A-MYB and BRDT-dependent RNA Polymerase II pause release orchestrates transcriptional regulation in mammalian meiosis.

During meiotic prophase I, spermatocytes must balance transcriptional activation with homologous recombination and chromosome synapsis, biological processes requiring extensive changes to chromatin state. We explored the interplay between chromatin accessibility and transcription through prophase I of mammalian meiosis by measuring genome-wide patterns of chromatin accessibility, nascent transcription, and processed mRNA. We find that Pol II is loaded on chromatin and maintained in a paused state early during prophase I. In later stages, paused Pol II is released in a coordinated transcriptional burst mediated by the transcription factors A-MYB and BRDT, resulting in ~3-fold increase in transcription. Transcriptional activity is temporally and spatially segregated from key steps of meiotic recombination: double strand breaks show evidence of chromatin accessibility earlier during prophase I and at distinct loci from those undergoing transcriptional activation, despite shared chromatin marks. Our findings reveal mechanisms underlying chromatin specialization in either transcription or recombination in meiotic cells.

GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1.

BACKGROUND: Rearranged during transfection (RET) tyrosine kinase signaling has been previously implicated in endocrine resistant breast cancer, however the mechanism by which this signaling cascade promotes resistance is currently not well described. We recently reported that glial cell-derived neurotrophic factor (GDNF)-RET signaling appears to promote a positive feedback loop with the transcription factor early growth response 1 (EGR1). Here we investigate the mechanism behind this feedback loop and test the hypothesis that GDNF-RET signaling forms a regulatory loop with EGR1 to upregulate cyclin D1 (CCND1) transcription, leading to cell cycle progression and tamoxifen resistance. METHODS: To gain a better understanding of the GDNF-RET-EGR1 resistance mechanism, we studied the GDNF-EGR1 positive feedback loop and the role of GDNF and EGR1 in endocrine resistance by modulating their transcription levels using CRISPR-dCAS9 in tamoxifen sensitive (TamS) and tamoxifen resistant (TamR) MCF-7 cells. Additionally, we performed kinetic studies using recombinant GDNF (rGDNF) treatment of TamS cells. Finally, we performed cell proliferation assays using rGDNF, tamoxifen (TAM), and Palbociclib treatments in TamS cells. Statistical significance for qPCR and chromatin immunoprecipitation (ChIP)-qPCR experiments were determined using a student's paired t-test and statistical significance for the cell viability assay was a one-way ANOVA. RESULTS: GDNF-RET signaling formed a positive feedback loop with EGR1 and also downregulated estrogen receptor 1 (ESR1) transcription. Upregulation of GDNF and EGR1 promoted tamoxifen resistance in TamS cells and downregulation of GDNF promoted tamoxifen sensitivity in TamR cells. Additionally, we show that rGDNF treatment activated GDNF-RET signaling in TamS cells, leading to recruitment of phospho-ELK-1 to the EGR1 promoter, upregulation of EGR1 mRNA and protein, binding of EGR1 to the GDNF and CCND1 promoters, increased GDNF protein expression, and subsequent upregulation of CCND1 mRNA levels. We also show that inhibition of cyclin D1 with Palbociclib, in the presence of rGDNF, decreases cell proliferation and resensitizes cells to TAM. CONCLUSION: Outcomes from these studies support the hypotheses that GDNF-RET signaling forms a positive feedback loop with the transcription factor EGR1, and that GDNF-RET-EGR1 signaling promotes endocrine resistance via signaling to cyclin D1. Inhibition of components of this signaling pathway could lead to therapeutic insights into the treatment of endocrine resistant breast cancer.

SETD2 Haploinsufficiency Enhances Germinal Center-Associated AICDA Somatic Hypermutation to Drive B-cell Lymphomagenesis.

SETD2 is the sole histone methyltransferase responsible for H3K36me3, with roles in splicing, transcription initiation, and DNA damage response. Homozygous disruption of SETD2 yields a tumor suppressor effect in various cancers. However, SETD2 mutation is typically heterozygous in diffuse large B-cell lymphomas. Here we show that heterozygous Setd2 deficiency results in germinal center (GC) hyperplasia and increased competitive fitness, with reduced DNA damage checkpoint activity and apoptosis, resulting in accelerated lymphomagenesis. Impaired DNA damage sensing in Setd2-haploinsufficient germinal center B (GCB) and lymphoma cells associated with increased AICDA-induced somatic hypermutation, complex structural variants, and increased translocations including those activating MYC. DNA damage was selectively increased on the nontemplate strand, and H3K36me3 loss was associated with greater RNAPII processivity and mutational burden, suggesting that SETD2-mediated H3K36me3 is required for proper sensing of cytosine deamination. Hence, Setd2 haploinsufficiency delineates a novel GCB context-specific oncogenic pathway involving defective epigenetic surveillance of AICDA-mediated effects on transcribed genes. SIGNIFICANCE: Our findings define a B cell-specific oncogenic effect of SETD2 heterozygous mutation, which unleashes AICDA mutagenesis of nontemplate strand DNA in the GC reaction, resulting in lymphomas with heavy mutational burden. GC-derived lymphomas did not tolerate SETD2 homozygous deletion, pointing to a novel context-specific therapeutic vulnerability. This article is highlighted in the In This Issue feature, p. 1599.

