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"Morgan, Michael T."
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Structural basis for histone H2B deubiquitination by the SAGA DUB module
Monoubiquitinated histone H2B plays multiple roles in transcription activation. H2B is deubiquitinated by the Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator, which contains a four-protein subcomplex known as the deubiquitinating (DUB) module. The crystal structure of the Ubp8/Sgf11/Sus1/Sgf73 DUB module bound to a ubiquitinated nucleosome reveals that the DUB module primarily contacts H2A/H2B, with an arginine cluster on the Sgf11 zinc finger domain docking on the conserved H2A/H2B acidic patch. The Ubp8 catalytic domain mediates additional contacts with H2B, as well as with the conjugated ubiquitin. We find that the DUB module deubiquitinates H2B both in the context of the nucleosome and in H2A/H2B dimers complexed with the histone chaperone, FACT, suggesting that SAGA could target H2B at multiple stages of nucleosome disassembly and reassembly during transcription.
Journal Article
Measuring DNA mechanics on the genome scale
Mechanical deformations of DNA such as bending are ubiquitous and have been implicated in diverse cellular functions
1
. However, the lack of high-throughput tools to measure the mechanical properties of DNA has limited our understanding of how DNA mechanics influence chromatin transactions across the genome. Here we develop ‘loop-seq’—a high-throughput assay to measure the propensity for DNA looping—and determine the intrinsic cyclizabilities of 270,806 50-base-pair DNA fragments that span
Saccharomyces cerevisiae
chromosome V, other genomic regions, and random sequences. We found sequence-encoded regions of unusually low bendability within nucleosome-depleted regions upstream of transcription start sites (TSSs). Low bendability of linker DNA inhibits nucleosome sliding into the linker by the chromatin remodeller INO80, which explains how INO80 can define nucleosome-depleted regions in the absence of other factors
2
. Chromosome-wide, nucleosomes were characterized by high DNA bendability near dyads and low bendability near linkers. This contrast increases for deeper gene-body nucleosomes but disappears after random substitution of synonymous codons, which suggests that the evolution of codon choice has been influenced by DNA mechanics around gene-body nucleosomes. Furthermore, we show that local DNA mechanics affect transcription through TSS-proximal nucleosomes. Overall, this genome-scale map of DNA mechanics indicates a ‘mechanical code’ with broad functional implications.
A high-throughput, chromosome-wide analysis of DNA looping reveals its contribution to the organization of chromatin, and provides insight into how nucleosomes are deposited and organised de novo.
Journal Article
Crystal structure of human thymine DNA glycosylase bound to DNA elucidates sequence-specific mismatch recognition
Cytosine methylation at CpG dinucleotides produces m⁵CpG, an epigenetic modification that is important for transcriptional regulation and genomic stability in vertebrate cells. However, m⁵C deamination yields mutagenic G·T mispairs, which are implicated in genetic disease, cancer, and aging. Human thymine DNA glycosylase (hTDG) removes T from G·T mispairs, producing an abasic (or AP) site, and follow-on base excision repair proteins restore the G·C pair. hTDG is inactive against normal A·T pairs, and is most effective for G·T mispairs and other damage located in a CpG context. The molecular basis of these important catalytic properties has remained unknown. Here, we report a crystal structure of hTDG (catalytic domain, hTDGcat) in complex with abasic DNA, at 2.8 Å resolution. Surprisingly, the enzyme crystallized in a 2:1 complex with DNA, one subunit bound at the abasic site, as anticipated, and the other at an undamaged (nonspecific) site. Isothermal titration calorimetry and electrophoretic mobility-shift experiments indicate that hTDG and hTDGcat can bind abasic DNA with 1:1 or 2:1 stoichiometry. Kinetics experiments show that the 1:1 complex is sufficient for full catalytic (base excision) activity, suggesting that the 2:1 complex, if adopted in vivo, might be important for some other activity of hTDG, perhaps binding interactions with other proteins. Our structure reveals interactions that promote the stringent specificity for guanine versus adenine as the pairing partner of the target base and interactions that likely confer CpG sequence specificity. We find striking differences between hTDG and its prokaryotic ortholog (MUG), despite the relatively high (32%) sequence identity.
Journal Article
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 cause different phenotypes and alter the transcription of different genes. We show that Ubp10 has poor activity on yeast nucleosomes, but that the 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.
