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Paf1 and Ctr9 subcomplex formation is essential for Paf1 complex assembly and functional regulation.

ABSTRACT: The evolutionarily conserved multifunctional polymerase-associated factor 1 (Paf1) complex (Paf1C), which is composed of at least five subunits (Paf1, Leo1, Ctr9, Cdc73, and Rtf1), plays vital roles in gene regulation and has connections to development and human diseases. Here, we report two structures of each of the human and yeast Ctr9/Paf1 subcomplexes, which assemble into heterodimers with very similar conformations, revealing an interface between the tetratricopeptide repeat module in Ctr9 and Paf1. The structure of the Ctr9/Paf1 subcomplex may provide mechanistic explanations for disease-associated mutations in human PAF1 and CTR9. Our study reveals that the formation of the Ctr9/Paf1 heterodimer is required for the assembly of yeast Paf1C, and is essential for yeast viability. In addition, disruption of the interaction between Paf1 and Ctr9 greatly affects the level of histone H3 methylation in vivo. Collectively, our results shed light on Paf1C assembly and functional regulation.

SUBMITTER: Xie Y

PROVIDER: S-EPMC6143631 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Paf1 and Ctr9 subcomplex formation is essential for Paf1 complex assembly and functional regulation.

Xie Ying Y Zheng Minying M Chu Xinlei X Chen Yue Y Xu Huisha H Wang Jiawei J Zhou Hao H Long Jiafu J

Nature communications 20180918 1

The evolutionarily conserved multifunctional polymerase-associated factor 1 (Paf1) complex (Paf1C), which is composed of at least five subunits (Paf1, Leo1, Ctr9, Cdc73, and Rtf1), plays vital roles in gene regulation and has connections to development and human diseases. Here, we report two structures of each of the human and yeast Ctr9/Paf1 subcomplexes, which assemble into heterodimers with very similar conformations, revealing an interface between the tetratricopeptide repeat module in Ctr9 ...[more]

PMID: 30228257

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Project description:Spores are the major infectious particle of the Gram-positive nosocomial pathogen Clostridioides difficile (formerly Clostridium difficile), but the molecular details of how this organism forms these metabolically dormant cells remain poorly characterized. The composition of the spore coat in C. difficile differs markedly from that defined in the well-studied organism Bacillus subtilis, with only 25% of the ?70 spore coat proteins being conserved between the two organisms and with only 2 of 9 coat assembly (morphogenetic) proteins defined in B. subtilis having homologs in C. difficile We previously identified SipL as a clostridium-specific coat protein essential for functional spore formation. Heterologous expression analyses in Escherichia coli revealed that SipL directly interacts with C. difficile SpoIVA, a coat-morphogenetic protein conserved in all spore-forming organisms, through SipL's C-terminal LysM domain. In this study, we show that SpoIVA-SipL binding is essential for C. difficile spore formation and identify specific residues within the LysM domain that stabilize this interaction. Fluorescence microscopy analyses indicate that binding of SipL's LysM domain to SpoIVA is required for SipL to localize to the forespore while SpoIVA requires SipL to promote encasement of SpoIVA around the forespore. Since we also show that clostridial LysM domains are functionally interchangeable at least in C. difficile, the basic mechanism for SipL-dependent assembly of clostridial spore coats may be conserved.IMPORTANCE The metabolically dormant spore form of the major nosocomial pathogen Clostridioides difficile is its major infectious particle. However, the mechanisms controlling the formation of this resistant cell type are not well understood, particularly with respect to its outermost layer, the spore coat. We previously identified two spore-morphogenetic proteins in C. difficile: SpoIVA, which is conserved in all spore-forming organisms, and SipL, which is conserved only in the clostridia. Both SpoIVA and SipL are essential for heat-resistant spore formation and directly interact through SipL's C-terminal LysM domain. In this study, we demonstrate that the LysM domain is critical for SipL and SpoIVA function, likely by helping recruit SipL to the forespore during spore morphogenesis. We further identified residues within the LysM domain that are important for binding SpoIVA and, thus, functional spore formation. These findings provide important insight into the molecular mechanisms controlling the assembly of infectious C. difficile spores.

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Paf1 and Ctr9 subcomplex formation is essential for Paf1 complex assembly and functional regulation.

Publications

Paf1 and Ctr9 subcomplex formation is essential for Paf1 complex assembly and functional regulation.

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