Chromatin run-on sequencing analysis finds that ECM remodeling plays an important role in canine hemangiosarcoma pathogenesis.

BACKGROUND: Canine visceral hemangiosarcoma (HSA) is a highly aggressive cancer of endothelial origin that closely resembles visceral angiosarcoma in humans, both clinically and histopathologically. Currently there is an unmet need for new diagnostics and therapies for both forms of this disease. The goal of this study was to utilize Chromatin run-on sequencing (ChRO-seq) and immunohistochemistry (IHC) to identify gene and protein expression signatures that may be important drivers of HSA progression. RESULTS: ChRO-seq was performed on tissue isolated from 17 HSA samples and 4 normal splenic samples. Computational analysis was then used to identify differentially expressed genes and these factors were subjected to gene ontology analysis. ChRO-seq analysis revealed over a thousand differentially expressed genes in HSA tissue compared with normal splenic tissue (FDR < 0.005). Interestingly, the majority of genes overexpressed in HSA tumor tissue were associated with extracellular matrix (ECM) remodeling. This observation correlated well with our histological analysis, which found that HSA tumors contain a rich and complex collagen network. Additionally, we characterized the protein expression patterns of two highly overexpressed molecules identified in ChRO-seq analysis, podoplanin (PDPN) and laminin alpha 4 (LAMA4). We found that the expression of these two ECM-associated factors appeared to be largely limited to transformed endothelial cells within the HSA lesions. CONCLUSION: Outcomes from this study suggest that ECM remodeling plays an important role in HSA progression. Additionally, our study identified two potential novel biomarkers of HSA, PDPN and LAMA4. Interestingly, given that function-blocking anti-PDPN antibodies have shown anti-tumor effects in mouse models of canine melanoma, our studies raise the possibility that these types of therapeutic strategies could potentially be developed for treating canine HSA.

Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme.

The human genome encodes a variety of poorly understood RNA species that remain challenging to identify using existing genomic tools. We developed chromatin run-on and sequencing (ChRO-seq) to map the location of RNA polymerase for almost any input sample, including samples with degraded RNA that are intractable to RNA sequencing. We used ChRO-seq to map nascent transcription in primary human glioblastoma (GBM) brain tumors. Enhancers identified in primary GBMs resemble open chromatin in the normal human brain. Rare enhancers that are activated in malignant tissue drive regulatory programs similar to the developing nervous system. We identified enhancers that regulate groups of genes that are characteristic of each known GBM subtype and transcription factors that drive them. Finally we discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study characterizes the transcriptional landscape of GBM and introduces ChRO-seq as a method to map regulatory programs that contribute to complex diseases.

ER-positive breast cancer cells are poised for RET-mediated endocrine resistance.

The RET tyrosine kinase signaling pathway is involved in the development of endocrine resistant ER+ breast cancer. However, we know little about how ER+ cells activate RET signaling and initiate an endocrine resistant phenotype. Here we show that both ER+ endocrine resistant and sensitive breast cancers have a functional RET tyrosine kinase signaling pathway, but that endocrine sensitive breast cancer cells lack RET ligands that are necessary to drive endocrine resistance. Transcription of one RET ligand, GDNF, is necessary and sufficient to confer resistance in the ER+ MCF-7 cell line. Endogenous GDNF produced by endocrine resistant cells is translated, secreted into the media, and activates RET signaling in nearby cells. In patients, RET ligand expression predicts responsiveness to endocrine therapies and correlates with survival. Collectively, our findings show that ER+ tumor cells are "poised" for RET mediated endocrine resistance, expressing all components of the RET signaling pathway, but endocrine sensitive cells lack high expression of RET ligands that are necessary to initiate the resistance phenotype.

A bi-stable feedback loop between GDNF, EGR1, and ERα contribute to endocrine resistant breast cancer.

Discovering regulatory interactions between genes that specify the behavioral properties of cells remains an important challenge. We used the dynamics of transcriptional changes resolved by PRO-seq to identify a regulatory network responsible for endocrine resistance in breast cancer. We show that GDNF leads to endocrine resistance by switching the active state in a bi-stable feedback loop between GDNF, EGR1, and the master transcription factor ERα. GDNF stimulates MAP kinase, activating the transcription factors SRF and AP-1. SRF initiates an immediate transcriptional response, activating EGR1 and suppressing ERα. Newly translated EGR1 protein activates endogenous GDNF, leading to constitutive GDNF and EGR1 up-regulation, and the sustained down-regulation of ERα. Endocrine resistant MCF-7 cells are constitutively in the GDNF-high/ ERα-low state, suggesting that the state in the bi-stable feedback loop may provide a 'memory' of endocrine resistance. Thus, we identified a regulatory network switch that contributes to drug resistance in breast cancer.