Yeast-based screening of cancer mutations in the DNA damage response protein Mre11 demonstrates importance of conserved capping domain residues.

DNA damage response (DDR) pathways are initiated to prevent mutations from being passed on in the event of DNA damage. Mutations in DDR proteins can contribute to the development and maintenance of cancer cells, but many mutations observed in human tumors have not been functionally characterized. Because a proper response to DNA damage is fundamental to living organisms, DDR proteins and processes are often highly conserved. The goal of this project was to use Saccharomyces cerevisiae as a model for functional screening of human cancer mutations in conserved DDR proteins. After comparing the cancer mutation frequency and conservation of DDR proteins, Mre11 was selected for functional screening. A subset of mutations in conserved residues was analyzed by structural modeling and screened for functional effects in yeast Mre11. Yeast expressing wild type or mutant Mre11 were then assessed for DNA damage sensitivity using hydroxyurea (HU) and methyl methanesulfonate (MMS). The results were further validated in human cancer cells. The N-terminal point mutations tested in yeast Mre11 do not confer sensitivity to DNA damage sensitivity, suggesting that these residues are dispensable for yeast Mre11 function and may have conserved sequence without conserved function. However, a mutation near the capping domain associated with breast and colorectal cancers compromises Mre11 function in both yeast and human cells. These results provide novel insight into the function of this conserved capping domain residue and demonstrate a framework for yeast-based screening of cancer mutations.

HDAC inhibition results in widespread alteration of the histone acetylation landscape and BRD4 targeting to gene bodies.

Histone acetylation levels are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) that antagonistically control the overall balance of this post-translational modification. HDAC inhibitors (HDACi) are potent agents that disrupt this balance and are used clinically to treat diseases including cancer. Despite their use, little is known about their effects on chromatin regulators, particularly those that signal through lysine acetylation. We apply quantitative genomic and proteomic approaches to demonstrate that HDACi robustly increases a low-abundance histone 4 polyacetylation state, which serves as a preferred binding substrate for several bromodomain-containing proteins, including BRD4. Increased H4 polyacetylation occurs in transcribed genes and correlates with the targeting of BRD4. Collectively, these results suggest that HDAC inhibition functions, at least in part, through expansion of a rare histone acetylation state, which then retargets lysine-acetyl readers associated with changes in gene expression, partially mimicking the effect of bromodomain inhibition.

PBRM1 bromodomains variably influence nucleosome interactions and cellular function.

Chromatin remodelers use bromodomains (BDs) to recognize histones. Polybromo 1 (PBRM1 or BAF180) is hypothesized to function as the nucleosome-recognition subunit of the PBAF chromatin-remodeling complex and is frequently mutated in clear cell renal cell carcinoma (ccRCC). Previous studies have applied in vitro methods to explore the binding specificities of the six individual PBRM1 BDs. However, BD targeting to histones and the influence of neighboring BD on nucleosome recognition have not been well characterized. Here, using histone microarrays and intact nucleosomes to investigate the histone-binding characteristics of the six PBRM1 BDs individually and combined, we demonstrate that BD2 and BD4 of PBRM1 mediate binding to acetylated histone peptides and to modified recombinant and cellular nucleosomes. Moreover, we show that neighboring BDs variably modulate these chromatin interactions, with BD1 and BD5 enhancing nucleosome interactions of BD2 and BD4, respectively, whereas BD3 attenuated these interactions. We also found that binding pocket missense mutations in BD4 observed in ccRCC disrupt PBRM1-chromatin interactions and that these mutations in BD4, but not similar mutations in BD2, in the context of full-length PBRM1, accelerate ccRCC cell proliferation. Taken together, our biochemical and mutational analyses have identified BD4 as being critically important for maintaining proper PBRM1 function and demonstrate that BD4 mutations increase ccRCC cell growth. Because of the link between PBRM1 status and sensitivity to immune checkpoint inhibitor treatment, these data also suggest the relevance of BD4 as a potential clinical target.

Histone peptide microarray screen of chromo and Tudor domains defines new histone lysine methylation interactions.

