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SummaryMany membrane-less organelles form through liquid-liquid phase separation, but how their size is controlled and whether size is linked to function remain poorly understood. The Histone Locus Body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of histone mRNAs. Here, we show thatDrosophilaHLBs form through phase separation of the scaffold protein multi-sex combs (Mxc). The size of HLBs is controlled in a precise and dynamic manner that is dependent on the cell cycle and zygotic gene activation. Control of HLB growth is achieved by a mechanism integrating nascent mRNAs at the histone locus, which catalyzes phase separation, and the nuclear concentration of Mxc, which is controlled by the activity of cyclin-dependent kinases. Reduced Cdk2 activity results in smaller HLBs and the appearance of nascent, misprocessed histone mRNAs. Our experiments thus identify a mechanism linking nuclear body growth and size with gene expression.

Concentrating factors in nuclear bodies is thought to promote efficient gene expression. Tatomer et al. show that the histone locus body (HLB) concentrates pre-mRNA processing factors at replication-dependent histone genes, resulting in optimal 3′ end formation of histone mRNAs coupled with transcription termination., The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3′ processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3′ end processing with transcription termination.

Posttranslational modifications (PTMs) on microtubules differentiate these cytoskeletal elements for a variety of cellular functions. We recently identified SETD2 as a dual-function histone and microtubule methyltransferase, and methylation as a new microtubule PTM that occurs on lysine 40 of α-tubulin, which is trimethylated (α-TubK40me3) by SETD2. In the course of these studies, we generated polyclonal (α-TubK40me3 pAb) and monoclonal (α-TubK40me3 mAb) antibodies to a methylated α-tubulin peptide (GQMPSD-Kme3-TIGGGDC). Here, we characterize these antibodies, and the specific mono-, di- or tri-methylated lysine residues they recognize. While both the pAb and mAb antibodies recognized lysines methylated by SETD2 on microtubules and histones, the clone 18 mAb was more specific for methylated microtubules, with little cross-reactivity for methylated histones. The clone 18 mAb recognized specific subsets of microtubules during mitosis and cytokinesis, and lacked the chromatin staining seen by immunocytochemistry with the pAb. Western blot analysis using these antibodies revealed that methylated α-tubulin migrated faster than unmethylated α-tubulin, suggesting methylation may be a signal for additional processing of α-tubulin and/or microtubules. As the first reagents that specifically recognize methylated α-tubulin, these antibodies are a valuable tool for studying this new modification of the cytoskeleton, and the function of methylated microtubules.

Mammalian cell identity or cell fate is governed by transcription factors that recruit co-activators to regulate cell-specific gene expression programs. The histone acetyltransferase (HAT) paralogs EP300 and CREB binding protein (CBP) are master transcriptional co-activators that catalyze acetylation of key histones that mark active enhancers, thereby operating at a fundamental step of transcriptional control. Enhancers are often co-opted by cancer cells to control expression of oncogenic transcriptional programs. Identifying cancer-specific dependencies will greatly help prioritize clinical indications that are uniquely vulnerable to HAT inhibition, allowing for precise targeting of cancer cells while sparing normal tissue to maximize the therapeutic window and clinical efficacy. Estrogen receptor (ER) signaling is a validated clinical pathway in over 70% of breast cancer. ER-directed therapies are foundational for the treatment of ER+ breast cancer. However, these therapies are limited by residual ER signaling, which remains online through late-stage disease in a significant fraction of patients. EP300/CBP are critical components of the core ER signaling complex, which is recruited to target DNA by ER and other coactivators, to potentiate ER signaling. Furthermore, CBP and EP300 are dependency factors in the context of ESR1 mutations that arise in metastatic disease to facilitate disease progression. We report for the first time the activity of a novel, potent, selective and in vivo active EP300/CBP HAT inhibitor for the treatment of ER-positive (ER+) breast cancer. CPI-1 has pM biochemical and nM activity in ER+ breast cancer cell lines. Analysis of nascent and steady-state RNA levels facilitated by PRO-seq and RNA-seq technologies revealed a time and dose-dependent suppression of the ER transcriptome. Dynamic gene regulation was coupled with enhancer RNA (eRNA) transcription and enhancer acetylation, to identify enhancers that were hypersensitive to CBP/EP300 HAT inhibition. CPI-1 demonstrates significant tumor growth inhibition in a xenograft mouse model of ER+ breast cancer as a monotherapy and together with an ER targeting agent, the combination displays superior efficacy to either treatment alone. These results highlight the potential of targeting key oncogenic transcriptional nodes with complementary therapeutic mechanisms to achieve superior results and reveals the potential utility of inhibiting the HAT domain of EP300/CBP in molecularly refined cancer populations. Citation Format: Archana Bommi-Reddy, Jonathan Wilson, Annissa J. Huhn, Esteban Terzo, Florence Poy, Sungmi Park, Michael J. Steinbaugh, Barbara M. Bryant, Andrew R. Conery, Richard Cummings, Julian Levell, Robert J. Sims. Efficacy of a novel EP300/CBP histone acetyltransferase inhibitor in hormone responsive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4722.

The Drosophila Multi Sex Combs (Mxc) protein is necessary for the recruitment of histone mRNA biosynthetic factors to the histone locus body (HLB). Mxc contains multiple domains required for HLB assembly and histone mRNA biosynthesis. Two N-terminal domains of Mxc are essential for promoting HLB assembly via a self-interaction., Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. We studied the Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus. We coupled biochemical analysis with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel self-interaction facilitator (SIF) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or the SIF domain severely impairs Mxc function in vivo, resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Finally, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA-processing factor, to the HLB. We conclude that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis.