Marc A. Morgan

The MLL3/MLL4 branches of the COMPASS family function as major histone H3K4 monomethylases at enhancers

ABSTRACT Histone H3 lysine 4 (H3K4) can be mono-, di-, and trimethylated by members of the COMPASS (complex of proteins associated with Set1) family from Saccharomyces cerevisiae to humans, and these modifications can be found at distinct regions of the genome. Monomethylation of histone H3K4 (H3K4me1) is relatively more enriched at metazoan enhancer regions compared to trimethylated histone H3K4 (H3K4me3), which is enriched at transcription start sites in all eukaryotes. Our recent studies of Drosophila melanogaster demonstrated that the Trithorax-related (Trr) branch of the COMPASS family regulates enhancer activity and is responsible for the implementation of H3K4me1 at these regions. There are six COMPASS family members in mammals, two of which, MLL3 (GeneID 58508) and MLL4 (GeneID 8085), are most closely related to Drosophila Trr. Here, we use chromatin immunoprecipitation-sequencing (ChIP-seq) of this class of COMPASS family members in both human HCT116 cells and mouse embryonic stem cells and find that MLL4 is preferentially found at enhancer regions. MLL3 and MLL4 are frequently mutated in cancer, and indeed, the widely used HCT116 cancer cell line contains inactivating mutations in the MLL3 gene. Using HCT116 cells in which MLL4 has also been knocked out, we demonstrate that MLL3 and MLL4 are major regulators of H3K4me1 in these cells, with the greatest loss of monomethylation at enhancer regions. Moreover, we find a redundant role between Mll3 (GeneID 231051) and Mll4 (GeneID 381022) in enhancer H3K4 monomethylation in mouse embryonic fibroblast (MEF) cells. These findings suggest that mammalian MLL3 and MLL4 function in the regulation of enhancer activity and that mutations of MLL3 and MLL4 that are found in cancers could exert their properties through malfunction of these Trr/MLL3/MLL4-specific (Trrific) enhancers.

The Mll2 branch of the COMPASS family regulates bivalent promoters in mouse embryonic stem cells

Promoters of many developmentally regulated genes, in the embryonic stem cell state, have a bivalent mark of H3K27me3 and H3K4me3, proposed to confer precise temporal activation upon differentiation. Although Polycomb repressive complex 2 is known to implement H3K27 trimethylation, the COMPASS family member responsible for H3K4me3 at bivalently marked promoters was previously unknown. Here, we identify Mll2 (KMT2b) as the enzyme catalyzing H3K4 trimethylation at bivalentlymarked promoters in embryonic stem cells. Although H3K4me3 at bivalent genes is proposed to prime future activation, we detected no substantial defect in rapid transcriptional induction after retinoic acid treatment in Mll2-depleted cells. Our identification of the Mll2 complex as the COMPASS family member responsible for H3K4me3 marking bivalent promoters provides an opportunity to reevaluate and experimentally test models for the function of bivalency in the embryonic stem cell state and in differentiation.

Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling

Chromatin mutations disrupt development Histone proteins form the core packaging material for our genomic DNA, and covalent modifications to amino acid residues in their structure play an important role in the epigenetic control of gene expression. Herz et al. show that specific mutations in the residues that are normally modified to regulate expression cause severe disruption of normal development in the fruit fly. Similar mutations are known to be involved in a subtype of aggressive pediatric brain cancers. Insights into the epigenetic regulatory pathways disrupted by these mutations in Drosophila may suggest possible treatments for human cancers. Science, this issue p. 1065 Mutations in the DNA packaging material disrupt fruit fly development and reveal epigenetic regulatory pathways. Histone H3 lysine27-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. We established a Drosophila animal model for the pathogenic histone H3K27M mutation and show that its overexpression resembles polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing derepression of PRC2 target genes and developmental perturbations. Similarly, an H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M-containing nucleosomes, and its misregulation in Drosophila results in changes of H3K9 methylation levels and heterochromatic silencing defects. We have established histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin signaling pathways.

Chromatin signatures of cancer

Changes in the pattern of gene expression play an important role in allowing cancer cells to acquire their hallmark characteristics, while genomic instability enables cells to acquire genetic alterations that promote oncogenesis. Chromatin plays central roles in both transcriptional regulation and the maintenance of genomic stability. Studies by cancer genome consortiums have identified frequent mutations in genes encoding chromatin regulatory factors and histone proteins in human cancer, implicating them as major mediators in the pathogenesis of both hematological malignancies and solid tumors. Here, we review recent advances in our understanding of the role of chromatin in cancer, focusing on transcriptional regulatory complexes, enhancer-associated factors, histone point mutations, and alterations in heterochromatin-interacting factors.

Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise. A heterozygous point mutation of histone H3 occurs in more than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M). Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3), and this is hypothesized to be a driving event of DIPG oncogenesis. Despite a major loss of H3K27me3, PRC2 activity is still detected in DIPG cells positive for H3K27M. To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis, we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation (H3K27ac). In accordance with previous biochemical data, the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes, whereas PRC2 is excluded from these regions; this suggests that H3K27M does not sequester PRC2 on chromatin. Residual PRC2 activity is required to maintain DIPG proliferative potential, by repressing neuronal differentiation and function. Finally, to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells, we performed treatments in vivo with BET bromodomain inhibitors and demonstrate that they efficiently inhibit tumor progression, thus identifying this class of compounds as potential therapeutics in DIPG.

Context dependency of Set1/COMPASS-mediated histone H3 Lys4 trimethylation

The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. Here, we used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. We also performed detailed screens and identified yeast strains lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. Our study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASSs H3K4me3 activity at promoter-proximal regions in a context-dependent manner.

Drosophila SETs Its Sights on Cancer: Trr/MLL3/4 COMPASS-Like Complexes in Development and Disease

The COMPASS family, which functions in the regulation of developmental gene expression, is a group of histone H3 lysine 4 (H3K4) methylases that is evolutionarily conserved from Saccharomyces cerevisiae (yeast) to human (1). Although there is only one Set1/COMPASS in yeast, Drosophila cells possess three yeast Set1-related proteins: dSet1, Trithorax (Trx), and Trithorax-related (Trr), all found within COMPASS-like compositions (1). Mammalian cells possess two representatives for each of the three subclasses found in Drosophila for a total of six COMPASS family members: SET1A and SET1B (related to dSet1); MLL1 and MLL2 (related to Trx); and MLL3 and MLL4 (related to Trr). Expansion of this family over evolutionary time implies a diversification in the function of H3K4 methylation, and studies into the distinct roles of the different branches of the COMPASS family support this notion. Drosophila and mammalian Set1 complexes mediate the bulk of genomic H3K4 diand trimethylation (2–4). In contrast, the Trx/MLL1/2 complexes act in a highly gene-specific manner, in particular, controlling expression of distinct homeotic genes, including those within the Hox gene clusters (1, 5). MLL1 has been extensively studied in mouse models and human cells, as MLL1 translocations cause aggressive infant leukemias (6–8). Trr/ MLL3/4 complexes are involved in nuclear hormone receptor signaling in both Drosophila and mammals (9, 10), and inactivating mutations have recently been implicated in human cancer (11– 16). Mammalian MLL3/4 are large proteins (approximately 5,000 amino acids), whereas Drosophila Trr is homologous to the carboxy-terminal PHD, FYRN, FYRC, and SET domain of MLL3/4. A separate gene, LPT (Lost PHDs of Trr), encodes a protein homologous to the MLL3/4 amino terminus (3, 17). Moreover, Trr and LPT associate in the same complex, suggesting that a gene fission event had occurred in an ancestral gene in the Drosophila lineage (3). Set1/COMPASS in yeast is unique in its ability to mono-, di-, and trimethylate its nucleosomal substrate (1, 18). The pattern of localization of histone H3K4 trimethylation (H3K4me3) and COMPASS on chromatin was first demonstrated to strongly correlate with transcriptionally active promoters in yeast (19), and this role of H3K4me3 in marking actively transcribed genes is highly conserved across the eukaryotes and is indeed used as a landmark for finding active promoters (20, 21). In contrast to H3K4me3, H3K4 monomethylation (H3K4me1) is found on poised and/or active enhancers (22, 23). Given that there are six COMPASS family members in mammalian cells, it was not clear until recently which COMPASS family member is involved in implementing H3K4me1 on enhancers. Recent work has now uncovered an unexpected role for Trr/MLL3/4 in gene regulation through enhancer-promoter communication. It was demonstrated that Trr functions as a major H3K4 monomethylase targeting enhancers in Drosophila (24). Moreover, loss of Trr impairs long-range enhancer function during Drosophila wing development. Given the strong association of H3K4me1 with enhancers (22) and the emerging connections between MLL3/4 and human disease, the relationship between Trr/MLL3/4 methylase activity and gene regulation is an area of burgeoning interest. In this issue, Kanda and coworkers from the Hariharan laboratory (25) report the use of elegant genetic tools in Drosophila to shed light on Trr function during development and draw a striking parallel between Drosophila Trr and MLL3/4 mutations in human cancer. Using genetic mosaics, Kanda et al. demonstrate that during Drosophila eye development, cells lacking Trr have a clonal growth advantage over their wild-type counterparts. In agreement with recent work identifying Trr as a major H3K4 monomethylase involved in enhancer function (24), they observed a dramatic loss of H3K4me1 in trr mutant tissue accompanied by altered activity of key developmental signaling pathways, namely, Notch, Dpp/BMP, and receptor tyrosine kinases (RTK). In stark contrast to the growth advantage conferred by Trr deficiency, Trx mutant clones fail to proliferate and display increased apoptosis, mirroring the phenotypes observed in mammalian Mll1/2 loss-of-function studies (26, 27). Quite remarkably, these distinct Trx (growth-promoting) versus Trr (growth-suppressing) functions may be conserved in mammals. Mll1 knockout mice lack hematopoetic stem cells and display embryonic proliferation defects, whereas gain-of-function Mll1 fusions cause aggressive leukemia (6, 27, 28). Similarly, Mll2 mutant embryos are severely growth retarded at early developmental stages and display widespread apoptosis (26). In contrast, mice lacking the Mll3 SET domain are viable but develop ureteric tumors, demonstrating a tumor suppressor function (29). Moreover, a series of genome-wide studies have identified loss-of-function mutations in MLL3 and MLL4 and in their cofactor, UTX, in diverse human cancers (11–16). Consistent with this, Drosophila Utx mutant clones also display an overgrowth phenotype (30). As for many of the Drosophila trr alleles characterized by the Hariharan laboratory, many cancer-associated MLL3 and MLL4 mutations result in truncation of point mutations in the catalytic SET domain (Fig. 1). Intriguingly, chromatin profiling in human cancer suggests a key role for H3K4me1. The genome-wide distribution of H3K4me1 undergoes a consistent alteration in colon cancer, often resulting in the loss of intestinal crypt-specific H3K4me1 marks (31). Collectively, these data provide evidence

