Chromatin reader L(3)mbt requires the Myb–MuvB/ DREAM transcriptional regulatory complex for chromosomal recruitment Daniel P. Blancharda, Daphne Georlettea,1, Lisa Antoszewskib, and Michael R. Botchana,2 aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; and bDepartment of Biology, Canisius College, Buffalo, NY 14208 Contributed by Michael R. Botchan, August 25, 2014 (sent for review July 16, 2014; reviewed by Robert J. Duronio and J. Robert Manak) Lethal malignant brain tumors (lmbt) result from the loss of the of its second MBT repeat (14–16). To establish a contributing conserved transcriptional repressor l(3)mbt, in Drosophila mela- mode for vertebrate L(3)mbt recruitment, several studies have nogaster. Similar mutations in the human homolog L3MBTL1 correlate provided evidence consistent with L(3)mbt preferentially, albeit with some cancers. The protein’s C-terminal MBT repeats bind mono promiscuously, interacting with most mono- and dimethylated and dimethylated histones in vitro, which could influence recruitment lysine residues of histones, with very low affinity for the non- or of L3MBTL1 to its target sites. The L(3)mbt chromatin targeting mech- trimethylated forms (15, 20, 21). The available crystal structures anism, however, is controversial and several studies suggest insuffi- provide evidence of how this methylated-lysine histone binding may ciency or a minor role for histone methylation in determining the site be mediated and suggest that the arrangement of MBT repeats is specificity for recruitment. We report that L(3)mbt colocalizes with required for protein stability, even if only one repeat performs the – actual binding (15, 16, 22). However, biochemical experiments have core members of the Myb MuvB/DREAM (MMB/DREAM) transcrip- – tional regulatory complex genome-wide, and that L(3)mbt-mediated shown the methylated histone L(3)mbt interaction to be weak, repression requires this complex in salivary glands and larval brains. suggesting that it may only be partly responsible for recruitment (17). Loss of l(3)mbt or of MMB components through mutation cause sim- Moreover, ChIP-sequencing (ChIP-Seq) experiments on Drosophila ilar spurious expression of genes, including the transposon regulatory larval brains suggest a close genomic binding correlation be- tween several chromatin insulators (especially the 190KD gene piwi, in terminally differentiated cells. The DNA-binding MMB centrosomal protein, CP190) and L(3)mbt (23), raising the pos- core component Mip120 (Lin54) is required for L(3)mbt recruitment to sibility that their recruitment may be interdependent, and that en- chromosomes, whereas Mip130 (Lin9) (an MMB core protein) and E2f2 gagement of repressive functions mediated in part by l(3)mbt may be (an MMB transcriptional repressor) are not, but are essential for re- complex. In further support of this latter notion, a recently identified pression. Cytolocalization experiments suggest the presence of site- “LINT” complex was biochemically shown to harbor L(3)mbt, par- specific differential composition of MMB in polytene chromosomes tially colocalize with it, genome-wide, and is required for L(3)mbt- where some loci were bound by a Myb-containing or alternatively, mediated repression. In Drosophila,L(3)mbt’s ability to function an E2f2 and L(3)mbt form of the complex. at many sites is independent of the histone H4K20 modification status (12), whereas the contrary has been emphasized for human epigenetics | bar coding | tumorigenesis L3MBTL1 function (17). However, the H4K20 monomethyl mark, set by the histone methyltransferase PRSet7, is stabilized by ethal 3 malignant brain tumor [l(3)mbt] was discovered in L(3)mbt binding in a process that is likely important for proper LDrosophila, where mutations in this gene lead to the de- chromatin organization (24). Taken together, the available data velopment of optical neural ganglion malignant tumors in late third instar larvae, with a terminal phenotype preceding pupa- Significance tion (1, 2). L(3)mbt homologs are found throughout the phylo- genetic tree, including humans, where hL(3)MBTL1 has been Histone binding proteins are critical for chromosome function, correlated with several types of cancer (3–5). L(3)mbt contains and mechanisms targeting them to nucleosomes are crucial. three repeated sequence elements dubbed MBT repeats, the The Drosophila tumor suppressor L(3)mbt binds to methylated presence of which is a common feature shared by the MBT large family of proteins (reviewed in ref. 6). The Drosophila ge- lysines of nucleosomal histones repressing gene transcription. We show that L(3)mbt chromosomal targeting requires proteins nome contains two MBT repeat encoding genes in addition to – l(3)mbt; dsFMBT and dSCM, with four and two MBT repeats, of a site-specific DNA binding complex [Myb MuvB (MMB)/ respectively (7, 8). Sex comb in midleg (dSCM) is involved in DREAM] and Mip120, an MMB/DREAM core component, is crit- regulation by repression of