes by Recruitment of a Histone Methyltransferase Complex

Affiliations

Pax7 Activates Myogenic Genes by Recruitment of a Histone Methyltransferase Complex

Iain W McKinnell et al. Nat Cell Biol.

Abstract

Satellite cells purified from adult skeletal muscle can participate extensively in muscle regeneration and can also re-populate the satellite cell pool, suggesting that they have direct therapeutic potential for treating degenerative muscle diseases. The paired-box transcription factor Pax7 is required for satellite cells to generate committed myogenic progenitors. In this study we undertook a multi-level approach to define the role of Pax7 in satellite cell function. Using comparative microarray analysis, we identified several novel and strongly regulated targets; in particular, we identified Myf5 as a gene whose expression was regulated by Pax7. Using siRNA, fluorescence-activated cell sorting (FACS) and chromatin immunoprecipitation (ChIP) studies we confirmed that Myf5 is directly regulated by Pax7 in myoblasts derived from satellite cells. Tandem affinity purification (TAP) and mass spectrometry were used to purify Pax7 together with its co-factors. This revealed that Pax7 associates with the Wdr5-Ash2L-MLL2 histone methyltransferase (HMT) complex that directs methylation of histone H3 lysine 4 (H3K4, refs 4-10). Binding of the Pax7-HMT complex to Myf5 resulted in H3K4 tri-methylation of surrounding chromatin. Thus, Pax7 induces chromatin modifications that stimulate transcriptional activation of target genes to regulate entry into the myogenic developmental programme.

Figures

Figure 1
Figure 1
Candidate target genes are specifically activated by Pax7 in C2C12 myoblasts and are only weakly responsive to Pax3. (a) Pax7 strongly activated PlagL1, Cipar1, Lix1, Mest and Trim54 in C2C12 myoblasts whereas Pax3 was far less effective. Syne2 was only weakly increased by Pax7. Expression was normalized to GAPDH transcript levels and is shown relative to the empty-vector (Puro) controls. (b) PlagL1, and particularly Lix1, were both strongly induced by Pax7 in non-muscle 10T1/2 fibroblasts but were weakly induced or unresponsive to Pax3. Cipar, Trim54 and Syne2 transcripts are also increased by Pax7d in 10T1/2 cells. (c) Myf5 protein levels were also increased by Pax7–FLAG and Pax3–FLAG expression in C2C12 myoblasts whereas MyoD protein levels remained unaffected; α-tubulin was included as a control. (d) Real-time PCR and western blot analysis demonstrated that Myf5 RNA and protein, respectively, were increased in 10T1/2 cells expressing Pax7-FLAG (error bars are standard error (s.e.m.)). Full-length scans of western blot data can be found in Supplementary Information S4.
Figure 2
Figure 2
Identification of Pax7-interacting co-factors. (a) A TAP tag, consisting of six histidine and three FLAG epitopes, separated by a TEV cleavage site, was fused to the C-terminus of Pax7 to create a Pax7–CTAP (His–TEV–FLAG) construct. A construct expressing only the tag (referred to as HisFLAG-tag) was used as a negative control. (b) High yield purification of the Pax7–CTAP protein as shown by detection of the fusion protein in the initial cell lysate, in the eluate following TEV cleavage, and in the final elution from Ni+-resin. (c,) TAP of Pax7–CTAP- compared with HisFLAG-tag-associated proteins from C2C12 cells. Protein matches were generated from multiple data sets and were identified following MALDI–TOF (analysed via Mascot). Pax7–CTAP-interacting proteins were compared with those identified in HisFLAG-tag purifications to unequivocally identify those that were Pax7-specific (versus those that represented contaminants). (d) Pax7 co-immunoprecipitated with Wdr5 and Ash2L, two conserved units of a HMT complex. As a negative control, the Pax7-immunoprecipitate was also probed with an antibody to a non-native-Pax7 interacting nuclear protein, ERK1/2. (e) Co-purification of Pax7 and MLL2 immunoprecipitated from primary myoblast nuclear extract. (f) Pax7-deletion constructs used to map Pax7 binding to the HMT complex. (g) Loss of the paired domain was observed to almost entirely abolish binding between Pax7 and Wdr5. (h) Pax7–FLAG truncations where observed to localize to the nucleus (DAPI-blue, Pax7–FLAG deletion constructs-red, scale bar = 20 μm). Full length scans of western blot data can be found in Supplementary Information S4.
Figure 3
Figure 3
Pax7 immunocomplex has HMT activity and associates with sites of H3K4 methylation. (a) Pax7 or IgG immunoprecipitates from differentiated myotubes (Pax7 negative) or primary myoblasts were incubated with core histones and 3H-SAM. Pax7 immunocomplexes from primary myoblasts were consistently observed to induce a markedly higher level of histone methylation than IgG controls (error bars represent s.e.m.). (b) Fluorography of histones incubated with Pax7 immunocomplexes from primary myoblasts indicated that the methyltransferase activity associated with the Pax7 immunocomplex were directed at histone H3. (c) Western blot analysis with antisera directed against dimethylated H3K4 (H3K42me) and trimethylated H3K4 (H3K43me) indicate that histones incubated with the Pax7 immunocomplexes show a markedly increased level of H3K43me, with a concurrent but lesser increase in H3K42me. (d) IP-western blot analysis revealed that Pax7 was highly associated with chromatin isolated from primary myoblasts using antisera reactive with H3K42me and H3K43me, but not at all with chromatin isolated with antisera reactive with dimethylated H3K9. Full-length scans of western blot data can be found in Supplementary Information S4.
Figure 4
Figure 4
Pax7 regulates Myf5 expression directly in satellite cell-derived myoblasts. (a) Over-expression of Pax7 resulted in elevated levels of Myf5 RNA and protein. (b) siRNA knockdown of Pax7 in satellite cell-derived myoblasts results in concurrent knockdown of Myf5 expression. (c) FACS sorted satellite cells (Integrin α-7+; Lin: CD31−, CD45−, CD11−, Sca1−) from Pax7−/− mice show complete absence of Myf5 expression via both (d) RNA (n = 3 animals per group, P =: 2.9 × 10−5 for Pax7 and 1.7 × 10−5, for Myf5, * = P < 0.001), and (e) immunofluorescence (Syndecan 4-red, (Syn4) Myf5-green (Myf5), scale bar = 20 μm). Expression of LacZ was assayed as the Pax7 knockout was created via the insertion of a LacZ sequence into the Pax7 locus and as such all satellite cells should be expressing LacZ in place of Pax7. (f, g) ChIP demonstrated that Pax7 was bound directly to the −57.5 kb region of Myf5 in complex with Ash2L (f) and in a site-specific manner (g). The downstream −315 bp loci, which does not contain a binding site and hence did not show any enrichment, is shown for comparison purposes. (h) Antisera directed against H3K4 trimethylation ChIP demonstrated that, accordingly, the coding region of Myf5 showed a marked increase in activation as measured by trimethylation of H3K4 (error bars = s.e.m.). Full-length scans of western blot data can be found in Supplementary Information S4.

Similar articles

See all similar articles

Cited by 137 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback