{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:33:15Z","timestamp":1760059995501,"version":"build-2065373602"},"reference-count":55,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2025,7,23]],"date-time":"2025-07-23T00:00:00Z","timestamp":1753228800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"VCU start-up package"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>The histone H2A variant H2AZ plays pivotal roles in shaping chromatin architecture and regulating gene expression. We recently identified H2AZ.2 in histone H2A lysine 119 ubiquitination (H2AK119ub)-enriched nucleosomes, but it is not known whether its highly related isoform H2AZ.1 also regulates this modification. In this study, we employed isoform-specific epitope-tagged knock-in mouse embryonic stem cell (ESC) lines to dissect the roles of each isoform in Polycomb Repressive Complex 1 (PRC1)-mediated H2AK119ub. Our results show that H2AZ.1 and H2AZ.2 share highly overlapping genomic binding profiles, both co-localizing extensively with H2AK119ub-enriched loci. The knockdown of either isoform led to reduced H2AK119ub levels; however, the two isoforms appear to function through distinct mechanisms. H2AZ.1 facilitates the recruitment of Ring1B, the catalytic subunit of PRC1, thereby promoting the deposition of H2AK119ub. In contrast, H2AZ.2 does not significantly affect Ring1B recruitment but instead functions as a structural component that stabilizes H2AK119ub-modified nucleosomes. In vitro ubiquitination assays indicate that H2AZ.1-containing nucleosomes serve as more efficient substrates for PRC1-mediated ubiquitination compared to those containing H2AZ.2. Thus, these findings define the distinct mechanisms of the two H2AZ variants in regulated PRC1-mediated H2AK119 ubiquitination and highlight a functional division of labor in epigenetic regulation.<\/jats:p>","DOI":"10.3390\/cells14151133","type":"journal-article","created":{"date-parts":[[2025,7,23]],"date-time":"2025-07-23T10:49:17Z","timestamp":1753267757000},"page":"1133","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Divergent Mechanisms of H2AZ.1 and H2AZ.2 in PRC1-Mediated H2A Ubiquitination"],"prefix":"10.3390","volume":"14","author":[{"given":"Xiangyu","family":"Shen","sequence":"first","affiliation":[{"name":"School of Life Sciences, Henan University, Kaifeng 475004, China"},{"name":"Department of Internal Medicine, Division of Hematology, Oncology and Palliative Care, Massey Comprehensive Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chunxu","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, Division of Hematology, Oncology and Palliative Care, Massey Comprehensive Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amanda E.","family":"Jones","sequence":"additional","affiliation":[{"name":"Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-5975-2116","authenticated-orcid":false,"given":"Xiaokun","family":"Jian","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Henan University, Kaifeng 475004, China"},{"name":"Department of Internal Medicine, Division of Hematology, Oncology and Palliative Care, Massey Comprehensive Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gengsheng","family":"Cao","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Henan University, Kaifeng 475004, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2414-0609","authenticated-orcid":false,"given":"Hengbin","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, Division of Hematology, Oncology and Palliative Care, Massey Comprehensive Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1038\/nrm3941","article-title":"Histone exchange, chromatin structure and the regulation of transcription","volume":"16","author":"Venkatesh","year":"2015","journal-title":"Nat. Rev. Mol. Cell. Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1007\/s11427-015-4817-4","article-title":"Histone variants: The artists of eukaryotic chromatin","volume":"58","author":"Li","year":"2015","journal-title":"Sci. China. Life. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"103301","DOI":"10.1016\/j.dnarep.2022.103301","article-title":"Histone variants: The unsung guardians of the genome","volume":"112","author":"Phillips","year":"2022","journal-title":"DNA. Repair"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1038\/s41576-024-00759-1","article-title":"Multifunctional histone variants in genome function","volume":"26","author":"Wong","year":"2024","journal-title":"Nat. Rev. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Henikoff, S., and Smith, M.M. (2015). Histone variants and epigenetics. Cold. Spring. Harb. Perspect. Biol., 7.","DOI":"10.1101\/cshperspect.a019364"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Talbert, P.B., and Henikoff, S. (2021). Histone variants at a glance. J. Cell. Sci., 134.","DOI":"10.1242\/jcs.244749"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2100178118","DOI":"10.1073\/pnas.2100178118","article-title":"Replication-dependent histone biosynthesis is coupled to cell-cycle commitment","volume":"118","author":"Armstrong","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Hegazy, Y.A., Dhahri, H., El Osmani, N., George, S., Chandler, D.P., and Fondufe-Mittendorf, Y.N. (2024). Histone variants: The bricks that fit differently. J. Biol. Chem., 301.","DOI":"10.1016\/j.jbc.2024.108048"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1016\/j.tibs.2022.04.014","article-title":"H2A. Z\u2019s \u2018social\u2019 network: Functional partners of an enigmatic histone variant","volume":"47","author":"Kreienbaum","year":"2022","journal-title":"Trends. Biochem. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.semcdb.2022.03.011","article-title":"Histone H2A variants: Diversifying chromatin to ensure genome integrity","volume":"135","author":"Oberdoerffer","year":"2022","journal-title":"Semin. Cell. Dev. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1016\/S0092-8674(00)00133-1","article-title":"Histone H2A.Z Regulates Transcription and Is Partially Redundant with Nucleosome Remodeling Complexes","volume":"103","author":"Santisteban","year":"2000","journal-title":"Cell"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Updike, D.L., and Mango, S.E. (2006). Temporal regulation of foregut development by HTZ-1\/H2A.Z and PHA-4\/FoxA. PLoS Genet., 2.","DOI":"10.1371\/journal.pgen.0020161"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"7487","DOI":"10.1093\/nar\/16.15.7487","article-title":"Drosophila has a single copy of the gene encoding a highly conserved histone H2A variant of the H2A.F\/Z type","volume":"16","author":"White","year":"1988","journal-title":"Nucleic Acids Res."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Cheema, M.S., Good, K.V., Kim, B., Soufari, H., O\u2019sullivan, C., Freeman, M.E., Stefanelli, G., Casas, C.R., Zengeler, K.E., and Kennedy, A.J. (2020). Deciphering the Enigma of the Histone H2A.Z-1\/H2A.Z-2 Isoforms: Novel Insights and Remaining Questions. Cells, 9.","DOI":"10.3390\/cells9051167"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Lamaa, A., Humbert, J., Aguirrebengoa, M., Cheng, X., Nicolas, E., C\u00f4t\u00e9, J., and Trouche, D. (2020). Integrated analysis of H2A.Z isoforms function reveals a complex interplay in gene regulation. eLife, 9.","DOI":"10.7554\/eLife.53375"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3698","DOI":"10.1093\/nar\/gkf496","article-title":"DNA double-strand break-induced phosphorylation of Drosophila histone variant H2Av helps prevent radiation-induced apoptosis","volume":"30","author":"Madigan","year":"2002","journal-title":"Nucleic. Acids. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1007\/s00412-013-0409-x","article-title":"The variant histone H2A.V of Drosophila\u2013Three roles, two guises","volume":"122","author":"Baldi","year":"2013","journal-title":"Chromosoma"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Dryhurst, D., Ishibashi, T., Rose, K.L., Eir\u00edn-L\u00f3pez, J.M., McDonald, D., Silva-Moreno, B., and Ausi\u00f3, J. (2009). Characterization of the histone H2A.Z-1 and H2A.Z-2 isoforms in vertebrates. BMC. Biol., 7.","DOI":"10.1186\/1741-7007-7-86"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"5951","DOI":"10.1093\/nar\/gks267","article-title":"H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization","volume":"40","author":"Schneider","year":"2012","journal-title":"Nucleic. Acids. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1186\/s13072-019-0274-9","article-title":"The histone variant H2A.Z in gene regulation","volume":"12","author":"Giaimo","year":"2019","journal-title":"Epigenetics Chromatin"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.cell.2005.10.002","article-title":"Histone variant H2A.Z marks the 5\u2032 ends of both active and inactive genes in euchromatin","volume":"123","author":"Raisner","year":"2005","journal-title":"Cell"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Gaillard, H., Ciudad, T., Aguilera, A., Wellinger, R.E., and Gordenin, D.A. (2024). Histone variant H2A.Z is needed for efficient transcription-coupled NER and genome integrity in UV challenged yeast cells. PLOS. Genet., 20.","DOI":"10.1101\/2024.05.13.593813"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"7507","DOI":"10.1073\/pnas.1504868112","article-title":"Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair","volume":"112","author":"Ayrapetov","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_24","first-page":"1","article-title":"The evolutionary differentiation of two histone H2A.Z variants in chordates (H2A.Z-1 and H2A.Z-2) is mediated by a stepwise mutation process that affects three amino acid residues","volume":"9","author":"Dryhurst","year":"2009","journal-title":"BMC. Evol. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1016\/j.tig.2021.10.003","article-title":"The H2A.Z-nucleosome code in mammals: Emerging functions","volume":"38","author":"Clark","year":"2022","journal-title":"Trends. Genet."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e52061","DOI":"10.15252\/embr.202052061","article-title":"Non-redundant functions of H2A.Z.1 and H2A.Z.2 in chromosome segregation and cell cycle progression","volume":"22","author":"Kommer","year":"2021","journal-title":"Embo. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"736","DOI":"10.1016\/j.ijrobp.2014.03.031","article-title":"Reorganization of Damaged Chromatin by the Exchange of Histone Variant H2A.Z-2","volume":"89","author":"Nishibuchi","year":"2014","journal-title":"Int. J. Radiat. Oncol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.molcel.2015.05.009","article-title":"Histone Variant H2A.Z.2 Mediates Proliferation and Drug Sensitivity of Malignant Melanoma","volume":"59","author":"Vardabasso","year":"2015","journal-title":"Mol. Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"11412","DOI":"10.18632\/oncotarget.7194","article-title":"Oncogenic potential of histone-variant H2A.Z.1 and its regulatory role in cell cycle and epithelial-mesenchymal transition in liver cancer","volume":"7","author":"Yang","year":"2016","journal-title":"Oncotarget"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Gonz\u00e1lez, J., Bosch-Presegu\u00e9, L., Marazuela-Duque, A., Guitart-Solanes, A., Espinosa-Alcantud, M., Fernandez, A.F., Brown, J.P., Ausi\u00f3, J., Vazquez, B.N., and Singh, P.B. (2023). A complex interplay between H2A.Z and HP1 isoforms regulates pericentric heterochromatin. Front. Cell. Dev. Biol., 11.","DOI":"10.3389\/fcell.2023.1293122"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1421","DOI":"10.1016\/j.cell.2019.08.002","article-title":"Single Amino Acid Change Underlies Distinct Roles of H2A.Z Subtypes in Human Syndrome","volume":"178","author":"Greenberg","year":"2019","journal-title":"Cell"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1016\/j.cell.2008.09.056","article-title":"H2AZ Is Enriched at Polycomb Complex Target Genes in ES Cells and Is Necessary for Lineage Commitment","volume":"135","author":"Creyghton","year":"2008","journal-title":"Cell"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"6457","DOI":"10.1128\/MCB.00241-07","article-title":"Monoubiquitylation of H2A.Z Distinguishes Its Association with Euchromatin or Facultative Heterochromatin","volume":"27","author":"Sarcinella","year":"2007","journal-title":"Mol. Cell Biol."},{"key":"ref_34","first-page":"1","article-title":"The histone H2A deubiquitinase Usp16 regulates embryonic stem cell gene expression and lineage commitment","volume":"5","author":"Yang","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Shen, X., Chen, C., Wang, Y., Zheng, W., Zheng, J., Jones, A.E., and Wang, H. (2024). Role of histone variants H2BC1 and H2AZ.2 in H2AK119ub nucleosome organization and Polycomb gene silencing. bioRxiv.","DOI":"10.1101\/2024.01.16.575234"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"W242","DOI":"10.1093\/nar\/gky354","article-title":"CRISPOR: Intuitive guide selection for CRISPR\/Cas9 genome editing experiments and screens","volume":"46","author":"Concordet","year":"2018","journal-title":"Nucleic. Acids. Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"941","DOI":"10.1038\/nmeth.1733","article-title":"Surrogate reporters for enrichment of cells with nuclease-induced mutations","volume":"8","author":"Kim","year":"2011","journal-title":"Nat. Methods"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1038\/s41586-019-1711-4","article-title":"Search-and-replace genome editing without double-strand breaks or donor DNA","volume":"576","author":"Anzalone","year":"2019","journal-title":"Nature"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1151","DOI":"10.1038\/s41587-022-01613-7","article-title":"Predicting prime editing efficiency and product purity by deep learning","volume":"41","author":"Mathis","year":"2023","journal-title":"Nat. Biotechnol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2431","DOI":"10.1038\/s41596-022-00724-4","article-title":"Designing and executing prime editing experiments in mammalian cells","volume":"17","author":"Doman","year":"2022","journal-title":"Nat. Protoc."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1038\/s41586-024-07259-6","article-title":"Improving prime editing with an endogenous small RNA-binding protein","volume":"628","author":"Yan","year":"2024","journal-title":"Nature"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3264","DOI":"10.1038\/s41596-020-0373-x","article-title":"Efficient low-cost chromatin profiling with CUT & Tag","volume":"15","author":"Janssens","year":"2020","journal-title":"Nat. Protoc."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1068","DOI":"10.1038\/nature06256","article-title":"Regulation of cell cycle progression and gene expression by H2A deubiquitination","volume":"449","author":"Joo","year":"2007","journal-title":"Nature"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"873","DOI":"10.1038\/nature02985","article-title":"Role of histone H2A ubiquitination in Polycomb silencing","volume":"431","author":"Wang","year":"2004","journal-title":"Nature"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.ymeth.2011.03.003","article-title":"Purification of histone ubiquitin ligases from HeLa cells","volume":"54","author":"Jones","year":"2011","journal-title":"Methods"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1038\/nature13890","article-title":"Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome","volume":"514","author":"McGinty","year":"2014","journal-title":"Nature"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-021-22688-x","article-title":"Multiple roles of H2A.Z in regulating promoter chromatin architecture in human cells","volume":"12","author":"Cole","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Moos, H.K., Patel, R., Flaherty, S.K., Loverde, S.M., and Nikolova, E.N. (2025). H2A.Z facilitates Sox2\u2013nucleosome interaction by promoting DNA and histone H3 tail mobility. bioRxiv.","DOI":"10.1101\/2025.03.06.641691"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1016\/j.stem.2012.11.003","article-title":"H2A.Z Facilitates Access of Active and Repressive Complexes to Chromatin in Embryonic Stem Cell Self-Renewal and Differentiation","volume":"12","author":"Hu","year":"2013","journal-title":"Cell. Stem. Cell"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"E7949","DOI":"10.1073\/pnas.1711158114","article-title":"Role of remodeling and spacing factor 1 in histone H2A ubiquitination-mediated gene silencing","volume":"114","author":"Zhang","year":"2017","journal-title":"Proc. Natl. Acad. Sci."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"4645","DOI":"10.1074\/jbc.M109.072348","article-title":"Chromatin Remodeling by Imitation Switch (ISWI) Class ATP-dependent Remodelers Is Stimulated by Histone Variant H2A.Z","volume":"285","author":"Goldman","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"723","DOI":"10.1016\/j.molcel.2012.09.026","article-title":"Histone H2A.Z Controls a Critical Chromatin Remodeling Step Required for DNA Double-Strand Break Repair","volume":"48","author":"Xu","year":"2012","journal-title":"Mol. Cell"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"2431","DOI":"10.1107\/S090744491302252X","article-title":"Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z","volume":"69","author":"Horikoshi","year":"2013","journal-title":"Acta. Crystallogr. D. Biol. Crystallogr."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"11379","DOI":"10.1093\/nar\/gkab907","article-title":"Structural basis of chromatin regulation by histone variant H2A.Z","volume":"49","author":"Lewis","year":"2021","journal-title":"Nucleic. Acids. Res."},{"key":"ref_55","first-page":"2954","article-title":"Single-molecule tools elucidate H2A.Z nucleosome composition","volume":"125","author":"Chen","year":"2012","journal-title":"J. Cell. Sci."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/14\/15\/1133\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:14:29Z","timestamp":1760033669000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/14\/15\/1133"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,23]]},"references-count":55,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2025,8]]}},"alternative-id":["cells14151133"],"URL":"https:\/\/doi.org\/10.3390\/cells14151133","relation":{},"ISSN":["2073-4409"],"issn-type":[{"type":"electronic","value":"2073-4409"}],"subject":[],"published":{"date-parts":[[2025,7,23]]}}}