A large-scale cancer-specific protein-DNA interaction network.

Cancer development and progression are generally associated with gene dysregulation, often resulting from changes in the transcription factor (TF) sequence or expression. Identifying key TFs involved in cancer gene regulation provides a framework for potential new therapeutics. This study presents a large-scale cancer gene TF-DNA interaction network, as well as an extensive promoter clone resource for future studies. Highly connected TFs bind to promoters of genes associated with either good or poor cancer prognosis, suggesting that strategies aimed at shifting gene expression balance between these two prognostic groups may be inherently complex. However, we identified potential for oncogene-targeted therapeutics, with half of the tested oncogenes being potentially repressed by influencing specific activators or bifunctional TFs. Finally, we investigate the role of intrinsically disordered regions within the key cancer-related TF ESR1 in DNA binding and transcriptional activity, and found that these regions can have complex trade-offs in TF function. Altogether, our study broadens our knowledge of the TFs involved in cancer gene regulation and provides a valuable resource for future studies and therapeutics.

Widespread variation in molecular interactions and regulatory properties among transcription factor isoforms.

Most human Transcription factors (TFs) genes encode multiple protein isoforms differing in DNA binding domains, effector domains, or other protein regions. The global extent to which this results in functional differences between isoforms remains unknown. Here, we systematically compared 693 isoforms of 246 TF genes, assessing DNA binding, protein binding, transcriptional activation, subcellular localization, and condensate formation. Relative to reference isoforms, two-thirds of alternative TF isoforms exhibit differences in one or more molecular activities, which often could not be predicted from sequence. We observed two primary categories of alternative TF isoforms: "rewirers" and "negative regulators", both of which were associated with differentiation and cancer. Our results support a model wherein the relative expression levels of, and interactions involving, TF isoforms add an understudied layer of complexity to gene regulatory networks, demonstrating the importance of isoform-aware characterization of TF functions and providing a rich resource for further studies.

A large-scale cancer-specific protein-DNA interaction network.

Cancer development and progression are generally associated with dysregulation of gene expression, often resulting from changes in transcription factor (TF) sequence or expression. Identifying key TFs involved in cancer gene regulation provides a framework for potential new therapeutics. This study presents a large-scale cancer gene TF-DNA interaction network as well as an extensive promoter clone resource for future studies. Most highly connected TFs do not show a preference for binding to promoters of genes associated with either good or poor cancer prognosis, suggesting that emerging strategies aimed at shifting gene expression balance between these two prognostic groups may be inherently complex. However, we identified potential for oncogene targeted therapeutics, with half of the tested oncogenes being potentially repressed by influencing specific activator or bifunctional TFs. Finally, we investigate the role of intrinsically disordered regions within the key cancer-related TF estrogen receptor ɑ (ESR1) on DNA binding and transcriptional activity, and found that these regions can have complex trade-offs in TF function. Altogether, our study not only broadens our knowledge of TFs involved in the cancer gene regulatory network but also provides a valuable resource for future studies, laying a foundation for potential therapeutic strategies targeting TFs in cancer.

Transcription factors in the development and treatment of immune disorders.

Immune function is highly controlled at the transcriptional level by the binding of transcription factors (TFs) to promoter and enhancer elements. Several TF families play major roles in immune gene expression, including NF-κB, STAT, IRF, AP-1, NRs, and NFAT, which trigger anti-pathogen responses, promote cell differentiation, and maintain immune system homeostasis. Aberrant expression, activation, or sequence of isoforms and variants of these TFs can result in autoimmune and inflammatory diseases as well as hematological and solid tumor cancers. For this reason, TFs have become attractive drug targets, even though most were previously deemed "undruggable" due to their lack of small molecule binding pockets and the presence of intrinsically disordered regions. However, several aspects of TF structure and function can be targeted for therapeutic intervention, such as ligand-binding domains, protein-protein interactions between TFs and with cofactors, TF-DNA binding, TF stability, upstream signaling pathways, and TF expression. In this review, we provide an overview of each of the important TF families, how they function in immunity, and some related diseases they are involved in. Additionally, we discuss the ways of targeting TFs with drugs along with recent research developments in these areas and their clinical applications, followed by the advantages and disadvantages of targeting TFs for the treatment of immune disorders.

Paired yeast one-hybrid assays to detect DNA-binding cooperativity and antagonism across transcription factors.

Cooperativity and antagonism between transcription factors (TFs) can drastically modify their binding to regulatory DNA elements. While mapping these relationships between TFs is important for understanding their context-specific functions, existing approaches either rely on DNA binding motif predictions, interrogate one TF at a time, or study individual TFs in parallel. Here, we introduce paired yeast one-hybrid (pY1H) assays to detect cooperativity and antagonism across hundreds of TF-pairs at DNA regions of interest. We provide evidence that a wide variety of TFs are subject to modulation by other TFs in a DNA region-specific manner. We also demonstrate that TF-TF relationships are often affected by alternative isoform usage and identify cooperativity and antagonism between human TFs and viral proteins from human papillomaviruses, Epstein-Barr virus, and other viruses. Altogether, pY1H assays provide a broadly applicable framework to study how different functional relationships affect protein occupancy at regulatory DNA regions.

Widespread perturbation of ETS factor binding sites in cancer.

Although >90% of somatic mutations reside in non-coding regions, few have been reported as cancer drivers. To predict driver non-coding variants (NCVs), we present a transcription factor (TF)-aware burden test based on a model of coherent TF function in promoters. We apply this test to NCVs from the Pan-Cancer Analysis of Whole Genomes cohort and predict 2555 driver NCVs in the promoters of 813 genes across 20 cancer types. These genes are enriched in cancer-related gene ontologies, essential genes, and genes associated with cancer prognosis. We find that 765 candidate driver NCVs alter transcriptional activity, 510 lead to differential binding of TF-cofactor regulatory complexes, and that they primarily impact the binding of ETS factors. Finally, we show that different NCVs within a promoter often affect transcriptional activity through shared mechanisms. Our integrated computational and experimental approach shows that cancer NCVs are widespread and that ETS factors are commonly disrupted.

Intragenic proviral elements support transcription of defective HIV-1 proviruses.

HIV-1 establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV proviruses has revealed that greater than 90% of integrated HIV genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, we observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription droplet digital PCR identified env and nef transcripts which lacked 5' untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5'UTR-deficient env transcripts were also identified in a cohort of people living with HIV (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5' rapid amplification of cDNA ends (RACE), we mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH.

Comprehensive mapping of the human cytokine gene regulatory network.

Proper cytokine gene expression is essential in development, homeostasis and immune responses. Studies on the transcriptional control of cytokine genes have mostly focused on highly researched transcription factors (TFs) and cytokines, resulting in an incomplete portrait of cytokine gene regulation. Here, we used enhanced yeast one-hybrid (eY1H) assays to derive a comprehensive network comprising 1380 interactions between 265 TFs and 108 cytokine gene promoters. Our eY1H-derived network greatly expands the known repertoire of TF-cytokine gene interactions and the set of TFs known to regulate cytokine genes. We found an enrichment of nuclear receptors and confirmed their role in cytokine regulation in primary macrophages. Additionally, we used the eY1H-derived network as a framework to identify pairs of TFs that can be targeted with commercially-available drugs to synergistically modulate cytokine production. Finally, we integrated the eY1H data with single cell RNA-seq and phenotypic datasets to identify novel TF-cytokine regulatory axes in immune diseases and immune cell lineage development. Overall, the eY1H data provides a rich resource to study cytokine regulation in a variety of physiological and disease contexts.

Prediction of genome-wide effects of single nucleotide variants on transcription factor binding.

Single nucleotide variants (SNVs) located in transcriptional regulatory regions can result in gene expression changes that lead to adaptive or detrimental phenotypic outcomes. Here, we predict gain or loss of binding sites for 741 transcription factors (TFs) across the human genome. We calculated 'gainability' and 'disruptability' scores for each TF that represent the likelihood of binding sites being created or disrupted, respectively. We found that functional cis-eQTL SNVs are more likely to alter TF binding sites than rare SNVs in the human population. In addition, we show that cancer somatic mutations have different effects on TF binding sites from different TF families on a cancer-type basis. Finally, we discuss the relationship between these results and cancer mutational signatures. Altogether, we provide a blueprint to study the impact of SNVs derived from genetic variation or disease association on TF binding to gene regulatory regions.

Discovering human transcription factor physical interactions with genetic variants, novel DNA motifs, and repetitive elements using enhanced yeast one-hybrid assays.

Identifying transcription factor (TF) binding to noncoding variants, uncharacterized DNA motifs, and repetitive genomic elements has been technically and computationally challenging. Current experimental methods, such as chromatin immunoprecipitation, generally test one TF at a time, and computational motif algorithms often lead to false-positive and -negative predictions. To address these limitations, we developed an experimental approach based on enhanced yeast one-hybrid assays. The first variation of this approach interrogates the binding of >1000 human TFs to repetitive DNA elements, while the second evaluates TF binding to single nucleotide variants, short insertions and deletions (indels), and novel DNA motifs. Using this approach, we detected the binding of 75 TFs, including several nuclear hormone receptors and ETS factors, to the highly repetitive Alu elements. Further, we identified cancer-associated changes in TF binding, including gain of interactions involving ETS TFs and loss of interactions involving KLF TFs to different mutations in the TERT promoter, and gain of a MYB interaction with an 18-bp indel in the TAL1 superenhancer. Additionally, we identified TFs that bind to three uncharacterized DNA motifs identified in DNase footprinting assays. We anticipate that these enhanced yeast one-hybrid approaches will expand our capabilities to study genetic variation and undercharacterized genomic regions.

Comparison of the diagnostic performance of the 2017 ACR TI-RADS guideline to the Kwak guideline in children with thyroid nodules.

BACKGROUND: The Kwak Thyroid Imaging Reporting and Data System (Kwak-TI-RADS) guideline (2011) and American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS) guideline (2017) were developed as ultrasound (US) risk stratification tools for detecting thyroid malignancy in adults. OBJECTIVE: The purpose of this study was to investigate the inter-rater reliability and diagnostic performance of the ACR TI-RADS guideline in the pediatric population and compare it to the Kwak guideline. MATERIALS AND METHODS: This retrospective study comprised 75 children who underwent thyroid US at a tertiary-level pediatric hospital. Three pediatric radiologists and one pediatric radiology fellow graded the US findings using the Kwak-TI-RADS and ACR TI-RADS guidelines. We assessed reliability of radiologists' ratings using percentage inter-rater agreement, and intra-class correlation coefficients (ICC2,1). We assessed area-under-the-receiver-operating-characteristic curve (AUROCC) to compare the discriminative diagnostic ability of the Kwak-TI-RADS and ACR TI-RADS scoring systems against histopathology/cytology, or stability on US over a 2-year follow-up period for cases without tissue diagnosis. RESULTS: The inter-rater agreement was significantly better for the ACR TI-RADS level compared to the Kwak-TI-RADS level (P<0.001) using the percentage pairwise agreement. The ROC curves for assessing the diagnostic performance of the two methods showed no significant difference between the methods. The AUROCCs for the Kwak-TI-RADS and ACR TI-RADS levels were 0.74 (95% confidence interval [CI] 0.67-0.82) and 0.72 (95% CI 0.61-0.82), respectively. CONCLUSION: Both the Kwak-TI-RADS and ACR TI-RADS guidelines provide moderate malignancy risk stratification for thyroid nodules in the pediatric population, with better inter-rater agreement for the ACR TI-RADS guideline. Further work to adjust the recommendations for pediatric patients is necessary.

