PLS3 missense variants affecting the actin-binding domains cause X-linked congenital diaphragmatic hernia and body-wall defects.

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with an X-linked condition characterized by diaphragm defects, variable anterior body-wall anomalies, and/or facial dysmorphism. Using linkage analysis and exome or genome sequencing, we found that missense variants in plastin 3 (PLS3), a gene encoding an actin bundling protein, co-segregate with disease in all families. Loss-of-function variants in PLS3 have been previously associated with X-linked osteoporosis (MIM: 300910), so we used in silico protein modeling and a mouse model to address these seemingly disparate clinical phenotypes. The missense variants in individuals with CDH are located within the actin-binding domains of the protein but are not predicted to affect protein structure, whereas the variants in individuals with osteoporosis are predicted to result in loss of function. A mouse knockin model of a variant identified in one of the CDH-affected families, c.1497G>C (p.Trp499Cys), shows partial perinatal lethality and recapitulates the key findings of the human phenotype, including diaphragm and abdominal-wall defects. Both the mouse model and one adult human male with a CDH-associated PLS3 variant were observed to have increased rather than decreased bone mineral density. Together, these clinical and functional data in humans and mice reveal that specific missense variants affecting the actin-binding domains of PLS3 might have a gain-of-function effect and cause a Mendelian congenital disorder.

miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma.

Drug-tolerance is an acute defense response prior to a fully drug-resistant state and tumor relapse, however there are few therapeutic agents targeting drug-tolerance in the clinic. Here we show that miR-147b initiates a reversible tolerant-state to the EGFR inhibitor osimertinib in non-small cell lung cancer. With miRNA-seq analysis we find that miR-147b is the most upregulated microRNA in osimertinib-tolerant and EGFR mutated lung cancer cells. Whole transcriptome analysis of single-cell derived clones reveals a link between osimertinib-tolerance and pseudohypoxia responses irrespective of oxygen levels. Further metabolomics and genetic studies demonstrate that osimertinib-tolerance is driven by miR-147b repression of VHL and succinate dehydrogenase linked to the tricarboxylic acid cycle and pseudohypoxia pathways. Finally, pretreatment with a miR-147b inhibitor delays osimertinib-associated drug tolerance in patient-derived three-dimensional (3D) structures. This link between miR-147b and tricarboxylic acid cycle may provide promising targets for preventing tumor relapse.

Identifying Indicators of National Council Licensure Examination for Registered Nurses (NCLEX-RN) Success in Nursing Graduates in Newfoundland & Labrador.

In Canada in 2015, the pass rates on the National Council Licensure Examination (NCLEX-RN) were considerably lower than pass rates on the Canadian Registered Nurse Examination (CRNE) causing nurse educators to express concern regarding the NCLEX-RN. The purpose of this study was to examine the relationship between candidate variables (e. g. academic performance, demographics) on their NCLEX-RN outcome (pass/fail). A cross-sectional data linkage design was employed using multiple sources of data on nursing graduates who wrote the NCLEX-RN in 2015, 2016 and 2017 (n = 259). Results showed that fewer questions answered on the NCLEX-RN and higher grades in various nursing courses (e. g. Introduction to Nursing, Statistics) predicted higher odds of passing the NCLEX-RN. To improve pass rates, nurse educators must integrate diverse methods of testing into existing curricula that mimic the NCLEX-RN exam, specifically computer adaptive exams. Further research is needed to determine other possible challenges for countries considering adopting the NCLEX-RN.

Temporal dynamics of the developing lung transcriptome in three common inbred strains of laboratory mice reveals multiple stages of postnatal alveolar development.

