Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation

Abstract

Background Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts. Results We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes. Conclusion Our results provide an evolutionary perspective on H3.3’s conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.

RebL1 is required for macronuclear structure stability and gametogenesis in Tetrahymena thermophila

“…RebL1 in this region might be involved in transcription repression or chromatin remodeling. RebL1-FZZ localizes in the MAC and micronuclear localization disappears with changes in the cell cycle (Nabeel-Shah et al 2023 ). However, we found that the signal of RebL1-HA did not disappear in MIC, rather it performed positional shifts during the cell cycle.…”

Independent and Complementary Functions of Caf1b and Hir1 for Chromatin Assembly in Tetrahymena thermophila

“…Although Caf1b and Hir1 interacting proteins were detected previously, specific interactors for Hir1 and Caf1b under different development stages were less clear [49]. To further investigate the function of Caf1b and Hir1, interactors of Caf1b and Hir1 were identified.…”