Publications by authors named "Jacques Cote"

92 Publications

The deubiquitylase Ubp15 couples transcription to mRNA export.

Elife 2020 11 23;9. Epub 2020 Nov 23.

Institut de recherches cliniques de Montréal, Montréal, Canada.

Nuclear export of messenger RNAs (mRNAs) is intimately coupled to their synthesis. pre-mRNAs assemble into dynamic ribonucleoparticles as they are being transcribed, processed, and exported. The role of ubiquitylation in this process is increasingly recognized but, while a few E3 ligases have been shown to regulate nuclear export, evidence for deubiquitylases is currently lacking. Here we identified deubiquitylase Ubp15 as a regulator of nuclear export in Ubp15 interacts with both RNA polymerase II and the nuclear pore complex, and its deletion reverts the nuclear export defect of E3 ligase Rsp5 mutants. The deletion of leads to hyper-ubiquitylation of the main nuclear export receptor Mex67 and affects its association with THO, a complex coupling transcription to mRNA processing and involved in the recruitment of mRNA export factors to nascent transcripts. Collectively, our data support a role for Ubp15 in coupling transcription to mRNA export.
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http://dx.doi.org/10.7554/eLife.61264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682988PMC
November 2020

De Novo KAT5 Variants Cause a Syndrome with Recognizable Facial Dysmorphisms, Cerebellar Atrophy, Sleep Disturbance, and Epilepsy.

Am J Hum Genet 2020 09 20;107(3):564-574. Epub 2020 Aug 20.

Sainte-Justine Hospital Research Center, University of Montreal, Montreal, QC H3T 1C5, Canada. Electronic address:

KAT5 encodes an essential lysine acetyltransferase, previously called TIP60, which is involved in regulating gene expression, DNA repair, chromatin remodeling, apoptosis, and cell proliferation; but it remains unclear whether variants in this gene cause a genetic disease. Here, we study three individuals with heterozygous de novo missense variants in KAT5 that affect normally invariant residues, with one at the chromodomain (p.Arg53His) and two at or near the acetyl-CoA binding site (p.Cys369Ser and p.Ser413Ala). All three individuals have cerebral malformations, seizures, global developmental delay or intellectual disability, and severe sleep disturbance. Progressive cerebellar atrophy was also noted. Histone acetylation assays with purified variant KAT5 demonstrated that the variants decrease or abolish the ability of the resulting NuA4/TIP60 multi-subunit complexes to acetylate the histone H4 tail in chromatin. Transcriptomic analysis in affected individual fibroblasts showed deregulation of multiple genes that control development. Moreover, there was also upregulated expression of PER1 (a key gene involved in circadian control) in agreement with sleep anomalies in all of the individuals. In conclusion, dominant missense KAT5 variants cause histone acetylation deficiency with transcriptional dysregulation of multiples genes, thereby leading to a neurodevelopmental syndrome with sleep disturbance, cerebellar atrophy, and facial dysmorphisms, and suggesting a recognizable syndrome.
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http://dx.doi.org/10.1016/j.ajhg.2020.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477011PMC
September 2020

JMJD6 participates in the maintenance of ribosomal DNA integrity in response to DNA damage.

PLoS Genet 2020 06 29;16(6):e1008511. Epub 2020 Jun 29.

LBCMCP, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France.

Ribosomal DNA (rDNA) is the most transcribed genomic region and contains hundreds of tandem repeats. Maintaining these rDNA repeats as well as the level of rDNA transcription is essential for cellular homeostasis. DNA damages generated in rDNA need to be efficiently and accurately repaired and rDNA repeats instability has been reported in cancer, aging and neurological diseases. Here, we describe that the histone demethylase JMJD6 is rapidly recruited to nucleolar DNA damage and is crucial for the relocalisation of rDNA in nucleolar caps. Yet, JMJD6 is dispensable for rDNA transcription inhibition. Mass spectrometry analysis revealed that JMJD6 interacts with the nucleolar protein Treacle and modulates its interaction with NBS1. Moreover, cells deficient for JMJD6 show increased sensitivity to nucleolar DNA damage as well as loss and rearrangements of rDNA repeats upon irradiation. Altogether our data reveal that rDNA transcription inhibition is uncoupled from rDNA relocalisation into nucleolar caps and that JMJD6 is required for rDNA stability through its role in nucleolar caps formation.
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http://dx.doi.org/10.1371/journal.pgen.1008511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351224PMC
June 2020

Structural Basis for EPC1-Mediated Recruitment of MBTD1 into the NuA4/TIP60 Acetyltransferase Complex.

Cell Rep 2020 03;30(12):3996-4002.e4

Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, PR China; Structural Genomics Consortium and Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

MBTD1, a H4K20me reader, has recently been identified as a component of the NuA4/TIP60 acetyltransferase complex, regulating gene expression and DNA repair. NuA4/TIP60 inhibits 53BP1 binding to chromatin through recognition of the H4K20me mark by MBTD1 and acetylation of H2AK15, blocking the ubiquitination mark required for 53BP1 localization at DNA breaks. The NuA4/TIP60 non-catalytic subunit EPC1 enlists MBTD1 into the complex, but the detailed molecular mechanism remains incompletely explored. Here, we present the crystal structure of the MBTD1-EPC1 complex, revealing a hydrophobic C-terminal fragment of EPC1 engaging the MBT repeats of MBTD1 in a site distinct from the H4K20me binding site. Different cellular assays validate the physiological significance of the key residues involved in the MBTD1-EPC1 interaction. Our study provides a structural framework for understanding the mechanism by which MBTD1 recruits the NuA4/TIP60 acetyltransferase complex to influence transcription and DNA repair pathway choice.
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http://dx.doi.org/10.1016/j.celrep.2020.03.003DOI Listing
March 2020

Integrated analysis of H2A.Z isoforms function reveals a complex interplay in gene regulation.

Elife 2020 Feb 28;9. Epub 2020 Feb 28.

LBCMCP, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, Toulouse, France.

The H2A.Z histone variant plays major roles in the control of gene expression. In human, H2A.Z is encoded by two genes expressing two isoforms, H2A.Z.1 and H2A.Z.2 differing by three amino acids. Here, we undertook an integrated analysis of their functions in gene expression using endogenously-tagged proteins. RNA-Seq analysis in untransformed cells showed that they can regulate both distinct and overlapping sets of genes positively or negatively in a context-dependent manner. Furthermore, they have similar or antagonistic function depending on genes. H2A.Z.1 and H2A.Z.2 can replace each other at Transcription Start Sites, providing a molecular explanation for this interplay. Mass spectrometry analysis showed that H2A.Z.1 and H2A.Z.2 have specific interactors, which can mediate their functional antagonism. Our data indicate that the balance between H2A.Z.1 and H2A.Z.2 at promoters is critically important to regulate specific gene expression, providing an additional layer of complexity to the control of gene expression by histone variants.
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http://dx.doi.org/10.7554/eLife.53375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048395PMC
February 2020

Molecular Basis for the PZP Domain of BRPF1 Association with Chromatin.

Structure 2020 01 8;28(1):105-110.e3. Epub 2019 Nov 8.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address:

The assembly of human histone acetyltransferase MOZ/MORF complexes relies on the scaffolding bromodomain plant homeodomain (PHD) finger 1 (BRPF1) subunit. The PHD-zinc-knuckle-PHD module of BRPF1 (BRPF1) has been shown to associate with the histone H3 tail and DNA; however, the molecular mechanism underlying recognition of H3 and the relationship between the histone and DNA-binding activities remain unclear. In this study, we report the crystal structure of BRPF1 bound to the H3 tail and characterize the role of the bipartite interaction in the engagement of BRPF1 with the nucleosome core particle (NCP). We find that although both interactions of BRPF1 with the H3 tail and DNA are required for tight binding to NCP and for acetyltransferase function of the BRPF1-MORF-ING5-MEAF6 complex, binding to extranucleosomal DNA dominates. Our findings suggest that functionally active BRPF1 might be important in stabilization of the MOZ/MORF complexes at chromatin with accessible DNA.
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http://dx.doi.org/10.1016/j.str.2019.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949404PMC
January 2020

Gcn5 and Esa1 function as histone crotonyltransferases to regulate crotonylation-dependent transcription.

J Biol Chem 2019 12 7;294(52):20122-20134. Epub 2019 Nov 7.

Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands

Histone post-translational modifications (PTMs) are critical for processes such as transcription. The more notable among these are the nonacetyl histone lysine acylation modifications such as crotonylation, butyrylation, and succinylation. However, the biological relevance of these PTMs is not fully understood because their regulation is largely unknown. Here, we set out to investigate whether the main histone acetyltransferases in budding yeast, Gcn5 and Esa1, possess crotonyltransferase activity. studies revealed that the Gcn5-Ada2-Ada3 (ADA) and Esa1-Yng2-Epl1 (Piccolo NuA4) histone acetyltransferase complexes have the capacity to crotonylate histones. Mass spectrometry analysis revealed that ADA and Piccolo NuA4 crotonylate lysines in the N-terminal tails of histone H3 and H4, respectively. Functionally, we show that crotonylation selectively affects gene transcription in a manner dependent on Gcn5 and Esa1. Thus, we identify the Gcn5- and Esa1-containing ADA and Piccolo NuA4 complexes as crotonyltransferases that promote crotonylation-dependent transcription.
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http://dx.doi.org/10.1074/jbc.RA119.010302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937567PMC
December 2019

Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation.