To characterize temporal patterns of transcriptional activity during normal lung development, we generated genome wide gene expression data for 26 pre- and post-natal time points in three common inbred strains of laboratory mice (C57BL/6J, A/J, and C3H/HeJ). Using Principal Component Analysis and least squares regression modeling, we identified both strain-independent and strain-dependent patterns of gene expression. The 4,683 genes contributing to the strain-independent expression patterns were used to define a murine Developing Lung Characteristic Subtranscriptome (mDLCS). Regression modeling of the Principal Components supported the four canonical stages of mammalian embryonic lung development (embryonic, pseudoglandular, canalicular, saccular) defined previously by morphology and histology. For postnatal alveolar development, the regression model was consistent with four stages of alveolarization characterized by episodic transcriptional activity of genes related to pulmonary vascularization. Genes expressed in a strain-dependent manner were enriched for annotations related to neurogenesis, extracellular matrix organization, and Wnt signaling. Finally, a comparison of mouse and human transcriptomics from pre-natal stages of lung development revealed conservation of pathways associated with cell cycle, axon guidance, immune function, and metabolism as well as organism-specific expression of genes associated with extracellular matrix organization and protein modification. The mouse lung development transcriptome data generated for this study serves as a unique reference set to identify genes and pathways essential for normal mammalian lung development and for investigations into the developmental origins of respiratory disease and cancer. The gene expression data are available from the Gene Expression Omnibus (GEO) archive (GSE74243). Temporal expression patterns of mouse genes can be investigated using a study specific web resource (http://lungdevelopment.jax.org).

A Phase I study of olaparib and irinotecan in patients with colorectal cancer: Canadian Cancer Trials Group IND 187.

Background Olaparib is an orally available inhibitor of PARP-1. In pre-clinical studies, olaparib was shown to potentiate anti-tumor effects of irinotecan in colon cancer cell lines. This phase I study was conducted to evaluate the safety and tolerability of olaparib in combination with irinotecan. Patients and Methods Patients with advanced colorectal cancer whose disease progressed after at least one systemic therapy regimen were enrolled. Dose escalation and de-escalation were based on toxicity assessment. Pharmacokinetic samples were collected in Cycle 1 for olaparib, irinotecan and SN-38. Results Twenty-five patients were enrolled, 11 patients on a schedule of continuous olaparib and irinotecan every 3 weeks (Part A) and 14 patients on a schedule of intermittent olaparib and irinotecan every 2 weeks (Part B). Continuous olaparib administration was associated with higher than expected toxicities and was not considered to be tolerable. Intermittent olaparib administration was better tolerated, and the recommended phase 2 doses were olaparib 50 mg p.o twice daily days 1-5 and irinotecan 125 mg/m(2) i.v. every 2 weeks. Common toxicities included fatigue, anorexia, diarrhea, nausea, vomiting, neutropenia, thrombocytopenia and abdominal pain. Nine patients had stable disease as the best response, 2 from Part A (3 and 9 months respectively), and 7 from Part B (median duration: 7.4 months; range: 4 to 13 months). There was no pharmacokinetic interaction between olaparib and irinotecan. Conclusions Olaparib can be combined with irinotecan if administered intermittently. Both olaparib and irinotecan required significant dose reductions. The lack of anti-tumor efficacy observed in this trial makes this combination of little interest for further clinical development. Trial Registration ID NCT00535353.

Molecular pathogenesis of congenital diaphragmatic hernia revealed by exome sequencing, developmental data, and bioinformatics.

Congenital diaphragmatic hernia (CDH) is a common and severe birth defect. Despite its clinical significance, the genetic and developmental pathways underlying this disorder are incompletely understood. In this study, we report a catalog of variants detected by a whole exome sequencing study on 275 individuals with CDH. Predicted pathogenic variants in genes previously identified in either humans or mice with diaphragm defects are enriched in our CDH cohort compared with 120 size-matched random gene sets. This enrichment was absent in control populations. Variants in these critical genes can be found in up to 30.9% of individuals with CDH. In addition, we filtered variants by using genes derived from regions of recurrent copy number variations in CDH, expression profiles of the developing diaphragm, protein interaction networks expanded from the known CDH-causing genes, and prioritized genes with ultrarare and highly disruptive variants, in 11.3% of CDH patients. These strategies have identified several high priority genes and developmental pathways that likely contribute to the CDH phenotype. These data are valuable for comparison of candidate genes generated from whole exome sequencing of other CDH cohorts or multiplex kindreds and provide ideal candidates for further functional studies. Furthermore, we propose that these genes and pathways will enhance our understanding of the heterogeneous molecular etiology of CDH.

Phase I clinical trial of Exherin (ADH-1) in patients with advanced solid tumors.

