Our findings here demonstrated that the MBT domain containing protein L3MBTL2 interacted with a number of PcG proteins previously identified in PRC1-like (PRC1L) complexes. Interestingly, these PRC1L complexes purified from human cells contain overlapping but not identical subunit compositions (). RING1 and RING2 are invariant components found in all PRC1L complexes and are required to mediate H2AK119ub1 (de Napoles et al., 2004
). We suggest that a major determinant for the classification of human PRC1L complexes be the presence of only one of six RING domain containing human homologs of Drosophila
Psc, termed BMI1/PCGF4, MEL18/PCGF2, MBLR/PCGF6, PCGF1, PCGF3 and PCGF5. The L3MBTL2-complex purified here contained several PRC1 subunits including RING1, RING2 and the Psc homolog MBLR, and exhibited H2AK119ub1 E3-ligase activity. We therefore termed this complex PRC1L4 (). Complexes containing BMI1/PCGF4, MEL18/PCGF2 and PCGF1 have been reported earlier () and we predict that at least two more PRC1L complexes will be discovered, being characterized by the presence of PCGF3 and PCGF5, respectively (). This claim is supported by a recent proteomics study focused on identifying RING2 interaction partners in which all six PCGF homologous proteins were recovered (Sanchez et al., 2007
Polycomb Repressive Complex 1 (PRC1) variations in human cells
The direct physical interaction between L3MBTL2 and multiple PcG proteins suggests a tight functional cooperation, and given that RING1, RING2 and E2F6 null
mice showed similar developmental defects (del Mar Lorente et al., 2000
; Storre et al., 2002
; Voncken et al., 2003
), we anticipate that mice null
for L3MBTL2 will also exhibit abnormalities during embryogenesis. Previously, a larger protein assembly containing L3MBTL2 was shown to repress genes in quiescent cells (Ogawa et al., 2002
). Importantly, we have shown here that L3MBTL2 and PRC1L4 also play a gene regulatory role in actively dividing cells which is consistent with earlier studies that implicated E2F6 in gene regulatory events at particular cell cycle stages (Giangrande et al., 2004
). Prior to our study, E2F6 was shown to interact with a number of different PcG proteins (Attwooll et al., 2005
; Deshpande et al., 2007
; Ogawa et al., 2002
), and we speculate that such interactions might arise as a function of and/or be specific to cell cycle stages and/or differentiation states of the cell.
In this study and a previous one (Trojer et al., 2007
), we investigated two MBT-domain containing proteins L3MBTL2, and -L1, as to their functional import in gene regulation. Our results showed negligible genomic co-occupancy of L3MBTL1 with L3MBTL2 (, S3A
), suggesting that the two disparate MBT-domain containing proteins function independently and in a non-redundant manner. H2AK119ub1 was present on all L3MBTL2 target genes tested. Its catalysis by PRCIL4 likely bears directly on L3MBTL2-mediated repression given that L3MBTL2 down-regulation not only resulted in up-regulated gene expression, but also in loss of H2AK119ub1 (). Since L3MBTL2 did not stimulate RING2 E3-ligase activity in vitro
(data not shown), we conclude that L3MBTL2 is an important factor in recruiting RING1 and RING2 on PRC1L4 genomic binding regions.
In earlier reports, the removal of H2AK119ub1 correlated with an increase in productive transcript but did not change overall RNA polymerase II levels on target promoters, suggesting that H2AK119ub1 affects the establishment of a mature elongation complex (Stock et al., 2007
; Zhou et al., 2008
). Consistently, studies established that H2AK119ub1 suppresses ongoing transcription proximal to double strand breaks (DSB) in an ATM-dependent manner. DSB repair leads to rapid transcriptional de-repression and coincides with H2AK119ub1 loss (Shanbhag et al., 2010
). However, the molecular mechanism of H2AK119ub1-mediated transcriptional repression remains unknown such that it is still possible to consider that H2AK119ub1 might directly obstruct elongation factor dependent chromatin disassembly or prevent transcription initiation (Nakagawa et al., 2008
). Interestingly, we have found a correlation of PRC1L4 occupancy with H3K4me3 (, S4C-F
). This finding supports H2AK119ub1 functioning as a roadblock in elongation on the H3K4me3-marked subset of PRC1L4 target genes, given that H3K4me3 is placed co-transcriptionally and not prior to transcription initiation (Pavri et al., 2006
) and that H3K4me3 marked genes might lack productive transcripts but have an already initiated and probably stalled RNA polymerase II transcription complex in their 5’-region. A recent study suggested that PRC1L complexes repress transcription of Hox genes independently of H2AK119ub1 since both a Ring1b (the mouse homolog of human RING2) wild type and E3-ligase deficient mutant could rescue Hox gene cluster chromatin decondensation in mouse embryonic stem cells (Eskeland et al., 2010
). It is likely that loss of RING2 would destabilize the entire PRC1L complex, and thus up-regulate gene expression. More mechanistic studies are required to examine the direct effect of H2AK119ub1 on chromatin structure.
Surprisingly, we did not find any correlation of L3MBTL2 binding sites and the occurrence of repressive histone methylation marks in our genome-wide ChIP-seq analyses. The lack of H3K27me3 on PRC1L4 target genes clearly indicated that the recruitment and repressive function of PRC1L4 is independent of the H3K27me3. Moreover, the lack of H3K9me3 on PRC1L4 target genes suggested that PRC1L4 recruitment is not dependent of HP1γ-mediated binding to H3K9me3. These findings are also consistent with our earlier data from Ntera2 cells in that E2F6 binding sites did not correlate with the presence of H3K9me3 or H3K27me3 (Xu et al., 2007
Previously, the four MBT domains of L3MBTL2 were shown to bind to H4K20me1 and H3K9me1 histone peptides by isothermal calorimetry (ITC) and a crystallographic analysis determined multiple aromatic residues in the fourth MBT domain to be critical for histone methyl-lysine binding (Guo et al., 2009
). Surprisingly, we did not find any correlation of H4K20me1 and H3K9me1 with L3MBTL2 binding sites on a genome scale (), suggesting that these two marks do not play a major role in L3MBTL2 recruitment to chromatin. Recombinant, full-length L3MBTL2 bound to unmodified and mono- and di-methylated histone peptides in a similar manner (). Also peculiar is the lack of binding to H3K27 methylated peptides (), while such binding was previously detected by ITC. This could be explained by the weak binding affinity (KD
=40-60 μM) towards H3K27me1 and -me2. Alternatively, the peptide length and the position of the methylated lysine within the peptide sequence might contribute to the observed differences in binding. Regardless, we found that L3MBTL2 could bind equally effectively to recombinant and native histones (), and its histone binding was not dependent on the N-terminal H3 and H4 sequences (). We concluded that in the context of full-length L3MBTL2 histone recognition did not require histone lysine methylation. Formally, it is still possible that L3MBTL2 has a binding preference for methyl marks on the linker histone, for instance H1.4K26 methylation.
While L3MBTL1 required H1.4K26 methylation for chromatin compaction, L3MBTL2 interacted with chromatin devoid of histone H1 or histone modifications (). Electron microscopy allowed us to visualize chromatin compaction resultant to L3MBTL2 binding (). Thus, L3MBTL2 likely functions like the Psc subunit of PRC1 (Francis et al., 2004
), that mediates chromatin compaction in a histone modification-independent manner.