Nat Commun 2019 10 17;10(1):4724. Epub 2019 Oct 17.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, 80045, USA.

Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORF). The crystal structure of MORF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.
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http://dx.doi.org/10.1038/s41467-019-12551-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797804PMC
October 2019

Pre-existing H4K16ac levels in euchromatin drive DNA repair by homologous recombination in S-phase.

Commun Biol 2019 5;2:253. Epub 2019 Jul 5.

1Department of Radiation Oncology, The Houston Methodist Research Institute, Houston, TX 77030 USA.

The homologous recombination (HR) repair pathway maintains genetic integrity after DNA double-strand break (DSB) damage and is particularly crucial for maintaining fidelity of expressed genes. Histone H4 acetylation on lysine 16 (H4K16ac) is associated with transcription, but how pre-existing H4K16ac directly affects DSB repair is not known. To answer this question, we used CRISPR/Cas9 technology to introduce I-SceI sites, or repair pathway reporter cassettes, at defined locations within gene-rich (high H4K16ac/euchromatin) and gene-poor (low H4K16ac/heterochromatin) regions. The frequency of DSB repair by HR is higher in gene-rich regions. Interestingly, artificially targeting H4K16ac at specific locations using gRNA/dCas9-MOF increases HR frequency in euchromatin. Finally, inhibition/depletion of RNA polymerase II or Cockayne syndrome B protein leads to decreased recruitment of HR factors at DSBs. These results indicate that the pre-existing H4K16ac status at specific locations directly influences the repair of local DNA breaks, favoring HR in part through the transcription machinery.
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http://dx.doi.org/10.1038/s42003-019-0498-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611875PMC
May 2020

Measurement and Analysis of Lysine Acetylation by KAT Complexes In Vitro and In Vivo.

Methods Mol Biol 2019 ;1983:57-77

St. Patrick Research Group in Basic Oncology, Quebec City, QC, Canada.

The acetylation of the ε-amine of lysine residues has significant impacts on the cellular functions of proteins. Through the combination of unbiased and targeted analysis of acetylated proteins, biological insights on lysine acetylation are now routinely generated. To help in this endeavor, we describe detailed protocols for the identification of acetylated lysine residues and the preparation of multiple reagents for the characterization of these sites in order to obtain functional insights on this widespread modification.
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http://dx.doi.org/10.1007/978-1-4939-9434-2_5DOI Listing
January 2020

The Tumor Suppressor PALB2: Inside Out.

Trends Biochem Sci 2019 03 10;44(3):226-240. Epub 2019 Jan 10.

CHU de Québec Research Center, Oncology Division, 9 McMahon, Québec City, QC, G1R 3S3, Canada; Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Québec City, QC, G1V 0A6, Canada. Electronic address:

Partner and Localizer of BRCA2 (PALB2) has emerged as an important and versatile player in genome integrity maintenance. Biallelic mutations in PALB2 cause Fanconi anemia (FA) subtype FA-N, whereas monoallelic mutations predispose to breast, and pancreatic familial cancers. Herein, we review recent developments in our understanding of the mechanisms of regulation of the tumor suppressor PALB2 and its functional domains. Regulation of PALB2 functions in DNA damage response and repair occurs on multiple levels, including homodimerization, phosphorylation, and ubiquitylation. With a molecular emphasis, we present PALB2-associated cancer mutations and their detailed analysis by functional assays.
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http://dx.doi.org/10.1016/j.tibs.2018.10.008DOI Listing
March 2019

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures.

Mol Cell 2018 10 27;72(2):250-262.e6. Epub 2018 Sep 27.

LBCMCP, Centre de Biologie Integrative (CBI), CNRS, Université de Toulouse, UT3, Toulouse 31062, France. Electronic address:

Double-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.
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http://dx.doi.org/10.1016/j.molcel.2018.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202423PMC
October 2018

Phospho-dependent recruitment of the yeast NuA4 acetyltransferase complex by MRX at DNA breaks regulates RPA dynamics during resection.

Proc Natl Acad Sci U S A 2018 10 17;115(40):10028-10033. Epub 2018 Sep 17.

St. Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Centre de Recherche du Centre Hospitalier Universitaire de Québec-Axe Oncologie, Québec City, QC G1R 3S3, Canada;

The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair.
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http://dx.doi.org/10.1073/pnas.1806513115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176631PMC
October 2018

Architecture of the Saccharomyces cerevisiae NuA4/TIP60 complex.

