2022 journal article

Densely methylated DNA traps Methyl-CpG-binding domain protein 2 but permits free diffusion by Methyl-CpG-binding domain protein 3

JOURNAL OF BIOLOGICAL CHEMISTRY, 298(10).

By: G. Leighton *, E. Irvin n, P. Kaur n, M. Liu n, C. You *, D. Bhattaram *, J. Piehler *, R. Riehn n  ...

MeSH headings : CpG Islands; DNA Methylation; DNA-Binding Proteins / metabolism; Mi-2 Nucleosome Remodeling and Deacetylase Complex / genetics; Mi-2 Nucleosome Remodeling and Deacetylase Complex / metabolism; Nucleosomes; Protein Binding; Transcription Factors / metabolism; Humans; Single Molecule Imaging
TL;DR: 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. (via Semantic Scholar)
UN Sustainable Development Goal Categories
14. Life Below Water (OpenAlex)
Source: Web Of Science
Added: October 24, 2022

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.N. Williams Jr., D.C. Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches.Nucleic Acids Res. 2014; 42: 11218-11232Crossref PubMed Scopus (24) Google Scholar, 6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 7Otani J. Arita K. Kato T. Kinoshita M. Kimura H. Suetake I. et al.Structural basis of the versatile DNA recognition ability of the methyl-CpG binding domain of methyl-CpG binding domain protein 4.J. Biol. Chem. 2013; 288: 6351-6362Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Liu K. Xu C. Lei M. Yang A. Loppnau P. Hughes T.R. et al.Structural basis for the ability of MBD domains to bind methyl-CG and TG sites in DNA.J. Biol. Chem. 2018; 293: 7344-7354Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 9Liu K. Lei M. Wu Z. Gan B. Cheng H. Li Y. et al.Structural analyses reveal that MBD3 is a methylated CG binder.FEBS J. 2019; 286: 3240-3254Crossref PubMed Scopus (12) Google Scholar). However, biologically relevant differential DNA methylation occurs within regions of the genome that contain tens to hundreds of CpG sites (CpG islands) (10Deaton A.M. Bird A. CpG islands and the regulation of transcription.Genes Dev. 2011; 25: 1010-1022Crossref PubMed Scopus (2147) Google Scholar, 11Portela A. Liz J. Nogales V. Setien F. Villanueva A. Esteller M. DNA methylation determines nucleosome occupancy in the 5'-CpG islands of tumor suppressor genes.Oncogene. 2013; 32: 5421-5428Crossref PubMed Scopus (33) Google Scholar, 12Collings C.K. Anderson J.N. Links between DNA methylation and nucleosome occupancy in the human genome.Epigenetics Chromatin. 2017; 10: 18Crossref PubMed Scopus (41) Google Scholar, 13Collings C.K. Waddell P.J. Anderson J.N. Effects of DNA methylation on nucleosome stability.Nucleic Acids Res. 2013; 41: 2918-2931Crossref PubMed Scopus (68) Google Scholar, 14Doi A. Park I.H. Wen B. Murakami P. Aryee M.J. Irizarry R. et al.Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts.Nat. Genet. 2009; 41: 1350-1353Crossref PubMed Scopus (965) Google Scholar). Furthermore, methylation of CpG islands in promoters and enhancers correlates with nucleosome occupancy, chromatin compaction, and associated gene silencing. Hence, we have investigated how MBD proteins bind and diffuse along these CpG islands to better understand the functional consequences in a more biologically relevant context. In the current work, we focus on the structure and dynamics of the MBD2 and MBD3 proteins. These two highly homologous proteins arose from a duplication of the ancestral MBD present across the animal kingdom (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, 15Cramer J.M. Pohlmann D. Gomez F. Mark L. Kornegay B. Hall C. et al.Methylation specific targeting of a chromatin remodeling complex from sponges to humans.Sci. Rep. 2017; 740674Crossref Scopus (20) Google Scholar). They contribute to the structure and function of the Nucleosome Remodeling and Deacetylase (NuRD) (16Le Guezennec X. Vermeulen M. Brinkman A.B. Hoeijmakers W.A. Cohen A. Lasonder E. et al.MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties.Mol. Cell. Biol. 2006; 26: 843-851Crossref PubMed Scopus (266) Google Scholar) complex that can reposition nucleosomes, deacetylate histones, and modify gene expression. The NuRD complex (17Torrado M. Low J.K.K. Silva A.P.G. Schmidberger J.W. Sana M. Sharifi Tabar M. et al.Refinement of the subunit interaction network within the nucleosome remodelling and deacetylase (NuRD) complex.FEBS J. 2017; 284: 4216-4232Crossref PubMed Scopus (39) Google Scholar, 18Allen H.F. Wade P.A. Kutateladze T.G. The NuRD architecture.Cell. Mol. Life Sci. 2013; 70: 3513-3524Crossref PubMed Scopus (126) Google Scholar, 19Zhang W. Aubert A. Gomez de Segura J.M. Karuppasamy M. Basu S. Murthy A.S. et al.The nucleosome remodeling and deacetylase complex NuRD is built from preformed catalytically active sub-modules.J. Mol. Biol. 2016; 428: 2931-2942Crossref PubMed Scopus (47) Google Scholar, 20Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (937) Google Scholar) consists of a least six additional proteins, each of which has multiple paralogs that provide histone deacetylase activity (HDAC1/2), histone binding, and chromatin remodeling function (CHD3/4), and protein–protein interactions (GATAD2A/B, RBBP4/7, MTA1/2/3, CDK2AP1). The MBD2 and MBD3 proteins form distinct NuRD complexes that appear to have unique functional roles (16Le Guezennec X. Vermeulen M. Brinkman A.B. Hoeijmakers W.A. Cohen A. Lasonder E. et al.MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties.Mol. Cell. Biol. 2006; 26: 843-851Crossref PubMed Scopus (266) Google Scholar, 21Leighton G. Williams Jr., D.C. The methyl-CpG-binding domain 2 and 3 proteins and formation of the nucleosome remodeling and deacetylase complex.J. Mol. Biol. 2020; 432: 1624-1639Crossref Scopus (18) Google Scholar, 22Yu X. Azzo A. Bilinovich S.M. Li X. Dozmorov M. Kurita R. et al.Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells.Haematologica. 2019; 104: 2361-2371Crossref PubMed Scopus (32) Google Scholar). The two proteins show different levels of selectivity for mCpGs attributable primarily to a single amino acid change from tyrosine (MBD2) to phenylalanine (MBD3) within the DNA-binding site (Fig. 1) (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, 6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 24Saito M. Ishikawa F. The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2.J. Biol. Chem. 2002; 277: 35434-35439Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). MBD2 shows up to 100-fold selectivity for a fully mCpG dinucleotide compared to an unmethylated 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, 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, 25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar, 26Hashimoto H. Liu Y. Upadhyay A.K. Chang Y. Howerton S.B. Vertino P.M. et al.Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.Nucleic Acids Res. 2012; 40: 4841-4849Crossref PubMed Scopus (356) Google Scholar). In contrast, MBD3 binds DNA with an overall much lower affinity and shows no or slight (3–5 fold) selectivity for an mCpG (6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 26Hashimoto H. Liu Y. Upadhyay A.K. Chang Y. Howerton S.B. Vertino P.M. et al.Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.Nucleic Acids Res. 2012; 40: 4841-4849Crossref PubMed Scopus (356) Google Scholar). Consistent with this binding difference, previous genomic localization studies found that MBD2-NuRD predominantly binds at heavily mCpG islands associated with silenced genes, whereas MBD3 localizes to methylated and unmethylated CpG islands associated with expressed genes (23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 27Menafra R. Brinkman A.B. Matarese F. Franci G. Bartels S.J. Nguyen L. et al.Genome-wide binding of MBD2 reveals strong preference for highly methylated loci.PLoS One. 2014; 9: e99603Crossref PubMed Scopus (37) Google Scholar, 28Shimbo T. Du Y. Grimm S.A. Dhasarathy A. Mav D. Shah R.R. et al.MBD3 localizes at promoters, gene bodies and enhancers of active genes.Plos Genet. 2013; 9e1004028Crossref PubMed Scopus (82) Google Scholar, 29Gunther K. Rust M. Leers J. Boettger T. Scharfe M. Jarek M. et al.Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences.Nucleic Acids Res. 2013; 41: 3010-3021Crossref PubMed Scopus (90) Google Scholar). However, recent data suggests the alternative interpretation that both MBD2 and MBD3 depend on methylation for proper localization across the genome (30Hainer S.J. McCannell K.N. Yu J. Ee L.-S. Zhu L.J. Rando O.J. et al.DNA methylation directs genomic localization of Mbd2 and Mbd3 in embryonic stem cells.Elife. 2016; 5: e21964Crossref PubMed Scopus (21) Google Scholar). Therefore, how methylation selectivity of the MBD2 and MBD3 proteins impacts this localization and function remains an open question in the field. In previous work, we used single-molecule analyses to study the behavior of the isolated MBD from MBD2 on various DNA substrates (31Pan H. Bilinovich S.M. Kaur P. Riehn R. Wang H. Williams Jr., D.C. CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level.Nucleic Acids Res. 2017; 45: 9164-9177Crossref PubMed Scopus (19) Google Scholar). Consistent with NMR and bulk biochemical studies, we found a remarkable difference in DNA bending and sliding of the MBD from MBD2 (MBD2MBD) on methylated and unmethylated DNA containing CpG islands. MBD2MBD is mostly restricted to the mCpG islands, while it freely diffuses across unmethylated CpG islands. Furthermore, we also uncovered a novel role for the intrinsically disordered region (IDR) of MBD2 in DNA bending (25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar). The DNA-bending angle induced by both the MBD and a small portion of the IDR (MBD2MBD+IDR) on unmethylated CpG-rich DNA is larger than observed for CpG-free and further increases upon binding mCpG-rich DNA. In separate structural studies of MBD3, we found that the MBD from MBD3 shows only weak selectivity for a single mCpG within a small (17 bp) dsDNA fragment (6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Based on a combination of chemical shift analyses, mutagenesis, and residual dipolar coupling measurements, we showed that MBD3 exchanges rapidly between CpG-specific and nonspecific binding modes, leading to chemical shift averaging between these two states. Hence, MBD3 recognizes an mCpG site, as evidenced by significant chemical shift changes, but does not strongly localize to this site when bound to DNA. This difference between the DNA-binding dynamics of MBD2 and MBD3 correlates with the prior localization studies that show both MBD2 and MBD3 localize to unmethylated CpG islands, while MBD2 more exclusively localizes to mCpG islands (23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 27Menafra R. Brinkman A.B. Matarese F. Franci G. Bartels S.J. Nguyen L. et al.Genome-wide binding of MBD2 reveals strong preference for highly methylated loci.PLoS One. 2014; 9: e99603Crossref PubMed Scopus (37) Google Scholar, 28Shimbo T. Du Y. Grimm S.A. Dhasarathy A. Mav D. Shah R.R. et al.MBD3 localizes at promoters, gene bodies and enhancers of active genes.Plos Genet. 2013; 9e1004028Crossref PubMed Scopus (82) Google Scholar, 29Gunther K. Rust M. Leers J. Boettger T. Scharfe M. Jarek M. et al.Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences.Nucleic Acids Res. 2013; 41: 3010-3021Crossref PubMed Scopus (90) Google Scholar). Despite these recent studies, we do not know how the remaining, largely unstructured regions of MBD2 and MBD3 influence diffusion along DNA. Furthermore, it is unclear how reduced selectivity and binding affinity of MBD3 modifies its distribution and sliding on methylated and unmethylated CpG islands, which contain many CpG sites. To address these questions, in the current studies, we use a combination of biophysical techniques, including atomic force microscopy (AFM) imaging (32Benarroch-Popivker D. Pisano S. Mendez-Bermudez A. Lototska L. Kaur P. Bauwens S. et al.TRF2-Mediated control of telomere DNA topology as a mechanism for chromosome-end protection.Mol. Cell. 2016; 61: 274-286Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 33Wang H. Tessmer I. Croteau D.L. Erie D.A. Van Houten B. Functional characterization and atomic force microscopy of a DNA repair protein conjugated to a quantum dot.Nano Lett. 2008; 8: 1631-1637Crossref PubMed Scopus (52) Google Scholar, 34Wang H. Yang Y. Schofield M.J. Du C. Fridman Y. Lee S.D. et al.DNA bending and unbending by MutS govern mismatch recognition and specificity.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14822-14827Crossref PubMed Scopus (164) Google Scholar), DNA tightrope assays (35Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (24) Google Scholar, 36Kad N.M. Wang H. Kennedy G.G. Warshaw D.M. Van Houten B. Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single- molecule imaging of quantum-dot-labeled proteins.Mol. Cell. 2010; 37: 702-713Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 37Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. et al.TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (51) Google Scholar), and NMR (5Walavalkar N.M. Cramer J.M. Buchwald W.A. Scarsdale J.N. Williams Jr., D.C. Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches.Nucleic Acids Res. 2014; 42: 11218-11232Crossref PubMed Scopus (24) Google Scholar, 6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) to measure the binding and sliding of MBD2 and MBD3 on methylated and unmethylated DNA substrates. In the DNA tightrope assay, DNA molecules are stretched under hydrodynamic flow inside a flow cell. Anchoring of stretched DNA between poly-L-lysine–coated silica microspheres leads to the formation of DNA tightropes at an elongation of ∼90% of the DNA contour length (Fig. 2A). The spatial resolution of the DNA tightrope assay was estimated to be 16 nm (37Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. et al.TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (51) Google Scholar). Uniquely, DNA tightropes created using tandemly ligated DNA allow us to directly correlate DNA-binding events with the underlying specific DNA sequences or structures such as three-stranded R-loops (31Pan H. Bilinovich S.M. Kaur P. Riehn R. Wang H. Williams Jr., D.C. CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level.Nucleic Acids Res. 2017; 45: 9164-9177Crossref PubMed Scopus (19) Google Scholar, 32Benarroch-Popivker D. Pisano S. Mendez-Bermudez A. Lototska L. Kaur P. Bauwens S. et al.TRF2-Mediated control of telomere DNA topology as a mechanism for chromosome-end protection.Mol. Cell. 2016; 61: 274-286Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 37Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. et al.TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (51) Google Scholar, 38Pan H. Jin M. Ghadiyaram A. Kaur P. Miller H.E. Ta H.M. et al.Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA.Nucleic Acids Res. 2020; 48: 5639-5655Crossref PubMed Google Scholar). To study MBD proteins diffusion, we ligated linear DNA fragments to form DNA tightropes with CpG free or alternating CpG-free and CpG-rich regions (Fig. 2B and S8A). The results from our previous study revealed that the isolated MBD from MBD2, with or without a small portion of the adjacent IDR (MBD2MBD and MBD2MBD+IDR), carry out unbiased 1D diffusion on CpG-rich DNA but undergoes subdiffusion on CpG-free DNA. In contrast, both proteins stably and statically bind to mCpG regions. In this study, we purified a construct that contains almost the entire length of the MBD2b isoform plus the coiled-coil region from GATAD2A (MBD2sc) to investigate how these additional regions impact DNA binding and diffusion (Experimental Procedures, Fig. 1A). We previously found that the MBD2sc binds DNA with an approximately 100× higher affinity than MBD2MBD (25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar). Consistent with these results, fluorescence anisotropy experiments showed that MBD2sc binds to DNA containing unmethylated, methylated, or no CpG site with equilibrium dissociation constants of 2.8 (±0.1 μM), 0.007 (±0.002 μM), or 9.1 (±0.4 μM), respectively (Fig. 2C). Hence, we questioned whether the additional binding affinity provided by the IDR in MBD2 would modify DNA binding and sliding. We directly addressed this question using the DNA tightrope assay. For the DNA tightrope assay, we first generated DNA substrates by tandemly ligating linear DNA fragments containing CpG-free or unmethylated CpG-free–rich sequences (Fig. 2B). Further, we conjugated His-tagged MBD2sc to streptavidin-coated quantum dots (SAv-QDs) through the multivalent chelator tris-nitrilotriacetic acid linker (Fig. 2A) (39Reichel A. Schaible D. Al Furoukh N. Cohen M. Schreiber G. Piehler J. Noncovalent, site-specific biotinylation of histidine-tagged proteins.Anal. Chem. 2007; 79: 8590-8600Crossref PubMed Scopus (47) Google Scholar). Following the formation of the DNA tightropes, we introduced QD-labeled MBD2sc into the flow cell. Analysis of MBD2sc on DNA tightropes revealed two populations (Table 1): apparently immobile throughout data acquisition and mobile molecules (Fig. 2D). To exclude that this apparently immobile population reflects aggregation, we categorized particles as a single protein or cluster based on their individual QD blinking rate (Table S1) which shows that only a small fraction comprises multiple proteins. To obtain diffusion coefficients for mobile MBD2sc on DNA tightropes, we tracked the position of MBD2sc-QDs on DNA by Gaussian fitting to kymographs (particle position versus time plots) (36Kad N.M. Wang H. Kennedy G.G. Warshaw D.M. Van Houten B. Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single- molecule imaging of quantum-dot-labeled proteins.Mol. Cell. 2010; 37: 702-713Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 40Dunn A.R. Kad N.M. Nelson S.R. Warshaw D.M. Wallace S.S. Single Qdot-labeled glycosylase molecules use a wedge amino acid to probe for lesions while scanning along DNA.Nucleic Acids Res. 2011; 39: 7487-7498Crossref PubMed Scopus (98) Google Scholar). We obtained diffusion coefficients and alpha exponents by fitting the mean square displacement (MSD) versus time. An alpha exponent of 1 indicates an unbiased random walk, and a value less than 1 indicates subdiffusion (41Saxton M.J. Single-particle tracking: The distribution of diffusion coefficients.Biophys. J. 1997; 72: 1744-1753Abstract Full Text PDF PubMed Scopus (479) Google Scholar). The diffusion coefficients displayed by MBD2sc on the CpG-free DNA tightrope were significantly slower than those on DNA tightropes containing CpG sites (CpG-free–rich) (Fig. 2E). In addition, on the CpG-free–rich tightropes, MBD2sc displayed alpha exponents close to 1 (1.0 ± 0.2), indicating largely unbiased 1D diffusion on DNA (Fig. 2F, Table 2). As compared to CpG-free–rich DNA, the alpha exponents for MBD2sc on CpG-free DNA tightropes were slightly (p < 0.001) reduced (0.8 ± 0.2, Fig. 2F, Table 2). Overall, MBD2sc displayed slightly different diffusion ranges on CpG-free DNA tightropes compared to CpG-free–rich DNA tightropes containing multiple CpG sites (Fig. S3). In summary, MBD2sc shows more rapid and extensive 1D diffusion on CpG-free–rich DNA than CpG-free sequences.Table 1Fraction of statically bound MBD2sc and MBD3sc on unmethylated- and methylated-DNA tightropesDNAMBD2scMBD3scStatic binding (%)NStatic binding (%)NCpG-free–rich23 ± 614747 ± 7123CpG-free19 ± 633354 ± 8240mCpG-free–rich96 ± 324652 ± 10345mCpG-mini90 ± 149550 ± 8158The values represent mean ± SD from 2 to 3 experiments for each data set. Open table in a new tab Table 2Diffusion coefficient of MBD2sc and MBD3sc on different DNA substratesDNAMBD2scMBD3scD (μm2/s)α exponentND (μm2/s)α exponentNCpG-free0.04 ± 0.020.8 ± 0.2990.04 ± 0.030.8 ± 0.3199CpG-free–rich0.15 ± 0.051.0 ± 0.21000.04 ± 0.010.9 ± 0.2144mCpG-free–richNA.NA.0.09 ± 0.021.0 ± 0.2174mCpG-miniNA.NA.0.05 ± 0.020.9 ± 0.170The values represent mean ± SD from 2 to 4 experiments for each data set. Open table in a new tab The values represent mean ± SD from 2 to 3 experiments for each data set. The values represent mean ± SD from 2 to 4 experiments for each data set. To evaluate how DNA methylation affects the dynamics of MBD2sc on DNA, we imaged QD-labeled MBD2sc on the CpG-free–rich DNA tightropes after methylation. Linear DNA substrates were methylated before ligation using CpG Methyltransferase (M.SssI) with SAM as a cofactor (Experimental procedures). We confirmed methylation of the linear CpG-free–rich DNA substrate by digestion with the methylation-sensitive HpaII restriction endonuclease (Fig. S1). We then tandemly ligated the mCpG-free–rich (mCpG-free-rich, Fig. 3A) and used it to form DNA tightropes between silica beads. Compared to unmethylated CpG-free–rich and CpG-free DNA, the binding density of MBD2sc increased approximately 4× on mCpG-free–rich DNA tightropes (Fig. 3B, S4A). This result is consistent with the preferential binding of MBD2sc to mCpG sites, as supported by binding affinity measurements (Fig. 2C). Notably, while the majority of MBD2sc observed on unmethylated CpG-free–rich DNA tightropes was mobile (77%), on mCpG-free–rich tightropes, the majority of the protein bound was apparently immobile (96%) throughout data acquisition (Fig. 3B and Table 1). Furthermore, the distance between adjacent proteins on the mCpG-free–rich DNA tightropes is Gaussian distributed, with the peak centered at 2.3 (±0.3 μm) (Fig. 3C). This spacing matches the calculated distances between adjacent mCpG-rich regions on DNA tightropes (Fig. 3A), considering that they are stretched to ∼90% of their contour length. Taken together, fluorescence imaging of MBD2sc on DNA tightropes establishes that MBD2sc recognizes mCpG islands through stable and apparently static binding. To evaluate whether MBD2s