Method for identifying histone tail proteolysis

Abstract

Although limited proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during mammalian differentiation, the specific genomic sites targeted for H3NT proteolysis and the functional significance of H3NT cleavage remain largely unknown. Here Applicant reports the first method to identify and examine H3NT-cleaved regions in mammals, called ChIP of acetylated chromatin (ChIPac). By applying ChIPac-Seq to an established cell model of osteoclast differentiation, Applicant discovered that H3NT proteolysis is selectively targeted near transcription start sites of a small group of genes and that most H3NT-cleaved genes displayed significant expression changes during osteoclastogenesis. Applicant also discovered that the principal H3NT protease of osteoclastogenesis is matrix metalloproteinase 9 (MMP-9). In contrast to other known H3NT proteases, MMP-9 primarily cleaved H3K18-Q19 in vitro and in cells. Furthermore, Applicant's results support CBP/p300-mediated acetylation of H3K18 as a central regulator of MMP-9 H3NT protease activity both in vitro and at H3NT-cleavage sites during osteoclastogenesis. Importantly, Applicant found that abrogation of H3NT proteolysis impaired osteoclastogenic gene activation concomitant with defective osteoclast differentiation. Applicant's collective results support the necessity of MMP-9-dependent H3NT proteolysis in regulating gene pathways required for proficient osteoclastogenesis.

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