A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, we performed genomewide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq in two different embryonic cell lines and found that AGO2 localizes to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. We propose that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.

Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type–specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. Here we identify the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila. Mutation of shep improves gypsy-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. We developed a novel, quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP–seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator.

Translation and transcription are frequently dysregulated in cancer. These two processes are generally regulated by distinct sets of factors. The CBFB gene, which encodes a transcription factor, has recently emerged as a highly mutated driver in a variety of human cancers including breast cancer. Here we report a noncanonical role of CBFB in translation regulation. RNA immunoprecipitation followed by deep sequencing (RIP-seq) reveals that cytoplasmic CBFB binds to hundreds of transcripts and regulates their translation. CBFB binds to mRNAs via hnRNPK and enhances translation through eIF4B, a general translation initiation factor. Interestingly, the RUNX1 mRNA, which encodes the transcriptional partner of CBFB, is bound and translationally regulated by CBFB. Furthermore, nuclear CBFB/RUNX1 complex transcriptionally represses the oncogenic NOTCH signaling pathway in breast cancer. Thus, our data reveal an unexpected function of CBFB in translation regulation and propose that breast cancer cells evade translation and transcription surveillance simultaneously through downregulating CBFB.

Highly repetitive and transposable element rich regions of the genome must be stabilized by the presence of heterochromatin. A direct role for RNA interference in the establishment of heterochromatin has been demonstrated in fission yeast. In metazoans, which possess multiple RNA–silencing pathways that are both functionally distinct and spatially restricted, whether RNA silencing contributes directly to heterochromatin formation is not clear. Previous studies in Drosophila melanogaster have suggested the involvement of both the AGO2-dependent endogenous small interfering RNA (endo-siRNA) as well as Piwi-interacting RNA (piRNA) silencing pathways. In order to determine if these Argonaute genes are required for heterochromatin formation, we utilized transcriptional reporters and chromatin immunoprecipitation of the critical factor Heterochromatin Protein 1 (HP1) to monitor the heterochromatic state of piRNA clusters, which generate both endo-siRNAs and the bulk of piRNAs. Surprisingly, we find that mutation of AGO2 or piwi increases silencing at piRNA clusters corresponding to an increase of HP1 association. Furthermore, loss of piRNA production from a single piRNA cluster results in genome-wide redistribution of HP1 and reduction of silencing at a distant heterochromatic site, suggesting indirect effects on HP1 recruitment. Taken together, these results indicate that heterochromatin forms independently of endo-siRNA and piRNA pathways.

Purpose: TGFbs are overexpressed in many advanced cancers and promote cancer progression through mechanisms that include suppression of immunosurveillance. Multiple strategies to antagonize the TGFb pathway are in early-phase oncology trials. However, TGFbs also have tumor-suppressive activities early in tumorigenesis, and the extent to which these might be retained in advanced disease has not been fully explored. Experimental Design: A panel of 12 immunocompetent mouse allograft models of metastatic breast cancer was tested for the effect of neutralizing anti-TGFb antibodies on lung metastatic burden. Extensive correlative biology analyses were performed to assess potential predictive biomarkers and probe underlying mechanisms. Results: Heterogeneous responses to anti-TGFb treatment were observed, with 5 of 12 models (42%) showing suppression of metastasis, 4 of 12 (33%) showing no response, and 3 of 12 (25%) showing an undesirable stimulation (up to 9-fold) of metastasis. Inhibition of metastasis was immune-dependent, whereas stimulation of metastasis was immune-independent and targeted the tumor cell compartment, potentially affecting the cancer stem cell. Thus, the integrated outcome of TGFb antagonism depends on a complex balance between enhancing effective antitumor immunity and disrupting persistent tumor-suppressive effects of TGFb on the tumor cell. Applying transcriptomic signatures derived from treatment-na€ ve mouse primary tumors to human breast cancer datasets suggested that patients with breast cancer with high-grade, estrogen receptor-negative disease are most likely to benefit from anti-TGFb therapy. Conclusions: Contrary to dogma, tumor-suppressive responses to TGFb are retained in some advanced metastatic tumors. Safe deployment of TGFb antagonists in the clinic will require good predictive biomarkers.

Main text: 5772 (excluding acknowledgments, references and figure legends) Number of Figures: 5 Number of Tables: 1

Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.

Breast cancer accounts for >500,000 deaths annually worldwide. Despite therapeutic advances, treated patients often relapse with metastases. Emerging research indicates that relapse may be driven by a subpopulation of tumor cells termed cancer stem cells (CSCs), that are uniquely capable of self-renewal and have enhanced drug resistance. Understanding the properties of these CSCs is critical for development of more effective therapies. Peptidyl arginine deiminase 4 (PADI4) is an enzyme that catalyzes deimination of arginine to citrulline. Citrullination of histones modulates expression of key stem cell transcription factors in embryonic stem cells (Christophorou, et al., 2014, Nature 507:104), so we hypothesized that PADI4 could be involved in CSC regulation. We showed that PADI4 mRNA and protein are expressed at varying levels across a panel of breast cancer cell lines, and we knocked down PADI4 in two high expressing lines, MCF10Ca1h and MDAMB231-LM2. PADI4 knockdown did not alter tumor cell proliferation in vitro, but it enhanced migration and invasion and increased cell survival. Importantly, PADI4 knockdown increased clonogenicity and enhanced tumorsphere formation, suggesting that it specifically increases the CSC population. In vivo studies showed higher tumor initiation efficiency following PADI4 knockdown, and increased lung metastasis. PADI4 is predicted to have multiple isoforms with unknown biological activity that complicate PADI4 expression-based analysis in patient data sets. We circumvented this issue by pharmacologically inhibiting PADI4 in MCF10Ca1h to generate a transcriptomic signature reflecting PADI4 gene activity. This PADI4 gene activity signature was shown to correlate with lower tumor grade and better disease outcome in transcriptomic datasets from human ER+ breast cancers. In conclusion, our findings suggest that PADI4 functions as a tumor suppressor in breast cancer, in part through effects on the CSC. Citation Format: Humberto J. Ochoa, Nellie Moshkovich, Binwu Tang, Howard H. Yang, Maxwell P. Lee, Lalage M. Wakefield. Novel tumor suppressive role of Peptidylarginine deiminase IV involving cancer stem cell regulation in breast cancer models [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 3676.