Abstract
BackgroundSelective proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during eukaryotic development, generating a cleaved histone H3 (H3cl) product within a small, but significant, portion of the genome. Although increasing evidence supports a regulatory role for H3NT proteolysis in gene activation, the nuclear H3NT proteases and the biological significance of H3NT proteolysis remain largely unknown.ResultsIn this study, established cell models of skeletal myogenesis were leveraged to investigate H3NT proteolysis. These cells displayed a rapid and progressive accumulation of a single H3cl product within chromatin during myoblast differentiation. Using conventional approaches, we discovered that the canonical extracellular matrix (ECM) protease, matrix metalloproteinase 2 (MMP-2), is the principal H3NT protease of myoblast differentiation that cleaves H3 between K18-Q19. Gelatin zymography demonstrated progressive increases in nuclear MMP-2 activity, concomitant with H3cl accumulation, during myoblast differentiation. RNAi-mediated depletion of MMP-2 impaired H3NT proteolysis and resulted in defective myogenic gene activation and myoblast differentiation. Supplementation of MMP-2 ECM activity in MMP-2-depleted cells was insufficient to rescue defective H3NT proteolysis and myogenic gene activation.ConclusionsThis study revealed that MMP-2 is a novel H3NT protease and the principal H3NT protease of myoblast differentiation. The results indicate that myogenic signaling induces MMP-2-dependent H3NT proteolysis at early stages of myoblast differentiation. Importantly, the results support the necessity of nuclear MMP-2 H3NT protease activity, independent of MMP-2 activity in the ECM, for myogenic gene activation and proficient myoblast differentiation.
Highlights
Developmental signaling cascades converge in the nucleus to induce broad, yet precise, epigenomic changes that facilitate the activation of lineage-specific factors necessary for differentiation
Myoblast differentiation induces N‐terminal proteolysis of chromatin‐bound histone H3 To investigate the timing of H3 N-terminal tail (H3NT) proteolysis during myoblast differentiation, mouse C2C12 cells were expanded to confluence in growth media before replacement with differentiation media to induce synchronous myoblast differentiation [11]
The fast-migrating H3 band is indicative of a chromatin-bound cleaved H3 (H3cl) product generated by proteolysis of the histone H3 N-terminus (H3NT) [14]
Summary
Developmental signaling cascades converge in the nucleus to induce broad, yet precise, epigenomic changes that facilitate the activation of lineage-specific factors necessary for differentiation. Rice et al Epigenetics & Chromatin (2021) 14:23 indicate that H3NT proteolysis is a consistent feature of eukaryotic developmental programs including yeast sporulation, mouse spermatogenesis, mouse and human embryonic stem cell differentiation, and differentiation of specialized tissues including breast, bone and skeletal muscle [11,12,13,14,15] These collective studies support H3NT proteolysis as an evolutionarily conserved event in development, the functional and biological significance of this epigenetic modification remain largely unknown. One obvious functional role of H3NT proteolysis is the “erasure” of existing H3NT post-translational modifications (PTMs) and their associated binding proteins/ complexes that likely expedites robust changes in gene expression observed during differentiation This mechanism is consistent with observations reported in yeast where H3NT proteolysis “erased” repressive H3NT PTMs and resulted in gene activation [15]. Increasing evidence supports a regulatory role for H3NT proteolysis in gene activation, the nuclear H3NT proteases and the biological significance of H3NT proteolysis remain largely unknown
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