scholarly journals 4277 Functional consequences of the juvenile idiopathic arthritis risk variant at 1q24.3

2020 ◽  
Vol 4 (s1) ◽  
pp. 95-96
Author(s):  
Halima Moncrieffe ◽  
Katelyn Dunn ◽  
Yongbo Huang ◽  
Xiaoting Chen ◽  
Carl D. Langefeld ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Juvenile Idiopathic Arthritis ◽  
Transcription Factor Binding ◽  
Luciferase Reporter ◽  
Common Disease ◽  
Reference Allele ◽  
Enhancer Activity ◽  
Risk Variant ◽  
Factor Binding

OBJECTIVES/GOALS: Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatologic disease childhood and a cause of pain and potential disability. JIA has a strong genetic component and no known cure. The goal of this study is to evaluate allele-dependent effects of a novel JIA risk variant at 1q24.3. METHODS/STUDY POPULATION: JIA patients meeting criteria for the two most common disease subtypes (oligoarticular and RF neg polyarthritis) were genotyped using the Immunochip, an Illumina array with dense coverage of the HLA region and 186 other loci previously reported in autoimmune diseases. Phase I association findings (Hinks, 2013) and Phase II analysis (unpublished) of an expanded cohort (4,271 JIA and 14,390 controls) identified new risk loci, including rs78037977 at 1q24.3. We prioritized rs78037977 and predicted possible impacted mechanisms based on Bayesian predictions of attributable risk, the surrounding chromatin landscape, and transcription factor binding data. A luciferase reporter assay was used to assess allele-dependent enhancer activity. RESULTS/ANTICIPATED RESULTS: rs78037977 is located between FASLG and TNFSF18 at chromosome 1q24.3 is associated with JIA (p = 6.3x10−09), and explains 94% of the posterior probability at this locus; no other SNPs in linkage disequilibrium (r2>0.6). The chromatin landscape around rs78037977 contains H3K4Me1 and H3K27Ac marks, which are indicative of enhancer activity. Further, >160 transcription factors have chromatin immunoprecipitation followed by sequencing (ChIP-seq) peaks overlapping rs78037977 in various cellular contexts. In luciferase reporter assays, the region around rs78037977 containing the reference A allele had ~2-fold increased enhancer activity compared to the non-reference allele. DISCUSSION/SIGNIFICANCE OF IMPACT: This work provides in vitro evidence to support allele-dependent enhancer activity of a novel JIA-risk variant at 1q24.3. Our ongoing work investigates the effect of the DNA-containing region of rs78037977 on gene expression and differential transcription factor binding at rs78037977.

The g.763G>C SNP of the bovine FASN gene affects its promoter activity via Sp-mediated regulation: implications for the bovine lactating mammary gland

2008 ◽  
Vol 34 (2) ◽  
pp. 144-148 ◽  
Author(s):  
Laura Ordovás ◽  
Rosa Roy ◽  
Sandra Pampín ◽  
Pilar Zaragoza ◽  
Rosario Osta ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mammary Gland ◽  
Milk Fat ◽  
Promoter Activity ◽  
Transcription Factor Binding ◽  
Fat Content ◽  
Luciferase Reporter ◽  
Binding Ability ◽  
Factor Binding ◽  
Lactating Mammary Gland

Fatty acid synthase (FASN) is an enzyme that catalyzes de novo synthesis of fatty acids in cells. The bovine FASN gene maps to BTA 19, where several quantitative trait loci for fat-related traits have been described. Our group recently reported the identification of a single nucleotide polymorphism (SNP), g.763G>C, in the bovine FASN 5′ flanking region that was significantly associated with milk fat content in dairy cattle. The g.763G>C SNP was part of a GC-rich region that may constitute a cis element for members of the Sp transcription factor family. Thus the SNP could alter the transcription factor binding ability of the FASN promoter and consequently affect the promoter activity of the gene. However, the functional consequences of the SNP on FASN gene expression are unknown. The present study was therefore directed at elucidating the underlying molecular mechanism that could explain the association of the SNP with milk fat content. Three cellular lines (3T3L1, HepG2, and MCF-7) were used to test the promoter and the transcription factor binding activities by luciferase reporter assays and electrophoretic mobility shift assays, respectively. Band shift assays were also carried out with nuclear extracts from lactating mammary gland (LMG) to further investigate the role of the SNP in this tissue. Our results demonstrate that the SNP alters the bovine FASN promoter activity in vitro and the Sp1/Sp3 binding ability of the sequence. In bovine LMG, the specific binding of Sp3 may account for the association with milk fat content.

scholarly journals Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance

2021 ◽  
Author(s):  
Amir Shahein ◽  
Maria L&oacutepez-Malo ◽  
Ivan Istomin ◽  
Evan J. Olson ◽  
Shiyu Cheng ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcription Factor Binding ◽  
Affinity Binding ◽  
Factor Binding ◽  
Functional Relevance

