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INTRODUCTION: Supracondylar humerus fractures are the most common surgically treated fracture in children. National trends have demonstrated an increase in transfers of supracondylar fractures to pediatric hospitals due to the perception that supracondylar fractures need to be treated by pediatric specialists. The objectives of the study are to compare the outcomes of surgically treated pediatric supracondylar humerus fractures (PSCHF) between a pediatric orthopedic surgeon and a non-pediatric orthopedic surgeon at a single academic medical center; to assess radiographic reduction, the number of pins used, surgical time, Flynn criteria outcomes, and complications associated with PSCHF treatment by both types of surgeons; to determine if there is a significant difference in outcomes between pediatric and non-pediatric orthopedic surgeons in the treatment of PSCHF. METHODS: Forty-seven consecutive pediatric patients, with an average age of 5.5 years old, who had undergone surgical correction for supracondylar humerus fractures during 2019 were included in this study. The intervention performed was closed reduction and percutaneous pinning. The main outcome measured: radiographic reduction on the AP and lateral X-ray view, number of K wires used, use of a medial K wire, time of surgery, the Flynn criteria, and complications. The Human Research Protection Program (HRPP) at Penn State approval was obtained. RESULTS: Radiographic reductions as measured by Bauman's angle and the position of the anterior humeral line were excellent and similar between surgeons. The pediatric orthopedic surgeon used more medial K wires (p=0.0007), fewer K wires (p=0.0065), and the length of surgery was shorter (p=0.019). The Flynn criteria were similar with equal excellent and good results. For both surgeons, no complications such as loss of reduction, infection, iatrogenic nerve injury, compartment syndrome, or cubitus varus occurred. CONCLUSIONS: Outcomes of treatment of displaced PSCHF by the pediatric and non-pediatric orthopedic surgeons were equal. The results of this study reaffirm the assertion that both pediatric and non-pediatric orthopedic surgeons have sufficient training and skill to treat these common fractures, thereby contributing to a more informed decision-making process in clinical practice.
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BACKGROUND: Identifying the DNA-binding specificities of transcription factors (TF) is central to understanding gene networks that regulate growth and development. Such knowledge is lacking in oomycetes, a microbial eukaryotic lineage within the stramenopile group. Oomycetes include many important plant and animal pathogens such as the potato and tomato blight agent Phytophthora infestans, which is a tractable model for studying life-stage differentiation within the group. RESULTS: Mining of the P. infestans genome identified 197 genes encoding proteins belonging to 22 TF families. Their chromosomal distribution was consistent with family expansions through unequal crossing-over, which were likely ancient since each family had similar sizes in most oomycetes. Most TFs exhibited dynamic changes in RNA levels through the P. infestans life cycle. The DNA-binding preferences of 123 proteins were assayed using protein-binding oligonucleotide microarrays, which succeeded with 73 proteins from 14 families. Binding sites predicted for representatives of the families were validated by electrophoretic mobility shift or chromatin immunoprecipitation assays. Consistent with the substantial evolutionary distance of oomycetes from traditional model organisms, only a subset of the DNA-binding preferences resembled those of human or plant orthologs. Phylogenetic analyses of the TF families within P. infestans often discriminated clades with canonical and novel DNA targets. Paralogs with similar binding preferences frequently had distinct patterns of expression suggestive of functional divergence. TFs were predicted to either drive life stage-specific expression or serve as general activators based on the representation of their binding sites within total or developmentally-regulated promoters. This projection was confirmed for one TF using synthetic and mutated promoters fused to reporter genes in vivo. CONCLUSIONS: We established a large dataset of binding specificities for P. infestans TFs, representing the first in the stramenopile group. This resource provides a basis for understanding transcriptional regulation by linking TFs with their targets, which should help delineate the molecular components of processes such as sporulation and host infection. Our work also yielded insight into TF evolution during the eukaryotic radiation, revealing both functional conservation as well as diversification across kingdoms.
