TRPS1 modulates chromatin accessibility to regulate estrogen receptor alpha (ER) binding and ER target gene expression in luminal breast cancer cells.

Common genetic variants in the repressive GATA-family transcription factor (TF) TRPS1 locus are associated with breast cancer risk, and luminal breast cancer cell lines are particularly sensitive to TRPS1 knockout. We introduced an inducible degron tag into the native TRPS1 locus within a luminal breast cancer cell line to identify the direct targets of TRPS1 and determine how TRPS1 mechanistically regulates gene expression. We acutely deplete over 80 percent of TRPS1 from chromatin within 30 minutes of inducing degradation. We find that TRPS1 regulates transcription of hundreds of genes, including those related to estrogen signaling. TRPS1 directly regulates chromatin structure, which causes estrogen receptor alpha (ER) to redistribute in the genome. ER redistribution leads to both repression and activation of dozens of ER target genes. Downstream from these primary effects, TRPS1 depletion represses cell cycle-related gene sets and reduces cell doubling rate. Finally, we show that high TRPS1 activity, calculated using a gene expression signature defined by primary TRPS1-regulated genes, is associated with worse breast cancer patient prognosis. Taken together, these data suggest a model in which TRPS1 modulates the genomic distribution of ER, both activating and repressing transcription of genes related to cancer cell fitness.

The Androgen Receptor Does Not Directly Regulate the Transcription of DNA Damage Response Genes.

The clinical success of combined androgen deprivation therapy (ADT) and radiotherapy (RT) in prostate cancer created interest in understanding the mechanistic links between androgen receptor (AR) signaling and the DNA damage response (DDR). Convergent data have led to a model where AR both regulates, and is regulated by, the DDR. Integral to this model is that the AR regulates the transcription of DDR genes both at a steady state and in response to ionizing radiation (IR). In this study, we sought to determine which immediate transcriptional changes are induced by IR in an AR-dependent manner. Using PRO-seq to quantify changes in nascent RNA transcription in response to IR, the AR antagonist enzalutamide, or the combination of the two, we find that enzalutamide treatment significantly decreased expression of canonical AR target genes but had no effect on DDR gene sets in prostate cancer cells. Surprisingly, we also found that the AR is not a primary regulator of DDR genes either in response to IR or at a steady state in asynchronously growing prostate cancer cells. IMPLICATIONS: Our data indicate that the clinical benefit of combining ADT with RT is not due to direct AR regulation of DDR gene transcription, and that the field needs to consider alternative mechanisms for this clinical benefit.

TRPS1 modulates chromatin accessibility to regulate estrogen receptor (ER) binding and ER target gene expression in luminal breast cancer cells.

Breast cancer is the most frequently diagnosed cancer in women. The most common subtype is luminal breast cancer, which is typically driven by the estrogen receptor α (ER), a transcription factor (TF) that activates many genes required for proliferation. Multiple effective therapies target this pathway, but individuals often develop resistance. Thus, there is a need to identify additional targets that regulate ER activity and contribute to breast tumor progression. TRPS1 is a repressive GATA-family TF that is overexpressed in breast tumors. Common genetic variants in the TRPS1 locus are associated with breast cancer risk, and luminal breast cancer cell lines are particularly sensitive to TRPS1 knockout. However, we do not know how TRPS1 regulates target genes to mediate these breast cancer patient and cellular outcomes. We introduced an inducible degron tag into the native TRPS1 locus within a luminal breast cancer cell line to identify the direct targets of TRPS1 and determine how TRPS1 mechanistically regulates gene expression. We acutely deplete over eighty percent of TRPS1 from chromatin within 30 minutes of inducing degradation. We find that TRPS1 regulates transcription of hundreds of genes, including those related to estrogen signaling. TRPS1 directly regulates chromatin structure, which causes ER to redistribute in the genome. ER redistribution leads to both repression and activation of dozens of ER target genes. Downstream from these primary effects, TRPS1 depletion represses cell cycle-related gene sets and reduces cell doubling rate. Finally, we show that high TRPS1 activity, calculated using a gene expression signature defined by primary TRPS1-regulated genes, is associated with worse breast cancer patient prognosis. Taken together, these data suggest a model in which TRPS1 modulates the activity of other TFs, both activating and repressing transcription of genes related to cancer cell fitness.

