Journal: Nucleic Acids Research

Article Title: Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation

doi: 10.1093/nar/gky104

Figure Lengend Snippet: Molecular determinants of DNMT1 binding to ubiquitin and UBL domain of UHRF1. ( A ) Domain organization of DNMT1. Three DNMT1 fragments were prepared for ubiquitin binding analysis. ( B and C ) 15 N HSQC spectra show that ubiquitin binds to DNMT1 RFTS domain (aa: 351–600) but not DNMT1_621 (aa: 621–1616), as illustrated by strong selective line broadening effect induced by addition of DNMT1 RFTS domain but not DNMT1_621. ( D ) Domain organization of DNMT1 fragments that were prepared for UHRF1 UBL binding. ( E and F ) NMR titration indicates UHRF1 UBL binds to DNMT1 RFTS domain as well as DNMT1_621, as illustrated by strong line broadening of certain resonances induced by the binding of DNMT1 RFTS (E) and DNMT1_621 fragment (F). ( G and H ) ITC confirms the molecular interaction of DNMT1 RFTS domain with ubiquitin and UHRF1 UBL domain. The top panel shows experimental ITC curve of titrating DNMT1 RFTS domain into ubiquitin (G) and UHRF1 UBL (H) respectively. The lower panel shows fitted curves of calorimetric titrations. The measured dissociation constant ( K d ) is shown.

Article Snippet: Myc-DDK-tagged human DNMT1 (RC226414) and UHRF1 (RC214251) plasmids were purchased from OriGene.

Techniques: Binding Assay, Titration

Journal: Nucleic Acids Research

Article Title: Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation

doi: 10.1093/nar/gky104

Figure Lengend Snippet: Crystal structure of DNMT1 RFTS domain in complex with ubiquitin. ( A ) 1:2 DNMT1 RFTS domain-ubiquitin contents of the asymmetric unit are shown as ribbon diagrams. DNMT1 RFTS domain is colored in green, two ubiquitin molecules are colored in marine and cyan respectively. ( B and C ) Close-up view of the interactions between the RFTS domain and ubiquitin 1 (Ubq1). ( D and E ) Detailed view of the interaction network of the RFTS domain with ubiquitin 2 (Ubq2). Residues that form the binding interface are depicted as stick models and labeled. Hydrogen bonds and salt bridges are shown in magenta dashes. ( F ) DNMT1 RFTS domain undergoes conformational changes upon ubiquitin binding. The α-helix (aa: 493–518) of RFTS domain adopts as a straight α-helical conformation in DNMT1 (PDB ID: 4WXX) but bends about 32° in the middle of α-helix (aa: 493–518) at the position of Met502 in RFTS/ubiquitin complex. Ubiquitin binding caused the bending of RFTS α-helix (aa: 493–518) that connects the preceding β-barrel (aa: 400–490) to the α-helical bundle, this eventually leads to large changes in the relative orientation of the β-barrel with respect to α-helical bundle.

Article Snippet: Myc-DDK-tagged human DNMT1 (RC226414) and UHRF1 (RC214251) plasmids were purchased from OriGene.

Techniques: Binding Assay, Labeling

Journal: Nucleic Acids Research

Article Title: Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation

doi: 10.1093/nar/gky104

Figure Lengend Snippet: Experimental measurement of DNMT1 RFTS domain binding to ubiquitin or UHRF1 UBL domain. ( A ) ITC fitting curves for binding of DNMT1 RFTS domain and its mutants to ubiquitin, the insert lists the calculated dissociation constant ( K d ). ND, not detectable. ( B ) ITC fitting curves for binding of DNMT1 RFTS domain to ubiquitin wild-type and mutants, along with the calculated K d . ( C ) ITC fitting curves for binding of DNMT1 RFTS domain and its mutants with UHRF1 UBL domain with dissociation constant ( K d ) values indicated. ( D and E ) In vitro DNA methylation reactions were performed as a function of time and the concentration of ubiquitin or UHRF1 UBL. (D) DNMT1_351 (aa: 351–1616) or ( E ) DNMT1_621 (aa: 621–1616) was used as the enzyme, and a 30-bp long hemimethylated DNA fragment as the substrate. ( F and G ) In vitro DNA methylation reactions were performed as a function of time and the concentration of ubiquitin, histone H3 or H3ub2. (F) DNMT1_351 (aa: 351–1616) or ( G) DNMT1_621 (aa: 621–1616) was used as the enzyme.

Article Snippet: Myc-DDK-tagged human DNMT1 (RC226414) and UHRF1 (RC214251) plasmids were purchased from OriGene.

Techniques: Binding Assay, In Vitro, DNA Methylation Assay, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation

doi: 10.1093/nar/gky104

Figure Lengend Snippet: Assessing nuclear localization and DNA methylation status in DNMT1 −/− mouse embryonic stem cells stably expressing DNMT1 wild-type or mutants. ( A ) Flag-Myc-tagged wild-type DNMT1, DNMT1 E384A, E397A, E384A/E397A and Y399A mutants were stably expressed in DNMT1 −/− mouse ESCs, as analyzed by western blot using antibodies that recognize DNMT1. The expression level of these exogenous proteins similar to endogenous DNMT1 was selected for the subsequent study. β-actin was selected as a loading control. ( B ) Immunofluorescence analysis of DNMT1 focal staining pattern in DNMT1 wild-type or knockout mouse ES cells or DNMT1 −/− ES cells stably expressing DNMT1 wild-type, E384A or E397A or Y339G single point mutant, or E384A/E397A double point mutant respectively. Scale bars, 10 μm. ( C ) Immunostaining using an antibody against 5mC in control and DNMT1 −/− mouse ES cells after genetic complementation with DNMT1 wild-type or various mutants. 5mC fluorescence signals from ∼50 cells were quantified and normalized against the wild-type cells, the mean value with a standard error has been provided. ( D ) The DNA methylation status of LINE1 and IAP was analyzed by bisulfite sequencing in control, DNMT1 −/− ESCs and DNMT1 −/− ESCs stably expressing DNMT1 wild-type, E384A, E397A, E384A/E397A and Y399A mutants. The percentage of 5mC was calculated and shown.

Article Snippet: Myc-DDK-tagged human DNMT1 (RC226414) and UHRF1 (RC214251) plasmids were purchased from OriGene.

Techniques: DNA Methylation Assay, Stable Transfection, Expressing, Western Blot, Immunofluorescence, Staining, Knock-Out, Mutagenesis, Immunostaining, Fluorescence, Methylation Sequencing

Journal: Nucleic Acids Research

Article Title: Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation

doi: 10.1093/nar/gky104

Figure Lengend Snippet: Both UHRF1 UBL and RING finger are critical for DNMT1 proper nuclear localization and maintenance DNA methylation in mouse embryonic stem cells. ( A ) UHRF1 −/− mouse ES cell lines stably expressing Flag-Myc-tagged UHRF1 wild-type or various mutants were analyzed by western blot using antibodies that recognize UHRF1. Flag-Myc-tagged UHRF1 or mutants expressed at a level similar to endogenous UHRF1 were selected for the study. β-actin was selected as a loading control. ( B ) Immunofluorescence analysis of DNMT1 focal staining pattern in UHRF1 wild-type, UHRF1 knockout mouse ES cells and UHRF1 −/− ES cells transfected with UHRF1-ΔUBL or UHRF1-ΔRING truncated mutant. Exogenous expression of UHRF1 and mutants was detected by Flag antibodies. Scale bars, 10 μm. ( C ) Immunostaining using an antibody against 5mC in control and UHRF1 −/− mouse ES cells after genetic complementation with wild-type or UHRF1-ΔUBL or UHRF1-ΔRING. The 5mC levels relative to wild-type ESCs were shown. Error bars represent ± s.e.m. ( D ) The DNA methylation status of LINE1 and IAP was analyzed by bisulfite sequencing in wild-type ESCs (as control), UHRF1 −/− ESCs and UHRF1 −/- ESCs stably expressing UHRF1 wild-type, or UHRF1-ΔUBL or UHRF1-ΔRING mutants. The percentage of 5mC was calculated and shown.

Article Snippet: Myc-DDK-tagged human DNMT1 (RC226414) and UHRF1 (RC214251) plasmids were purchased from OriGene.

Techniques: DNA Methylation Assay, Stable Transfection, Expressing, Western Blot, Immunofluorescence, Staining, Knock-Out, Transfection, Mutagenesis, Immunostaining, Methylation Sequencing