L3MBTL3 is a methyl-lysine reader that binds to mono- and dimethylated lysine. L3MBTL3 interacts with the Notch (see 190198) coactivator RBPJ (147183) and switches it to a transcriptional repressor of Notch target genes via KDM1A (609132)-mediated histone demethylation (Xu et al., 2017). L3MBTL3 also regulates proteolysis of methylated SOX2 (184429) (Zhang et al., 2019).

Arai and Miyazaki (2005) cloned mouse and human L3MBTL3, which they called MBT1. The 780-amino acid human protein contains an N-terminal nuclear localization signal (NLS), followed by 3 MBT motifs and a C-terminal sterile-alpha motif (SAM)/SPM domain. Mouse Mbt1 has a glutamine-rich domain that is absent in human MBT1. RT-PCR analysis showed that hematopoietic cells in mouse fetal liver and adult bone marrow had comparable levels of Mbt1 expression throughout development. All types of hematopoietic progenitor cells in mouse fetal liver expressed Mbt1. Northern blot analysis showed that Mbt1 was widely expressed in mouse tissues. FLAG-tagged MBT1 protein localized to nucleus in transfected Chinese hamster ovarian carcinoma cells.

Xu et al. (2017) showed that the human L3MBTL3 protein contains a C2C2 zinc finger domain just N-terminal to the MBT motifs and a C2H2 zinc finger domain just C-terminal to the MBT motifs.

By database analysis, Arai and Miyazaki (2005) mapped the MBT1 gene to chromosome 6q23.

Using RT-PCR analysis, Arai and Miyazaki (2005) showed that MBT1 expression increased significantly in response to induction of differentiation in human leukemia cells, then was rapidly downregulated, displaying lowest expression during a late stage of differentiation. Yeast 2-hybrid screening and coimmunoprecipitation analysis identified RNF2 (608985) as a binding partner of MBT1.

By yeast 2-hybrid, coimmunoprecipitation, and pull-down analyses, Xu et al. (2017) found that L3MBTL3 interacted directly with RBPJ. Mutation analysis showed that the interaction required the N-terminal region of L3MBTL3 and the beta-trefoil domain (BTD) of RBPJ. The Notch intracellular domain (ICD) also interacted with the BTD of RBPJ, allowing competition between L3MBTL3 and the Notch ICD for RBPJ binding. Thermodynamic analysis showed that the Notch ICD could outcompete L3MBTL3 for binding to RBPJ. However, in the absence of Notch signaling, interaction with L3MBTL3 allowed RBPJ to recruit L3MBTL3 on chromatin to repress expression of Notch target genes. L3MBTL3 also interacted with KDM1A, a histone demethylase, and linked KDM1A to Notch-responsive elements. KDM1A interacted with RBPJ and promoted demethylation of dimethylated lys4 of histone H3 (see 602810), resulting in repression of Notch target gene expression. Genetic analysis in Drosophila and C. elegans demonstrated that the RBPJ-L3MBTL3 interaction was evolutionarily conserved in metazoans.

Zhang et al. (2019) found that human L3MBTL3 preferentially bound monomethylated lys42 in SOX2 and regulated stability of the SOX2 protein, which was sensitive to loss of both LSD1 (KDM1A) and PHF20L1, in human PA-1 teratocarcinoma cells. L3MBTL3 also interacted with DCAF5 (603812), a subunit of a CRL4 ubiquitin E3 ligase complex (see CUL4A, 603137), and cooperatively targeted methylated SOX2 for polyubiquitination-dependent proteolysis. Similarly, L3mbtl3 interacted with Sox2 and destabilized the Sox2 protein in mouse embryonic stem (ES) cells. Loss of L3mbtl3 stabilized Sox2 and restored self-renewal and pluripotency in Lsd1- or Phf20l1-knockdown mouse ES cells. Methylated Sox2 was a critical target of Lsd1 in mouse ES cells, and it appeared that methylation at both lys42 and lys117 was important for Lsd1 to maintain self-renewal and pluripotency of mouse ES cells. Induction of mouse ES cell differentiation enhanced proteolytic degradation of Sox2, which also depended on methylation of both lys42 and lys117 in Sox2.

Arai and Miyazaki (2005) found that loss of Mbt1 in mice disturbed definitive erythropoiesis, resulting in embryonic lethality due to anemia. Deletion of Mbt1 led to defective myelopoiesis, where immature progenitor cells proliferated normally but were unable to differentiate. As a result, not only were mature erythrocytes in peripheral blood of Mbt1 -/- embryos decreased in a cell-autonomous manner, but other types of mature myeloid lineage cells were decreased in Mbt1 -/- fetal liver at a late embryonic stage. Mbt1 -/- common myeloid progenitor cells displayed inefficient maturation transition and had to undergo a larger number of cell divisions to achieve some maturation. Gene expression profiling revealed that Mbt1 regulated myeloid progenitor cell maturation by inducing cell cycle arrest by enhancing expression of p57(Kip2) (CDKN1C; 600856).