Correction of transposase sequence bias in ATAC-seq data with rule ensemble modeling.

Chromatin accessibility assays have revolutionized the field of transcription regulation by providing single-nucleotide resolution measurements of regulatory features such as promoters and transcription factor binding sites. ATAC-seq directly measures how well the Tn5 transposase accesses chromatinized DNA. Tn5 has a complex sequence bias that is not effectively scaled with traditional bias-correction methods. We model this complex bias using a rule ensemble machine learning approach that integrates information from many input k-mers proximal to the ATAC sequence reads. We effectively characterize and correct single-nucleotide sequence biases and regional sequence biases of the Tn5 enzyme. Correction of enzymatic sequence bias is an important step in interpreting chromatin accessibility assays that aim to infer transcription factor binding and regulatory activity of elements in the genome.

ANKLE1 cleaves mitochondrial DNA and contributes to cancer risk by promoting apoptosis resistance and metabolic dysregulation.

Alleles within the chr19p13.1 locus are associated with increased risk of both ovarian and breast cancer and increased expression of the ANKLE1 gene. ANKLE1 is molecularly characterized as an endonuclease that efficiently cuts branched DNA and shuttles between the nucleus and cytoplasm. However, the role of ANKLE1 in mammalian development and homeostasis remains unknown. In normal development ANKLE1 expression is limited to the erythroblast lineage and we found that ANKLE1's role is to cleave the mitochondrial genome during erythropoiesis. We show that ectopic expression of ANKLE1 in breast epithelial-derived cells leads to genome instability and mitochondrial DNA (mtDNA) cleavage. mtDNA degradation then leads to mitophagy and causes a shift from oxidative phosphorylation to glycolysis (Warburg effect). Moreover, mtDNA degradation activates STAT1 and expression of epithelial-mesenchymal transition (EMT) genes. Reduction in mitochondrial content contributes to apoptosis resistance, which may allow precancerous cells to avoid apoptotic checkpoints and proliferate. These findings provide evidence that ANKLE1 is the causal cancer susceptibility gene in the chr19p13.1 locus and describe mechanisms by which higher ANKLE1 expression promotes cancer risk.

Kinetic networks identify TWIST2 as a key regulatory node in adipogenesis.

Adipocytes contribute to metabolic disorders such as obesity, diabetes, and atherosclerosis. Prior characterizations of the transcriptional network driving adipogenesis have overlooked transiently acting transcription factors (TFs), genes, and regulatory elements that are essential for proper differentiation. Moreover, traditional gene regulatory networks provide neither mechanistic details about individual regulatory element-gene relationships nor temporal information needed to define a regulatory hierarchy that prioritizes key regulatory factors. To address these shortcomings, we integrate kinetic chromatin accessibility (ATAC-seq) and nascent transcription (PRO-seq) data to generate temporally resolved networks that describe TF binding events and resultant effects on target gene expression. Our data indicate which TF families cooperate with and antagonize each other to regulate adipogenesis. Compartment modeling of RNA polymerase density quantifies how individual TFs mechanistically contribute to distinct steps in transcription. The glucocorticoid receptor activates transcription by inducing RNA polymerase pause release, whereas SP and AP-1 factors affect RNA polymerase initiation. We identify Twist2 as a previously unappreciated effector of adipocyte differentiation. We find that TWIST2 acts as a negative regulator of 3T3-L1 and primary preadipocyte differentiation. We confirm that Twist2 knockout mice have compromised lipid storage within subcutaneous and brown adipose tissue. Previous phenotyping of Twist2 knockout mice and Setleis syndrome Twist2 -/- patients noted deficiencies in subcutaneous adipose tissue. This network inference framework is a powerful and general approach for interpreting complex biological phenomena and can be applied to a wide range of cellular processes.

Epidemiology and survival factors of appendicular myxofibrosarcoma: a SEER-retrospective study.