Identification of Single Nucleotide Non-coding Driver Mutations in Cancer.

Recent whole-genome sequencing studies have identified millions of somatic variants present in tumor samples. Most of these variants reside in non-coding regions of the genome potentially affecting transcriptional and post-transcriptional gene regulation. Although a few hallmark examples of driver mutations in non-coding regions have been reported, the functional role of the vast majority of somatic non-coding variants remains to be determined. This is because the few driver variants in each sample must be distinguished from the thousands of passenger variants and because the logic of regulatory element function has not yet been fully elucidated. Thus, variants prioritized based on mutational burden and location within regulatory elements need to be validated experimentally. This is generally achieved by combining assays that measure physical binding, such as chromatin immunoprecipitation, with those that determine regulatory activity, such as luciferase reporter assays. Here, we present an overview of in silico approaches used to prioritize somatic non-coding variants and the experimental methods used for functional validation and characterization.

Currarino Syndrome in a Fetus, Infant, Child, and Adolescent: Spectrum of Clinical Presentations and Imaging Findings.

In 1981, Currarino et al described a triad of findings that consist of partial sacral dysgenesis, presacral mass (anterior meningocele, enteric cyst, or presacral teratoma) and anorectal malformation. Currarino syndrome exhibits variable expressivity and the clinical presentation tends to vary with the age of the subject such as spinal anomaly detected in the fetus, imperforate anus in the newborn, and intractable constipation or neurologic symptoms in the infant and older child. At any age, meningitis can be the presenting symptom and imaging is required for proper investigation. Meningitis, sepsis, urinary tract infections, and, rarely, malignant transformation of a teratoma are serious potential complications. This pictorial review describes the imaging findings, clinical history, surgical interventions, and genetic background in 5 children with this syndrome who presented in our hospital in the interval of 1 year.

Formula-feeding and hypertrophic pyloric stenosis: is there an association? A case-control study.

BACKGROUND: The etiology of infantile hypertrophic pyloric stenosis (HPS) is not fully understood. The objective of this study was to determine whether formula-feeding is associated with increased incidence. METHODS: This case-control study included HPS cases and controls admitted between 1992 and 2012. Demographic data including feeding method were collected from patient charts and analyzed. RESULTS: We identified 882 HPS cases and 955 controls. The highest incidence of HPS presentation was in summer (P=0.0028). Infants with HPS were more likely to have been exclusively formula-fed, have a family history of HPS, and be male compared to infants in the control group (P<0.001); they were also more likely to live in rural areas, although not significantly so. After adjusting for family history, sex, place of residence, and season of presentation, exclusively formula-fed infants were 1.36 times more likely to develop HPS compared with exclusively breastfed infants (RR 1.36, 95% CI 1.18-1.57, P<0.005). CONCLUSIONS: Formula-feeding is associated with significantly increased risk of HPS. Further investigation may help to determine the components of formula that simulate hypertrophy of the pylorus muscle, or the components of breast milk that are protective, as well as other influencing factors. LEVEL OF EVIDENCE: 3b.

Histologic inflammatory activity of the rectal margin as a predictor of postoperative complication in ileoanal anastomosis (J-pouch) procedure in children with refractory ulcerative colitis.

BACKGROUND: Postoperative complication from the ileoanal anastomosis (J-pouch) procedure for surgical management of refractory ulcerative colitis (UC) increases the risk of pouch dysfunction. The purpose of this study was to determine if histologic inflammatory activity of the rectal margin is an independent predictor of complication after controlling for other variables. METHODS: A retrospective chart review was performed to identify all pediatric patients with UC who underwent a J-pouch procedure between 1995 and 2014. Univariate and multivariate regression analyses were performed on the following variables: age at surgery, body mass index, comorbidities, time between colectomy and pouch, mucosectomy, protective ileostomy status, length of pouch, and histologic inflammatory activity in the intestinal epithelium of the rectal margin. RESULTS: Nineteen complicated and 23 uncomplicated cases were included. Histologic inflammatory activity was significantly higher among the complicated group (9.3±3.1 vs. 4.1±3.1, p=0.02). No significant difference was found regarding other variables. In a multivariate regression, histologic inflammation of the rectal margin remained significantly associated with complication (p=0.04) after adjusting for other factors. CONCLUSION: After controlling for potential confounders, histologic inflammatory activity at the rectal margin was found to be a significant predictor of postoperative complication in the J-pouch procedure for refractory UC. LEVEL OF EVIDENCE: 2b.

Effect of delivery approach on outcomes in fetuses with gastroschisis.

BACKGROUND/PURPOSE: There is considerable controversy regarding optimal mode and timing of delivery for fetuses with gastroschisis. Our objectives were to describe the variation in institutional approach regarding these factors, and to evaluate the effect of timing of delivery on outcomes in fetuses with gastroschesis. METHODS: Members of the maternal-fetal medicine community across Canada were surveyed regarding their personal and institutional approach of delivery. Data from the Canadian Pediatric Surgery Network (CAPSnet) were analyzed. RESULTS: The survey showed significant variability in delivery approach between institutions, although no center routinely performs cesarean section. Infants delivered vaginally (VD) were categorized into three groups: Group 1, VD <36 weeks (n=114); Group 2, VD 36-37 weeks (n=218); and Group 3, VD ≥38 weeks (n=75). Score of Neonatal Acute Physiology, complication rates, length of time on total parenteral nutrition (TPN), and length of hospital stay (LOS) were higher in Group 1; bowel matting was greater in Group 3. There were no differences between the groups regarding other complications. CONCLUSIONS: Our data suggest that preterm delivery was associated with more complications, longer time on TPN, and longer LOS; delivery ≥38 weeks was associated with increased bowel matting. These outcomes should be considered when determining institutional protocol.

Thoracoscopic vs open resection of congenital lung lesions: a meta-analysis.

BACKGROUND: Several comparative studies are published evaluating both the open and the minimally invasive approaches for congenital lung lesions with inconsistent results. Our objective was to compare both procedures using systematic review and meta-analysis methodology. METHODS: All publications describing both interventions were reviewed. The statistical analysis was performed using RevMan 5 software (Cochrane library). MAIN RESULTS: No randomized trials were identified. Six retrospective studies were identified and were included in this study. There was no significant difference in overall complication rates between both techniques. Lengths of hospital stay as well as days with chest tube in place were longer with the open approach. There was no difference in the duration of surgery. Postoperative pain management was heterogeneous between studies. No study looked at long-term follow-up. Subgroup analysis for congenital cystic adenomatoid malformation of the lung was done. CONCLUSIONS: Our results suggest no differences between thoracotomy versus thoracoscopy for congenital lung lesions with respect to overall complications and the duration of surgery. However, length of hospital stay and days with chest tube in place were longer after the open approach. Thoracoscopic resection is a safe and feasible alternative to open resection of congenital lung lesions in experienced hands.

Analysis of the clinical significance and cost associated with the routine pathological analysis of pediatric inguinal hernia sacs.

PURPOSE: Pediatric inguinal and scrotal surgeries for inguinal hernia, cryptorchidism and hydrocele are common and usually involve the excision of a hernia sac. Groups at many centers send hernia sacs for pathological analysis to identify occult disease as well as structures that may have been erroneously resected. We hypothesized that, since the incidence of significant findings is low and the associated health care costs are significant, the routine pathological analysis of inguinal hernia sacs is unnecessary. MATERIALS AND METHODS: After receiving institutional review board approval we retrospectively reviewed pathology reports at our institution of patients who underwent surgery with an inguinal hernia sac sent for pathological analysis from January 2000 to September 2009. The primary outcome was to determine the incidence of clinically significant structures in hernia sac specimens. The secondary outcome was to evaluate the costs associated with analyzing these specimens. RESULTS: A total of 2,287 boys and 441 girls underwent some form of inguinal or scrotal surgery during the study. In the 2,287 boys a total of 2,657 hernia sac specimens were analyzed, of which 2 (0.08%) contained clusters of epididymal-like tubules. Most unexpected findings were likely clinically insignificant, including mesothelial proliferation in 5.6% of cases, genital duct remnants in 0.8%, lipoma in 0.23% and adrenocortical rests in 0.04%. The average cost of analyzing hernia sac specimens at our institution was approximately $7,100 Canadian annually. CONCLUSIONS: Routine analysis of inguinal hernia sacs is unnecessary and costly, and should be reserved for cases in which resection of important structures such as the vas deferens is suspected.

Central venous catheter database: an important issue in quality assurance.

BACKGROUND/PURPOSE: The purpose of this study was to analyze the factors that affect the longevity of central venous catheters. METHODS: Comprehensive clinical data recorded during insertion and removal of totally implantable devices (TID) and tunneled lines (TL) from October 1988 to January 2009 were analyzed. Univariate and multivariate Cox proportional hazards regression models were used to identify clinical factors that predict catheter longevity. RESULTS: Information was available for 1167 central venous catheter insertions in 858 patients, 648 TID and 509 TL. Univariate analysis detected longer device longevity in the following: TID longer than TL (P < .0001), catheter tip in the superior vena cava (SVC)/right atrial junction (P < .0001), and right side greater than left (P = .002). Shorter device longevity was observed in lines used for total parenteral nutrition (P < .0001) and young age (P < .0001). Multivariate model detected the following: hazard of removal for TID is 0.304 that of TL (P < .0001) and SVC is 0.525 that of other locations (P = .0005). Hazard decreases by 5.4% for every 1-year increase in patient age (P < .0004). CONCLUSION: Multiple confounding factors were encountered. However, the single most important factor in catheter longevity that is influenced by the surgeon is tip location in the SVC/right atrial junction.

Flagellin delays spontaneous human neutrophil apoptosis.

Neutrophils are short-lived cells that rapidly undergo apoptosis. However, their survival can be regulated by signals from the environment. Flagellin, the primary component of the bacterial flagella, is known to induce neutrophil activation. In this study we examined the ability of flagellin to modulate neutrophil apoptosis. Neutrophils cultured for 12 and 24 h in the presence of flagellin from Salmonella typhimurium at concentrations found in pathological situations underwent a marked prevention of apoptosis. In contrast, Helicobacter pylori flagellin did not affect neutrophil survival, suggesting that Salmonella flagellin exerts the antiapoptotic effect by interacting with TLR5. The delaying in apoptosis mediated by Salmonella flagellin was coupled to higher expression levels of the antiapoptotic protein Mcl-1 and lower levels of activated caspase-3. Analysis of the signaling pathways indicated that Salmonella flagellin induced the activation of the p38 and ERK1/2 MAPK pathways as well as the PI3K/Akt pathway. Furthermore, it also stimulated IkappaBalpha degradation and the phosphorylation of the p65 subunit, suggesting that Salmonella flagellin also triggers NF-kappaB activation. Moreover, the pharmacological inhibition of ERK1/2 pathway and NF-kappaB activation partially prevented the antiapoptotic effects exerted by flagellin. Finally, the apoptotic delaying effect exerted by flagellin was also evidenced when neutrophils were cultured with whole heat-killed S. typhimurium. Both a wild-type and an aflagellate mutant S. typhimurium strain promoted neutrophil survival; however, when cultured in low bacteria/neutrophil ratios, the flagellate bacteria showed a higher capacity to inhibit neutrophil apoptosis, although both strains showed a similar ability to induce neutrophil activation. Taken together, our results indicate that flagellin delays neutrophil apoptosis by a mechanism partially dependent on the activation of ERK1/2 MAPK and NF-kappaB. The ability of flagellin to delay neutrophil apoptosis could contribute to perpetuate the inflammation during infections with flagellated bacteria.