Histone Crotonylation as a Novel Chromatin Mark Regulating Spermatogenesis Genes
DOI:
https://doi.org/10.5281/zenodo.22757457Keywords:
histone crotonylation, H3K9cr, BRDT, spermatogenesis, round spermatid, ACSS2, testis chromatinAbstract
Histone crotonylation is a short-chain acylation mark enriched at active promoters in male germ cells, where it has
been proposed to mark a distinct set of post-meiotic spermatogenesis genes recognised by the testis-specific
tandem bromodomain protein BRDT, yet the genome-wide distribution of this mark across spermatogenic stages
and its functional dependence on BRDT recognition have not been directly resolved in the same system. Here we
used CUT&RUN for H3K9 crotonylation (H3K9cr) across four purified spermatogenic cell populations, mass
spectrometry-based quantification of crotonyl-CoA, and a CRISPR-generated BRDT bromodomain point mutation
deficient in crotonyl-lysine recognition to dissect the role of histone crotonylation in spermatogenesis gene
regulation. CUT&RUN showed that H3K9cr was low in spermatogonia and pachytene spermatocytes but increased
sharply in round spermatids, where it was enriched at the promoters of a defined set of post-meiotic spermiogenesis
genes distinct from the constitutively H3K9-acetylated housekeeping gene set. Mass spectrometry showed a
corresponding round spermatid-specific increase in crotonyl-CoA abundance, consistent with substrate availability
contributing to the stage-specific gain of the mark. Mice carrying the BRDT bromodomain point mutation showed
preserved H3K9cr deposition at round spermatid promoters but a significant reduction in expression of the
H3K9cr-marked spermiogenesis gene set, together with impaired chromatin compaction during the
histone-to-protamine transition and reduced sperm count, without a corresponding change in the expression of
genes lacking H3K9cr enrichment. These results establish that histone crotonylation is deposited in a
stage-specific, substrate-linked manner during spermatogenesis and that its recognition by BRDT is functionally
required for expression of the post-meiotic gene set it marks and for normal completion of spermiogenesis,
identifying crotonylation-BRDT reading as a distinct regulatory layer operating alongside conventional histone
acetylation during male germ cell differentiation.
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