UNLABELLED: ADH-1 (Exherin™) is a pentapeptide, which competitively inhibits N-cadherin, resulting in vascular disruptive effect of tumor vasculature in preclinical models. This study was designed to assess the toxicity of ADH-1 and to determine the maximal tolerated dose (MTD). PATIENTS AND METHODS: Adult patients with advanced measurable solid tumors were stratified according to their tumor N-cadherin status. ADH-1 was administered as a short infusion, every six weeks. Assessment of response was done every 6 weeks. PK parameters included: estimated volume of distribution of the central compartment, the α and ß phase half-lives, area under the plasma concentration- time curve (AUC), clearance, and volume of distribution. Target lesions were assessed by dynamic contrast enhancing- magnetic resonance imaging (DCE-MRI). RESULTS: 46 patients were enrolled, 25 (54%) had N-cadherin positive status. The doses administered ranged from 50 mg/m2 to 1000 mg/m2, and the MTD was not reached. The PK analysis of the concentration-time data displayed a biphasic profile. Most of the toxicities were grade 1 and 2 with fatigue, nausea, chest pain and dysgeusia being the most common. Eleven patients had disease control, the single patient who had partial response had N-cadherin positive tumor. CONCLUSION: ADH-1 is a well tolerated drug with a modest anti tumor effect in tumors which express N-cadherin.

Congenital diaphragmatic hernia candidate genes derived from embryonic transcriptomes.

Congenital diaphragmatic hernia (CDH) is a common (1 in 3,000 live births) major congenital malformation that results in significant morbidity and mortality. The discovery of CDH loci using standard genetic approaches has been hindered by its genetic heterogeneity. We hypothesized that gene expression profiling of developing embryonic diaphragms would help identify genes likely to be associated with diaphragm defects. We generated a time series of whole-transcriptome expression profiles from laser captured embryonic mouse diaphragms at embryonic day (E)11.5 and E12.5 when experimental perturbations lead to CDH phenotypes, and E16.5 when the diaphragm is fully formed. Gene sets defining biologically relevant pathways and temporal expression trends were identified by using a series of bioinformatic algorithms. These developmental sets were then compared with a manually curated list of genes previously shown to cause diaphragm defects in humans and in mouse models. Our integrative filtering strategy identified 27 candidates for CDH. We examined the diaphragms of knockout mice for one of the candidate genes, pre-B-cell leukemia transcription factor 1 (Pbx1), and identified a range of previously undetected diaphragmatic defects. Our study demonstrates the utility of genetic characterization of normal development as an integral part of a disease gene identification and prioritization strategy for CDH, an approach that can be extended to other diseases and developmental anomalies.

ADH-1 in the treatment of metastatic adrenocortical carcinoma--case report.

Adrenocortical Carcinoma (ACC) is rare with an annual incidence of 0.5-2 cases per million worldwide. Some ACC tumors over express N-cadherin, which correlates with metastatic potential. ADH-1 (Exherin™) is a competitive inhibitor of N-cadherin, resulting in rapid onset of tumor vascular angiolysis and apoptosis in preclinical models. Targeting N-cadherin may cause direct anti-tumor and anti-vascular effects. We report a case of ACC with benefit from ADH-1 therapy. A 24 year old woman with an N-cadherin expressing metastatic ACC was treated on a phase I trial and treated with ADH-1 subsequently received additional doses through a special access program. The patient presented with cushingoid features from cortisol over-secretion and was diagnosed with metastatic ACC in January 2003. Tumor progression followed treatment with a combination of doxorubicin, cisplatin and mitotane. In October 2003, as a part of a phase I clinical trial she was treated with as a single dose of ADH-1 at 150 mg/m(2). This resulted in transient normalization of cortisol, tumor necrosis on CT imaging, and reduction in tumor perfusion on DCE-MRI. Following progression on several additional lines of chemotherapy, she was again treated with ADH-1 under a Special Access Program (SAP). After 33 weekly doses (22 with 150 mg/m(2) and 11 with 300 mg/m(2)) radiographic tumor progression was demonstrated and treatment discontinued. She survived 40 months with metastatic disease, dying 12 months after her last dose of ADH-1. This observation merits consideration for prospectively evaluating the efficacy of ADH-1 in patients with cortisol secreting ACC that over express N-cadherin.

The predominant WT1 isoform (+KTS) encodes a DNA-binding protein targeting the planar cell polarity gene Scribble in renal podocytes.

