AzidoTMT Enables Direct Enrichment and Highly Multiplexed Quantitation of Proteome-Wide Functional Residues.

Recent advances in targeted covalent inhibitors have aroused significant interest for their potential in drug development for difficult therapeutic targets. Proteome-wide profiling of functional residues is an integral step of covalent drug discovery aimed at defining actionable sites and evaluating compound selectivity in cells. A classical workflow for this purpose is called IsoTOP-ABPP, which employs an activity-based probe and two isotopically labeled azide-TEV-biotin tags to mark, enrich, and quantify proteome from two samples. Here we report a novel isobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP, that significantly expands multiplexing power as compared to the original isoTOP-ABPP. We demonstrate its application in identifying cysteine on- and off-targets using a KRAS G12C covalent inhibitor ARS-1620. However, changes in some of these hits can be explained by modulation at the protein and post-translational levels. Thus, it would be crucial to interrogate site-level bona fide changes in concurrence to proteome-level changes for corroboration. In addition, we perform a multiplexed covalent fragment screening using four acrylamide-based compounds as a proof-of-concept. This study identifies a diverse set of liganded cysteine residues in a compound-dependent manner with an average hit rate of 0.07% in intact cell. Lastly, we screened 20 sulfonyl fluoride-based compounds to demonstrate that the AT-MAPP assay is flexible for noncysteine functional residues such as tyrosine and lysine. Overall, we envision that 11plex-AzidoTMT will be a useful addition to the current toolbox for activity-based protein profiling and covalent drug development.

Super-enhancer acquisition drives oncogene expression in triple negative breast cancer.

Triple Negative Breast Cancer (TNBC) is a heterogeneous disease lacking known molecular drivers and effective targeted therapies. Cytotoxic chemotherapy remains the mainstay of treatment for TNBCs, which have significantly poorer survival rates compared to other breast cancer subtypes. In addition to changes within the coding genome, aberrant enhancer activity is a well-established contributor to tumorigenesis. Here we use H3K27Ac chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map the active cis-regulatory landscape in TNBC. We identify distinct disease subtypes associated with specific enhancer activity, and over 2,500 unique superenhancers acquired by tumor cells but absent from normal breast tissue. To identify potential actionable disease drivers, we probed the dependency on genes that associate with tumor-specific enhancers by CRISPR screening. In this way we identify a number of tumor-specific dependencies, including a previously uncharacterized dependency on the TGFß pseudo-receptor BAMBI.

Enhancer Activity Requires CBP/P300 Bromodomain-Dependent Histone H3K27 Acetylation.

Acetylation of histone H3 at lysine 27 is a well-defined marker of enhancer activity. However, the functional impact of this modification at enhancers is poorly understood. Here, we use a chemical genetics approach to acutely block the function of the cAMP response element binding protein (CREB) binding protein (CBP)/P300 bromodomain in models of hematological malignancies and describe a consequent loss of H3K27Ac specifically from enhancers, despite the continued presence of CBP/P300 at chromatin. Using this approach to dissect the role of H3K27Ac at enhancers, we identify a critical role for this modification in the production of enhancer RNAs and transcription of enhancer-regulated gene networks.

Therapeutic Targeting of the CBP/p300 Bromodomain Blocks the Growth of Castration-Resistant Prostate Cancer.

Resistance invariably develops to antiandrogen therapies used to treat newly diagnosed prostate cancers, but effective treatments for castration-resistant disease remain elusive. Here, we report that the transcriptional coactivator CBP/p300 is required to maintain the growth of castration-resistant prostate cancer. To exploit this vulnerability, we developed a novel small-molecule inhibitor of the CBP/p300 bromodomain that blocks prostate cancer growth in vitro and in vivo Molecular dissection of the consequences of drug treatment revealed a critical role for CBP/p300 in histone acetylation required for the transcriptional activity of the androgen receptor and its target gene expression. Our findings offer a preclinical proof of concept for small-molecule therapies to target the CBP/p300 bromodomain as a strategy to treat castration-resistant prostate cancer. Cancer Res; 77(20); 5564-75. ©2017 AACR.

Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform.

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-L-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l(-1)) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

Timing of transcriptional quiescence during gametogenesis is controlled by global histone H3K4 demethylation.

Gametes are among the most highly specialized cells produced during development. Although gametogenesis culminates in transcriptional quiescence in plants and animals, regulatory mechanisms controlling this are unknown. Here, we confirm that gamete differentiation in the single-celled yeast Saccharomyces cerevisiae is accompanied by global transcriptional shutoff following the completion of meiosis. We show that Jhd2, a highly conserved JARID1-family histone H3K4 demethylase, activates protein-coding gene transcription in opposition to this programmed transcriptional shutoff, sustaining the period of productive transcription during spore differentiation. Moreover, using genome-wide nucleosome, H3K4me, and transcript mapping experiments, we demonstrate that JHD2 globally represses intergenic noncoding transcription during this period. The widespread transcriptional defects of JHD2 mutants are associated with precocious differentiation and the production of stress-sensitive spores, demonstrating that Jhd2 regulation of the global postmeiotic transcriptional program is critical for the production of healthy meiotic progeny.