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Evolución Molecular , Filogenia , Phytophthora infestans , Factores de Transcripción , Phytophthora infestans/genética , Phytophthora infestans/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Sitios de Unión , Unión ProteicaRESUMEN
INTRODUCTION: The Greulich & Pyle (G&P) Radiographic Atlas of Skeletal Development uses hand x-rays obtained between 1926 and 1942 on children of Caucasian ancestry. Our study uses modern Caucasian, Black, Hispanic, and Asian children to investigate patterns of development as a function of percent final height (PFH). METHODS: A retrospective review, at a single institution, was conducted using children who received a hand x-ray, a height measurement taken within 60 days of that x-ray, and a final height. BA and CA were compared between races. PFH was calculated by dividing height at the time of the x-ray by their final height. To further evaluate differences between races in CA or BA, PFH was then modeled as a function of CA or BA using a fifth-degree polynomial regression, and mean ages at the 85th PFH were compared. Patients were then divided into Sanders stages 1, 2-4, and 5-8 and the mean PFH, CA, and BA of the Asian, Black, and Hispanic children were compared with the White children using Student t test. P values less than 0.05 were considered significant. RESULTS: We studied 498 patients, including 53 Asian, 83 Black, 190 Hispanic, and 172 White patients. Mean BA was significantly greater than CA in Black males (1.27 y) and females (1.36 y), Hispanic males (1.12 y) and females (1.29 y), and White females (0.74 y). Hispanic and Black patients were significantly more advanced in BA than White patients (P<0.001). At the 85th PFH, White and Hispanic males were older than Black males by at least 7 months (P<0.001), and White females were significantly older than Hispanic females by 6.4 months (P<0.001). At 85th PFH for males, Hispanic and Black males had greater BA than White males by at least 5 months (P<0.001), and Asian females had a greater BA than Black females by at least 5 months (P<0.001). Compared with White children, Hispanic children were significantly younger at Sanders 2-4 than White children, and Black children were skeletally older at Sanders 5-8. CONCLUSIONS: BA was greater than CA by ≥1 year in Black and Hispanic children, and that these children had a significantly greater BA than their White counterparts. Black males and Hispanic females reached their 85th PFH at younger ages, and Hispanic males and Asian females were the most skeletally mature at their 85th PFH. Our results suggest that BA and CA may vary as a function of race, and further studies evaluating growth via the 85th PFH may be necessary. LEVEL OF EVIDENCE: Therapeutic Study - Level IV.
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AIM: To examine patients with cerebral palsy (CP) undergoing open reduction and internal fixation (ORIF) for ankle fractures. METHOD: This was a retrospective study of adult patients undergoing ankle fracture ORIF for closed, isolated ankle fractures identified in between 2010 and 2021 in the Q1 PearlDiver M151 database. Patients with CP were identified with International Classification of Diseases (ICD)-9 and ICD-10 codes, and were matched to those without 1:10 on age, sex, and Elixhauser comorbidity index (ECI). Ninety-day adverse events were assessed with multivariable logistic regression. RESULTS: A total of 148 993 patients with isolated ankle fracture ORIF were identified, of whom 407 (0.27%) had CP. After matching, 3863 without CP were compared to 389 with CP. Patients with CP were at increased odds of: 90-day urinary tract infection (odds ratios [OR] 6.26), pneumonia (OR 3.50), minor adverse events (OR 3.46), sepsis (OR 3.30), any adverse events (OR 3.04), emergency department visits (OR 2.28), serious adverse events (OR 1.77), and prolonged length of stay more than 4 days (OR 22.44) (p < 0.001 for all). INTERPRETATION: Patients with CP undergoing ORIF for isolated, closed ankle fractures are at increased odds of several 90-day adverse events and prolonged length of stay compared to matched patients without CP.
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Mammalian mRNA and lncRNA exons are often small compared to introns. The exon definition model predicts that exons splice autonomously, dependent on proximal exon sequence features, explaining their delineation within large introns. This model has not been examined on a genome-wide scale, however, leaving open the question of how often mRNA and lncRNA exons are autonomous. It is also unknown how frequently such exons can arise by chance. Here, we directly assayed large fragments (500-1000 bp) of the human genome by exon trapping, which detects exons spliced into a heterologous transgene, here designed with a large intron context. We define the trapped exons as "autonomous." We obtained â¼1.25 million trapped exons, including most known mRNA and well-annotated lncRNA internal exons, demonstrating that human exons are predominantly autonomous. mRNA exons are trapped with the highest efficiency. Nearly a million of the trapped exons are unannotated, most located in intergenic regions and antisense to mRNA, with depletion from the forward strand of introns. These exons are not conserved, suggesting they are nonfunctional and arose from random mutations. They are nonetheless highly enriched with known splicing promoting sequence features that delineate known exons. Novel autonomous exons are more numerous than annotated lncRNA exons, and computational models also indicate they will occur with similar frequency in any randomly generated sequence. These results show that most human coding exons splice autonomously, and provide an explanation for the existence of many unconserved lncRNAs, as well as a new annotation and inclusion levels of spliceable loci in the human genome.
