Scientific Reports (Sep 2024)

Comprehensive posttranslational modifications in the testis-specific histone variant H3t protein validated in tagged knock-in mice

  • Takayuki Kawaguchi,
  • Michihiro Hashimoto,
  • Reiko Nakagawa,
  • Ryunosuke Minami,
  • Masahito Ikawa,
  • Jun-ichi Nakayama,
  • Jun Ueda

DOI
https://doi.org/10.1038/s41598-024-72362-7
Journal volume & issue
Vol. 14, no. 1
pp. 1 – 12

Abstract

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Abstract During the development of multicellular organisms and cell differentiation, the chromatin structure in the cell nucleus undergoes extensive changes, and the nucleosome structure is formed by a combination of various histone variants. Histone variants with diverse posttranslational modifications are known to play crucial roles in different regulatory functions. We have previously reported that H3t, a testis-specific histone variant, is essential for spermatogenesis. To elucidate the function of this chromatin molecule in vivo, we generated knock-in mice with a FLAG tag attached to the carboxyl terminus of H3t. In the present study, we evaluated the utility of the generated knock-in mice and comprehensively analyzed posttranslational modifications of canonical H3 and H3t using mass spectrometry. Herein, we found that H3t-FLAG was incorporated into spermatogonia and meiotic cells in the testes, as evidenced by immunostaining of testicular tissue. According to the mass spectrometry analysis, the overall pattern of H3t-FLAG posttranslational modification was comparable to that of the control H3, while the relative abundances of certain specific modifications differed between H3t-FLAG and the control bulk H3. The generated knock-in mice could be valuable for analyzing the function of histone variants in vivo.

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