Biology subjects

McCullough, L.

Publications and source records attributed to McCullough, L..

4 recordsLinked to original sources

bioRxiv2024

Tom1p ubiquitin ligase structure, interaction with Spt6p, and function in maintaining normal transcript levels and the stability of chromatin in promoters

Phosphorylation-dependent binding of the S. cerevisiae Spt6p tSH2 domain (Spt6ptSH2) to the Rbp1p subunit of RNA polymerase II supports efficient transcription. Here, we report that Spt6ptSH2 also binds the HECT-family E3 ubiquitin ligase Tom1p, a homolog of human HUWE1. Tom1p/HUWE1 have been implicated in targeting many small basic proteins for degradation, including excess ribosomal subunits and histones, although the mechanism of substrate recognition is not known. Our cryo-EM data revealed that Tom1p can adopt a compact -solenoidal "basket" similar to the previously described structure of HUWE1, with the central cavity partially occupied by a disordered acidic domain. Sub-regions of this acidic domain supported binding to Spt6p or histones/nucleosomes in vitro, and the histone-binding region was important for Tom1p function in vivo. We also visualized Tom1p in more extended forms, and speculate that transitions among these forms could be important for substrate selection and ubiquitylation. Genomic analyses provided additional support for the previously observed role for Tom1p in maintaining ribosomal protein pools, and also demonstrated a role in maintaining chromatin structure near genes. This suggests that the interaction with Spt6ptSH2 affects substrate specificity by anchoring Tom1p to localized environments where histone ubiquitylation alters chromatin architecture.

molecular biology↗

bioRxiv2021

ATP-Independent Nucleosome Unfolding by FACT: Electron Microscopy Analysis

FACT is a histone chaperone that unfolds nucleosomes without ATP hydrolysis. We used electron microscopy to study FACT and FACT:nucleosome complexes, and found that both adopt broad ranges of configurations, indicating high flexibility. We found unexpectedly that the DNA binding protein Nhp6 also binds to the C-terminal tails of FACT subunits, inducing more open geometries of FACT even in the absence of nucleosomes. Nhp6 therefore supports nucleosome unfolding by altering both FACT structure and nucleosome properties. Complexes formed with FACT, Nhp6, and nucleosomes also produced a broad range of structures, revealing a large number of potential intermediates along a proposed unfolding pathway. The data suggest that Nhp6 has multiple roles before and during nucleosome unfolding by FACT, and that the process proceeds through a series of energetically similar intermediate structures, ultimately leading to an extensively unfolded form. One Sentence SummaryElectron microscopy reveals the pathway of ATP-Independent nucleosome unfolding by histone chaperone FACT.

molecular biology↗

bioRxiv2018

FACT and Ubp10 collaborate to modulate H2B deubiquitination and nucleosome dynamics

Monoubiquitination of histone H2B (H2B-Ub) plays a role in transcription and DNA replication, and is required for normal localization of the histone chaperone, FACT. In yeast, H2B-Ub is deubiquitinated by Ubp8, a subunit of SAGA, and Ubp10. Although they target the same substrate, loss of Ubp8 and Ubp10 causes different phenotypes and alters the transcription of different genes. We show that Ubp10 has poor activity on yeast nucleosomes, but that addition of FACT stimulates Ubp10 activity on nucleosomes and not on other substrates. Consistent with a role for FACT in deubiquitinating H2B in vivo, a FACT mutant strain shows elevated levels of H2B-Ub. Combination of FACT mutants with deletion of Ubp10, but not Ubp8, confers increased sensitivity to hydroxyurea and activates a cryptic transcription reporter, suggesting that FACT and Ubp10 may coordinate nucleosome assembly during DNA replication and transcription. Our findings reveal unexpected interplay between H2B deubiquitination and nucleosome dynamics.

biochemistry↗

bioRxiv2018

FACT activity and histone H3-K56 acetylation promote optimal establishment of chromatin architecture independent of ongoing transcription in Saccharomyces cerevisiae

FACT is a histone chaperone that can destabilize or assemble nucleosomes. Acetylation of histone H3-K56 weakens a histone:DNA contact that is central to FACT activity, suggesting that this modification could affect FACT functions. We tested this by asking how mutations of H3-K56 and FACT affect nucleosome structure, chromatin integrity, and transcription output. Mimics of unacetylated or permanently acetylated H3-K56 had different effects on FACT in vitro and in vivo as expected, but H3-K56 and FACT mutations caused surprisingly similar changes in transcription of individual genes. Notably, neither the changes in transcript levels nor the effects on nucleosome occupancy resulting from mutations conformed to the model that FACT is needed to overcome nucleosomal barriers during transcription initiation or elongation. Instead, the results suggest that both FACT and H3-K56ac are involved in establishing chromatin architecture prior to transcription and restoring it afterwards. They contribute to a process that optimizes transcription frequency, especially at conditionally expressed genes, and restores chromatin integrity after transcription, especially at the +1 nucleosome to block antisense transcription, but FACT appears to be less involved than expected in directly promoting transcription.

genomics↗