homeotic developmental patterning ical for recruitment. Surprisingly, chromosome association of and thus classified as a polycomb gene (9). Akin to dSCM, the L(3)mbt is insufficient for repression, as other MMB/DREAM available data suggests that l(3)mbt functions through the re- members are required for L(3)mbt-mediated repression but not pression of developmentally regulated genes (10–13), although the its chromosome targeting. Loss of l(3)mbt leads to lethal ma- inhibitory mechanism remains an unresolved question. A potential lignant brain tumors. We discuss our findings in the context of clue into how repression might be exerted comes from biochem- complex mechanisms where specific genes activated by loss of ical studies by Reinberg and coworkers, stating that the human l(3)mbt may help tumor progression, whereas deletion of MMB L(3)mbt can, once bound to DNA, act as a chromatin compaction genes may suppress this phenotype. agent; the bound protein condenses chromatin and thus prevents the expression of the underlying genes (14). This model, supported Author contributions: D.P.B., D.G., and M.R.B. designed research; D.P.B. and D.G. per- by studies in the homolog L3MBTL1, elaborates that the formed research; D.P.B. and L.A. contributed new reagents/analytic tools; D.P.B. and MBT repeats may form higher order structures with nucleo- M.R.B. analyzed data; and D.P.B. and M.R.B. wrote the paper. somes through the binding to modified histone tails (15–17). Reviewers: R.J.D., University of North Carolina; and J.R.M., University of Iowa. MBT repeats have sequence homology to the “Royal” family The authors declare no conflict of interest. of chromatin binding proteins (18), which are known to selec- 1Present address: Meso Scale Diagnostics, Rockville, MD 20850. tively bind to histones (and histone modifications) and thus act 2To whom correspondence should be addressed. Email: [email protected]. “ ” as chromatin readers (reviewed in ref. 19). L(3)mbt has in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. trinsic chromatin binding capabilities, which require the integrity 1073/pnas.1416321111/-/DCSupplemental. E4234–E4243 | PNAS | Published online September 23, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1416321111 Downloaded by guest on September 29, 2021 suggest that the affinity of L(3)mbt to mono- and dimethylated teins alternate through the cell cycle in partnership with common PNAS PLUS histones, although key for function, likely does not play a driving core members of each complex (28, 30). role in direct recruitment to specific loci. Further direct studies are therefore required to address specific recruitment for gene Results repression and an overall clarification as to the role of methylated L(3)mbt Localization on Polytene Chromosomes and Correlation with histones in L(3)mbt biology. In this study, we compiled mono- and Histone Modifications. Multiple lines of evidence, including crys- dimethylated histone genome-wide patterns and compared them tallographic studies, have established that L(3)mbt preferentially with those of L(3)mbt. We found little overlap with any specific binds mono- and dimethylated lysine residues in histones, albeit histone mono- or dimethylated lysine mark. with low affinity (15–17, 20, 21). These data and the structural Biochemical data from our laboratory identified L(3)mbt as homology of the MBT repeats to bona fide “chromatin readers” a substoichiometric member of a large transcription and repli- cation repressor and activator complex named Myb–MuvB or have propelled the notion that mono- and dimethylation histones MMB (also called DREAM or LINC), which is highly conserved might function as L(3)mbt site-specific recruiters (14). To ana- from nematodes to humans (13, 25–29). Several of the MMB lyze the correlation between L(3)mbt and overall methylated complex members have intrinsic DNA binding properties, and histone binding, we compared the genome-wide localization of previous studies indicate that this protein complex binds widely, several mono- and dimethylated histones, previously obtained by yet specifically, throughout all Drosophila and human chromo- the ModEncode project using ChIP-Seq on chromatin from third somes (10, 30). Moreover, RNAi-mediated l(3)mbt or MMB instar Dm larvae(31),withthatofL(3)mbtinDrosophila depletion has shown a significant derepression overlap (10, 13), melanogaster (Dm) larval brains, also analyzed by ChIP-Seq suggesting a potential role for the MMB in specific recruitment (23). As can be seen in the Venn diagrams, there is only a very of the nucleosome binding factor. partial overlap with any specific mono- or dimethylated histone We have applied a combination of fluorescent cytolocalization signal (Fig. 1A), including those with which L(3)mbt has the studies with genetic and molecular experiments and report that reported highest affinity, H3K9 and H4K20 mono- and dimethyl L(3)mbt function and localization to specific regions of the chro- (Fig.