WT1 encodes a tumor suppressor first identified by its inactivation in Wilms' Tumor. Although one WT1 splicing variant encodes a well-characterized zinc finger transcription factor, little is known about the function of the most prevalent WT1 isoform, whose DNA binding domain is disrupted by a three-amino acid (KTS) insertion. Using cells that conditionally express WT1(+KTS), we undertook a genome-wide chromatin immunoprecipitation and cloning analysis to identify candidate WT1(+KTS)-regulated promoters. We identified the planar cell polarity gene Scribble (SCRB) as the first WT1(+KTS) target gene in podocytes of the kidney. WT1 and SCRB expression patterns overlap precisely in developing renal glomeruli of mice, and WT1(+KTS) binds to a 33-nucleotide region within the Scribble promoter in mouse and human cell lines and kidneys. Together, our results support a role for the predominant WT1(+KTS) isoform in transcriptional regulation and suggest a link between the WT1-dependent tumor suppressor pathway and a key component of the planar cell polarity pathway.

Using siRNA knockdown in HaCaT cells to study transcriptional control of epidermal proliferation potential.

Compared to primary keratinocytes, HaCaT cells are easier to transfect and yet still maintain at least some features of normal epidermal proliferation and differentiation. This chapter describes methods used in our laboratory to maintain HaCaT cells in an undifferentiated state and to use the siRNA technology to efficiently deplete a gene product of interest from these cells. We also provide protocols on how to couple siRNA knockdown with a clonal assay to examine keratinocyte proliferation potential and a luciferase reporter assay to examine promoter regulation.

Ovol2 suppresses cell cycling and terminal differentiation of keratinocytes by directly repressing c-Myc and Notch1.

Ovol2 belongs to the Ovo family of evolutionarily conserved zinc finger transcription factors that act downstream of key developmental signaling pathways including Wg/Wnt and BMP/TGF-beta. We previously reported Ovol2 expression in the basal layer of epidermis, where epidermal stem/progenitor cells reside. In this work, we use HaCaT human keratinocytes to investigate the cellular and molecular functions of Ovol2. We show that depletion of Ovol2 leads to transient cell expansion but a loss of cells with long term proliferation potential. Mathematical modeling and experimental findings suggest that both faster cycling and precocious withdrawal from the cell cycle underlie this phenotype. Ovol2 depletion also accelerates extracellular signal-induced terminal differentiation in two- and three-dimensional culture models. By chromatin immunoprecipitation, luciferase reporter, and functional rescue assays, we demonstrate that Ovol2 directly represses two critical downstream targets, c-Myc and Notch1, thereby suppressing keratinocyte transient proliferation and terminal differentiation, respectively. These findings shed light on how an epidermal cell maintains a proliferation-competent and differentiation-resistant state.

The tumor suppressor WTX shuttles to the nucleus and modulates WT1 activity.

WTX encodes a tumor suppressor gene inactivated in Wilms tumor and recently implicated in WNT signaling through enhancement of cytoplasmic beta-catenin (CTNNB1) degradation. Here, we report that WTX translocates to the nucleus, a property that is modified by an endogenous splicing variant and is modulated by a nuclear export inhibitor. WTX is present in distinct subnuclear structures and co-localizes with the paraspeckle marker p54NRB/NONO, suggesting a role in transcriptional regulation. Notably, WTX binds WT1, another Wilms tumor suppressor and stem cell marker that encodes a zinc-finger transcription factor, and enhances WT1-mediated transcription of Amphiregulin, an endogenous target gene. Together, these observations suggest a role for WTX in nuclear pathways implicated in the transcriptional regulation of cellular differentiation programs.

Phase I study of cediranib in combination with oxaliplatin and infusional 5-Fluorouracil in patients with advanced colorectal cancer.

