A MOZ-TIF2 leukemia mouse model displays KAT6-dependent H3K23 propionylation and overexpression of a set of active developmental genes.

Aberrant regulation of chromatin modifiers is a common occurrence across many cancer types, and a key priority is to determine how specific alterations of these proteins, often enzymes, can be targeted therapeutically. MOZ, a histone acyltransferase, is recurrently fused to coactivators CBP, p300, and TIF2 in cases of acute myeloid leukemia (AML). Using either pharmacological inhibition or targeted protein degradation in a mouse model for MOZ-TIF2-driven leukemia, we show that KAT6 (MOZ/MORF) enzymatic activity and the MOZ-TIF2 protein are necessary for indefinite proliferation in cell culture. MOZ-TIF2 directly regulates a small subset of genes encoding developmental transcription factors, augmenting their high expression. Furthermore, transcription levels in MOZ-TIF2 cells positively correlate with enrichment of histone H3 propionylation at lysine 23 (H3K23pr), a recently appreciated histone acylation associated with gene activation. Unexpectedly, we also show that MOZ-TIF2 and MLL-AF9 regulate transcription of unique gene sets, and their cellular models exhibit distinct sensitivities to multiple small-molecule inhibitors directed against AML pathways. This is despite the shared genetic pathways of wild-type MOZ and MLL. Overall, our data provide insight into how aberrant regulation of MOZ contributes to leukemogenesis. We anticipate that these experiments will inform future work identifying targeted therapies in the treatment of AML and other diseases involving MOZ-induced transcriptional dysregulation.

Cell state-dependent chromatin targeting in NUT carcinoma.

Aberrant transcriptional programming and chromatin dysregulation are common to most cancers. Whether by deranged cell signaling or environmental insult, the resulting oncogenic phenotype is typically manifested in transcriptional changes characteristic of undifferentiated cell growth. Here we analyze targeting of an oncogenic fusion protein, BRD4-NUT, composed of two normally independent chromatin regulators. The fusion causes the formation of large hyperacetylated genomic regions or megadomains, mis-regulation of c-MYC , and an aggressive carcinoma of squamous cell origin. Our previous work revealed largely distinct megadomain locations in different NUT carcinoma patient cell lines. To assess whether this was due to variations in individual genome sequences or epigenetic cell state, we expressed BRD4-NUT in a human stem cell model and found that megadomains formed in dissimilar patterns when comparing cells in the pluripotent state with the same cell line following induction along a mesodermal lineage. Thus, our work implicates initial cell state as the critical factor in the locations of BRD4-NUT megadomains. These results, together with our analysis of c-MYC protein-protein interactions in a patient cell line, are consistent with a cascade of chromatin misregulation underlying NUT carcinoma.

Cell state-dependent chromatin targeting in NUT carcinoma.

Aberrant transcriptional programming and chromatin dysregulation are common to most cancers. Whether by deranged cell signaling or environmental insult, the resulting oncogenic phenotype is typically manifested in transcriptional changes characteristic of undifferentiated cell growth. Here we analyze targeting of an oncogenic fusion protein, BRD4-NUT, composed of 2 normally independent chromatin regulators. The fusion causes the formation of large hyperacetylated genomic regions or megadomains, mis-regulation of c-MYC, and an aggressive carcinoma of squamous cell origin. Our previous work revealed largely distinct megadomain locations in different NUT carcinoma patient cell lines. To assess whether this was due to variations in individual genome sequences or epigenetic cell state, we expressed BRD4-NUT in a human stem cell model and found that megadomains formed in dissimilar patterns when comparing cells in the pluripotent state with the same cell line following induction along a mesodermal lineage. Thus, our work implicates initial cell state as the critical factor in the locations of BRD4-NUT megadomains. These results, together with our analysis of c-MYC protein-protein interactions in a patient cell line, are consistent with a cascade of chromatin misregulation underlying NUT carcinoma.

Variant Polycomb complexes in Drosophila consistent with ancient functional diversity.

Polycomb group (PcG) mutants were first identified in Drosophila on the basis of their failure to maintain proper Hox gene repression during development. The proteins encoded by the corresponding fly genes mainly assemble into one of two discrete Polycomb repressive complexes: PRC1 or PRC2. However, biochemical analyses in mammals have revealed alternative forms of PRC2 and multiple distinct types of noncanonical or variant PRC1. Through a series of proteomic analyses, we identify analogous PRC2 and variant PRC1 complexes in Drosophila, as well as a broader repertoire of interactions implicated in early development. Our data provide strong support for the ancient diversity of PcG complexes and a framework for future analysis in a longstanding and versatile genetic system.

Dynamic Competition of Polycomb and Trithorax in Transcriptional Programming.

Predicting regulatory potential from primary DNA sequences or transcription factor binding patterns is not possible. However, the annotation of the genome by chromatin proteins, histone modifications, and differential compaction is largely sufficient to reveal the locations of genes and their differential activity states. The Polycomb Group (PcG) and Trithorax Group (TrxG) proteins are the central players in this cell type-specific chromatin organization. PcG function was originally viewed as being solely repressive and irreversible, as observed at the homeotic loci in flies and mammals. However, it is now clear that modular and reversible PcG function is essential at most developmental genes. Focusing mainly on recent advances, we review evidence for how PcG and TrxG patterns change dynamically during cell type transitions. The ability to implement cell type-specific transcriptional programming with exquisite fidelity is essential for normal development.

Bivalent complexes of PRC1 with orthologs of BRD4 and MOZ/MORF target developmental genes in Drosophila.

Regulatory decisions in Drosophila require Polycomb group (PcG) proteins to maintain the silent state and Trithorax group (TrxG) proteins to oppose silencing. Since PcG and TrxG are ubiquitous and lack apparent sequence specificity, a long-standing model is that targeting occurs via protein interactions; for instance, between repressors and PcG proteins. Instead, we found that Pc-repressive complex 1 (PRC1) purifies with coactivators Fs(1)h [female sterile (1) homeotic] and Enok/Br140 during embryogenesis. Fs(1)h is a TrxG member and the ortholog of BRD4, a bromodomain protein that binds to acetylated histones and is a key transcriptional coactivator in mammals. Enok and Br140, another bromodomain protein, are orthologous to subunits of a mammalian MOZ/MORF acetyltransferase complex. Here we confirm PRC1-Br140 and PRC1-Fs(1)h interactions and identify their genomic binding sites. PRC1-Br140 bind developmental genes in fly embryos, with analogous co-occupancy of PRC1 and a Br140 ortholog, BRD1, at bivalent loci in human embryonic stem (ES) cells. We propose that identification of PRC1-Br140 "bivalent complexes" in fly embryos supports and extends the bivalency model posited in mammalian cells, in which the coexistence of H3K4me3 and H3K27me3 at developmental promoters represents a poised transcriptional state. We further speculate that local competition between acetylation and deacetylation may play a critical role in the resolution of bivalent protein complexes during development.

Sex comb on midleg (Scm) is a functional link between PcG-repressive complexes in Drosophila.

The Polycomb group (PcG) proteins are key regulators of development in Drosophila and are strongly implicated in human health and disease. How PcG complexes form repressive chromatin domains remains unclear. Using cross-linked affinity purifications of BioTAP-Polycomb (Pc) or BioTAP-Enhancer of zeste [E(z)], we captured all PcG-repressive complex 1 (PRC1) or PRC2 core components and Sex comb on midleg (Scm) as the only protein strongly enriched with both complexes. Although previously not linked to PRC2, we confirmed direct binding of Scm and PRC2 using recombinant protein expression and colocalization of Scm with PRC1, PRC2, and H3K27me3 in embryos and cultured cells using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing). Furthermore, we found that RNAi knockdown of Scm and overexpression of the dominant-negative Scm-SAM (sterile α motif) domain both affected the binding pattern of E(z) on polytene chromosomes. Aberrant localization of the Scm-SAM domain in long contiguous regions on polytene chromosomes revealed its independent ability to spread on chromatin, consistent with its previously described ability to oligomerize in vitro. Pull-downs of BioTAP-Scm captured PRC1 and PRC2 and additional repressive complexes, including PhoRC, LINT, and CtBP. We propose that Scm is a key mediator connecting PRC1, PRC2, and transcriptional silencing. Combined with previous structural and genetic analyses, our results strongly suggest that Scm coordinates PcG complexes and polymerizes to produce broad domains of PcG silencing.

BioTAP-XL: Cross-linking/Tandem Affinity Purification to Study DNA Targets, RNA, and Protein Components of Chromatin-Associated Complexes.

In order to understand how chromatin complexes function in the nucleus, it is important to obtain a comprehensive picture of their protein, DNA, and RNA components, as well as their mutual interactions. This unit presents a chromatin cross-linking approach (BioTAP-XL) that utilizes a special BioTAP-tagged transgenic protein bait along with mass spectrometry to identify protein complex components, and high-throughput sequencing to identify RNA components and DNA binding sites. Full protocols are provided for Drosophila cells and for human cells in culture, along with an additional protocol for Drosophila embryos as the source material. A key element of the approach in all cases is the generation of control data from input chromatin samples.

Are we there yet? Initial targeting of the Male-Specific Lethal and Polycomb group chromatin complexes in Drosophila.

Chromatin-binding proteins must navigate the complex nuclear milieu to find their sites of action, and a constellation of protein factors and other properties are likely to influence targeting specificity. Despite considerable progress, the precise rules by which binding specificity is achieved have remained elusive. Here, we consider early targeting events for two groups of chromatin-binding complexes in Drosophila: the Male-Specific Lethal (MSL) and the Polycomb group (PcG) complexes. These two serve as models for understanding targeting, because they have been extensively studied and play vital roles in Drosophila, and their targets have been documented at high resolution. Furthermore, the proteins and biochemical properties of both complexes are largely conserved in multicellular organisms, including humans. While the MSL complex increases gene expression and PcG members repress genes, the two groups share many similarities such as the ability to modify their chromatin environment to create active or repressive domains, respectively. With legacies of in-depth genetic, biochemical and now genomic approaches, the MSL and PcG complexes will continue to provide tractable systems for understanding the recruitment of multiprotein chromatin complexes to their target loci.

Chromatin insulators specifically associate with different levels of higher-order chromatin organization in Drosophila.

Chromatin insulators are required for proper temporal and spatial expression of genes in metazoans. Here, we have analyzed the distribution of insulator proteins on the 56F-58A region of chromosome 2R in Drosophila polytene chromosomes to assess the role of chromatin insulators in shaping genome architecture. Data show that the suppressor of Hairy-wing protein [Su(Hw)] is found in three structures differentially associated with insulator proteins: bands, interbands, and multi-gene domains of coexpressed genes. Results show that bands are generally formed by condensation of chromatin that belongs to genes containing one or more Su(Hw) binding sites, whereas, in interbands, Su(Hw) sites appear associated with open chromatin. In addition, clusters of coexpressed genes in this region form bands characterized by the lack of CP190 and BEAF-32 insulator proteins. This pattern correlates with the distribution of specific chromatin marks and is conserved in nurse cells, suggesting that this organization may not be limited to one cell type but represents the basic organization of interphasic chromosomes.