{"doi":"10.1016/j.jbc.2022.102428","title":"Densely methylated DNA traps Methyl-CpG–binding domain protein 2 but permits free diffusion by Methyl-CpG–binding domain protein 3","abstract":"The methyl-CpG–binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status. The methyl-CpG–binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status. The methyl-CpG–binding domain (MBD) family of proteins binds methylated DNA through a conserved domain that recognizes the symmetrically related methylcytosines in a cytosine-guanosine dinucleotide (CpG) (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar). The structure of this domain bound to a single methylated CpG (mCpG) site has been determined for most members of the MBD family (2Ohki I. Shimotake N. Fujita N. Jee J. Ikegami T. Nakao M. et al.Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA.Cell. 2001; 105: 487-497Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 3Ho K.L. McNae I.W. Schmiedeberg L. Klose R.J. Bird A.P. Walkinshaw M.D. MeCP2 binding to DNA depends upon hydration at methyl-CpG.Mol. Cell. 2008; 29: 525-531Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 4Scarsdale J.N. Webb H.D. Ginder G.D. Williams Jr., D.C. Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence.Nucleic Acids Res. 2011; 39: 6741-6752Crossref PubMed Scopus (80) Google Scholar, 5Walavalkar N.M. Cramer J.M. Buchwald W.A. Scarsdale J","journal":"Journal of Biological Chemistry","year":2022,"id":255659,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":20,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9495,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":903874,"name":"Elizabeth Marie Irvin","orcid":"0000-0001-8274-7113","position":1,"is_corresponding":false},{"id":310417,"name":"Parminder Kaur","orcid":"0000-0001-9183-4315","position":2,"is_corresponding":false},{"id":903875,"name":"Ming Liu","orcid":"0000-0002-8451-3238","position":3,"is_corresponding":false},{"id":258119,"name":"Changjiang You","orcid":"0000-0002-7839-6397","position":4,"is_corresponding":false},{"id":765638,"name":"Dhruv Bhattaram","orcid":"0000-0002-1002-5506","position":5,"is_corresponding":false},{"id":248038,"name":"Jacob Piehler","orcid":"0000-0002-2143-2270","position":6,"is_corresponding":false},{"id":310421,"name":"Robert Riehn","orcid":"0000-0002-4408-3560","position":7,"is_corresponding":false},{"id":237050,"name":"Hong Wang","orcid":"0000-0003-0165-3559","position":8,"is_corresponding":false},{"id":310415,"name":"Hai Pan","orcid":"0000-0001-6660-9052","position":9,"is_corresponding":false},{"id":308205,"name":"David C. Williams","orcid":"0000-0002-6536-4038","position":10,"is_corresponding":false},{"id":374766,"name":"Gage O. Leighton","orcid":"0000-0003-4636-0370","position":0,"is_corresponding":true}],"reference_count":57,"raw_metadata":null,"created_at":"2026-07-19T00:25:17.224217Z","pmid":"36037972","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}