Nat Commun 2018 03 20;9(1):1147. Epub 2018 Mar 20.

Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science & Technology of China, Hefei, 230026, China.

The NuA4/TIP60 acetyltransferase complex is required for gene regulation, DNA repair and cell cycle progression. The limited structural information impeded understanding of NuA4/TIP60 assembly and regulatory mechanism. Here, we report the 4.7 Å cryo-electron microscopy (cryo-EM) structure of a NuA4/TIP60 TEEAA assembly (Tra1, Eaf1, Eaf5, actin and Arp4) and the 7.6 Å cryo-EM structure of a TEEAA-piccolo assembly (Esa1, Epl1, Yng2 and Eaf6). The Tra1 and Eaf1 constitute the assembly scaffold. The Eaf1 SANT domain tightly binds to the LBE and FATC domains of Tra1 by ionic interactions. The actin/Arp4 peripherally associates with Eaf1 HSA domain. The Eaf5/7/3 (TINTIN) and piccolo modules largely pack against the FAT and HEAT repeats of Tra1 and their association depends on Eaf1 N-terminal and HSA regions, respectively. These structures elucidate the detailed architecture and molecular interactions between NuA4 subunits and offer exciting insights into the scaffolding and regulatory mechanisms of Tra1 pseudokinase.
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http://dx.doi.org/10.1038/s41467-018-03504-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861120PMC
March 2018

Molecular Architecture of the Essential Yeast Histone Acetyltransferase Complex NuA4 Redefines Its Multimodularity.

Mol Cell Biol 2018 05 16;38(9). Epub 2018 Apr 16.

Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada

Conserved from yeast to humans, the NuA4 histone acetyltransferase is a large multisubunit complex essential for cell viability through the regulation of gene expression, genome maintenance, metabolism, and cell fate during development and stress. How the different NuA4 subunits work in concert with one another to perform these diverse functions remains unclear, and addressing this central question requires a comprehensive understanding of NuA4's molecular architecture and subunit organization. We have determined the structure of fully assembled native yeast NuA4 by single-particle electron microscopy. Our data revealed that NuA4 adopts a trilobal overall architecture, with each of the three lobes constituted by one or two functional modules. By performing cross-linking coupled to mass spectrometry analysis and protein interaction studies, we further mapped novel intermolecular interfaces within NuA4. Finally, we combined these new data with other known structural information of NuA4 subunits and subassemblies to construct a multiscale model to illustrate how the different NuA4 subunits and modules are spatially arranged. This model shows that the multiple chromatin reader domains are clustered together around the catalytic core, suggesting that NuA4's multimodular architecture enables it to engage in multivalent interactions with its nucleosome substrate.
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http://dx.doi.org/10.1128/MCB.00570-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902594PMC
May 2018

The scaffolding protein JADE1 physically links the acetyltransferase subunit HBO1 with its histone H3-H4 substrate.

J Biol Chem 2018 03 30;293(12):4498-4509. Epub 2018 Jan 30.

From the Department of Biochemistry and Biophysics,

The human enzyme histone acetyltransferase binding to ORC1 (HBO1) regulates DNA replication, cell proliferation, and development. HBO1 is part of a multiprotein histone acetyltransferase (HAT) complex that also contains inhibitor of growth family member (ING) 4/5, MYST/Esa1-associated factor (MEAF) 6, and the scaffolding proteins Jade family PHD finger (JADE) 1/2/3 or bromodomain and PHD finger-containing protein (BRPF) 2/3 to acetylate histone H4 H4K5/8/12 or H3K14, respectively. Within this four-protein complex, JADE1 determines histone H4 substrate specificity of the HBO1-HAT complex. However, the mechanism by which JADE1 controls the H4-specific acetyltransferase activity of HBO1 is unknown. Here we used recombinant proteins to dissect the specific regions and activities of HBO1 and JADE1 that mediate histone H3-H4 acetylation via the HBO1-HAT domain. We found that JADE1 increases the catalytic efficiency of HBO1 acetylation of an H3-H4 substrate by about 5-fold through an N-terminal, 21-residue HBO1- and histone-binding domain and a nearby second histone core-binding domain. We also demonstrate that HBO1 contains an N-terminal histone-binding domain (HBD) that makes additional contacts with H3-H4 independent of JADE1 interactions with histones and that the HBO1 HBD does not significantly contribute to HBO1's overall HAT activity. Experiments with JADE1 deletions recapitulated these interactions and their roles in HBO1 histone acetylation activity. Together, these results indicate that the N-terminal region of JADE1 functions as a platform that brings together the catalytic HBO1 subunit with its cognate H3-H4 substrate for histone acetylation.
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http://dx.doi.org/10.1074/jbc.RA117.000677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868276PMC
March 2018

Yaf9 subunit of the NuA4 and SWR1 complexes targets histone H3K27ac through its YEATS domain.

Nucleic Acids Res 2018 01;46(1):421-430

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA.

Yaf9 is an integral part of the NuA4 acetyltransferase and the SWR1 chromatin remodeling complexes. Here, we show that Yaf9 associates with acetylated histone H3 with high preference for H3K27ac. The crystal structure of the Yaf9 YEATS domain bound to the H3K27ac peptide reveals that the sequence C-terminal to K27ac stabilizes the complex. The side chain of K27ac inserts between two aromatic residues, mutation of which abrogates the interaction in vitro and leads in vivo to phenotypes similar to YAF9 deletion, including loss of SWR1-dependent incorporation of variant histone H2A.Z. Our findings reveal the molecular basis for the recognition of H3K27ac by a YEATS reader and underscore the importance of this interaction in mediating Yaf9 function within the NuA4 and SWR1 complexes.
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http://dx.doi.org/10.1093/nar/gkx1151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758897PMC
January 2018

Novel mechanism of PCNA control through acetylation of its sliding surface.

Mol Cell Oncol 2017 13;4(2):e1279724. Epub 2017 Jan 13.

St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Centre de Recherche du CHU de Québec-Axe Oncologie , Quebec City, QC, Canada.

Recent findings revealed a new unexpected regulatory mechanism that controls the proliferating cell nuclear antigen (PCNA). Multiple positively-charged lysine residues located on the ring inner surface are neutralized by acetylation and required for cellular resistance to Desoxyribonucleic acid (DNA) damage. Here, we summarize the key observations, discuss implications, and perspectives linked to cancer, as well as challenges for future work.
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http://dx.doi.org/10.1080/23723556.2017.1279724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5383363PMC
January 2017

Recognition of Histone H3K14 Acylation by MORF.

Structure 2017 04 9;25(4):650-654.e2. Epub 2017 Mar 9.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address:

The monocytic leukemia zinc-finger protein-related factor (MORF) is a transcriptional coactivator and a catalytic subunit of the lysine acetyltransferase complex implicated in cancer and developmental diseases. We have previously shown that the double plant homeodomain finger (DPF) of MORF is capable of binding to acetylated histone H3. Here we demonstrate that the DPF of MORF recognizes many newly identified acylation marks. The mass spectrometry study provides comprehensive analysis of H3K14 acylation states in vitro and in vivo. The crystal structure of the MORF DPF-H3K14butyryl complex offers insight into the selectivity of this reader toward lipophilic acyllysine substrates. Together, our findings support the mechanism by which the acetyltransferase MORF promotes spreading of histone acylation.
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http://dx.doi.org/10.1016/j.str.2017.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415407PMC
April 2017

Acetylation of PCNA Sliding Surface by Eco1 Promotes Genome Stability through Homologous Recombination.

Mol Cell 2017 Jan 1;65(1):78-90. Epub 2016 Dec 1.

St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Centre de Recherche du CHU de Québec-Axe Oncologie, Quebec City, QC G1R 3S3, Canada. Electronic address:

During DNA replication, proliferating cell nuclear antigen (PCNA) adopts a ring-shaped structure to promote processive DNA synthesis, acting as a sliding clamp for polymerases. Known posttranslational modifications function at the outer surface of the PCNA ring to favor DNA damage bypass. Here, we demonstrate that acetylation of lysine residues at the inner surface of PCNA is induced by DNA lesions. We show that cohesin acetyltransferase Eco1 targets lysine 20 at the sliding surface of the PCNA ring in vitro and in vivo in response to DNA damage. Mimicking constitutive acetylation stimulates homologous recombination and robustly suppresses the DNA damage sensitivity of mutations in damage tolerance pathways. In comparison to the unmodified trimer, structural differences are observed at the interface between protomers in the crystal structure of the PCNA-K20ac ring. Thus, acetylation regulates PCNA sliding on DNA in the presence of DNA damage, favoring homologous recombination linked to sister-chromatid cohesion.
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http://dx.doi.org/10.1016/j.molcel.2016.10.033DOI Listing
January 2017

Purification of Yeast Native Reagents for the Analysis of Chromatin Function-II: Multiprotein Complexes and Biochemical Assays.

Methods Mol Biol 2017 ;1528:53-67

St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, CHU de Québec Research Center- Oncology axis, Hôtel-Dieu de Québec, 9 McMahon Street, Quebec City, QC, Canada, G1R 3S3.

Post-translational modifications of histones play essential roles in regulating chromatin structure and function. These are tightly regulated in vivo and there is an intricate cross-talk between different marks as they are recognized by specific reader modules present in a large number of nuclear factors. In order to precisely dissect these processes in vitro native reagents like purified chromatin and histone modifying/remodeling enzymes are required to more accurately reproduce physiological conditions. The vast majority of these enzymes need to be part of stable multiprotein complexes with cofactors enabling them to act on chromatin substrates and/or read specific histone marks. In the accompanying chapter, we have described the protocol for purification of native chromatin from yeast cells (Chapter 3 ). Here, we present the methods to obtain highly purified native chromatin modifying complexes from Saccharomyces cerevisiae, based on Tandem Affinity Purification (TAP). We also present possible applications and useful functional assays that can be performed using these yeast native reagents.
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http://dx.doi.org/10.1007/978-1-4939-6630-1_4DOI Listing
January 2018

Purification of Yeast Native Reagents for the Analysis of Chromatin Function-I: Nucleosomes for Reconstitution and Manipulation of Histone Marks.

Methods Mol Biol 2017 ;1528:39-51

St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, CHU de Québec Research Center- Oncology axis, Hôtel-Dieu de Québec, 9 McMahon Street, Quebec City, QC, Canada, G1R 3S3.

Purification of native biological material provides powerful tools for the functional analysis of enzymes and proteins in chromatin. In particular, histone proteins harbor numerous post-translational modifications, which may differ between species, tissues, and growth conditions and are lacking on recombinant histones. Moreover, the physiological substrate of most enzymes that modify histones is chromatin and the majority of these enzymes need to be part of a multiprotein assembly to be able to act on chromatin. For the yeast Saccharomyces cerevisiae different chromatin purification protocols are available but often result in poor yields or rely on genetic manipulation. We present a simple purification protocol that can yield up to 150 μg of pure native chromatin per liter of yeast culture. The purified material can be obtained from mutant cells lacking specific histone modifications and can be used in in vitro chromatin assembly for biochemical studies. Based on the extremely high degree of conservation throughout eukaryotes, this modifiable native chromatin can be used in studies with factors from other organisms including humans.
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http://dx.doi.org/10.1007/978-1-4939-6630-1_3DOI Listing
January 2018

Combined Action of Histone Reader Modules Regulates NuA4 Local Acetyltransferase Function but Not Its Recruitment on the Genome.

Mol Cell Biol 2016 11 28;36(22):2768-2781. Epub 2016 Oct 28.

St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Centre de Recherche du CHU de Québec-Axe Oncologie, Hôtel-Dieu de Québec, Québec City, Quebec, Canada

Recognition of histone marks by reader modules is thought to be at the heart of epigenetic mechanisms. These protein domains are considered to function by targeting regulators to chromosomal loci carrying specific histone modifications. This is important for proper gene regulation as well as propagation of epigenetic information. The NuA4 acetyltransferase complex contains two of these reader modules, an H3K4me3-specific plant homeodomain (PHD) within the Yng2 subunit and an H3K36me2/3-specific chromodomain in the Eaf3 subunit. While each domain showed a close functional interaction with the respective histone mark that it recognizes, at the biochemical level, genetic level (as assessed with epistatic miniarray profile screens), and phenotypic level, cells with the combined loss of both readers showed greatly enhanced phenotypes. Chromatin immunoprecipitation coupled with next-generation sequencing experiments demonstrated that the Yng2 PHD specifically directs H4 acetylation near the transcription start site of highly expressed genes, while Eaf3 is important downstream on the body of the genes. Strikingly, the recruitment of the NuA4 complex to these loci was not significantly affected. Furthermore, RNA polymerase II occupancy was decreased only under conditions where both PHD and chromodomains were lost, generally in the second half of the gene coding regions. Altogether, these results argue that methylated histone reader modules in NuA4 are not responsible for its recruitment to the promoter or coding regions but, rather, are required to orient its acetyltransferase catalytic site to the methylated histone 3-bearing nucleosomes in the surrounding chromatin, cooperating to allow proper transition from transcription initiation to elongation.
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http://dx.doi.org/10.1128/MCB.00112-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5086519PMC
November 2016

The TIP60 Complex Regulates Bivalent Chromatin Recognition by 53BP1 through Direct H4K20me Binding and H2AK15 Acetylation.

