Joshua D. Stender

Genome-Wide Analysis of Estrogen Receptor α DNA Binding and Tethering Mechanisms Identifies Runx1 as a Novel Tethering Factor in Receptor-Mediated Transcriptional Activation

ABSTRACT Nuclear receptor estrogen receptor alpha (ERα) controls the expression of hundreds of genes responsible for target cell phenotypic properties, but the relative importance of direct versus tethering mechanisms of DNA binding has not been established. In this first report, we examine the genome-wide chromatin localization of an altered-specificity mutant ER with a DNA binding domain deficient in binding to estrogen response element (ERE)-containing DNA (DBDmut ER) versus wild-type ERα. Using high-throughput sequencing of ER chromatin immunoprecipitations (ChIP-Seq) and mRNA transcriptional profiling, we show that direct ERE binding is required for most of (75%) estrogen-dependent gene regulation and 90% of hormone-dependent recruitment of ER to genomic binding sites. De novo motif analysis of the chromatin binding regions in MDA-MB-231 human breast cancer cells defined unique transcription factor profiles responsible for genes regulated through tethering versus direct ERE binding, with Runx motifs enriched in ER-tethered sites. We confirmed a role for Runx1 in mediating ERα genomic recruitment and regulation of tethering genes. Our findings delineate the contributions of direct receptor ERE binding versus binding through response elements for other transcription factors in chromatin localization and ER-dependent gene regulation, paradigms likely to underlie the gene regulatory actions of other nuclear receptors as well.

Control of Proinflammatory Gene Programs by Regulated Trimethylation and Demethylation of Histone H4K20

Regulation of genes that initiate and amplify inflammatory programs of gene expression is achieved by signal-dependent exchange of coregulator complexes that function to read, write, and erase specific histone modifications linked to transcriptional activation or repression. Here, we provide evidence for the role of trimethylated histone H4 lysine 20 (H4K20me3) as a repression checkpoint that restricts expression of toll-like receptor 4 (TLR4) target genes in macrophages. H4K20me3 is deposited at the promoters of a subset of these genes by the SMYD5 histone methyltransferase through its association with NCoR corepressor complexes. Signal-dependent erasure of H4K20me3 is required for effective gene activation and is achieved by NF-κB-dependent delivery of the histone demethylase PHF2. Liver X receptors antagonize TLR4-dependent gene activation by maintaining NCoR/SMYD5-mediated repression. These findings reveal a histone H4K20 trimethylation/demethylation strategy that integrates positive and negative signaling inputs that control immunity and homeostasis.

Conserved role for autophagy in Rho1-mediated cortical remodeling and blood cell recruitment

The Atg1 Ser/Thr kinase, although now a well-established regulator of autophagy, was first identified genetically in C. elegans as a requirement for axonal elongation. However, possible connections between Atg1 functions in cellular morphogenesis and in autophagy were previously unaddressed. In the recent paper highlighted in this punctum, we reconciled these dual roles for Atg1, demonstrating a requirement for p62-mediated selective autophagy in the dynamic regulation of cell shape, in both fly and mammalian macrophages, with effects on immune cell functions. This work further strengthens the emerging importance of autophagy as a post-translational regulatory mechanism in diverse cell signaling contexts, including the cortical remodeling and function of immune cells.Dynamic regulation of cell shape underlies many developmental and immune functions. Cortical remodeling is achieved under the central control of Rho GTPase pathways that modulate an exquisite balance in the dynamic assembly and disassembly of the cytoskeleton and focal adhesions. Macroautophagy (autophagy), associated with bulk cytoplasmic remodeling through lysosomal degradation, has clearly defined roles in cell survival and death. Moreover, it is becoming apparent that proteins, organelles, and pathogens can be targeted for autophagic clearance by selective mechanisms, although the extent and roles of such degradation are unclear. Here we report a conserved role for autophagy specifically in the cortical remodeling of Drosophila blood cells (hemocytes) and mouse macrophages. Continuous autophagy was required for integrin-mediated hemocyte spreading and Rho1-induced cell protrusions. Consequently, hemocytes disrupted for autophagy were impaired in their recruitment to epidermal wounds. Cell spreading required ref(2)P, the Drosophila p62 multiadaptor, implicating selective autophagy as a novel mechanism for modulating cortical dynamics. These results illuminate a specific and conserved role for autophagy as a regulatory mechanism for cortical remodeling, with implications for immune cell function.

53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration

Meitinger et al. perform a genome-wide CRISPR/Cas9 screen for centrinone resistance and identify a 53BP1-USP28 module as critical for communicating mitotic challenges to the p53 circuit and TRIM37 as an enforcer of the singularity of centrosome assembly.

Epigenomic control of the innate immune response.

Toll-like receptors (TLRs) play important roles in initiation of innate immune responses and promotion of pathological forms of inflammation. Recent technological advances have enabled the visualization of transcription factor binding and histone modifications in response to TLR signaling at genome-wide levels. Findings emerging from these studies are beginning to provide a picture of how signal-dependent transcription factors regulate the inflammatory response in a cell-specific manner by controlling the recruitment of nucleosome remodeling factors and histone modifying enzymes. Of particular interest, new small molecule inhibitors have been developed that influence inflammatory responses by altering the reading or erasure of histone modifications required for inflammatory gene activation. These findings suggest new approaches for treatment of inflammatory diseases.

NFκB affects estrogen receptor expression and activity in breast cancer through multiple mechanisms

Estrogen receptor (ER) and NFκB are two widely expressed, pleiotropic transcription factors that have been shown to interact and affect one anothers activity. While the ability of ER to repress NFκB activity has been extensively studied and is thought to underlie the anti-inflammatory activity of estrogens, how NFκB signaling affects ER activity is less clear. This is a particularly important question in breast cancer since activation of NFκB in ER positive tumors is associated with failure of endocrine and chemotherapies. In this review, we provide an update on the multiple mechanisms by which NFκB can influence ER activity, including down-regulation of ER expression, enhanced ER recruitment to DNA, and increased transcriptional activity of both liganded and unliganded ER. Additionally, a novel example of NFκB potentiation of ER-dependent gene repression is reviewed. Together, these mechanisms can alter response to endocrine therapies and may underlie the poor outcome for women with ER positive tumors that have active NFκB signaling.

Pleckstrin homology domain leucine-rich repeat protein phosphatases set the amplitude of receptor tyrosine kinase output

Significance This work unveils a previously unidentified function of the tumor suppressor pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) in inhibiting oncogenic signaling by suppressing the steady-state levels of receptor tyrosine kinases such as the EGF receptor. Specifically, PHLPP modifies the histone code to control the transcription of receptor tyrosine kinases. This epigenetic function can account for the upregulation of receptor tyrosine kinases in the multiple cancer types where PHLPP function is compromised. Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical implications. Here we show that the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) suppresses receptor tyrosine kinase (RTK) signaling output by a previously unidentified epigenetic mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for the protein kinase AKT, protein kinase C, and S6 kinase. Specifically, we show that nuclear-localized PHLPP suppresses histone phosphorylation and acetylation, in turn suppressing the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a much broader role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: By suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the Rat sarcoma (RAS)/ERK pathways. Our data are consistent with a model in which PHLPP modifies the histone code to control the transcription of RTKs.

A Cell-Permeable Stapled Peptide Inhibitor of the Estrogen Receptor/Coactivator Interaction

We and others have proposed that coactivator binding inhibitors, which block the interaction of estrogen receptor and steroid receptor coactivators, may represent a potential class of new breast cancer therapeutics. The development of coactivator binding inhibitors has been limited, however, because many of the current molecules which are active in in vitro and biochemical assays are not active in cell-based assays. Our goal in this work was to prepare a coactivator binding inhibitor active in cellular models of breast cancer. To accomplish this, we used molecular dynamics simulations to convert a high-affinity stapled peptide with poor cell permeability into R4K1, a cell-penetrating stapled peptide. R4K1 displays high binding affinity for estrogen receptor α, inhibits the formation of estrogen receptor/coactivator complexes, and distributes throughout the cell with a high percentage of nuclear localization. R4K1 represses native gene transcription mediated by estrogen receptor α and inhibits proliferation of estradiol-stimulated MCF-7 cells. Using RNA-Seq, we demonstrate that almost all of the effects of R4K1 on global gene transcription are estrogen-receptor-associated. This chemical probe provides a significant proof-of-concept for preparing cell-permeable stapled peptide inhibitors of the estrogen receptor/coactivator interaction.

Diverse motif ensembles specify non-redundant DNA binding activities of AP-1 family members in macrophages

Mechanisms by which members of the AP-1 family of transcription factors play both redundant and non-redundant biological roles despite recognizing the same DNA sequence remain poorly understood. To address this question, we investigated the molecular functions and genome-wide DNA binding patterns of AP-1 family members in macrophages. ChIP-sequencing showed overlapping and distinct binding profiles for each factor that were remodeled following TLR4 ligation. Development of a machine learning approach that jointly weighs hundreds of DNA recognition elements yielded dozens of motifs predicted to drive factor-specific binding profiles. Machine learning-based predictions were confirmed by analysis of the effects of mutations in genetically diverse mice and by loss of function experiments. These findings provide evidence that non-redundant genomic locations of different AP-1 family members in macrophages largely result from collaborative interactions with diverse, locus-specific ensembles of transcription factors and suggest a general mechanism for encoding functional specificities of their common recognition motif.

Abstract LB-148: Inflammatory cytokines alter the sensitivity of breast cancer cells to endocrine treatments

In breast cancer, infiltration of macrophages establishes an inflammatory tumor microenvironment, which can foster an extremely aggressive and therapy resistant tumor. Although inflammatory cytokines are known to modulate the transcriptional potential of estrogens and contribute to an endocrine-resistant state in cell-based model systems, the underlying molecular mechanisms remain poorly defined. We have used the human breast cancer cell-line MCF-7, an estrogen receptor α(ERα) positive and inflammatory cytokine sensitive cell line, to define the global impact inflammatory cytokines on the ERα cistrome and ERα-dependent transcriptional activity using next-generation sequencing methods. IL-1β and TNFα treatments establish an ERα cistrome that substantially overlaps with the E2-dependent ERα cistrome and results in ERα-dependent, ligand-independent activation of gene expression. Cytokine stimulation of MCF-7 cells results in phosphorylation of ERα at S305 which drives the ligand-independent activation of ERα. Pharmacological inhibition of IKKα/β blocks cytokine-dependent S305 phosphorylation, inhibits the cytokine-dependent ERα cistome, and abolishes cytokine-dependent gene activation. Additionally, S305 phosphorylation by cytokine treatments blocks the ability of tamoxifen to inhibit the expression of a subset of E2-dependent target genes and mutation of S305 to alanine restores tamoxifen sensitivity in the presence of IL-1β or TNFα. Collectively, these results provide molecular details dictating how inflammatory cytokines activate the transcriptional potential of ERα and drive an endocrine-resistant state in human breast cancer cells. In addition, they highlight the potential for anti-inflammatory therapies to be utilized in the treatment of patients with tamoxifen-resistant breast cancer. Citation Format: Joshua D. Stender, Irida Kastrati, Maayan Yakir, Jonna Frasor, Christopher K. Glass. Inflammatory cytokines alter the sensitivity of breast cancer cells to endocrine treatments. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-148. doi:10.1158/1538-7445.AM2014-LB-148