SUMOylation of the polycomb group protein L3MBTL2 facilitates repression of its target genes
Christina Stielow
1
Bastian Stielow
1
Florian Finkernagel
1
Maren Scharfe
0
Michael Jarek
0
Guntram Suske
1
0
Helmholtz Centre for Infection Research (HZI)
, Inhoffenstrae 7, D-38124 Braunschweig,
Germany
1
Institute of Molecular Biology and Tumor Research, Philipps-University
, Emil-Mannkopff-Str. 2, D-35032 Marburg
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 derepressed 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.
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INTRODUCTION
Methylation of histone tails represents a crucial
posttranslational modification involved in transcriptional
regulation. Methylated histone tails are recognized by
chromatin-reading modules such as Chromo, Tudor,
PWWP and MBT (malignant brain tumor) domains,
together designated as the Royal family of chromatin
binding domains (1). MBT-domain proteins contain
arrays of two (2,3), three (46) or four (79) MBT
domains that form an interlocked substructure that bind
specifically to mono- and dimethylated lysine residues on
histone tails. In humans, the family of MBT-domain
proteins comprises nine members, which can be almost
invariably linked to one of the three Drosophila
MBTdomain proteins L(3)mbt, Scm and Sfmbt. All family
members fulfil functions in differentiation, regulation of
mitosis or tumor suppression (10).
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 acts as a transcriptional repressor (11,12) and
is involved in compaction of chromatin (12). Originally,
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). The majority of these proteins were also
found to be associated with L3mbtl2 in murine embryonic
stem cells (14). In addition, L3MBTL2 was identified as a
crucial subunit of the PRC1 subcomplexes PRC1L4 (12)
and polycomb repressive complex 1.6 (PRC1.6) (15).
Genome-wide binding studies in K562 cells revealed a
large overlap (>50%) between L3MBTL2- and
E2F6binding sites but no correlation with repressive histone
marks (12). Consistently, full-length L3MBTL2 bound
to histone tails and compacted nucleosomal arrays in a
histone methylation-independent manner (12). The
physiological relevance of L3mbtl2 was shown in mice,
where it is essential for embryonic development (14).
L3mbtl2 deficiency is embryonic lethal with failure in
gastrulation. Moreover, proliferation of L3mbtl2 /
embryonic-stem cells is strongly impaired due to a prolonged
G0/1 phase (14).
Many proteins regulating gene expression including
histones and chromatin-associated enzymes are reversibly
modified by SUMO thereby affecting gene expression
positively or negatively (16). SUMOylation occurs
through an enzymatic cascade including a heterodimeric
E1 enzyme (AOS1/UBA2), an E2 enzyme (UBC9) and an
E3 ligase, the latter enhancing the rate of SUMOylation,
and potentially contributing to specificity (17). Vertebrates
express three functional SUMO paralogs (SUMO1, 2 and
3) (18) of which SUMO2 and SUMO3 are 97% identical
and are therefore referred to as SUMO2/3. The covalent
attachment of SUMO occurs mainly at lysine residues
within the consensus sequence KXE (19) but
nonconsensus SUMO-attachment sites are also known (20).
Here, we report the identification of L3MBTL2 as a
novel SUMOylated protein that is specifically modified
with SUMO2/3 at the two C-terminal lysine residues
K675 and K700. SUMOylation of L3MBTL2 was not
required for its repressive activity in reporter gene
assays, its binding to histon (...truncated)