Abstract
Lineage potential is triggered by lineage-specific transcription factors in association with changes in the chromatin structure. Histone H3.3 variant is thought to play an important role in the regulation of lineage-specific genes. To elucidate the function of H3.3 in myogenic differentiation, we forced the expression of GFP-H3.1 to alter the balance between H3.1 and H3.3 in mouse C2C12 cells that could be differentiated into myotubes. GFP-H3.1 replaced H3.3 in the regulatory regions of skeletal muscle (SKM) genes and induced a decrease of H3K4 trimethylation (H3K4me3) and increase of H3K27 trimethylation (H3K27me3). Similar results were obtained by H3.3 knockdown. In contrast, MyoD-dependent H3.3 incorporation into SKM genes in fibroblasts induced an increase of H3K4me3 and H3K27me3. In mouse embryos, a bivalent modification of H3K4me3 and H3K27me3 was formed on H3.3-incorporated SKM genes before embryonic skeletal muscle differentiation. These results suggest that lineage potential is established through a selective incorporation of specific H3 variants that governs the balance of histone modifications.
Highlights
The development of multicellular organisms is accompanied by the acquisition of various differentiated cells
Dox-dependent expression of Green Fluorescence Protein (GFP)-H3.1 or GFP-H3.3 was confirmed by immunoblotting using whole cell lysate (Supplementary Figure S1B) and their incorporation into chromatin was supported by the association with HAP-purified nucleosomes (Supplementary Figure S1C; nucleosomal GFP-H3.1 and GFP-H3.3 were estimated to occupy ∼20 and ∼12% of the total H3, respectively) and by little fluorescence recovery after photobleaching in living cells (Supplementary Figure S1D)
Since H3.3 is incorporated into the promoter regions of skeletal muscle (SKM) genes before differentiation [13], we considered whether the exogenous expression of GFP-H3.1 could replace endogenous H3.3 at critical regulatory regions
Summary
The development of multicellular organisms is accompanied by the acquisition of various differentiated cells. Cells acquire lineage potential toward specific directions during cell fate decision, and the lineage potential can be established by marking genes prior to their expression after differentiation. One example is K4me in histone H3 (H3K4me3), which is localized around the transcription start sites (TSS) of actively transcribed genes. K27me in histone H3 (H3K27me3) is associated with transcriptionally repressed chromatin. Even though these two modifications function antagonistically, their coexistence (known as bivalent modification) has been shown in many promoter regions of genes important for developmental lineage regulation in mouse embryonic stem (mES) cells [2,3,4]. H3K4me and H3K27me may mark lineage specific genes prior to their expression in differentiation
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