Uncoupling histone modification crosstalk by engineering lysine demethylase LSD1.

Biochemical crosstalk between two or more histone modifications is often observed in epigenetic enzyme regulation, but its functional significance in cells has been difficult to discern. Previous enzymatic studies revealed that Lys14 acetylation of histone H3 can inhibit Lys4 demethylation by lysine-specific demethylase 1 (LSD1). In the present study, we engineered a mutant form of LSD1, Y391K, which renders the nucleosome demethylase activity of LSD1 insensitive to Lys14 acetylation.

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.

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.

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.

Variant Polycomb complexes in consistent with ancient functional diversity.

Polycomb group (PcG) mutants were first identified in on the basis of their failure to maintain proper 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.

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.

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.

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.

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

Acetylation of histone H4 at lysine 16 (H4K16) modulates nucleosome-nucleosome interactions and directly affects nucleosome binding by certain proteins. In , 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 containing different H4K16 mutations or lacking Mof protein.

"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 -fusion oncogenes resulting from chromosomal translocation. In most cases, the gene () is fused to bromodomain containing 4 () forming the oncogene. Here, a novel fusion partner to was discovered using next-generation sequencing and FISH from a young patient with an undifferentiated malignant round cell tumor.

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

Regulatory decisions in 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.

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.

, the 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 homolog of yeast Set3 and mammalian KMT2E (MLL5).

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.

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.

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.

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.

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.

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.

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.

Comparative analysis of metazoan chromatin organization.

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization.

Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs.

Heterochromatin protein 1 (HP1a) has conserved roles in gene silencing and heterochromatin and is also implicated in transcription, DNA replication, and repair. Here we identify chromatin-associated protein and RNA interactions of HP1a by BioTAP-XL mass spectrometry and sequencing from Drosophila S2 cells, embryos, larvae, and adults. Our results reveal an extensive list of known and novel HP1a-interacting proteins, of which we selected three for validation.

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.