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Chromatin accessibility is integral to the process by which transcription factors (TFs) read out cis-regulatory DNA sequences, but it is difficult to differentiate between TFs that drive accessibility and those that do not. Deep learning models that learn complex sequence rules provide an unprecedented opportunity to dissect this problem. Using zygotic genome activation in Drosophila as a model, we analyzed high-resolution TF binding and chromatin accessibility data with interpretable deep learning and performed genetic validation experiments. We identify a hierarchical relationship between the pioneer TF Zelda and the TFs involved in axis patterning. Zelda consistently pioneers chromatin accessibility proportional to motif affinity, whereas patterning TFs augment chromatin accessibility in sequence contexts where they mediate enhancer activation. We conclude that chromatin accessibility occurs in two tiers: one through pioneering, which makes enhancers accessible but not necessarily active, and the second when the correct combination of TFs leads to enhancer activation.
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Cilia allowed our protistan ancestors to sense and explore their environment, avoid predation, and capture bacterial prey.1,2,3 Regulated ciliogenesis was likely critical for early animal evolution,2,4,5,6 and in modern animals, deploying cilia in the right cells at the right time is crucial for development and physiology. Two transcription factors, RFX and FoxJ1, coordinate ciliogenesis in animals7,8,9 but are absent from the genomes of many other ciliated eukaryotes, raising the question of how the regulation of ciliogenesis in animals evolved.10,11 By comparing the genomes of animals with those of their closest living relatives, the choanoflagellates, we found that the genome of their last common ancestor encoded at least three RFX paralogs and a FoxJ1 homolog. Disruption of the RFX homolog cRFXa in the model choanoflagellate Salpingoeca rosetta resulted in delayed cell proliferation and aberrant ciliogenesis, marked by the collapse and resorption of nascent cilia. In cRFXa mutants, ciliogenesis genes and foxJ1 were significantly downregulated. Moreover, the promoters of S. rosetta ciliary genes are enriched for DNA motifs matching those bound by the cRFXa protein in vitro. These findings suggest that an ancestral cRFXa homolog coordinated ciliogenesis in the progenitors of animals and choanoflagellates and that the selective deployment of the RFX regulatory module may have been necessary to differentiate ciliated from non-ciliated cell types during early animal evolution.
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Proteínas de Unión al ADN , Factores de Transcripción , Animales , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción del Factor Regulador X/genética , Factores de Transcripción del Factor Regulador X/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Cilios/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismoRESUMEN
BACKGROUND CONTEXT: Pedicle screw fixation has become common in the treatment of adolescent idiopathic scoliosis (AIS). Malpositioned pedicle screws have significant complications and identifying surgical techniques to optimize screw placement accuracy is imperative. PURPOSE: To compare the rate of intraoperative revision, replacement, or removal of pedicle screws placed utilizing 3D printed guides compared with pedicle screws placed utilizing a freehand technique. STUDY DESIGN/SETTING: Retrospective cohort study/single academic center. PATIENT SAMPLE: Thirty-two patients aged 10 to 18 with AIS. OUTCOME MEASURES: Revision rate of pedicle screws and operative time between groups. METHODS: A retrospective study was performed on patients 10 to 18 years of age who underwent posterior spinal instrumented fusion for AIS from February 2021 to July 2022. The study received an IRB exemption. Patient demographics, intraoperative measures, and outcome variables were recorded. Intraoperatively, all patients underwent a 3-dimensional fluoroscopic "check scan," which included axial, sagittal, and coronal images, to assess for screw accuracy. A secondary outcome of operative time was compared between groups. The p-values <.05 were considered significant. RESULTS: A total of 32 patients were included in this study. There were 17 cases in the 3D guided and 15 cases in fluoroscopy-guided freehand cohort. There was a total of 254 pedicle screws using 3D guides and 402 screws using freehand technique. Between cohorts, there were no significant differences in a number of levels fused (p=.54) or length of surgery (p=.36). The total revision rate of 3D guided screw placement was 5.5% and that of the freehand technique was 8.5%. The freehand screw placement group had significantly higher revision rates per vertebral level compared with 3D guided (p=.0096). Notably, 3D printed guides had fewer screws that were removed/revised for being too anterior (7.1%) compared with freehand (23.5%). Surgical time was not significantly different between the 3D guided and freehand cohort (p=.35). CONCLUSIONS: 3D printed guides reduce intraoperative revision rate compared with freehand techniques. Total operative time is comparable to freehand technique.