PURPOSE: Cediranib is a potent oral inhibitor of the tyrosine kinase activity associated with all subtypes of vascular endothelial growth factor receptor. Purposes of this study were to determine the recommended phase II dose of cediranib in combination with standard doses of modified FOLFOX-6 (mFOLFOX-6), and the tolerability, safety, pharmacokinetics, and antitumor activity of this combination in patients with untreated metastatic colorectal cancer. EXPERIMENTAL DESIGN: Cediranib was administered daily orally at a starting dose of 30 mg and escalated to 45 mg daily, and mFOLFOX-6 was repeated every 14 days. Pharmacokinetic studies were done for oxaliplatin, 5-fluorouracil, and cediranib. Response was assessed by Response Evaluation Criteria in Solid Tumors every four cycles. RESULTS: Sixteen patients received 150 cycles of treatment (median, 6; range, 1-20 cycles). Of 9 patients enrolled at the 30-mg dose level, 1 patient experienced grade 3 diarrhea during cycle 1. No dose-limiting toxicity was observed in 7 patients at the 45-mg dose level. Common grade 3 toxicities related to cediranib included hypertension, diarrhea, fatigue, and anorexia. Of 14 patients evaluable for response, there were 6 partial responses (42.9%; 95% confidence interval, 17.7-71.1%) and 6 stable disease. The median progression-free survival was 9.3 months. There were no pharmacokinetic interactions between cediranib and 5-fluorouracil or free plasma intact oxaliplatin. CONCLUSIONS: Toxicities of this combination were manageable and consistent with previous studies. The recommended phase II dose is cediranib at 30 mg daily continuously in combination with standard doses of mFOLFOX-6. Cediranib and mFOLFOX-6 has promising antitumor activity and this combination warrants further investigation.

An X chromosome gene, WTX, is commonly inactivated in Wilms tumor.

Wilms tumor is a pediatric kidney cancer associated with inactivation of the WT1 tumor-suppressor gene in 5 to 10% of cases. Using a high-resolution screen for DNA copy-number alterations in Wilms tumor, we identified somatic deletions targeting a previously uncharacterized gene on the X chromosome. This gene, which we call WTX, is inactivated in approximately one-third of Wilms tumors (15 of 51 tumors). Tumors with mutations in WTX lack WT1 mutations, and both genes share a restricted temporal and spatial expression pattern in normal renal precursors. In contrast to biallelic inactivation of autosomal tumor-suppressor genes, WTX is inactivated by a monoallelic "single-hit" event targeting the single X chromosome in tumors from males and the active X chromosome in tumors from females.

Strain-dependent perinatal lethality of Ovol1-deficient mice and identification of Ovol2 as a downstream target of Ovol1 in skin epidermis.

Ovol1 encodes a zinc finger transcriptional repressor that is downstream of the LEF1/beta-catenin complex, nuclear effectors of canonical Wnt signaling. Previous gene knockout studies performed in a 129SvxC57BL/6 mixed genetic background revealed that Ovol1-deficient mice survive to adulthood but display multiple tissue defects. In this study, we describe a C57BL/6 strain-specific reduction in perinatal survival of Ovol1 mutant mice. The perinatal lethality is accompanied by kidney epithelial cysts of embryonic onset and delayed skin barrier acquisition. Genetic analysis suggests a partial functional compensation by Ovol2 for the loss of Ovol1. The expression of Ovol2 was up-regulated in Ovol1-deficient epidermis, and Ovol1 represses the activity of Ovol2 promoter in a DNA binding-dependent manner. Collectively, these studies uncover novel functions of Ovol1 in mouse development and identify Ovol2 as a downstream target of Ovol1.

Prostate cancer screening attitudes and continuing education needs of primary care physicians.

BACKGROUND: In May 2003, a survey questionnaire was distributed to all licensed primary care physicians in Newfoundland and Labrador. The objective was to examine the attitudes, self-reported practices, and continuing medical education (CME) preferences of primary care physicians as they pertain to prostate cancer screening. METHODS: Data was obtained from 485 primary care physicians using self-reports of prostate cancer screening practices, attitudes towards prostate cancer screening, and CME preferences. Respondents' characteristics were also collected (eg, gender, years of experience). RESULTS: A majority of respondents screen asymptomatic male patients for prostate cancer. Screening behaviour was related to high volume practice settings, fee-for-service and increased with patient age. Most common reasons for screening were family history, age of patient, and patient request. Majority of physicians agreed that prostate screening should be routinely performed on all men beginning at age 50, however half of physicians believe there is lack of evidence to support digital rectal examination (DRE) and one-third of physicians do not believe the prostate-specific antigen (PSA) nor DRE are accurate screening tests. Areas of greatest interest for CME included topics related to prostate cancer screening effectiveness, strategies for prevention, sexual dysfunction, available treatments and their side effects, and management options. CONCLUSION: Physicians are supportive of the value of screening, however the reliability of and evidence to support DRE and PSA as prostate cancer screening tests are in question. CME which addresses issues surrounding prostate screening and areas related to patient education and counselling are of greatest need.