Background: The low incidence of myxofibrosarcoma (MFS) makes high power studies difficult to perform. Demographic and prognostic factors for MFS and how they differ from all extremity soft tissue sarcomas (STS) are not well understood. The purpose of this study was to characterize a large cohort of patients with MFS and evaluate epidemiologic and survival factors when compared to all STS. Materials and methods: We performed a retrospective review of the Surveillance, Epidemiology, and End Results (SEER) database from 2000 to 2015 to identify 1,440 patients diagnosed with MFS and 12,324 with STS. Survival curves were creased using Kaplan-Meier, and Cox regression analyses were performed to identify hazard ratios (HRs). Results: Overall survival was greater for STS than MFS (79% vs. 67%). Patients with MFS had a higher average age at diagnosis than STS (62 vs. 56), and older age was strongly associated with decreased survivorship for MFS (HR = 7.94). A greater proportion of patients under 30 diagnosed with MFS were female when compared to STS (49.6% vs. 45.4%). The incidence of MFS and STS increased over the 15-year period, with MFS increasing at a greater rate than STS (1.25% vs. 2.59%). Survival increased for patients diagnosed after 2008 for both STS (9.4%) and MFS (13.2%). Conclusions: There are differences between patient demographics and survival factors when comparing MFS to all STS. Monitoring changes in demographic and survival trends for rare STS subtypes like MFS is important to improve diagnostic algorithms and treatment options.

Intrinsic bias estimation for improved analysis of bulk and single-cell chromatin accessibility profiles using SELMA.

Genome-wide profiling of chromatin accessibility by DNase-seq or ATAC-seq has been widely used to identify regulatory DNA elements and transcription factor binding sites. However, enzymatic DNA cleavage exhibits intrinsic sequence biases that confound chromatin accessibility profiling data analysis. Existing computational tools are limited in their ability to account for such intrinsic biases and not designed for analyzing single-cell data. Here, we present Simplex Encoded Linear Model for Accessible Chromatin (SELMA), a computational method for systematic estimation of intrinsic cleavage biases from genomic chromatin accessibility profiling data. We demonstrate that SELMA yields accurate and robust bias estimation from both bulk and single-cell DNase-seq and ATAC-seq data. SELMA can utilize internal mitochondrial DNA data to improve bias estimation. We show that transcription factor binding inference from DNase footprints can be improved by incorporating estimated biases using SELMA. Furthermore, we show strong effects of intrinsic biases in single-cell ATAC-seq data, and develop the first single-cell ATAC-seq intrinsic bias correction model to improve cell clustering. SELMA can enhance the performance of existing bioinformatics tools and improve the analysis of both bulk and single-cell chromatin accessibility sequencing data.

Distinct MUNC lncRNA structural domains regulate transcription of different promyogenic factors.

Many lncRNAs have been discovered using transcriptomic data; however, it is unclear what fraction of lncRNAs is functional and what structural properties affect their phenotype. MUNC lncRNA (also known as DRReRNA) acts as an enhancer RNA for the Myod1 gene in cis and stimulates the expression of other promyogenic genes in trans by recruiting the cohesin complex. Here, experimental probing of the RNA structure revealed that MUNC contains multiple structural domains not detected by prediction algorithms in the absence of experimental information. We show that these specific and structurally distinct domains are required for induction of promyogenic genes, for binding genomic sites and gene expression regulation, and for binding the cohesin complex. Myod1 induction and cohesin interaction comprise only a subset of MUNC phenotype. Our study reveals unexpectedly complex, structure-driven functions for the MUNC lncRNA and emphasizes the importance of experimentally determined structures for understanding structure-function relationships in lncRNAs.

Oncological Outcomes in Patients with Appendicular Myxofibrosarcomas: A Retrospective Study.

BACKGROUND: Myxofibrosarcoma (MFS) is notorious for its infiltrative growth pattern, making wide excisions difficult to achieve. Our objective was to assess the impact of surgical margins and other factors that affected rates of local recurrence (LR), distant metastasis (DM), and overall survival (OS) of individuals undergoing resection for MFS. METHODS: We retrospectively reviewed the medical records of 209 patients with appendicular soft tissue sarcomas between January 2012 and June 2018. Of these, 29 patients (14%) were diagnosed with myxofibrosarcoma. These patients underwent a total of 33 resections. The pathological analyses were conducted by an experienced musculoskeletal (MSK) pathologist. Demographics data, operative details, adjuvant therapy, and oncological outcomes were assessed. RESULTS: Of the 29 patients (33 resections), the overall LR rate was 24% (7/29) and the 2-year LR rate was 17% (5/29). Factors associated with negative oncological outcomes were as follows: tumor size ≤10 cm (2-year local recurrence-free rates (LRFRs), 65%; 95% CI, 44-86%; p=0.02) and positive surgical margins grouped with surgical margins ≤0.1 cm (hazard ratio (HR), 11.74; 95% CI, 1.41-97.74; p=0.02). Chemotherapy and radiotherapy together increased the 2-year LRFR (LRFR, 100%; 95% CI, 100%, p=0.001). Two-year DM and OS rates were 15% and 79%, respectively. Female gender was a predictor of distant metastasis. Local recurrence had a negative impact on overall survival. Intraoperative analysis of resection margin accuracy was 75% (12/16) when non-MSK pathologists were involved but 100% accurate (12/12) when analyzed by an MSK pathologist. CONCLUSION: Myxofibrosarcomas showed high LR rates after treatment. Close margins (≤0.1 cm) should be considered as a risk factor for LR, and LR is associated with negative overall survival. Neoadjuvant therapy in terms of combined chemotherapy and radiation therapy associates with decreased LR rates. If intraoperative assessment of margins is to be done, it should be performed by an experienced MSK pathologist.

PEPPRO: quality control and processing of nascent RNA profiling data.

Nascent RNA profiling is growing in popularity; however, there is no standard analysis pipeline to uniformly process the data and assess quality. Here, we introduce PEPPRO, a comprehensive, scalable workflow for GRO-seq, PRO-seq, and ChRO-seq data. PEPPRO produces uniformly processed output files for downstream analysis and assesses adapter abundance, RNA integrity, library complexity, nascent RNA purity, and run-on efficiency. PEPPRO is restartable and fault-tolerant, records copious logs, and provides a web-based project report. PEPPRO can be run locally or using a cluster, providing a portable first step for genomic nascent RNA analysis.

Responsible Return to Essential and Non-Essential Surgery During the COVID-19 Pandemic.

Non-essential surgery had largely been suspended during the COVID-19 Pandemic. Enormous amounts of resources were utilized to shift surgical practices to a "disaster footing" with most elective surgeons assuming new roles to offset the anticipated burden from surgical and medical personnel delivering acute care. As the number of COVID-19-infected patients began to plateau in the state of Ohio, a four-phase "Responsible Return to Surgery" approach was adopted in concert with the Ohio Department of Health and the Ohio Hospital Association. This approach was adopted understanding that a simple return to the status quo prior to the COVID-19 pandemic might be harmful to patients, providers, and staff. The discrete phases undertaken at our quaternary care institution for a responsible return to non-essential surgery are outlined with the goal of ensuring timely care, minimizing community transmission, and preserving personal protective equipment. Operationalizing these phases relied upon the widespread use of telehealth, systematic COVID-19 testing, and real-time monitoring of hospital and personal protective equipment resources.

Comparative interactomes of HSF1 in stress and disease reveal a role for CTCF in HSF1-mediated gene regulation.

Heat shock transcription factor 1 (HSF1) orchestrates cellular stress protection by activating or repressing gene transcription in response to protein misfolding, oncogenic cell proliferation, and other environmental stresses. HSF1 is tightly regulated via intramolecular repressive interactions, post-translational modifications, and protein-protein interactions. How these HSF1 regulatory protein interactions are altered in response to acute and chronic stress is largely unknown. To elucidate the profile of HSF1 protein interactions under normal growth and chronic and acutely stressful conditions, quantitative proteomics studies identified interacting proteins in the response to heat shock or in the presence of a poly-glutamine aggregation protein cell-based model of Huntington's disease. These studies identified distinct protein interaction partners of HSF1 as well as changes in the magnitude of shared interactions as a function of each stressful condition. Several novel HSF1-interacting proteins were identified that encompass a wide variety of cellular functions, including roles in DNA repair, mRNA processing, and regulation of RNA polymerase II. One HSF1 partner, CTCF, interacted with HSF1 in a stress-inducible manner and functions in repression of specific HSF1 target genes. Understanding how HSF1 regulates gene repression is a crucial question, given the dysregulation of HSF1 target genes in both cancer and neurodegeneration. These studies expand our understanding of HSF1-mediated gene repression and provide key insights into HSF1 regulation via protein-protein interactions.

A prospective observational cohort study to evaluate patients' experience during sequential cataract surgery under monitored anesthesia care and topical anesthesia.

Cataract surgery is the most common ambulatory surgery at our outpatient surgery center. Several studies have shown that patients with bilateral cataracts may experience different levels of anxiety, pain, and awareness during the first and second cataract extraction.A prospective observational cohort study was conducted at The Ohio State University Wexner Medical Center Eye and Ear Institute in order to compare anxiety, general comfort, awareness, and pain levels in patients undergoing sequential cataract surgeries. Likert and numerical rating scale were used to assess the outcomes. Patients receiving monitored anesthesia care and topical anesthesia were included.A total of 198 patients were enrolled in this study, 116 patients (59%) were female and 157 patients (78%) were Caucasians with a median age of 67 years among participants. Patients with rating "no anxiety" or feeling "somewhat anxious" were significantly higher during surgery 2 (P =< .001). Most of the patients felt "extremely comfortable" during surgery 1 when compared to surgery 2 (54% vs 42.9%; P = .08). No significant differences were found between surgeries regarding intraoperative awareness (P = .16). Overall, patients experienced mild pain during both procedures (92.4% in surgery 1 compared to 90.4% in surgery 2; P = .55). During the postoperative visit, 54% of the patients associated surgery 2 with less anxiety levels, 53% with no differences in general comfort, 60% felt more aware, and 59% had no differences in pain levels.Previous exposure to surgery could have been associated with a significant reduction in anxiety levels reported during surgery 2. Non-pharmacological strategies aiming to reduce perioperative anxiety may be considered an alternative or additional approach to premedication in patients undergoing consecutive cataract surgeries.

ARF-AID: A Rapidly Inducible Protein Degradation System That Preserves Basal Endogenous Protein Levels.

Inducible degron systems are widely used to specifically and rapidly deplete proteins of interest in cell lines and organisms. An advantage of inducible degradation is that the biological system under study remains intact and functional until perturbation, a feature that necessitates that the endogenous levels of the protein are maintained. However, endogenous tagging of genes with auxin-inducible degrons (AID) can result in chronic, auxin-independent proteasome-mediated degradation. The ARF-AID (auxin-response factor-auxin-inducible degron) system is a re-engineered auxin-inducible protein degradation system. The additional expression of the ARF-PB1 domain prevents chronic, auxin-independent degradation of AID-tagged proteins while preserving rapid auxin-induced degradation of tagged proteins. Here, we describe the protocol for engineering human cell lines to implement the ARF-AID system for specific and inducible protein degradation. These methods are adaptable and can be extended from cell lines to organisms. © 2020 The Authors. Basic Protocol 1: Generation of ARF-P2A-TIR1 progenitor cells Basic Protocol 2: Designing, cloning, and testing of a gene-specific sgRNA Basic Protocol 3: Design and amplification of a homology-directed repair construct (C-terminal tagging) Alternate Protocol 1: Design and amplification of a homology-directed repair construct (N-terminal tagging) Basic Protocol 4: Tagging of a gene of interest with AID Alternate Protocol 2: Establishment of an ARF-AID clamp system Basic Protocol 5: Testing of auxin-mediated degradation of the AID-tagged protein.

Defining data-driven primary transcript annotations with primaryTranscriptAnnotation in R.

SUMMARY: Nascent transcript measurements derived from run-on sequencing experiments are critical for the investigation of transcriptional mechanisms and regulatory networks. However, conventional mRNA gene annotations significantly differ from the boundaries of primary transcripts. New primary transcript annotations are needed to accurately interpret run-on data. We developed the primaryTranscriptAnnotation R package to infer the transcriptional start and termination sites of primary transcripts from genomic run-on data. We then used these inferred coordinates to annotate transcriptional units identified de novo. This package provides the novel utility to integrate data-driven primary transcript annotations with transcriptional unit coordinates identified in an unbiased manner. Highlighting the importance of using accurate primary transcript coordinates, we demonstrate that this new methodology increases the detection of differentially expressed transcripts and provides more accurate quantification of RNA polymerase pause indices. AVAILABILITY AND IMPLEMENTATION: https://github.com/WarrenDavidAnderson/genomicsRpackage/tree/master/primaryTranscriptAnnotation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion.