An engineered microbial platform for direct biofuel production from brown macroalgae.

Prospecting macroalgae (seaweeds) as feedstocks for bioconversion into biofuels and commodity chemical compounds is limited primarily by the availability of tractable microorganisms that can metabolize alginate polysaccharides. Here, we present the discovery of a 36-kilo-base pair DNA fragment from Vibrio splendidus encoding enzymes for alginate transport and metabolism. The genomic integration of this ensemble, together with an engineered system for extracellular alginate depolymerization, generated a microbial platform that can simultaneously degrade, uptake, and metabolize alginate. When further engineered for ethanol synthesis, this platform enables bioethanol production directly from macroalgae via a consolidated process, achieving a titer of 4.7% volume/volume and a yield of 0.281 weight ethanol/weight dry macroalgae (equivalent to ~80% of the maximum theoretical yield from the sugar composition in macroalgae).

Genomewide screen for negative regulators of sirtuin activity in Saccharomyces cerevisiae reveals 40 loci and links to metabolism.

Sirtuins are conserved proteins implicated in myriad key processes including gene control, aging, cell survival, metabolism, and DNA repair. In Saccharomyces cerevisiae, the sirtuin Silent information regulator 2 (Sir2) promotes silent chromatin formation, suppresses recombination between repeats, and inhibits senescence. We performed a genomewide screen for factors that negatively regulate Sir activity at a reporter gene placed immediately outside a silenced region. After linkage analysis, assessment of Sir dependency, and knockout tag verification, 40 loci were identified, including 20 that have not been previously described to regulate Sir. In addition to chromatin-associated factors known to prevent ectopic silencing (Bdf1, SAS-I complex, Rpd3L complex, Ku), we identified the Rtt109 DNA repair-associated histone H3 lysine 56 acetyltransferase as an anti-silencing factor. Our findings indicate that Rtt109 functions independently of its proposed effectors, the Rtt101 cullin, Mms1, and Mms22, and demonstrate unexpected interplay between H3K56 and H4K16 acetylation. The screen also identified subunits of mediator (Soh1, Srb2, and Srb5) and mRNA metabolism factors (Kem1, Ssd1), thus raising the possibility that weak silencing affects some aspect of mRNA structure. Finally, several factors connected to metabolism were identified. These include the PAS-domain metabolic sensor kinase Psk2, the mitochondrial homocysteine detoxification enzyme Lap3, and the Fe-S cluster protein maturase Isa2. We speculate that PAS kinase may integrate metabolic signals to control sirtuin activity.

Patterning chromatin: form and function for H2A.Z variant nucleosomes.

Although many histone variants are specific to higher eukaryotes, the H2A variant H2A.Z has been conserved during eukaryotic evolution. Genetic studies have demonstrated roles for H2A.Z in antagonizing gene-silencing, chromosome stability and gene activation. Biochemical work has identified a conserved chromatin-remodeling complex responsible for H2A.Z deposition. Recent studies have shown that two H2A.Z nucleosomes flank a nucleosome-free region containing the transcription initiation site in promoters of both active and inactive genes in Saccharomyces cerevisiae. This chromatin pattern is generated through the action of a DNA deposition signal and a specific pattern of histone tail acetylation.

Histone variant H2A.Z marks the 5' ends of both active and inactive genes in euchromatin.

In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread. We show that H2A.Z nucleosomes are found at promoter regions of nearly all genes in euchromatin. They generally occur as two positioned nucleosomes that flank a nucleosome-free region (NFR) that contains the transcription start site. Astonishingly, enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci. Mutagenesis of a typical promoter revealed a 22 bp segment of DNA sufficient to program formation of a NFR flanked by two H2A.Z nucleosomes. This segment contains a binding site of the Myb-related protein Reb1 and an adjacent dT:dA tract. Efficient deposition of H2A.Z is further promoted by a specific pattern of histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the Swr1 remodeling complex that deposits H2A.Z.

Evolution of a combinatorial transcriptional circuit: a case study in yeasts.

Developing new regulation of existing genes is likely a key mechanism by which organismal complexity arises in evolution. To examine plasticity of gene regulation over evolutionary timescales, we have determined the transcriptional circuit regulating mating type in the human fungal pathogen Candida albicans, and compared it to that of Saccharomyces cerevisiae. Since the two yeasts last shared an ancestor 100-800 million years ago, several major differences in circuitry have arisen. For example, a positive regulator of mating type was retained in C. albicans but lost in S. cerevisiae; this circuit branch was replaced by the modification of an existing negative regulator, thereby conserving the circuit output. We also characterize a tier of mating type transcriptional regulation that is present only in C. albicans, and likely results from the vastly different environmental selections imposed on the two yeasts--in this case, the pressure on C. albicans to survive in a mammalian host.