Successful reproduction requires an oocyte competent to sustain early embryo development. By the end of oogenesis, the oocyte has entered a transcriptionally silenced state, the mechanisms and significance of which remain poorly understood. Histone H3.3, a histone H3 variant, has unique cell cycle-independent functions in chromatin structure and gene expression. Here, we have characterised the H3.3 chaperone Hira/Cabin1/Ubn1 complex, showing that loss of function of any of these subunits causes early embryogenesis failure in mouse. Transcriptome and nascent RNA analyses revealed that transcription is aberrantly silenced in mutant oocytes. Histone marks, including H3K4me3 and H3K9me3, are reduced and chromatin accessibility is impaired in Hira/Cabin1 mutants. Misregulated genes in mutant oocytes include Zscan4d, a two-cell specific gene involved in zygote genome activation. Overexpression of Zscan4 in the oocyte partially recapitulates the phenotypes of Hira mutants and Zscan4 knockdown in Cabin1 mutant oocytes partially restored their developmental potential, illustrating that temporal and spatial expression of Zscan4 is fine-tuned at the oocyte-to-embryo transition. Thus, the H3.3 chaperone Hira complex has a maternal effect function in oocyte developmental competence and embryogenesis, through modulating chromatin condensation and transcriptional quiescence.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• chromatin metabolismus   MeSH
• embryonální vývoj genetika   MeSH
• genový knockdown MeSH
• histonové chaperony genetika   metabolismus   MeSH
• histony metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• oocyty růst a vývoj   metabolismus   MeSH
• oogeneze genetika   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• signální transdukce genetika   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zvířata MeSH
• zygota metabolismus   MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Transcription elongation factor Spt6 associates with RNA polymerase II (RNAP II) via a tandem SH2 (tSH2) domain. The mechanism and significance of the RNAP II-Spt6 interaction is still unclear. Recently, it was proposed that Spt6-tSH2 is recruited via a newly described phosphorylated linker between the Rpb1 core and its C-terminal domain (CTD). Here, we report binding studies with isolated tSH2 of Spt6 (Spt6-tSH2) and Spt6 lacking the first unstructured 297 residues (Spt6ΔN) with a minimal CTD substrate of two repetitive heptads phosphorylated at different sites. The data demonstrate that Spt6 also binds the phosphorylated CTD, a site that was originally proposed as a recognition epitope. We also show that an extended CTD substrate harboring 13 repetitive heptads of the tyrosine-phosphorylated CTD binds Spt6-tSH2 and Spt6ΔN with tighter affinity than the minimal CTD substrate. The enhanced binding is achieved by avidity originating from multiple phosphorylation marks present in the CTD. Interestingly, we found that the steric effects of additional domains in the Spt6ΔN construct partially obscure the binding of the tSH2 domain to the multivalent ligand. We show that Spt6-tSH2 binds various phosphorylation patterns in the CTD and found that the studied combinations of phospho-CTD marks (1,2; 1,5; 2,4; and 2,7) all facilitate the interaction of CTD with Spt6. Our structural studies reveal a plasticity of the tSH2 binding pockets that enables the accommodation of CTDs with phosphorylation marks in different registers.

• MeSH
• epitopy genetika   MeSH
• fosforylace genetika   MeSH
• genetická transkripce * MeSH
• histonové chaperony genetika   MeSH
• RNA-polymerasa II genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvence aminokyselin genetika   MeSH
• src homologní domény genetika   MeSH
• transkripční elongační faktory genetika   MeSH
• vazba proteinů genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH