Modulation of Dnmt3b function in vitro by interactions with Dnmt3L, Dnmt3a and Dnmt3b splice variants - PubMed (original) (raw)
. 2011 Jul;39(12):4984-5002.
doi: 10.1093/nar/gkr116. Epub 2011 Mar 4.
Affiliations
- PMID: 21378119
- PMCID: PMC3130282
- DOI: 10.1093/nar/gkr116
Modulation of Dnmt3b function in vitro by interactions with Dnmt3L, Dnmt3a and Dnmt3b splice variants
Beth O Van Emburgh et al. Nucleic Acids Res. 2011 Jul.
Abstract
DNA methylation, an essential regulator of transcription and chromatin structure, is established and maintained by the coordinated action of three DNA methyltransferases: DNMT1, DNMT3A and DNMT3B, and the inactive accessory factor DNMT3L. Disruptions in DNMT3B function are linked to carcinogenesis and genetic disease. DNMT3B is also highly alternatively spliced in a tissue- and disease-specific manner. The impact of intra-DNMT3 interactions and alternative splicing on the function of DNMT3 family members remains unclear. In the present work, we focused on DNMT3B. Using a panel of in vitro assays, we examined the consequences of DNMT3B splicing and mutations on its ability to bind DNA, interact with itself and other DNMT3's, and methylate DNA. Our results show that, while the C-terminal catalytic domain is critical for most DNMT3B functions, parts of the N-terminal region, including the PWWP domain, are also important. Alternative splicing and domain deletions also influence DNMT3B's cellular localization. Furthermore, our data reveal the existence of extensive DNMT3B self-interactions that differentially impact on its activity. Finally, we show that catalytically inactive isoforms of DNMT3B are capable of modulating the activity of DNMT3A-DNMT3L complexes. Our studies therefore suggest that seemingly 'inactive' DNMT3B isoforms may influence genomic methylation patterns in vivo.
Figures
Figure 1.
Assay systems, constructs and DNA templates used to assess Dnmt3b functions in vitro. (A) Schematic representation of Dnmt3b1 and the splice variants/deletions expressed and purified for in vitro studies. Critical domains and catalytic motifs are highlighted. SAM interaction sites are indicated by orange arrows. Numbers represent the amino acids of murine Dnmt3b1. Amino acids listed below represent ICF syndrome-associated mutations introduced into the murine sequence at homologous positions to human DNMT3B1 (
Supplementary Table S2
). Amino acids and red/green bars above Dnmt3b1 represent regions predicted to be important for protein–protein interaction based on Dnmt3a–Dnmt3L structural studies (29). (B) Representative coomassie blue stained gel showing a subset of the in vitro expressed purified 6XHis-tagged recombinant proteins used in this study. (C) Box chart of methodology employed to analyze functional effects of splice variants/deletions/ICF mutations. (D) Plot of CpG and HpaII sites in the plasmid containing the satellite 2 (SAT2) sequence used as the template DNA for in vitro activity and protein–DNA interaction assays. CpG sites analyzed by bisulfite genomic sequencing (BGS) and pyrosequencing (denoted by thick horizontal lines) are enlarged and shown below.
Figure 2.
Functional effects of Dnmt3b alternative splicing on protein–DNA binding, interaction with Dnmt3L, and enzyme activity. (A) Top panel: representative SYBR green stained EMSA gels with increasing amounts of the Dnmt3 proteins indicated (0–700 nM). Arrowhead—free probe. Bottom panels: percent shift quantitated from EMSA gels. Curves were fit using the Hill equation. Left: Dnmt3b’s and Dnmt3a. Right: effect of Dnmt3L (or GST) on DNMT3B1 binding to DNA. (B) GST pull down analysis of protein–protein interactions between the indicated Dnmt3 constructs and GST-Dnmt3L. Western blotting with anti-His antibody was used to detect interactions. +: pull down with GST-Dnmt3L, –: pull down with GST, low and high refer to the 100 and 150 mM KCl used in binding reaction/washes, respectively. The 6X-His fusion protein and GST-fusion protein loading inputs for each reaction are shown below the pull downs to illustrate equal loading. Reactions identical to the pull downs, without washing, were run along with all pull down reactions and proteins detected by western blotting with antibodies against the 6XHis or GST tags. Arrowhead—specific protein, asterisk—non-specific band. (C) Representative HpaII restriction digests of SAT2-containing plasmid following the methyltransferase activity assay. –, Dnmt3 enzyme alone; +, Dnmt3 and Dnmt3L. Numbers below indicate percentage methylation quantified from changes in band patterns resulting from inhibition of HpaII cutting due to methylation. (D) Methylation of individual CpG sites in SAT2 (Figure 1) by Dnmt3s with and without Dnmt3L analyzed by pyrosequencing. Error bars indicate standard deviation. (E) Linear correlation of methylation of individual CpGs between Dnmt3b1 with Dnmt3L and the other indicated Dnmt3s with Dnmt3L using SAT2 pyrosequencing data. Dnmt3b2 and Dnmt3L R: 1, R2: 1; Dnmt3a and Dnmt3L R: 0.61, R2: 0.37; DNMT3B1 and Dnmt3L R: 1, R2: 1. (F) SAT2 BGS results for Dnmt3s with and without Dnmt3L. Overall percentage methylation is indicated in parentheses. White circles: unmethylated CpG, black circles: methylated CpG. Each row represents one sequenced clone.