Journal Article
Mechanism of USP21 autoinhibition and histone H2AK119 deubiquitination
Monoubiquitinated histone H2A lysine 119 (H2AK119ub) is a signature modification associated with transcriptional silencing and heterochromatin formation. Ubiquitin-specific protease 21 (USP21), one of four major deubiquitinating enzymes (DUBs) that target H2AK119ub, plays critical roles in diverse cellular processes1–4. The molecular mechanisms by which USP21 specifically deubiquitinates H2AK119ub and is regulated is unknown. USP21 contains a C-terminal USP catalytic domain, preceded by an N-terminal intrinsically disordered region (IDR). We determined the cryo-EM structure of the USP21 catalytic domain bound to an H2AK119ub nucleosome, which reveals a recognition mode that differs from that of two other H2AK119-specific DUBs, Polycomb repressive complex5 and USP166. We unexpectedly discovered that the N-terminal intrinsically disordered region (IDR) of USP21 inhibits the enzyme’s activity. Using AlphaFold-Multimer to perform a virtual screen of USP21 interactors, we identified kinases that phosphorylate the USP21 IDR and thereby relieve autoinhibition. Modeling of USP21 using AlphaFold3 suggests a structural model explaining the mechanism of autoinhibition. AlphaFold analysis of other ubiquitin-specific proteases suggests that phosphorylation-regulated autoinhibition may be a feature of multiple USP enzymes. These findings shed light on the molecular mechanisms of H2AK119 deubiquitination and reveal a novel mode of phosphorylation-dependent DUB autoregulation.
Paper
Potent macrocycle inhibitors of the human SAGA deubiquitinating module
The SAGA complex is a transcriptional coactivator that plays multiple roles in activating transcription and is conserved from yeast to humans. One of SAGAs activities is the removal of ubiquitin from histone H2B-K120 by the deubiquitinating module (DUBm), a four-protein subcomplex containing the catalytic subunit, USP22, bound to three proteins that are required for catalytic activity and targeting to nucleosomes. Overexpression of USP22 is correlated with cancers with a poor prognosis that are resistant to available therapies. We used the RaPID (Random non-standard Peptides Integrated Discovery) system to identify cyclic peptides that are potent and highly specific inhibitors of USP22. Peptide binding did not impact the overall integrity of the DUBm complex as judged by small-angle x-ray scattering, indicating that the inhibitors do not disrupt subunit interactions required for USP22 activity. Cells treated with peptide had increased levels of H2B monoubiquitination, demonstrating the ability of the cyclic peptides to enter human cells and inhibit H2B deubiquitination. The macrocycle inhibitors we have identified in this work thus exhibit favorable drug-like properties and constitute, to our knowledge, the first reported inhibitors of USP22/SAGA DUB module. Competing Interest Statement H. Suga is on the board of directors of MiraBiologics. Footnotes * This revised manuscript has additional data testing inhibitor specificity over a broader range of deubiquitinating enzymes.
Paper
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.
Paper
Measuring DNA mechanics on the genome scale
Mechanical deformations of DNA such as bending are ubiquitous and implicated in diverse cellular functions. However, the lack of high-throughput tools to directly measure the mechanical properties of DNA limits our understanding of whether and how DNA sequences modulate DNA mechanics and associated chromatin transactions genome-wide. We developed an assay called loop-seq to measure the intrinsic cyclizability of DNA, a proxy for DNA bendability, in high throughput. We measured the intrinsic cyclizabilities of 270,806 50 bp DNA fragments that span the entire length of S. cerevisiae chromosome V and other genomic regions, and also include random sequences. We discovered sequence-encoded regions of unusually low bendability upstream of Transcription Start Sites (TSSs). These regions disfavor the sharp DNA bending required for nucleosome formation and are co-centric with known Nucleosome Depleted Regions (NDRs). We show biochemically that low bendability of linker DNA located about 40 bp away from a nucleosome edge inhibits nucleosome sliding into the linker by the chromatin remodeler INO80. The observation explains how INO80 can create promoter-proximal nucleosomal arrays in the absence of any other factors by reading the DNA mechanical landscape. We show that chromosome wide, nucleosomes are characterized by high DNA bendability near dyads and low bendability near the linkers. This contrast increases for nucleosomes deeper into gene bodies, suggesting that DNA mechanics plays a previously unappreciated role in organizing nucleosomes far from the TSS, where nucleosome remodelers predominate. Importantly, random substitution of synonymous codons does not preserve this contrast, suggesting that the evolution of codon choice has been impacted by selective pressure to preserve sequence-encoded mechanical modulations along genes. We also provide evidence that transcription through the TSS-proximal nucleosomes is impacted by local DNA mechanics. Overall, this first genome-scale map of DNA mechanics hints at a mechanical code with broad functional implications. Competing Interest Statement The authors have declared no competing interest.
Paper