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Cifosis , Tornillos Pediculares , Escoliosis , Fusión Vertebral , Humanos , Adolescente , Niño , Escoliosis/cirugía , Escoliosis/etiología , Tornillos Pediculares/efectos adversos , Estudios Retrospectivos , Cifosis/etiología , Fusión Vertebral/efectos adversos , Fusión Vertebral/métodos , Impresión TridimensionalRESUMEN
CASE: A 74-year-old man presented with septic shock with infection of his heart transplant and bilateral prosthetic knee joints simultaneously. He underwent bilateral knee resection arthroplasties with placement of articulating spacers. At 3-year follow-up, the patient was alive and ambulating independently. CONCLUSION: This case represents the first report of bilateral hematogenous prosthetic knee infections associated with concomitant enterococcal endocarditis of a heart transplant treated successfully and definitively with radical debridement and placement of articulating spacer with regular implants.
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Artritis Infecciosa , Artroplastia de Reemplazo de Rodilla , Prótesis de la Rodilla , Masculino , Humanos , Anciano , Antibacterianos/uso terapéutico , Reoperación , Articulación de la Rodilla/cirugía , Prótesis de la Rodilla/efectos adversos , Artritis Infecciosa/cirugíaRESUMEN
The BEN domain-containing transcription factors regulate transcription by recruiting chromatin-modifying factors to specific chromatin regions via their DNA-binding BEN domains. The BEN domain of BANP has been shown to bind to a CGCG DNA sequence or an AAA-containing matrix attachment regions DNA sequence. Consistent with these in vivo observations, we identified an optimal DNA-binding sequence of AAATCTCG by protein binding microarray, which was also confirmed by our isothermal titration calorimetry and mutagenesis results. We then determined crystal structures of the BANP BEN domain in apo form and in complex with a CGCG-containing DNA, respectively, which revealed that the BANP BEN domain mainly used the electrostatic interactions to bind DNA with some base-specific interactions with the TC motifs. Our isothermal titration calorimetry results also showed that BANP bound to unmethylated and methylated DNAs with comparable binding affinities. Our complex structure of BANP-mCGCG revealed that the BANP BEN domain bound to the unmethylated and methylated DNAs in a similar mode and cytosine methylation did not get involved in binding, which is also consistent with our observations from the complex structures of the BEND6 BEN domain with the CGCG or CGmCG DNAs. Taken together, our results further elucidate the elements important for DNA recognition and transcriptional regulation by the BANP BEN domain-containing transcription factor.
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Proteínas de Unión al ADN , Regulación de la Expresión Génica , Factores de Transcripción , Cromatina , ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/química , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/química , HumanosRESUMEN
Tibial tubercle fractures are uncommon injuries. The purpose of this study is to report the outcomes of surgical treatment of displaced tibial tubercle fractures in adolescents. This study was approved by the College of Medicine Institutional Review Board. A retrospective review was performed at our institution for patients who underwent surgical treatment of tibial tubercle fractures. Patient demographics, injury characteristics, and outcomes were recorded. A p-value of <0.05 was considered statistically significant. Nineteen male patients were identified. The average age was 14.6 years, and the average body mass index was 25.8. Basketball (63%) was the most common mechanism of injury. No patient was treated with bicortical screws. Two patients had preoperative computed tomography. One patient presented with acute compartment syndrome (ACS), and fasciotomy was performed. Twelve patients (63%) without clinical signs of ACS received anterior compartment fasciotomy on a case-by-case basis according to surgeon's preference. No growth injury, including growth arrest, angulation, or shortening occurred. All patients returned to preinjury activities at an average of 18.5 weeks. Displaced tibial tubercle fractures in this series occurred in male adolescents during athletic activity. Unicortical screws/pins were used with no loss of fixation. Routine use of advanced imaging was unnecessary. One patient (5%) underwent fasciotomy. No growth arrest occurred. All patients returned to preinjury athletic activities.
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Fijación Interna de Fracturas , Fracturas de la Tibia , Humanos , Masculino , Adolescente , Fijación Interna de Fracturas/métodos , Tibia , Fracturas de la Tibia/diagnóstico por imagen , Fracturas de la Tibia/cirugía , Fracturas de la Tibia/etiología , Estudios Retrospectivos , Clavos Ortopédicos , Resultado del TratamientoRESUMEN
The purpose of this study is to report the operative outcomes in a consecutive series of adolescent patients with symptomatic accessory navicular (AN). A retrospective review was conducted. Patient characteristics, operative techniques, and outcomes were recorded. Radiographs were used to identify the type of AN, skeletal maturity, and presence of concurrent pes planus. Twenty-two patients and 24 feet were studied. All 22 patients had an excision of the AN, and 19 patients had an additional reefing of the tibialis posterior tendon. At final follow up, 22 cases reported no pain, one had minimal pain, and one reported no change in pain. Symptomatic AN is more common in females. Surgery technique was not correlated with postoperative pain. Surgery eliminated pain in 91% of patients and can be safely performed in athletes with high rate of return to their previous athletic performance. (Journal of Surgical Orthopaedic Advances 31(1):053-055, 2022).
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Enfermedades del Pie , Huesos Tarsianos , Adolescente , Femenino , Humanos , Dolor Postoperatorio , Huesos Tarsianos/diagnóstico por imagen , Huesos Tarsianos/cirugía , Tendones/cirugía , Resultado del TratamientoRESUMEN
The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.
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Proteínas Represoras , Factores de Transcripción , Animales , Sitios de Unión , Drosophila/metabolismo , Mamíferos , Unión Proteica , Dominios Proteicos , Proteínas Represoras/genética , Factores de Transcripción/metabolismoRESUMEN
The human transcription factor (TF) CGGBP1 (CGG-binding protein) is conserved only in amniotes and is believed to derive from the zf-BED and Hermes transposase DNA-binding domains (DBDs) of a hAT DNA transposon. Here, we show that sequence-specific DNA-binding proteins with this bipartite domain structure have resulted from dozens of independent hAT domestications in different eukaryotic lineages. CGGBPs display a wide range of sequence specificity, usually including preferences for CGG or CGC trinucleotides, whereas some bind AT-rich motifs. The CGGBPs are almost entirely nonsyntenic, and their protein sequences, DNA-binding motifs, and patterns of presence or absence in genomes are uncharacteristic of ancestry via speciation. At least eight CGGBPs in the coelacanth Latimeria chalumnae bind distinct motifs, and the expression of the corresponding genes varies considerably across tissues, suggesting tissue-restricted function.
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Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Peces/genética , Animales , Proteínas de Unión al ADN/metabolismo , Peces/metabolismo , HumanosRESUMEN
The PWWP domain was first identified in the HDGF protein family and named after the conserved Proline-Tryptophan-Tryptophan-Proline motif in WHSC1. The PWWP domain-containing proteins play important roles in different biological processes, such as DNA replication, transcription, DNA repair, pre-mRNA processing by recognizing methylated histone and dsDNA simultaneously. Recently, how the HDGF family of PWWP domains recognize histone H3K36me3-modified nucleosome has been reported. In order to better understand the interactions between the PWWP domain and dsDNA, we carried out family-wide characterization of dsDNA binding abilities of human PWWP domains. Our binding assays confirmed that PWWP domains bind to dsDNA without sequence selectivity. Our crystal structure of the BRPF2 PWWP domain in complex with a 12-mer dsDNA reveals that the PWWP domain interacts with dsDNA by binding to its major groove, instead of the minor groove observed in the HDGF family of PWWP domains. Our study indicates that PWWP domains could bind to dsDNA in different modes.
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ADN/química , Histona Acetiltransferasas/química , Histonas/química , Nucleosomas/química , Cristalografía por Rayos X , ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Humanos , Nucleosomas/metabolismo , Unión ProteicaRESUMEN
We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15â bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.
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Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteínas Bacterianas/química , Secuencia de Bases , Sitios de Unión , Simulación por Computador , ADN/metabolismo , ADN Viral/metabolismo , Proteínas de Unión al ADN/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Factor Proteico para Inverción de Estimulación/química , Factor Proteico para Inverción de Estimulación/genética , Expresión Génica , Proteínas Mutantes/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Multimerización de Proteína/genética , Proteínas Virales/químicaRESUMEN
Transcription factor (TF) binding specificities (motifs) are essential for the analysis of gene regulation. Accurate prediction of TF motifs is critical, because it is infeasible to assay all TFs in all sequenced eukaryotic genomes. There is ongoing controversy regarding the degree of motif diversification among related species that is, in part, because of uncertainty in motif prediction methods. Here we describe similarity regression, a significantly improved method for predicting motifs, which we use to update and expand the Cis-BP database. Similarity regression inherently quantifies TF motif evolution, and shows that previous claims of near-complete conservation of motifs between human and Drosophila are inflated, with nearly half of the motifs in each species absent from the other, largely due to extensive divergence in C2H2 zinc finger proteins. We conclude that diversification in DNA-binding motifs is pervasive, and present a new tool and updated resource to study TF diversity and gene regulation across eukaryotes.
