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.

Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior.

Understanding the genetic control of human embryonic stem cell function is foundational for developmental biology and regenerative medicine. Here we describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. We identified a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance. We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification. Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers. Our results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks.

Chemical Screen Identifies Diverse and Novel Histone Deacetylase Inhibitors as Repressors of NUT Function: Implications for NUT Carcinoma Pathogenesis and Treatment.

NUT carcinoma (NC), characterized most commonly by the BRD4-NUTM1 fusion, is a rare, aggressive variant of squamous carcinoma with no effective treatment. BRD4-NUT drives growth and maintains the poorly differentiated state of NC by activating pro-growth genes such as MYC, through the formation of massive, hyperacetylated, superenhancer-like domains termed megadomains. BRD4-NUT-mediated hyperacetylation of chromatin is facilitated by the chromatin-targeting tandem bromodomains of BRD4, combined with NUT, which recruits the histone acetyltransferase, p300. Here, we developed a high-throughput small-molecule screen to identify inhibitors of transcriptional activation by NUT. In this dCAS9-based GFP-reporter assay, the strongest hits were diverse histone deacetylase (HDAC) inhibitors. Two structurally unrelated HDAC inhibitors, panobinostat and the novel compound, IRBM6, both repressed growth and induced differentiation of NC cells in proportion to their inhibition of NUT transcriptional activity. These two compounds repressed transcription of megadomain-associated oncogenic genes, such as MYC and SOX2, while upregulating pro-differentiation, non-megadomain-associated genes, including JUN, FOS, and key cell-cycle regulators, such as CDKN1A. The transcriptional changes correlate with depletion of BRD4-NUT from megadomains, and redistribution of the p300/CBP-associated chromatin acetylation mark, H3K27ac, away from megadomains toward regular enhancer regions previously populated by H3K27ac. In NC xenograft models, we demonstrated that suppression of tumor growth by panobinostat was comparable with that of bromodomain inhibition, and when combined they improved both survival and growth suppression. IMPLICATIONS: The findings provide mechanistic and preclinical rationale for the use of HDAC inhibitors, alone or combined with other agents, in the treatment of NUT carcinoma.

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.

Combination Targeting of the Bromodomain and Acetyltransferase Active Site of p300/CBP.

p300 and CBP are highly related histone acetyltransferase (HAT) enzymes that regulate gene expression, and their dysregulation has been linked to cancer and other diseases. p300/CBP is composed of a number of domains including a HAT domain, which is inhibited by the small molecule A-485, and an acetyl-lysine binding bromodomain, which was recently found to be selectively antagonized by the small molecule I-CBP112. Here we show that the combination of I-CBP112 and A-485 can synergize to inhibit prostate cancer cell proliferation. We find that the combination confers a dramatic reduction in p300 chromatin occupancy compared to the individual effects of blocking either domain alone. Accompanying this loss of p300 on chromatin, combination treatment leads to the reduction of specific mRNAs including androgen-dependent and pro-oncogenic prostate genes such as KLK3 (PSA) and c-Myc. Consistent with p300 directly affecting gene expression, mRNAs that are significantly reduced by combination treatment also exhibit a strong reduction in p300 chromatin occupancy at their gene promoters. The relatively few mRNAs that are up-regulated upon combination treatment show no correlation with p300 occupancy. These studies provide support for the pharmacologic advantage of concurrent targeting of two domains within one key epigenetic modification enzyme.

Sex-specific phenotypes of histone H4 point mutants establish dosage compensation as the critical function of H4K16 acetylation in Drosophila.

Acetylation of histone H4 at lysine 16 (H4K16) modulates nucleosome-nucleosome interactions and directly affects nucleosome binding by certain proteins. In Drosophila, H4K16 acetylation by the dosage compensation complex subunit Mof is linked to increased transcription of genes on the single X chromosome in males. Here, we analyzed Drosophila containing different H4K16 mutations or lacking Mof protein. An H4K16A mutation causes embryonic lethality in both sexes, whereas an H4K16R mutation permits females to develop into adults but causes lethality in males. The acetyl-mimic mutation H4K16Q permits both females and males to develop into adults. Complementary analyses reveal that males lacking maternally deposited and zygotically expressed Mof protein arrest development during gastrulation, whereas females of the same genotype develop into adults. Together, this demonstrates the causative role of H4K16 acetylation by Mof for dosage compensation in Drosophila and uncovers a previously unrecognized requirement for this process already during the onset of zygotic gene transcription.

"Z4" Complex Member Fusions in NUT Carcinoma: Implications for a Novel Oncogenic Mechanism.

Nuclear protein in testis (NUT) carcinoma (NC) is a rare, distinctly aggressive subtype of squamous carcinoma defined by the presence of NUT-fusion oncogenes resulting from chromosomal translocation. In most cases, the NUT gene (NUTM1) is fused to bromodomain containing 4 (BRD4) forming the BRD4-NUT oncogene. Here, a novel fusion partner to NUT was discovered using next-generation sequencing and FISH from a young patient with an undifferentiated malignant round cell tumor. Interestingly, the NUT fusion identified involved ZNF592, a zinc finger containing protein, which was previously identified as a component of the BRD4-NUT complex. In BRD4-NUT-expressing NC cells, wild-type ZNF592 and other associated "Z4" complex proteins, including ZNF532 and ZMYND8, colocalize with BRD4-NUT in characteristic nuclear foci. Furthermore, ectopic expression of BRD4-NUT in a non-NC cell line induces sequestration of Z4 factors to BRD4-NUT foci. Finally, the data demonstrate the specific dependency of NC cells on Z4 modules, ZNF532 and ZNF592. IMPLICATIONS: This study establishes the oncogenic role of Z4 factors in NC, offering potential new targeted therapeutic strategies in this incurable cancer.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/12/1826/F1.large.jpg.