Transcription factor binding to a single binding site and its functional consequence in a promoter context are beginning to be relatively well understood. However, binding to clusters of sites has yet to be characterized in depth, and the functional relevance of binding site clusters remains uncertain.We employed a high-throughput biochemical method to characterize transcription factor binding to clusters varying across a range of affinities and configurations. We found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Further-more, compared to an individual high-affinity binding site, small clusters with binding sites an order of magnitude lower in affinity give rise to higher mean occupancies at physiologically-relevant transcription factor concentrations in vitro. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters by inserting them into a synthetic minimal CYC1 and the native PHO5 S. cerevisiae promoter. In the minCYC1 promoter, clusters of low-affinity binding sites can generate transcriptional output comparable to a promoter containing three consensus binding sites. In the PHO5 promoter, replacing the native Pho4 binding sites with clusters of low-affinity binding sites recovered activation of these promoters as well. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters

scholarly journals Identification of a Novel Transcription Factor Binding Element Involved in the Regulation by Differentiation of the Human Telomerase (hTERT) Promoter

2000 ◽  
Vol 11 (12) ◽  
pp. 4381-4391 ◽  
Author(s):  
Maty Tzukerman ◽  
Catherine Shachaf ◽  
Yael Ravel ◽  
Ilana Braunstein ◽  
Orit Cohen-Barak ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Core Promoter ◽  
Embryonic Stem ◽  
Transcription Factor Binding ◽  
Telomerase Activity ◽  
Model Systems ◽  
Time Dependent ◽  
Luciferase Reporter ◽  
Factor Binding ◽  
The Core

Three different cell differentiation experimental model systems (human embryonic stem cells, mouse F9 cells, and human HL-60 promyelocytic cells) were used to determine the relationship between the reduction in telomerase activity after differentiation and the regulation of the promoter for the hTERT gene. Promoter constructs of three different lengths were subcloned into the PGL3-basic luciferase reporter vector. In all three experimental systems, all three promoter constructs drove high levels of reporter activity in the nondifferentiated state, with a marked and time-dependent reduction after the induction of differentiation. In all cases, the smallest core promoter construct (283 nt upstream of the ATG) gave the highest activity. Electrophoretic mobility shift assays revealed transcription factor binding to two E-box domains within the core promoter. There was also a marked time-dependent reduction in this binding with differentiation. In addition, a distinct and novel element was identified within the core promoter, which also underwent time-dependent reduction in transcription factor binding with differentiation. Site-directed mutagenesis of this novel element revealed a correlation between transcription factor binding and promoter activity. Taken together, the results indicate that regulation of overall telomerase activity with differentiation is mediated at least in part at the level of the TERT promoter and provides new information regarding details of the regulatory interactions that are involved in this process.

scholarly journals Acetylation of histone H4 plays a primary role in enhancing transcription factor binding to nucleosomal DNA in vitro.

1996 ◽  
Vol 15 (10) ◽  
pp. 2508-2518 ◽  
Author(s):  
M. Vettese-Dadey ◽  
P. A. Grant ◽  
T. R. Hebbes ◽  
C. Crane- Robinson ◽  
C. D. Allis ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factor Binding ◽  
Primary Role ◽  
Histone H4 ◽  
Factor Binding ◽  
Nucleosomal Dna

Rapid and easy method for in vitro determination of transcription factor binding core motifs

2019 ◽  
Vol 83 (12) ◽  
pp. 2276-2279
Author(s):  
Kyosuke Jinno ◽  
Wakana Kimura ◽  
Mina Komatsu ◽  
Maiko Miura ◽  
Satomi Sakaoka ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factor Binding ◽  
Easy Method ◽  
Factor Binding ◽  
Binding Core

scholarly journals RNA Polymerase I Transcription in a Brassica Interspecific Hybrid and Its Progenitors: Tests of Transcription Factor Involvement in Nucleolar Dominance

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 451-460
Author(s):  
Matthew Frieman ◽  
Z Jeffrey Chen ◽  
Julio Saez-Vasquez ◽  
L Annie Shen ◽  
Craig S Pikaard
Keyword(s):  
Transcription Factor ◽  
Cytosine Methylation ◽  
Transcription Factor Binding ◽  
The Other ◽  
Rrna Genes ◽  
Nucleolar Dominance ◽  
Specific Transcription Factor ◽  
Factor Binding ◽  
Species Specific

Abstract In interspecific hybrids or allopolyploids, often one parental set of ribosomal RNA genes is transcribed and the other is silent, an epigenetic phenomenon known as nucleolar dominance. Silencing is enforced by cytosine methylation and histone deacetylation, but the initial discrimination mechanism is unknown. One hypothesis is that a species-specific transcription factor is inactivated, thereby silencing one set of rRNA genes. Another is that dominant rRNA genes have higher binding affinities for limiting transcription factors. A third suggests that selective methylation of underdominant rRNA genes blocks transcription factor binding. We tested these hypotheses using Brassica napus (canola), an allotetraploid derived from B. rapa and B. oleracea in which only B. rapa rRNA genes are transcribed. B. oleracea and B. rapa rRNA genes were active when transfected into protoplasts of the other species, which argues against the species-specific transcription factor model. B. oleracea and B. rapa rRNA genes also competed equally for the pol I transcription machinery in vitro and in vivo. Cytosine methylation had no effect on rRNA gene transcription in vitro, which suggests that transcription factor binding was unimpaired. These data are inconsistent with the prevailing models and point to discrimination mechanisms that are likely to act at a chromosomal level.

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