BACKGROUND: Histone posttranslational modifications (PTMs) function to regulate chromatin structure and function in part through the recruitment of effector proteins that harbor specialized "reader" domains. Despite efforts to elucidate reader domain-PTM interactions, the influence of neighboring PTMs and the target specificity of many reader domains is still unclear. The aim of this study was to use a high-throughput histone peptide microarray platform to interrogate 83 known and putative histone reader domains from the chromo and Tudor domain families to identify their interactions and characterize the influence of neighboring PTMs on these interactions. RESULTS: Nearly a quarter of the chromo and Tudor domains screened showed interactions with histone PTMs by peptide microarray, revealing known and several novel methyllysine interactions. Specifically, we found that the CBX/HP1 chromodomains that recognize H3K9me also recognize H3K23me2/3-a poorly understood histone PTM. We also observed that, in addition to their interaction with H3K4me3, Tudor domains of the Spindlin family also recognized H4K20me3-a previously uncharacterized interaction. Several Tudor domains also showed novel interactions with H3K4me as well. CONCLUSIONS: These results provide an important resource for the epigenetics and chromatin community on the interactions of many human chromo and Tudor domains. They also provide the basis for additional studies into the functional significance of the novel interactions that were discovered.

Multivalent Histone and DNA Engagement by a PHD/BRD/PWWP Triple Reader Cassette Recruits ZMYND8 to K14ac-Rich Chromatin.

Elucidation of interactions involving DNA and histone post-translational-modifications (PTMs) is essential for providing insights into complex biological functions. Reader assemblies connected by flexible linkages facilitate avidity and increase affinity; however, little is known about the contribution to the recognition process of multiple PTMs because of rigidity in the absence of conformational flexibility. Here, we resolve the crystal structure of the triple reader module (PHD-BRD-PWWP) of ZMYND8, which forms a stable unit capable of simultaneously recognizing multiple histone PTMs while presenting a charged platform for association with DNA. Single domain disruptions destroy the functional network of interactions initiated by ZMYND8, impairing recruitment to sites of DNA damage. Our data establish a proof of principle that rigidity can be compensated by concomitant DNA and histone PTM interactions, maintaining multivalent engagement of transient chromatin states. Thus, our findings demonstrate an important role for rigid multivalent reader modules in nucleosome binding and chromatin function.

The Taf14 YEATS domain is a reader of histone crotonylation.

The discovery of new histone modifications is unfolding at startling rates; however, the identification of effectors capable of interpreting these modifications has lagged behind. Here we report the YEATS domain as an effective reader of histone lysine crotonylation, an epigenetic signature associated with active transcription. We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine-binding activity.

The essential role of acetyllysine binding by the YEATS domain in transcriptional regulation.

The YEATS domains of AF9 and Taf14 have recently been found to recognize the histone H3K9ac modification. In this commentary, we discuss the mechanistic and biological implications of this interaction. We compare structures of the YEATS-H3K9ac complexes the highlighting a novel mechanism for the acetyllysine recognition through the aromatic cage. We also summarize the latest findings underscoring a critical role of the acetyllysine binding function of AF9 and Taf14 in transcriptional regulation and DNA repair.

A course-based undergraduate research experience investigating p300 bromodomain mutations.

Course-based undergraduate research experiences (CUREs) provide an opportunity for students to engage in experiments with outcomes that are unknown to both the instructor and students. These experiences allow students and instructors to collaboratively bridge the research laboratory and classroom, and provide research experiences for a large number of students relative to traditional individual mentored research. Here, we describe a molecular biology CURE investigating the impact of clinically relevant mutations found in the bromodomain of the p300 transcriptional regulator on acetylated histone interaction. In the CURE, students identified missense mutations in the p300 bromodomain using the Catalogue of Somatic Mutations in Cancer (COSMIC) database and hypothesized the effects of the mutation on the acetyl-binding function of the domain. They cloned and purified the mutated bromodomain and performed peptide pulldown assays to define its potential to bind to acetylated histones. Upon completion of the course, students showed increased confidence performing molecular techniques and reported positively on doing a research project in class. In addition, results generated in the classroom were further validated in the research laboratory setting thereby providing a new model for faculty to engage in both course-based and individual undergraduate research experiences.

Prognostic significance of full-length estrogen receptor beta expression in stage I-III triple negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which there is a need to identify new therapeutic targets. Full-length estrogen receptor beta (ERß1) may be a possible target given its antiproliferative effects on breast cancer cells. The prognostic significance of ERß in breast cancer subtypes has remained elusive, and disparate results observed across previously published reports might be due to the detection of multiple ERß isoforms, the lack of specific antibodies and the use of different cutoffs to define ERßpositivity. The objective of this retrospective study was to determine the association between ERß1 expression and disease-free and overall survival, as well as Ki67 expression, in non-metastatic TNBC. Immunohistochemical protocols were optimized using xenograft tissues obtained from a breast cancer cell line with inducible ERß1 expression. ERß1 localization and expression were assessed in two cohorts of TNBC using the VECTRA(TM) platform. There was a close relationship between nuclear and cytoplasmic ERß1 expression. ERß1 was expressed in a subset of TNBCs, but its expression was significantly associated with Ki67 in only one of the cohorts. There was no significant association between ERß1 expression and disease-free and overall survival in either cohort. Although these results suggest that ERß1 expression alone may not be informative in TNBCs, this study provides a new strategy for optimizing and objectively measuring ERß1 expression in tissues, which may provide a standard for ERß1 immunohistochemistry in future large-scale clinical studies aimed at better understanding the role of ERß1 in breast cancer.

Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response.

The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14.

An Interactive Database for the Assessment of Histone Antibody Specificity.

Access to high-quality antibodies is a necessity for the study of histones and their posttranslational modifications (PTMs). Here we debut the Histone Antibody Specificity Database (http://www.histoneantibodies.com), an online and expanding resource cataloging the behavior of widely used, commercially available histone antibodies by peptide microarray. This interactive web portal provides a critical resource to the biological research community that routinely uses these antibodies as detection reagents for a wide range of applications.

Product binding enforces the genomic specificity of a yeast polycomb repressive complex.

We characterize the Polycomb system that assembles repressive subtelomeric domains of H3K27 methylation (H3K27me) in the yeast Cryptococcus neoformans. Purification of this PRC2-like protein complex reveals orthologs of animal PRC2 components as well as a chromodomain-containing subunit, Ccc1, which recognizes H3K27me. Whereas removal of either the EZH or EED ortholog eliminates H3K27me, disruption of mark recognition by Ccc1 causes H3K27me to redistribute. Strikingly, the resulting pattern of H3K27me coincides with domains of heterochromatin marked by H3K9me. Indeed, additional removal of the C. neoformans H3K9 methyltransferase Clr4 results in loss of both H3K9me and the redistributed H3K27me marks. These findings indicate that the anchoring of a chromatin-modifying complex to its product suppresses its attraction to a different chromatin type, explaining how enzymes that act on histones, which often harbor product recognition modules, may deposit distinct chromatin domains despite sharing a highly abundant and largely identical substrate-the nucleosome.

Research resource: global identification of estrogen receptor ß target genes in triple negative breast cancer cells.

Breast cancers that are negative for estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 are known as triple-negative breast cancers (TNBC). TNBCs are associated with an overall poor prognosis because they lack expression of therapeutic targets like ERα and are biologically more aggressive. A second estrogen receptor, ERß, has been found to be expressed in 50% to 90% of ERα-negative breast cancers, and ERß expression in TNBCs has been shown to correlate with improved disease-free survival and good prognosis. To elucidate the role of ERß in regulating gene expression and cell proliferation in TNBC cells, the TNBC cell line MDA-MB-468 was engineered with inducible expression of full-length ERß. In culture, ERß expression inhibited cell growth by inducing a G1 cell cycle arrest, which was further enhanced by 17ß-estradiol treatment. In xenografts, ERß expression also inhibited tumor formation and growth, and 17ß-estradiol treatment resulted in rapid tumor regression. Furthermore, genomic RNA sequencing identified both ligand-dependent and -independent ERß target genes, some of which were also regulated by ERß in other TNBC cell lines and correlated with ERß expression in a cohort of TNBCs from the Cancer Genome Atlas Network. ERß target genes were enriched in genes that regulate cell death and survival, cell movement, cell development, and growth and proliferation, as well as genes involved in the Wnt/ß-catenin and the G1/S cell cycle phase checkpoint pathways. In addition to confirming the anti-proliferative effects of ERß in TNBC cells, these data provide a comprehensive resource of ERß target genes and suggest that ERß may be targeted with ligands that can stimulate its growth inhibitory effects.

Differential action of monohydroxylated polycyclic aromatic hydrocarbons with estrogen receptors α and ß.

Polycyclic aromatic hydrocarbons (PAHs) are a diverse group of widespread environmental pollutants, some of which have been found to be estrogenic or antiestrogenic. Recent data have shown that hydroxylated PAH metabolites may be responsible for the estrogenic effects of some PAHs. The purpose of this study was to investigate the effects of several PAHs, as well as their monohydroxylated metabolites, on estrogen receptors (ERs), ERα and ERß. Three parent PAHs and their monohydroxylated metabolites were each evaluated using transcriptional reporter assays in isogenic stable cell lines to measure receptor activation, competitive binding assays to determine ligand binding, and bioluminescence resonance energy transfer assays to assess dimerization. Finally, the estrogenic effects of the hydroxylated metabolites were confirmed by quantitative real-time PCR of estrogen-responsive target genes. Although the parent PAHs did not induce ERα or ERß transcriptional activity, all of the monohydroxylated PAHs (1-OH naphthanol, 9-OH phenanthrene, 1-OH pyrene) selectively induced ERß transcriptional activity at the concentrations tested, while not activating ERα. Additionally, the monohydroxylated PAHs were able to competively bind ERß, induce ERß homodimers, and regulate ERß target genes. Although monohydroxylated PAHs appeared to have weak agonist activity to ERß, our results showed that they can elicit a biologically active response from ERß in human breast cancer cells and potentially interfere with ERß signaling pathways.