Rapid perturbation of protein function permits the ability to define primary molecular responses while avoiding downstream cumulative effects of protein dysregulation. The auxin-inducible degron (AID) system was developed as a tool to achieve rapid and inducible protein degradation in nonplant systems. However, tagging proteins at their endogenous loci results in chronic auxin-independent degradation by the proteasome. To correct this deficiency, we expressed the auxin response transcription factor (ARF) in an improved inducible degron system. ARF is absent from previously engineered AID systems but is a critical component of native auxin signaling. In plants, ARF directly interacts with AID in the absence of auxin, and we found that expression of the ARF PB1 (Phox and Bem1) domain suppresses constitutive degradation of AID-tagged proteins. Moreover, the rate of auxin-induced AID degradation is substantially faster in the ARF-AID system. To test the ARF-AID system in a quantitative and sensitive manner, we measured genome-wide changes in nascent transcription after rapidly depleting the ZNF143 transcription factor. Transcriptional profiling indicates that ZNF143 activates transcription in cis and regulates promoter-proximal paused RNA polymerase density. Rapidly inducible degradation systems that preserve the target protein's native expression levels and patterns will revolutionize the study of biological systems by enabling specific and temporally defined protein dysregulation.

Ambulatory Surgery Center Medical Director: Visionary Leader.

The ambulatory surgery center medical director is a physician leader who recognizes the need to develop a culture that encourages communication and empowerment of employees and professional staff, leading to engagement that optimize care through patient selection, safety and satisfaction requires vision and guidance from the medical director and is central to success of the ASC. Innovative thinking further improves patient care and long-term success by leveraging advances in technology and sustainable practices.

Genetic and epigenetic determinants establish a continuum of Hsf1 occupancy and activity across the yeast genome.

Heat shock factor 1 is the master transcriptional regulator of molecular chaperones and binds to the same cis-acting heat shock element (HSE) across the eukaryotic lineage. In budding yeast, Hsf1 drives the transcription of ∼20 genes essential to maintain proteostasis under basal conditions, yet its specific targets and extent of inducible binding during heat shock remain unclear. Here we combine Hsf1 chromatin immunoprecipitation sequencing (seq), nascent RNA-seq, and Hsf1 nuclear depletion to quantify Hsf1 binding and transcription across the yeast genome. We find that Hsf1 binds 74 loci during acute heat shock, and these are linked to 46 genes with strong Hsf1-dependent expression. Notably, Hsf1's induced DNA binding leads to a disproportionate (∼7.5-fold) increase in nascent transcription. Promoters with high basal Hsf1 occupancy have nucleosome-depleted regions due to the presence of "pioneer factors." These accessible sites are likely critical for Hsf1 occupancy as the activator is incapable of binding HSEs within a stably positioned, reconstituted nucleosome. In response to heat shock, however, Hsf1 accesses nucleosomal sites and promotes chromatin disassembly in concert with the Remodels Structure of Chromatin (RSC) complex. Our data suggest that the interplay between nucleosome positioning, HSE strength, and active Hsf1 levels allows cells to precisely tune expression of the proteostasis network.

Identification of Drivers of Aneuploidy in Breast Tumors.

Although aneuploidy is found in the majority of tumors, the degree of aneuploidy varies widely. It is unclear how cancer cells become aneuploid or how highly aneuploid tumors are different from those of more normal ploidy. We developed a simple computational method that measures the degree of aneuploidy or structural rearrangements of large chromosome regions of 522 human breast tumors from The Cancer Genome Atlas (TCGA). Highly aneuploid tumors overexpress activators of mitotic transcription and the genes encoding proteins that segregate chromosomes. Overexpression of three mitotic transcriptional regulators, E2F1, MYBL2, and FOXM1, is sufficient to increase the rate of lagging anaphase chromosomes in a non-transformed vertebrate tissue, demonstrating that this event can initiate aneuploidy. Highly aneuploid human breast tumors are also enriched in TP53 mutations. TP53 mutations co-associate with the overexpression of mitotic transcriptional activators, suggesting that these events work together to provide fitness to breast tumors.