Mol Cell 2016 05;62(3):409-421

St. Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Quebec City, QC G1R 3S3, Canada; Centre Hospitalier Universitaire de Québec Research Center and School of Medicine, Laval University, Quebec City, QC G1V 4G2, Canada. Electronic address:

The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here we identified MBTD1 as a stable subunit of the complex, and we reveal that, via a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double-strand breaks by homologous recombination. It was previously suggested that TIP60-dependent acetylation of H4 regulates binding of the non-homologous end joining factor 53BP1, which engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub. We find that the TIP60 complex regulates association of 53BP1 partly by competing for H4K20me2 and by regulating H2AK15ub. Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex to regulate 53BP1-dependent repair through competitive bivalent binding and modification of chromatin.
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http://dx.doi.org/10.1016/j.molcel.2016.03.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887106PMC
May 2016

EP400 Deposits H3.3 into Promoters and Enhancers during Gene Activation.

Mol Cell 2016 Jan 6;61(1):27-38. Epub 2015 Dec 6.

Department of Biological Chemistry, David Geffen School of Medicine, UCLA, 351A Biomedical Sciences Research Building, 615 Charles E. Young Drive South, Los Angeles, CA 90095-1737, USA; The Molecular Biology Institute, UCLA, Paul D. Boyer Hall, 611 Charles E. Young Drive South, Los Angeles, CA 90095-1570, USA. Electronic address:

Gene activation in metazoans is accompanied by the presence of histone variants H2AZ and H3.3 within promoters and enhancers. It is not known, however, what protein deposits H3.3 into chromatin or whether variant chromatin plays a direct role in gene activation. Here we show that chromatin containing acetylated H2AZ and H3.3 stimulates transcription in vitro. Analysis of the Pol II pre-initiation complex on immobilized chromatin templates revealed that the E1A binding protein p400 (EP400) was bound preferentially to and required for transcription stimulation by acetylated double-variant chromatin. EP400 also stimulated H2AZ/H3.3 deposition into promoters and enhancers and influenced transcription in vivo at a step downstream of the Mediator complex. EP400 efficiently exchanged recombinant histones H2A and H3.1 with H2AZ and H3.3, respectively, in a chromatin- and ATP-stimulated manner in vitro. Our data reveal that EP400 deposits H3.3 into chromatin alongside H2AZ and contributes to gene regulation after PIC assembly.
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http://dx.doi.org/10.1016/j.molcel.2015.10.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707986PMC
January 2016

Bivalent interaction of the PZP domain of BRPF1 with the nucleosome impacts chromatin dynamics and acetylation.

Nucleic Acids Res 2016 Jan 30;44(1):472-84. Epub 2015 Nov 30.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA

BRPF1 (bromodomain PHD finger 1) is a core subunit of the MOZ histone acetyltransferase (HAT) complex, critical for normal developmental programs and implicated in acute leukemias. BRPF1 contains a unique assembly of zinc fingers, termed a PZP domain, the physiological role of which remains unclear. Here, we elucidate the structure-function relationship of this novel epigenetic reader and detail the biological and mechanistic consequences of its interaction with nucleosomes. PZP has a globular architecture and forms a 2:1 stoichiometry complex with the nucleosome, bivalently interacting with histone H3 and DNA. This binding impacts the nucleosome dynamics, shifting the DNA unwrapping/rewrapping equilibrium toward the unwrapped state and increasing DNA accessibility. We demonstrate that the DNA-binding function of the BRPF1 PZP domain is required for the MOZ-BRPF1-ING5-hEaf6 HAT complex to be recruited to chromatin and to acetylate nucleosomal histones. Our findings reveal a novel link between chromatin dynamics and MOZ-mediated acetylation.
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http://dx.doi.org/10.1093/nar/gkv1321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705663PMC
January 2016

BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation.

EMBO J 2016 Jan 30;35(2):176-92. Epub 2015 Nov 30.

Biotech Research and Innovation Centre (BRIC) and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark

During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1-binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 recruitment, but not MCM2-7 loading, is impaired in BRPF3-depleted cells, identifying a BRPF3-dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3-HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby replication origins.
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http://dx.doi.org/10.15252/embj.201591293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718456PMC
January 2016

A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells.

Cell Rep 2015 Oct 8;13(3):621-633. Epub 2015 Oct 8.

Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Laval University, Quebec City, QC G1V 4G2, Canada. Electronic address:

Conventional affinity purification followed by mass spectrometry (AP-MS) analysis is a broadly applicable method used to decipher molecular interaction networks and infer protein function. However, it is sensitive to perturbations induced by ectopically overexpressed target proteins and does not reflect multilevel physiological regulation in response to diverse stimuli. Here, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. We used CRISPR/Cas9 and TAL effector nucleases (TALENs) to tag endogenous genes and purified several DNA repair and chromatin-modifying holoenzymes to near homogeneity. We uncovered subunits and interactions among well-characterized complexes and report the isolation of MCM8/9, highlighting the efficiency and robustness of the approach. These methods improve and simplify both small- and large-scale explorations of protein interactions as well as the study of biochemical activities and structure-function relationships.
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http://dx.doi.org/10.1016/j.celrep.2015.09.009DOI Listing
October 2015

Site specificity analysis of Piccolo NuA4-mediated acetylation for different histone complexes.