SUMO-1 modification of the Wilms' tumor suppressor WT1.

SUMO-1 conjugation modulates numerous cellular functions, including the subnuclear localization of its target proteins. The WT1 tumor suppressor encodes a four-zinc finger protein with distinct splicing isoforms. WT1(-KTS), encoding uninterrupted zinc fingers, functions as a transcription factor and has a diffusely nuclear distribution; WT1(+KTS), with an insertion of three amino acids (KTS) between zinc fingers three and four, localizes to discrete nuclear speckles, the function of which is unknown. Because the SUMO-1 E2-conjugating enzyme, Ubc9, interacts with WT1, we tested whether sumoylation modulates the cellular localization of WT1. We find here that both WT1 isoforms are directly sumoylated on lysine residues 73 and 177. Although RNA interference-mediated Ubc9 depletion effectively suppresses WT1 nuclear speckles, a SUMO-1-deficient WT1(+KTS)(K73, 177R) double mutant retains localization to speckles. Thus, direct sumoylation of WT1 is not responsible for its cellular localization, and other sumoylated proteins may target WT1 to these nuclear structures. Identification of other components of WT1-associated speckles is likely to provide clues to their function.

Identification of novel pRb binding sites using CpG microarrays suggests that E2F recruits pRb to specific genomic sites during S phase.

The retinoblastoma (Rb) tumor suppressor protein is an important regulator of cell proliferation and differentiation. Many studies have shown that pRb can negatively regulate the activity of the E2F family of transcription factors during G(0) and G(1) phases of the cell cycle, perhaps by serving as a bridge between the E2Fs and transcriptional repressors such as histone deacetylases and methylases. However, pRb has also been shown to localize to discrete DNA foci during S phase, a time at which pRb is thought to be dissociated from E2F. Numerous other DNA binding proteins have been shown to interact with pRb, suggesting that pRb may control progression through S phase by binding to sites in the genome distinct from E2F target gene promoters. To test this hypothesis, we have identified novel pRb binding sites within the human genome using an unbiased approach which relies upon a combination of chromatin immunoprecipitation and CpG microarray analysis. To provide the greatest opportunity of finding distinct sets of pRb binding sites, we examined pRb binding in chromatin obtained from human Raji cells synchronized in either G(0)/G(1) phase or S phase. These experiments have allowed us to identify a large set of new genomic binding sites for the pRb protein. We found that some sites are occupied by pRb only during G(0)/G(1) phase, as would be predicted from previous models of pRb function. We also identified sites to which pRb bound only during S phase and other sites which were bound constitutively by pRb. Surprisingly, we found that E2F1 was present at most of the CpG islands bound by pRb, independent of the phase of the cell cycle. Thus, although pRb has the potential to interact with numerous transcription factors, our data suggest that the majority of DNA-bound pRb is recruited to E2F target promoters during both G(0)/G(1) and S phases.

Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation.

In their article, A.S. Weinmann and P.J. Farnham (2002, Methods 26) described new techniques for isolating in vivo binding sites for any DNA-binding protein. In this article, we describe complementary methods for detailed in vivo characterizations of such identified protein-DNA interactions. First, we describe how formaldehyde crosslinking and chromatin immunoprecipitation (ChIP), in conjunction with transient transfections or the use of cell lines containing stably integrated constructs or episomes, can be employed to identify which specific nucleotides of a region of DNA are required for recruitment of a particular transcription factor. In contrast to in vivo footprinting, this method not only specifies which nucleotides are bound, but also identifies the protein(s) involved in binding. Next, we discuss the use of the ChIP assay to study how binding of a transcription factor is altered by passage through the cell cycle, by overexpression or deletion of another factor, or during tumorigenesis. Finally, a look toward the future suggests that the ChIP assay may be combined with Western blot analysis or mass spectrometry to identify additional proteins that interact with a transcription factor of interest.