Physical and functional interaction of DNA methyltransferase 3A with Mbd3 and Brg1 in mouse lymphosarcoma cells
Authors: Shoumei Bai
Kalpana Ghoshal
John W Crabb
Jhrana Datta
Huban Kutay
Samson T Jacob
Sarmila Majumder
David Spencer Smith
Journal: Cancer Research (2005)
Discipline: CLINICAL MEDICINE
Keywords: Dnmt3a; transcriptional repression; Mbd3; histone deacetylase 1; Brg1 complex
Affiliations: Department of Molecular and Cellular Biochemistry, Ohio State University, Columbus, Ohio 43210, USA.

Abstract:  Dnmt3a and Dnmt3b are de novo DNA methyltransferases that also act as transcriptional repressors independent of methyltransferase activity. To elucidate the underlying mechanism of transcriptional repression, Dnmt3a was purified from mouse lymphosarcoma cells (P1798) by extensive fractionation on five different chromatographic matrices followed by glycerol density gradient centrifugation. Liquid chromatography electrospray tandem mass spectrometry analysis of Dnmt3a-associated polypeptides identified the methyl CpG binding protein Mbd3, histone deacetylase 1(Hdac1), and components of Brg1 complex (Brg1, Baf155, and Baf57) in the purified preparation. Association of Dnmt3a with Mbd3 and Brg1 was confirmed by coimmunoprecipitation and coimmunolocalization studies. Glutathione S-transferase pulldown assay showed that the NH2-terminal ATRX homology domain of Dnmt3a interacts with the methyl CpG binding domain of Mbd3 and with both bromo and ATPase domains of Brg1. Chromatin immunoprecipitation assay revealed that all three proteins are associated with transcriptionally silent methylated metallothionein (MT-I) promoter in the mouse lymphosarcoma cells. To understand the functional significance of their association with the promoter, their role on the MT-I promoter activity was analyzed by transient transfection assay. The results showed that Mbd3 and Dnmt3a specifically inhibited the methylated promoter, and the catalytic activity of Dnmt3a was dispensable for the suppression. In contrast, the wild-type but not the ATPase-inactive mutant of Brg1 suppressed MT-I promoter irrespective of its methylation status, implicating involvement of ATP-dependent chromatin remodeling in the process. Coexpression of two of the three interacting proteins at a time augmented their repressor function. This study shows physical and functional interaction of Dnmt3a with components of nucleosome remodeling machinery. 

Retracted: No

PubPeer comments:
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 Figure 3(C).  Abrupt change in background between lanes 6 and 7 (from dithered to completely blank). Lanes 19-21 are unexpectedly similar to a stretched version of lanes 13-15. , 
 #4 Regarding Fig 3C: 1. Note that the image quality is better in the PDF version of the paper, where it easier to see some of the finer details. (See below) 2. I would not be surprised if lanes 1-2-3 are also a (vertically compressed) match for 13-14-15 and 19-20-21, but the dithered half-tone quality of lanes 1-6 obscures the details and makes it impossible to analyze. 3. Lanes 11 and 12 are mirror-images of each other, as illustrated here:  , 
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 Third author appears to currently be affiliated with University of Pittsburgh, and has the unfortunate distinction of appearing as coauthor of multiple papers with similar image concerns while at different institutions: https: pubpeer.com search?q=Shoumei+Bai  , 
 https: retractionwatch.com 2022 07 25 exclusive-osu-investigation-finds-dishonesty-and-permissive-culture-of-data-manipulation-in-cancer-research-lab ,