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Secuencia de Bases , Sitios de Unión , Evolución Molecular , Factores de Transcripción/metabolismo , Animales , Biología Computacional/métodos , Secuencia Conservada , Bases de Datos Genéticas , Regulación de la Expresión Génica , Humanos , Motivos de Nucleótidos , Unión ProteicaRESUMEN
Genome-wide association studies have identified 40 ovarian cancer risk loci. However, the mechanisms underlying these associations remain elusive. In this study, we conducted a two-pronged approach to identify candidate causal SNPs and assess underlying biological mechanisms at chromosome 9p22.2, the first and most statistically significant associated locus for ovarian cancer susceptibility. Three transcriptional regulatory elements with allele-specific effects and a scaffold/matrix attachment region were characterized and, through physical DNA interactions, BNC2 was established as the most likely target gene. We determined the consensus binding sequence for BNC2 in vitro, verified its enrichment in BNC2 ChIP-seq regions, and validated a set of its downstream target genes. Fine-mapping by dense regional genotyping in over 15,000 ovarian cancer cases and 30,000 controls identified SNPs in the scaffold/matrix attachment region as among the most likely causal variants. This study reveals a comprehensive regulatory landscape at 9p22.2 and proposes a likely mechanism of susceptibility to ovarian cancer. SIGNIFICANCE: Mapping the 9p22.2 ovarian cancer risk locus identifies BNC2 as an ovarian cancer risk gene.See related commentary by Choi and Brown, p. 439.
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Carcinoma Epitelial de Ovario/genética , Cromosomas Humanos Par 9 , Neoplasias Ováricas/genética , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Mapeo Cromosómico , Cistadenocarcinoma Seroso/genética , ADN de Neoplasias/genética , Proteínas de Unión al ADN/genética , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido SimpleRESUMEN
Here, we report that the Neurospora crassa FLB-3 protein, the ortholog of the Aspergillus nidulans FlbC transcription factor, is required for developmental control. Deletion of flb-3 leads to changes in hyphae morphology and affects sexual and asexual development. We identified, as putative FLB-3 targets, the N. crassa aba-1, wet-1 and vos-1 genes, orthologs of the ones involved in A. nidulans asexual development and that work downstream of FlbC (abaA, wetA and vosA). In N. crassa, these three genes require FLB-3 for proper expression; however, they appear not to be required for normal development, as demonstrated by gene expression analyses during vegetative growth and asexual development. Moreover, mutant strains in the three genes conidiate well and produce viable conidia. We also determined FLB-3 DNA-binding preferences via protein-binding microarrays (PBMs) and demonstrated by chromatin immunoprecipitation (ChIP) that FLB-3 binds the aba-1, wet-1 and vos-1 promoters. Our data support an important role for FLB-3 in N. crassa development and highlight differences between the regulatory pathways controlled by this transcription factor in different fungal species.
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Proteínas Fúngicas/fisiología , Neurospora crassa/crecimiento & desarrollo , Factores de Transcripción/fisiología , Proteínas Fúngicas/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Hifa/genética , Hifa/crecimiento & desarrollo , Neurospora crassa/genética , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Factores de Transcripción/genéticaRESUMEN
We studied adaptive evolution of gene expression using long-term experimental evolution of Saccharomyces cerevisiae in ammonium-limited chemostats. We found repeated selection for non-synonymous variation in the DNA binding domain of the transcriptional activator, GAT1, which functions with the repressor, DAL80 in an incoherent type-1 feedforward loop (I1-FFL) to control expression of the high affinity ammonium transporter gene, MEP2. Missense mutations in the DNA binding domain of GAT1 reduce its binding to the GATAA consensus sequence. However, we show experimentally, and using mathematical modeling, that decreases in GAT1 binding result in increased expression of MEP2 as a consequence of properties of I1-FFLs. Our results show that I1-FFLs, one of the most commonly occurring network motifs in transcriptional networks, can facilitate adaptive tuning of gene expression through modulation of transcription factor binding affinities. Our findings highlight the importance of gene regulatory architectures in the evolution of gene expression.