Figure 3.
Functional effects of Dnmt3b1 domain deletions on enzyme–DNA binding, interaction with Dnmt3L, and enzymatic activity. (A) Quantification of percent shifts from EMSA gels (not shown) as described in Figure 2A using the SAT2 DNA probe. (B) Western blot detection of protein–protein interactions between Dnmt3b1 deletions and GST-Dnmt3L following GST pull down analysis under low and moderate stringency conditions (100 and 150 mM KCl, respectively). Symbols are as described in Figure 2B. Input (I) represents the loading of each 6X-His fusion bait protein and is the same for low and high pull downs. (C) Overall methylation of the SAT2 sequence by Dnmt3b1 deletions and the Dnmt3b1 C657A catalytic mutant with and without Dnmt3L determined using pyrosequencing.
Figure 4.
Functional effects of ICF syndrome mutations on Dnmt3b1 protein–DNA interactions, interaction with Dnmt3L, and enzymatic activity. (A) Quantification of EMSA gels and Hill plots summarizing the binding of each Dnmt3b1 mutant (0–700 nM) to the SAT2 DNA probe. (B) Representative western blots showing protein–protein interactions of Dnmt3b1 mutants and GST-Dnmt3L by GST pull down as described in Figures 2B and 3B. (C) Pyrosequencing-based determination of overall percent methylation of the SAT2 sequence by mutant Dnmt3b1 isoforms with and without Dnmt3L.
Figure 5.
Impact of alternative splicing, domain deletions and ICF syndrome-associated mutations on Dnmt3b1 interaction with the catalytic domains (CD) of Dnmt3b1 and Dnmt3b3. (A) Top panel: schematic of the GST-tagged catalytic domain of Dnmt3b1 used for protein–protein interaction pull downs. Catalytic motifs are indicated by roman numerals. Numbering indicates amino acids of Dnmt3b present in the construct. Bottom panel: representative western blots using anti-6XHis tag antibody to study protein–protein interactions between the indicated Dnmt3′s and GST-Dnmt3b1 CD. +, pull down with GST-Dnmt3b1 CD;−,pull down with GST (negative control), low and high refer to the 100 and 150 mM KCl used in binding reaction/washes, respectively. (B) Top panel: schematic of the Dnmt3b3 catalytic domain-GST fusion protein used for pull downs. Numbering denotes amino acids of Dnmt3b present in the construct and the alternatively spliced region characteristic of Dnmt3b3. Bottom panel: representative western blots using anti-6XHis tag antibody to detect protein–protein interactions between the indicated Dnmt3 constructs and GST-Dnmt3b3 CD. (C) 6X-His fusion protein and (D) GST-fusion protein inputs used in the pull down reactions to demonstrate equal loading. Reactions identical to the pull downs (without washing) were run with the pull down reactions and proteins detected by western blotting with antibodies against the 6XHis or GST tags. Arrowhead—specific protein, asterisk—non-specific band.
Figure 6.
Effects of Dnmt3b alternative splicing, deletions and ICF syndrome-associated mutations on the enzymatic activity of Dnmt3a combined with Dnmt3L. (A) Representative HpaII restriction digests of SAT2-containing plasmid following methyltransferase activity assay combining Dnmt3a and Dnmt3L with the indicated Dnmt3b proteins. Lanes 8 (no Dnmt3) and 9 (no SAM) are negative controls. (B) Effects of select Dnmt3b constructs on methylation of the SAT2 sequence when mixed with Dnmt3a+Dnmt3L relative to Dnmt3a+Dnmt3L alone. Percent methylation is quantitated by HpaII digestion (black bars) and pyrosequencing at both the SAT2 and plasmid regions (light and dark gray bars, respectively), relative to a reaction with only Dnmt3a+Dnmt3L. Active 3b: +, construct has demonstrated activity alone; –, construct does not possess catalytic activity alone.
Figure 7.
Localization of Dnmt3b isoforms/mutations and Dnmt3L in transiently transfected HEK 293T cells. (A) Representative cells showing localization of indicated GFP-tagged Dnmt3b splice variants and DsRed-tagged Dnmt3L when transfected alone. (B) Representative cells showing colocalization of GFP-tagged Dnmt3b splice variants with DsRed-tagged Dnmt3L when cotransfected. (C) Representative cells showing colocalization of GFP-tagged Dnmt3b splice variants with DsRed-tagged Dnmt3b1when cotransfected. (D) Localization of GFP-tagged Dnmt3b1 C657A catalytic mutant, Dnmt3b1 V612A ICF syndrome mutant, and the Dnmt3b1 Δ227–429 deletion construct when transfected alone. Two patterns representative of the population of transfected cells are shown for the Δ227–429 construct. (E) Representative cells showing colocalization of GFP-tagged Dnmt3b1 C657A catalytic mutant, Dnmt3b1 V612A ICF syndrome mutant, and Dnmt3b1 Δ227–429 co-transfected with DsRed-tagged Dnmt3b1.
References
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