Dissecting the roles of MBD2 isoforms in regulating NuRD complex function during cellular differentiation

The Nucleosome Remodelling and Deacetylation (NuRD) complex is a crucial regulator of cellular differentiation. Two members of the Methyl-CpG-binding domain (MBD) protein family, MBD2 and MBD3, are known to be integral, but mutually exclusive subunits of the NuRD complex. Several MBD2 and MBD3 isoforms are present in mammalian cells, resulting in distinct MBD-NuRD complexes. If these different complexes serve distinct biochemical and/or functional activities during differentiation is not completely understood. Based on the essential role of MBD3 in lineage commitment, we systematically investigated a diverse set of MBD3 and MBD2 variants for their potential to rescue the differentiation block observed in mouse embryonic stem cells (ESCs) lacking MBD3. Our study reveals that while MBD3 is indeed crucial for ESC differentiation to neuronal cells, this function is independent of its MBD domain or binding to methylated DNA. While MBD3 isoforms are highly redundant, we identify that MBD2 isoforms vary in their potential to fully rescue the absence of MBD3 during lineage commitment. Full-length MBD2a only partially rescues the differentiation block; MBD2b, which lacks the N-terminal GR-rich repeat, fully rescues the differentiation block in MBD3 KO ES cells, and cells expressing the testis-specific isoform MBD2t that lacks the coiled-coil domain required for NuRD interactions are not able to generate any differentiated cells. In case of MBD2a, we further show that removing the m-CpG DNA binding capacity or the GR-rich repeat renders the protein fully redundant to MBD3, highlighting the requirements for these domains in diversifying NuRD complex function. In sum, our results highlight a partial redundancy of MBD2 and MBD3 during cellular differentiation and point to specific functions of distinct MBD2 isoforms and specific domains within the NuRD complex.