Knockdown of ALR (MLL2) Reveals ALR Target Genes and Leads to Alterations in Cell Adhesion and Growth

Abstract

ALR (MLL2) is a member of the human MLL family, which belongs to a larger SET1 family of histone methyltransferases. We found that ALR is present within a stable multiprotein complex containing a cohort of proteins shared with other SET1 family complexes and several unique components, such as PTIP and the jumonji family member UTX. Like other complexes formed by SET1 family members, the ALR complex exhibited strong H3K4 methyltransferase activity, conferred by the ALR SET domain. By generating ALR knockdown cell lines and comparing their expression profiles to that of control cells, we identified a set of genes whose expression is activated by ALR. Some of these genes were identified by chromatin immunoprecipitation as direct ALR targets. The ALR complex was found to associate in an ALR-dependent fashion with promoters and transcription initiation sites of target genes and to induce H3K4 trimethylation. The most characteristic features of the ALR knockdown cells were changes in the dynamics and mode of cell spreading/polarization, reduced migration capacity, impaired anchorage-dependent and -independent growth, and decreased tumorigenicity in mice. Taken together, our results suggest that ALR is a transcriptional activator that induces the transcription of target genes by covalent histone modification. ALR appears to be involved in the regulation of adhesion-related cytoskeletal events, which might affect cell growth and survival.

A phosphorylation switch on RbBP5 regulates histone H3 Lys4 methylation

“…These observations are also consistent with recent studies showing that deletion of MLL3 in NIH3T3-L1 cells results in a significant loss of H3K4me3 at the promoter region of the adipogenic marker gene aP2 . Moreover, B-cell-specific knockout of PTIP, a subunit associating with MLL3/MLL4 complexes (Cho et al 2007;Issaeva et al 2007), results in a loss of H3K4me3 at specific Igh switch regions upon LPS stimulation (Daniel et al 2010). These seemingly contrasting results potentially point to a model in which RbBP5 phosphorylation can act as a switch increasing MLL3 kinetics, facilitating the formation of H3K4me1 that can potentially be further methylated to ultimately form H3K4me2/3.…”

Regulation of Stem Cell Differentiation by Histone Methyltransferases and Demethylases

“…These results show that the coordinated regulation of histone modifications by a repressive complex, containing KMT and KDM activities, may be important for the transcriptional regulation of different genes during differentiation and development. Interestingly, similar biochemical structures containing KMT and KDM activities have also been reported for the TrxG-like MLL complex, where the MLL2 and ASH2 H3K4me3 KMTs are associated in a complex containing the H3K27me3-specific demethylase UTX Issaeva et al 2007;Lee et al 2007b). These findings indicate that these different enzymatic activities function together to coordinately regulate lysine methylation and transcription.…”

Kabuki syndrome revisited

“…Issaeva et al 8 determined the MLL2 protein to have a significant role in promoter and transcription initiation sites of target genes via the role as a histone modifier. Issaeva found that in MLL2-knockout mice models, there were 20 genes most significantly downregulated, which comprised various functions, including cell migration, growth adhesion and transcriptional regulation.…”

“…ENO3 is known to be responsible for glycolysis, muscle development and regeneration, TNNT2 for regulation of muscle contraction and development, whereas NPR3 involvement in skeletal development. 8 …”

“…Research with definitive conclusions is limited in the literature, and although the condition has been characterized since 1981, only over 30 years later had some ambiguity surrounding the etiology been resolved, which came in 2010 with the identification of potential causative defects within the 'MLL2 gene' (myeloid/lymphoid or mixed-lineage leukemia 2), further validated in July 2011. 7 The identification of such mutations in KS patients were not only promising because of its relatively high presence 66-74%, 7 but preexisting information on the MLL2 gene, 8,9 were already able to partly validate mutant MLL2 genes as a strong candidate. Although further research will undoubtedly be required, owing to the significant phenotypic overlap of KS with other congenital syndromes 10,11 these latest developments are able to potentially offer at least one test for accurate diagnoses; taking diagnosis from the potentially speculative, 7 to the molecularly validated.…”