Myogenin recruits the histone chaperone facilitates chromatin transcription (FACT) to promote nucleosome disassembly at muscle-specific genes

Abstract

Facilitates chromatin transcription (FACT) functions to reorganize nucleosomes by acting as a histone chaperone that destabilizes and restores nucleosomal structure. The FACT complex is composed of two subunits: SSRP1 and SPT16. We have discovered that myogenin interacts with the FACT complex. Transfection of FACT subunits with myogenin is highly stimulatory for endogenous muscle gene expression in 10T1/2 cells. We have also found that FACT subunits do not associate with differentiation-specific genes while C2C12 cells are proliferating but are recruited to muscle-specific genes as differentiation initiates and then dissociate as differentiation proceeds. The recruitment is dependent on myogenin, as knockdowns of myogenin show no recruitment of the FACT complex. These data suggest that FACT is involved in the early steps of gene activation through its histone chaperone activities that serve to open the chromatin structure and facilitate transcription. Consistent with this hypothesis, we find that nucleosomes are depleted at muscle-specific promoters upon differentiation and that this activity is dependent on the presence of FACT. Our results show that the FACT complex promotes myogenin-dependent transcription and suggest that FACT plays an important role in the establishment of the appropriate transcription profile in a differentiated muscle cell.

FIGURE 1.

Myogenin interacts with SSRP1 and SPT16. A, N-TAP myogenin is transcriptionally active. 10T1/2 cells were transfected with the indicated constructs and assayed for luciferase activity. The luciferase reporter constructs were Lmod2-luc, which contains the Lmod2 promoter, and Tcap-luc, which contains the Tcap promoter. All data were normalized to the pGL3 basic value, which was set to 1. Experiments were performed in triplicate. B and C, HEK cells were transfected with expression constructs for myogenin (EMSV-myog), SSRP1, or SPT16. B, extracts were immunoprecipitated (IP) with antibodies against myogenin (F5D) and SSRP1 and probed with antibodies against SSRP1 and myogenin. C, extracts were immunoprecipitated with antibodies against myogenin or SPT16 and probed with antibodies against SPT16 and myogenin. D, MyoD does not interact with SSRP1 or SPT16. HEK cells were transfected with expression constructs for MyoD, SSRP1, or SPT16 and immunoprecipitated for SSRP1 or SPT16 and probed with antibodies against MyoD and the reciprocal factor. E, SSRP1 and SPT16 interact with the C terminus of myogenin. HEK cells were transfected with expression constructs for the N terminus of myogenin-V5 (1–136), C terminus of myogenin-V5 (amino acids 59–224), full-length (FL) myogenin-V5 (amino acids 1–224) and either SSRP1 or SPT16. Extracts were immunoprecipitated with antibodies against V5 and probed with antibodies against SPT16 or SSRP1. Blots were also probed with V5 to confirm the immunoprecipitation. F, myogenin interact with SSRP1 and SPT16 in vivo. Extracts from primary myoblasts differentiated for 2 days were immunoprecipitated for myogenin and probed with antibodies against SPT16, SSRP1, and myogenin.

FIGURE 2.

SSRP1 and SPT16 do not activate plasmid-borne muscle-specific luciferase reporters in the presence of myogenin. 10T1/2 cells were transfected with the indicated constructs and assayed for luciferase activity. The luciferase reporter constructs were Lmod2-luc, which contains the Lmod2 promoter (A), and TRE-luc, a multimerized E box from the MCK promoter (B). All data were normalized to the pGL3 basic value, which was set to 1. Experiments were performed in triplicate.

FIGURE 3.

SSRP1 and SPT16 cooperatively promote endogenous myogenin dependent gene activation. 10T1/2 cells were transfected with expression constructs for myogenin (EMSV-myog), MyoD (pEMCIIS), SSRP1 (pcDNA Ssrp1 V5), and SPT16 (pCMV-4-SPT16) and assayed for muscle-specific gene expression by quantitative PCR. All data were normalized to Hprt. A, SSRP1 and SPT16 independently stimulate gene expression in the presence of myogenin. B, SSRP1 and SPT16 cooperatively stimulate gene expression in the presence of myogenin. C, SSRP1 and SPT16 also cooperatively stimulate myogenin-dependent gene expression in the presence of MyoD. D, MyoD stimulates myogenin expression in 10T1/2 cells. Myogenin expression was assayed in the experiment shown in C. E. SSRP1 and SPT16 do not activate a MyoD target gene that is not stimulated by myogenin.

FIGURE 4.

SSRP1 and SPT16 are expressed in proliferating cells but down-regulated during differentiation. A, C2C12 cells were differentiated for the indicated number of days and harvested for protein. Extracts were probed with antibodies against SSRP1, SPT16, MYOG, and GAPDH. UD, undifferentiated or proliferating cells; D1-D6, cells differentiated for the indicated number of days. B, primary myoblasts were differentiated as described in A. Extracts were probed with antibodies against SSRP1, SPT16, and GAPDH.

FIGURE 5.

FACT binds muscle-specific genes. A, the FACT complex associates with muscle-specific genes in the early stages of differentiation. ChIP assays were performed on C2C12 cells while proliferating (undifferentiated, UD), after differentiation for 2 days (D2), and after differentiation for 6 days (D6) with antibodies against SSRP1 and SPT16 and primers against the Tnni2 and Lmod2 promoters. All fold enrichment values are normalized to IgH. B, FACT associates with the promoter and coding region of Tnni2 at 2 days of differentiation (D2), coincident with the localization of RNAPII. ChIP assays were performed on C2C12 cells at D2 with antibodies against SSRP1, SPT16, and RNAPII and primers spanning the promoter and coding region of Tnni2. Data are presented as in A. C, FACT localization is coincident with the elongating form of RNAPII in the coding region of Tnni2. ChIP assays were performed as in B with antibodies against the serine 2 phosphorylated C-terminal domain of RBP1, SSRP1, and SPT16 and analyzed as in B.

FIGURE 6.

Myogenin is required for recruitment of SSRP1 to muscle-specific genes. A, stable cell lines expressing shRNA constructs were used to deplete myogenin. Depletion was confirmed by analysis of the level of RNA by quantitative real-time PCR (A) and protein by Western blotting (B). C, chromatin immunoprecipitation assays were performed on differentiated C2C12 cells (D2) transfected with scrambled shRNA constructs (scr) or shRNA constructs against myogenin with antibodies against SSRP1 and primers corresponding to the Tnni2 promoter.

FIGURE 7.

Histone occupancy at muscle-specific gene promoters is highly reduced during gene activation. ChIP assays were performed on C2C12 cells while proliferating (undifferentiated, UD), after differentiation for 2 days (D2), and after differentiation for 6 days (D6) with antibodies against H2A (A), H2B (B), and H3 (C) and primers against the Tnni2 and Lmod2 promoters. Data are shown as relative occupancy with the fold enrichment (normalized to IgH) set at 1 for the UD sample.

FIGURE 8.

SSRP1 is required for histone depletion at muscle-specific promoters. Stable cell lines expressing shRNA constructs were used to deplete SSRP1. Depletion was confirmed by analysis of the level of RNA by quantitative real-time PCR (A) and protein by Western blotting (B). C, histone levels were assayed by ChIP assays on C2C12 cells transfected with scrambled shRNA constructs (scr) or shRNA constructs against SSRP1 that were differentiated for 2 days. Antibodies against H2A and H2B and primers against the promoter regions of Tnni2 and Lmod2 were used. Data are shown as relative occupancy with the fold enrichment (normalized to IgH) set at 1 for the scr sample.

FIGURE 9.

SSRP1 promotes differentiation. A, SSRP1 is required for activation of myogenin-dependent muscle-specific genes. Gene expression for muscle specific genes was assayed by quantitative PCR in differentiated (D4) C2C12 cells transfected with scrambled shRNA constructs (scr) or shRNA constructs against SSRP1. Shown are data for Tnni2, Lmod2, and Acta1. All data were normalized to Hprt1. B, SSRP1 is required for myotube formation and MyHC expression. Stable cell lines expressing shRNA constructs targeting SSRP1 or a scrambled control (scr) were differentiated for 4 days and stained with antibodies against myosin heavy chain (MyHC) and DAPI. Fluorescent images are ×100 magnification. Scale bars = 50 μm. C, the fusion index for day 4 myotubes was calculated from the ratio of the number of nuclei in one field for ten random fields.