Biochem J 2015 Dec 29;472(2):239-48. Epub 2015 Sep 29.

Department of Cancer Biology, Fox Chase Cancer Center, Philadelphia, PA 19111, U.S.A.

We have a limited understanding of the site specificity of multi-subunit lysine acetyltransferase (KAT) complexes for histone-based substrates, especially in regards to the different complexes formed during nucleosome assembly. Histone complexes could be a major factor in determining the acetylation specificity of KATs. In the present study, we utilized a label-free quantitative MS-based method to determine the site specificity of acetylation catalysed by Piccolo NuA4 on (H3/H4)2 tetramer, tetramer bound DNA (tetrasome) and nucleosome core particle (NCP). Our results show that Piccolo NuA4 can acetylate multiple lysine residues on these three histone complexes, of which NCP is the most favourable, (H3/H4)2 tetramer is the second and tetrasome is the least favourable substrate for Piccolo NuA4 acetylation. Although Piccolo NuA4 preferentially acetylates histone H4 (H4K12), the site specificity of the enzyme is altered with different histone complex substrates. Our results show that before nucleosome assembly is complete, H3K14 specificity is almost equal to that of H4K12 and DNA-histone interactions suppress the acetylation ability of Piccolo NuA4. These data suggest that the H2A/H2B dimer could play a critical role in the increase in acetylation specificity of Piccolo NuA4 for NCP. This demonstrates that histone complex formation can alter the acetylation preference of Piccolo NuA4. Such findings provide valuable insight into regulating Piccolo NuA4 specificity by modulating chromatin dynamics and in turn manipulating gene expression.
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http://dx.doi.org/10.1042/BJ20150654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917874PMC
December 2015

Eaf1 Links the NuA4 Histone Acetyltransferase Complex to Htz1 Incorporation and Regulation of Purine Biosynthesis.

Eukaryot Cell 2015 Jun 3;14(6):535-44. Epub 2015 Apr 3.

St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Centre de Recherche du CHU de Québec-Axe Oncologie, Hôtel-Dieu de Québec, Quebec City, Quebec, Canada

Proper modulation of promoter chromatin architecture is crucial for gene regulation in order to precisely and efficiently orchestrate various cellular activities. Previous studies have identified the stimulatory effect of the histone-modifying complex NuA4 on the incorporation of the histone variant H2A.Z (Htz1) at the PHO5 promoter (A. Auger, L. Galarneau, M. Altaf, A. Nourani, Y. Doyon, R. T. Utley, D. Cronier, S. Allard, and J. Côté, Mol Cell Biol 28:2257-2270, 2008, http://dx.doi.org/10.1128/MCB.01755-07). In vitro studies with a reconstituted system also indicated an intriguing cross talk between NuA4 and the H2A.Z-loading complex, SWR-C (M. Altaf, A. Auger, J. Monnet-Saksouk, J. Brodeur, S. Piquet, M. Cramet, N. Bouchard, N. Lacoste, R. T. Utley, L. Gaudreau, J. Côté, J Biol Chem 285:15966-15977, 2010, http://dx.doi.org/10.1074/jbc.M110.117069). In this work, we investigated the role of the NuA4 scaffold subunit Eaf1 in global gene expression and genome-wide incorporation of Htz1. We found that loss of Eaf1 affects Htz1 levels mostly at the promoters that are normally highly enriched in the histone variant. Analysis of eaf1 mutant cells by expression array unveiled a relationship between NuA4 and the gene network implicated in the purine biosynthesis pathway, as EAF1 deletion cripples induction of several ADE genes. NuA4 directly interacts with Bas1 activation domain, a key transcription factor of adenine genes. Chromatin immunoprecipitation (ChIP) experiments demonstrate that nucleosomes on the inactive ADE17 promoter are acetylated already by NuA4 and enriched in Htz1. Upon derepression, these poised nucleosomes respond rapidly to activate ADE gene expression in a mechanism likely reminiscent of the PHO5 promoter, leading to nucleosome disassembly. These detailed molecular events depict a specific case of cross talk between NuA4-dependent acetylation and incorporation of histone variant Htz1, presetting the chromatin structure over ADE promoters for subsequent chromatin remodeling and activated transcription.
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http://dx.doi.org/10.1128/EC.00004-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452573PMC
June 2015