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.

Ectopic protein interactions within BRD4-chromatin complexes drive oncogenic megadomain formation in NUT midline carcinoma.

To investigate the mechanism that drives dramatic mistargeting of active chromatin in NUT midline carcinoma (NMC), we have identified protein interactions unique to the BRD4-NUT fusion oncoprotein compared with wild-type BRD4. Using cross-linking, affinity purification, and mass spectrometry, we identified the EP300 acetyltransferase as uniquely associated with BRD4 through the NUT fusion in both NMC and non-NMC cell types. We also discovered ZNF532 associated with BRD4-NUT in NMC patient cells but not detectable in 293T cells. EP300 and ZNF532 are both implicated in feed-forward regulatory loops leading to propagation of the oncogenic chromatin complex in BRD4-NUT patient cells. Adding key functional significance to our biochemical findings, we independently discovered a ZNF532-NUT translocation fusion in a newly diagnosed NMC patient. ChIP sequencing of the major players NUT, ZNF532, BRD4, EP300, and H3K27ac revealed the formation of ZNF532-NUT-associated hyperacetylated megadomains, distinctly localized but otherwise analogous to those found in BRD4-NUT patient cells. Our results support a model in which NMC is dependent on ectopic NUT-mediated interactions between EP300 and components of BRD4 regulatory complexes, leading to a cascade of misregulation.

upSET, the Drosophila homologue of SET3, Is Required for Viability and the Proper Balance of Active and Repressive Chromatin Marks.

Chromatin plays a critical role in faithful implementation of gene expression programs. Different post-translational modifications (PTMs) of histone proteins reflect the underlying state of gene activity, and many chromatin proteins write, erase, bind, or are repelled by, these histone marks. One such protein is UpSET, the Drosophila homolog of yeast Set3 and mammalian KMT2E (MLL5). Here, we show that UpSET is necessary for the proper balance between active and repressed states. Using CRISPR/Cas-9 editing, we generated S2 cells that are mutant for upSET We found that loss of UpSET is tolerated in S2 cells, but that heterochromatin is misregulated, as evidenced by a strong decrease in H3K9me2 levels assessed by bulk histone PTM quantification. To test whether this finding was consistent in the whole organism, we deleted the upSET coding sequence using CRISPR/Cas-9, which we found to be lethal in both sexes in flies. We were able to rescue this lethality using a tagged upSET transgene, and found that UpSET protein localizes to transcriptional start sites (TSS) of active genes throughout the genome. Misregulated heterochromatin is apparent by suppressed position effect variegation of the wm4 allele in heterozygous upSET-deleted flies. Using nascent-RNA sequencing in the upSET-mutant S2 lines, we show that this result applies to heterochromatin genes generally. Our findings support a critical role for UpSET in maintaining heterochromatin, perhaps by delimiting the active chromatin environment.

Dosage Compensation in Drosophila-a Model for the Coordinate Regulation of Transcription.

The sex chromosomes have special significance in the history of genetics. The chromosomal basis of inheritance was firmly established when Calvin Bridges demonstrated that exceptions to Mendel's laws of segregation were accompanied at the cytological level by exceptional sex chromosome segregation. The morphological differences between X and Y exploited in Bridges' experiments arose as a consequence of the evolution of the sex chromosomes. Originally a homologous chromosome pair, the degeneration of the Y chromosome has been accompanied by a requirement for increased expression of the single X chromosome in males. Drosophila has been a model for the study of this dosage compensation and has brought key strengths, including classical genetics, the exceptional cytology of polytene chromosomes, and more recently, comprehensive genomics. The impact of these studies goes beyond sex chromosome regulation, providing valuable insights into mechanisms for the establishment and maintenance of chromatin domains, and for the coordinate regulation of transcription.

The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns.

Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC), which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N) covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM) using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state.

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry.

Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.

The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains.

NUT midline carcinoma (NMC), a subtype of squamous cell cancer, is one of the most aggressive human solid malignancies known. NMC is driven by the creation of a translocation oncoprotein, BRD4-NUT, which blocks differentiation and drives growth of NMC cells. BRD4-NUT forms distinctive nuclear foci in patient tumors, which we found correlate with ∼100 unprecedented, hyperacetylated expanses of chromatin that reach up to 2 Mb in size. These "megadomains" appear to be the result of aberrant, feed-forward loops of acetylation and binding of acetylated histones that drive transcription of underlying DNA in NMC patient cells and naïve cells induced to express BRD4-NUT. Megadomain locations are typically cell lineage-specific; however, the cMYC and TP63 regions are targeted in all NMCs tested and play functional roles in tumor growth. Megadomains appear to originate from select pre-existing enhancers that progressively broaden but are ultimately delimited by topologically associating domain (TAD) boundaries. Therefore, our findings establish a basis for understanding the powerful role played by large-scale chromatin organization in normal and aberrant lineage-specific gene transcription.

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.

Dosage compensation in Drosophila.

Dosage compensation in Drosophila increases the transcription of genes on the single X chromosome in males to equal that of both X chromosomes in females. Site-specific histone acetylation by the male-specific lethal (MSL) complex is thought to play a fundamental role in the increased transcriptional output of the male X. Nucleation and sequence-independent spreading of the complex to active genes serves as a model for understanding the targeting and function of epigenetic chromatin-modifying complexes. Interestingly, two noncoding RNAs are key for MSL assembly and spreading to active genes along the length of the X chromosome.

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.