Journal: eNeuro
Article Title: Evidence That Dmrta2 Acts through Repression of Pax6 in Cortical Patterning and Identification of a Mutation Impairing DNA Recognition Associated with Microcephaly in Human
doi: 10.1523/ENEURO.0377-24.2025
Figure Lengend Snippet: The Dmrta2-Pax6 interaction requires the recruitment of the HDAC-NuRD complex by Zfp423. A , B , Coimmunoprecipitation assays as indicated with Zfp423, Flag-Dmrta2, and a Flag-Dmrta2 mutant lacking the DM domain (Flag-Dmrta2 ΔDM) overexpressed in HEK293T cells as indicated. Note in A that Flag-Dmrta2 pulled down Zpf423 (line 1–2) and that, conversely, Zpf423 coprecipitated Flag-Dmrta2 (line 3–4). Note in B that Flag-Dmrta2 ΔDM does not pull down Zfp423 (line 4). n = 3. Segments from the same blot have been spliced together to show side by side the results of the WT and the ΔDM Dmrta2 mutant. C , Full-length mouse Myc-tagged Zfp423 was synthesized in vitro and incubated with GST alone or with GST fusion proteins bound to glutathione-agarose beads as indicated. Bound Myc-tagged Zfp423 was detected by immunoblot with an anti-Myc antibody. A 2% input sample was loaded for comparison. The corresponding Coomassie-stained gel is shown. n = 2. D , Coimmunoprecipitation assays as indicated with HEK293T cells transfected with a Flag -Dmrta2 expression construct, alone or together with increasing doses of a Zfp423 expression construct. Note that Dmrta2 immunoprecipitates some NuRD subunits and that the binding of Zfp423 to Dmrta2 increases the amount of coimmunoprecipitated HDAC1/2 and MBD3. Densitometric quantification of the western blot results is shown in Extended Data . n = 3. E , Reporter assays in P19 cells transfected with a Pax6 E60 tk-luc reporter vector, or an “empty” tk-luc reporter vector as indicated, together with a pCS2Myc -Pax6 expression vector and/or a pCS2Flag -Dmrta2 expression vector as indicated, in the absence (white bars) or presence of increasing doses of the HDAC1 inhibitor romidepsin (gray bars). Note that romidepsin leads to a stronger increase of luciferase activity reaching significance in the presence of Dmrta2 but not in its absence. The mean activity of the Pax6 E60 enhancer reporter construct with cotransfected Pax6 is set to 1. NS, not significant. * p < 0.05, one-way ANOVA test. Results of similar reporter assays performed in P19 cells, in the presence or absence of Zfp423 are presented in Extended Data . F , Reporter assays in HEK293T cells show that both Gal4-Dmrta2 and the Gal4-Dmrta2 (126–531) fusion construct lacking the DM domain required for Zfp423 interaction has strong repression activity on the 5XUAS-tk-luc reporter construct and that Zfp423 slightly increases the repressive activity of Gal4-Dmrta2 but not of the Gal4-Dmrta2 (126–531) construct. In each condition, 200 ng of the 5XUAS-tk-luc reporter was transfected, together with 25 ng of the pCMV-Gal4-Dmrta2 or the pCMV-Gal4-Dmrta2 (126–531) and different doses (200, 400 and 600 ng) of pCDNA3-Myc -Zfp423 expression plasmids. Values represent the mean ± SD of one transfection done in triplicate. A Western blot showing the expression levels of the overexpressed factors is shown below. Reporter assays showing that the Gal4-Dmrta2 fusion protein represses in a UAS-dependent manner the activity of the 5XUAS-tk-luc reporter are presented in Extended Data . Reporter assays in HEK293T cells showing that Zfp423 does not increase the modest repression observed when an expression vector encoding the Gal4 DNA-binding domain alone is cotransfected with the 5XUAS-tk-luc reporter are presented in Extended Data .
Article Snippet: The HDAC class I inhibitor Romidepsin (MedChemExpress, catalog #HY-15149) was used at 0.005–0.01 μm.
Techniques: Mutagenesis, Synthesized, In Vitro, Incubation, Western Blot, Comparison, Staining, Transfection, Expressing, Construct, Binding Assay, Plasmid Preparation, Luciferase, Activity Assay