Generation of stable reporter breast cancer cell lines for the identification of ER subtype selective ligands.

Estrogen signaling is mediated by two estrogen receptors (ERs), ERα and ERß, which have unique roles in the regulation of breast cancer cell proliferation. ERα induces proliferation in response to estrogen and ERß inhibits proliferation in breast cancer cells, suggesting that ERß selective ligands may be beneficial for promoting the anti-proliferative action of ERß. Subtype selective ligands can be identified using transcriptional assays, but cell lines in which ERα or ERß are independently expressed are required. Of the available reporter cell lines, none have been generated in breast cancer cells to identify subtype selective ligands. Here we describe the generation of two isogenic breast cancer cell lines, Hs578T-ERαLuc and Hs578T-ERßLuc, with stable integration of an estrogen responsive luciferase reporter gene. Hs578T-ERαLuc and Hs578T-ERßLuc cell lines are highly sensitive to estrogenic chemicals and ER subtype selective ligands, providing a tool to characterize the transcriptional potency and subtype selectivity of estrogenic ligands in the context of breast cancer cells. In addition to measuring reporter activity, ERß target gene expression and growth inhibitory effects of ERß selective ligands can be determined as biological endpoints. The finding that activation of ERß by estrogen or ERß selective natural phytoestrogens inhibits the growth of Hs578T-ERß cells implies therapeutic potential for ERß selective ligands in breast cancer cells that express ERß.

Endocrine disrupting chemicals targeting estrogen receptor signaling: identification and mechanisms of action.

Many endocrine disrupting chemicals (EDCs) adversely impact estrogen signaling by interacting with two estrogen receptors (ERs): ERα and ERß. Though the receptors have similar ligand binding and DNA binding domains, ERα and ERß have some unique properties in terms of ligand selectivity and target gene regulation. EDCs that target ER signaling can modify genomic and nongenomic ER activity through direct interactions with ERs, indirectly through transcription factors such as the aryl hydrocarbon receptor (AhR), or through modulation of metabolic enzymes that are critical for normal estrogen synthesis and metabolism. Many EDCs act through multiple mechanisms as exemplified by chemicals that bind both AhR and ER, such as 3-methylcholanthrene. Other EDCs that target ER signaling include phytoestrogens, bisphenolics, and organochlorine pesticides, and many alter normal ER signaling through multiple mechanisms. EDCs can also display tissue-selective ER agonist and antagonist activities similar to selective estrogen receptor modulators (SERMs) designed for pharmaceutical use. Thus, biological effects of EDCs need to be carefully interpreted because EDCs can act through complex tissue-selective modulation of ERs and other signaling pathways in vivo. Current requirements by the U.S. Environmental Protection Agency require some in vitro and cell-based assays to identify EDCs that target ER signaling through direct and metabolic mechanisms. Additional assays may be useful screens for identifying EDCs that act through alternative mechanisms prior to further in vivo study.

Selectively targeting estrogen receptors for cancer treatment.

Estrogens regulate growth and development through the action of two distinct estrogen receptors (ERs), ERα and ERß, which mediate proliferation and differentiation of cells. For decades, ERα mediated estrogen signaling has been therapeutically targeted to treat breast cancer, most notably with the selective estrogen receptor modulator (SERM) tamoxifen. Selectively targeting ERs occurs at two levels: tissue selectivity and receptor subtype selectivity. SERMs have been developed with emphasis on tissue selectivity to target ER signaling for breast cancer treatment. Additionally, new approaches to selectively target the action of ERα going beyond ligand-dependent activity are under current investigation. As evidence of the anti-proliferative role of ERß accumulates, selectively targeting ERß is an attractive approach for designing new cancer therapies with the emphasis shifted to designing ligands with subtype selectivity. This review will present the mechanistic and structural features of ERs that determine tissue and subtype selectivity with an emphasis on current approaches to selectively target ERα and ERß for cancer treatment.