Abstract
Lethal(3) malignant brain tumour like 2 (L3MBTL2) is an integral component of the polycomb repressive complex 1.6 (PRC1.6) and has been implicated in transcriptional repression and chromatin compaction. Here, we show that L3MBTL2 is modified by SUMO2/3 at lysine residues 675 and 700 close to the C-terminus. SUMOylation of L3MBTL2 neither affected its repressive activity in reporter gene assays nor it’s binding to histone tails in vitro. In order to analyse whether SUMOylation affects binding of L3MBTL2 to chromatin, we performed ChIP-Seq analysis with chromatin of wild-type HEK293 cells and with chromatin of HEK293 cells stably expressing either FLAG-tagged SUMOylation-competent or SUMOylation-defective L3MBTL2. Wild-type FLAG-L3MBTL2 and the SUMOylation-defective FLAG-L3MBTL2 K675/700R mutant essentially occupied the same sites as endogenous L3MBTL2 suggesting that SUMOylation of L3MBTL2 does not affect chromatin binding. However, a subset of L3MBTL2-target genes, particularly those with low L3MBTL2 occupancy including pro-inflammatory genes, was de-repressed in cells expressing the FLAG-L3MBTL2 K675/700R mutant. Finally, we provide evidence that SUMOylation of L3MBTL2 facilitates repression of these PRC1.6-target genes by balancing the local H2Aub1 levels established by the ubiquitinating enzyme RING2 and the de-ubiquitinating PR–DUB complex.
Highlights
Methylation of histone tails represents a crucial posttranslational modification involved in transcriptional regulation
To test whether L3MBTL2 could be SUMOylated in vivo, we transiently transfected FLAG-tagged L3MBTL2 along with Histagged SUMO1 or His-tagged SUMO2 in HEK293 cells
We knocked down endogenous SUMO1 or SUMO2/3 by SUMO-isoform specific shRNAs followed by western-blot analysis of L3MBTL2
Summary
Methylation of histone tails represents a crucial posttranslational modification involved in transcriptional regulation. Lethal[3] Malignant Brain Tumor Like 2 (L3MBTL2) represents the human ortholog of the Drosophila polycomb group protein Sfmbt. It possesses a zinc finger domain at the N-terminal part and four centrally located MBT domains, of which the most C-terminal one mediates binding to methyl groups in vitro [8]. L3MBTL2 was described as a subunit of the E2F6.com-1 complex in HeLa cells along with E2F6, MGA, MAX, DP1, HP1g, G9a, GLP, RING1, RING2, PCGF6 and YAF2 [13]. Proliferation of L3mbtl2À/À embryonic-stem cells is strongly impaired due to a prolonged G0/1 phase [14]
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