Histone H3K4 monomethylation catalyzed by Trr and mammalian COMPASS-like proteins at enhancers is dispensable for development and viability

Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila melanogaster and MLL3 (encoded by KMT2C) and MLL4 (encoded by KMT2D) in mammals. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.

Inactivation of Ezh2 Upregulates Gfi1 and Drives Aggressive Myc-Driven Group 3 Medulloblastoma

The most aggressive of four medulloblastoma (MB) subgroups are cMyc-driven group 3 (G3) tumors, some of which overexpress EZH2, the histone H3K27 mono-, di-, and trimethylase of polycomb-repressive complex 2. Ezh2 has a context-dependent role in different cancers as an oncogene or tumor suppressor and retards tumor progression in a mouse model of G3 MB. Engineered deletions of Ezh2 in G3 MBs by gene editing nucleases accelerated tumorigenesis, whereas Ezh2 re-expression reversed attendant histone modifications and slowed tumor progression. Candidate oncogenic drivers suppressed by Ezh2 included Gfi1, a proto-oncogene frequently activated in human G3 MBs. Gfi1 disruption antagonized the tumor-promoting effects of Ezh2 loss; conversely, Gfi1 overexpression collaborated with Myc to bypass effects of Trp53 inactivation in driving MB progression in primary cerebellar neuronal progenitors. Although negative regulation of Gfi1 by Ezh2 may restrain MB development, Gfi1 activation can bypass these effects.

SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.

The homeotic (Hox) genes are highly conserved in metazoans, where they are required for various processes in development, and misregulation of their expression is associated with human cancer. In the developing embryo, Hox genes are activated sequentially in time and space according to their genomic position within Hox gene clusters. Accumulating evidence implicates both enhancer elements and noncoding RNAs in controlling this spatiotemporal expression of Hox genes, but disentangling their relative contributions is challenging. Here, we identify two cis-regulatory elements (E1 and E2) functioning as shadow enhancers to regulate the early expression of the HoxA genes. Simultaneous deletion of these shadow enhancers in embryonic stem cells leads to impaired activation of HoxA genes upon differentiation, while knockdown of a long noncoding RNA overlapping E1 has no detectable effect on their expression. Although MLL/COMPASS (complex of proteins associated with Set1) family of histone methyltransferases is known to activate transcription of Hox genes in other contexts, we found that individual inactivation of the MLL1-4/COMPASS family members has little effect on early Hox gene activation. Instead, we demonstrate that SET1A/COMPASS is required for full transcriptional activation of multiple Hox genes but functions independently of the E1 and E2 cis-regulatory elements. Our results reveal multiple regulatory layers for Hox genes to fine-tune transcriptional programs essential for development.