In this study, we investigated the mechanism by which MLL, a conserved component of the Trithorax-Polycomb system, perpetuates active chromatin states during cell division. While MLL is best known for influencing transcription through catalysis of H3K4 methylation, our findings revealed an additional epigenetic mechanism employed by this factor: a pervasive occupancy of promoter regions packaged into condensed mitotic chromosomes as they are inherited from mother to daughter cells. Our functional studies suggest that mitotic retention allows MLL to accelerate the kinetics of gene reactivation following the exit from mitosis. Surprisingly, this activity appears largely independent of H3K4 methylation on mitotic chromosomes. The precise mechanism through which MLL serves as a mitotic bookmark remains unclear, but might involve the action of key accessory proteins such as RbBP5, ASH2L, and Menin, which are tethered by MLL to select sites in mitotic chromatin.
An unexpected observation in this study was the vast reorganization of MLL occupancy observed during the cell-cycle. In contrast to the situation in interphase, MLL occupancy during mitosis is heavily biased towards genes with the highest levels of pre-mitotic activity. One explanation for this binding behavior is that highly-expressed genes undergo the profoundest transition from silent to active as cells exit mitosis. Such a rapid switch in transcriptional rate may require auxiliary factors like MLL to faithfully restore pre-mitotic expression levels. It is also interesting to note that a prior study demonstrated that MLL can be degraded and resynthesized at two stages of the cell cycle (M/G1 transition and G1/S transition), thus raising the possibility that the reorganization of MLL occupancy described here could be mediated via regulated proteolysis (Liu et al., 2007
). Importantly, the natural uncoupling of MLL’s interphase and mitotic occupancy allowed us to interrogate the functional relevance of its mitotic association independently of its interphase binding. Thus, our findings emphasize the extent to which the transcription factor landscape can be restructured during the cell cycle to facilitate perpetuation of transcriptional states.
A provocative question raised by our observations is regarding the relevant physical interactions utilized by MLL to remain associated with mitotic chromosomes. MLL contains several conserved domains that have been implicated in DNA or chromatin binding, such as several AT hooks, a CxxC domain, several PHD domains, and a BROMO domain. Menin has also been shown to cooperate with the PWWP protein, LEDGF, to target MLL to certain genomic regions (Yokoyama and Cleary, 2008
). MLL occupancy of mitotic chromatin correlates with the level of gene activity in interphase, thus, MLL retention could be mediated through a physical association with histone modifications or histone variants which are deposited co-transcriptionally during interphase. Since MLL is known to serve as a coactivator for sequence-specific DNA-binding proteins (Tyagi et al., 2007
), we speculate that the genome-wide return of DNA-binding transcriptional activators to their target sites following mitotic exit triggers the redistribution of MLL away from its mitotic locations to specific cis
elements in interphase. Future studies will be aimed at identifying the differential recruitment mechanisms utilized by MLL in interphase and mitosis and their relevance to mutant forms of MLL found in leukemia.
Surprisingly, the effects of MLL on post-mitotic gene reactivation seem to occur without a detectable influence on levels of H3K4 methylation. While prior studies found normal levels of global H3K4 methylation in MLL-deficient cells (Milne et al., 2002
; Wu et al., 2008
), it is notable that during mitosis H3K4 methylation is unaffected by MLL depletion, especially in light of the widespread occupancy pattern present in mitosis. This suggests that the mitotic stability of H3K4 methylation could be mediated by an alternative mechanism, such as eviction of H3K4 demethylases. As an example, we found that the H3K4 demethylase LSD1 is globally evicted from chromatin during mitosis. Nevertheless, the ostensible uncoupling of MLL’s bookmarking function from H3K4 methylation levels indicates that MLL carries out important functions independently of its enzymatic activity. This is supported by the observation that MLL’s H3K4 methyltransferase domain is largely dispensable for its essential functions in vivo
(Terranova et al., 2006
). Non-enzymatic mechanisms utilized by MLL might involve tethering RbBP5, Menin, and ASH2L to its binding sites in mitotic chromatin. During the transition into interphase, these components might be “handed-off” from MLL to the SETD1A/MLL2 complexes as they re-associate in G1. Alternatively, MLL-interacting proteins might engage in physical interactions that return the transcription machinery to a post-mitotic active state. Indeed, Menin has been shown to associate with the Rpb2 subunit of RNA polymerase II (Hughes et al., 2004
). MLL also contains a strong acidic activation domain at residues 2829–2883 as well as a demonstrated physical association with the C-terminal domain of RNA polymerase II (Milne et al., 2005
; Prasad et al., 1995
). These domains/interactions may provide a non-catalytic means by which MLL and its associated proteins deliver the transcriptional machinery back to genes as they reactivate at the end of mitosis.
Our study also highlights several important technical considerations when evaluating mitotic chromosome occupancy. First, it should be emphasized that IF visualizes the ratio of protein localized in the cytoplasm to the amount of protein localized to the chromosome. Thus, findings based on this assay alone can lead to misleading findings when the proportion of protein bound to mitotic chromosomes is low relative to the proportion present in the cytoplasm, as we observed for RbBP5, ASH2L, and Menin. In such instances, ChIP assays can more clearly ascertain whether a protein remains associated or not with mitotic chromatin. In addition, knockdown controls or use of multiple independent anti-sera is essential to verify antibody specificity.
Evidence in Drosophila
implicates Trithorax and Polycomb proteins in the maintenance of mitotically heritable “ON” and “OFF” states, respectively (Cavalli and Paro, 1998
; Maurange and Paro, 2002
). Both Polycomb and Trithorax proteins employ an assortment of self-reinforcing mechanisms to render an expression state immune to stochastic fluctuation or subversion by the opposing pathway. For example, Polycomb complexes include several chromatin-binding proteins and histone-modifying enzymes that establish a cooperative network of interactions that can stabilize a repressed expression state during the cell cycle (Francis et al., 2009
; Hansen et al., 2008
; Lee et al., 2007
; Wang et al., 2004
). In the case of Trithorax-related gene regulation in mammalian cells, heritable gene activity might also be mediated via the interplay among diverse mechanisms employed among multiple Trithorax-like and SET1 family proteins. While deposition of histone H3K4 methylation might play a role in this process, it alone is insufficient to account for all inheritance functions in vivo
(Terranova et al., 2006
). Thus, we wish to propose that mitotic retention of Trithorax-like proteins, when coupled with other self-perpetuating mechanisms functioning in interphase, contributes to a robust inheritance system for transcriptional programs in dividing metazoan tissues.