MBD was originally identified as a member of

MBD3 was originally identified as a member of the methyl-CpG binding domain (MBD) family of proteins (Hendrich and Bird, 1998). However, unlike MBD members MECP2 and MBD1, 2, and 4, MBD3 does not bind methylated DNA (Hendrich and Bird, 1998; Zhang et al., 1999). Three MBD3 isoforms (MBD3A, B, and C) are expressed in mouse ESCs, and only MBD3A has a full-length MBD (Kaji et al., 2006). Thus, the possibility exists for formation of multiple NuRD complexes of varying subunit combinations and functional specificities. Here, we have characterized a unique variant of the NuRD chromatin remodeling complex that harbors MBD3C, an ESC-specific isoform of MBD3, as well as the histone H3 binding protein WDR5. MBD3C is expressed almost exclusively in ESCs via an alternative CpG island (CGI)-containing promoter located in the second intron of the Mbd3 gene. We further show that MBD3C contains a unique 50-amino-acid N terminus that is necessary for WDR5 interaction. MBD3C interacts with the WDR5 H3 binding pocket through an arginine-containing motif also utilized by MLL1 for WDR5 binding. RNA sequencing (RNA-seq) analysis revealed that the three MBD3 isoforms are largely redundant for gene regulation, since knockout (KO) of all three isoforms had a more severe effect on gene expression than individual KO of Mbd3c or simultaneous KO of Mbd3a and Mbd3b. Importantly, the WDR5-interaction domain of Mbd3c is critical for its gene regulatory function, suggesting that WDR5 plays critical roles in MBD3C/NuRD complex.
Results and Discussion
Experimental Procedures
Author Contributions
Acknowledgments We thank Howard Chang for WDR5 binding domain mutant plasmids, Feng Wang and Yong Du for assistance in mouse tissue lysate preparation, and members of the T.G.F. laboratory for critical reading of the manuscript. This research was supported by NIH Grant HD072122 to T.G.F. and NSF Grant CLF1307367 to F.C. T.G.F. is a scholar of the Leukemia and Lymphoma Society. M.R.G. is an Investigator of the Howard Hughes Medical Institute.
Introduction Intercellular dialogue between tumor buy Pyridoxal isonicotinoyl hydrazone mediated by extracellular vesicles (EVs) is a powerful means of communication that facilitates exchange of active molecules (Bronisz et al., 2014; Skog et al., 2008). It is the linchpin of the molecular network, the invisible causeway on which the global cellular transcriptome hums. However, the culpability of EV communication for phenotypic and molecular diversity within heterogeneous tumors is not fully recognized. Intratumoral heterogeneity and invasiveness are the key characteristics of glioblastoma (GBM), the most common and most aggressive primary brain malignancy in adults, with a median survival of 14.2 months (Johnson and O\'Neill, 2012). The subpopulation of highly tumorigenic and therapy-resistant GBM stem-like cells (GSCs) (Schonberg et al., 2014) retains stem cell characteristics, including self-renewal and undifferentiated status, but also exhibits varying degrees of phenotypic and molecular polymorphism. Discovering whether the underlying cause of invasiveness is inherent or is a response to microenvironmental stimuli has important implications for better understanding of GBM pathobiology. The signatures of protein-coding gene expression and somatic copy-number alterations have revealed the existence of several distinct subtypes among GBM patients, known as mesenchymal, proneural, neural, and classical according to The Cancer Genome Atlas (TCGA) database (Phillips et al., 2006). The classification is further complicated by the fact that individual tumors contain a spectrum of subtypes and hybrid cellular states (Patel et al., 2014) and that GSC subpopulations retain transcriptome heterogeneity (Mao et al., 2013). These findings indicate that tissue-based classification likely shows merely characteristics of the predominant cellular component. Importantly, tissue and GSC subtype classification was also demonstrated by signatures of non-protein-coding genes, such as long non-coding RNAs (Du et al., 2013; Mineo et al., 2016). Significantly, microRNAs have not been shown to predict GBM classification and prognosis by global signature to date, while being strongly implicated as functionally deregulated in GBM as individual molecules (Godlewski et al., 2015).