Mechanism of allele specific assembly and disruption of master regulator transcription factor complexes of NF-KBp50, NF-KBp65 and HIF1a on a non-coding FAS SNP.
A challenging question in genetics is to understand the molecular function of non-coding variants of the genome. By using differential EMSA, ChIP and functional genome analysis, we have found that changes in transcription factors (TF) apparent binding affinity and dissociation rates are responsible for allele specific assembly or disruption of master TFs: we observed that NF-KBp50, NF-KBp65 and HIF1a bind with an affinity of up to 10 fold better to the C-allele than to the T-allele of rs7901656 both in vivo and in vitro. Furthermore, we showed that NF-KBp50, p65 and HIF1a form higher order heteromultimeric complexes overlapping rs7901656, implying synergism of action among TFs governing cellular response to infection and hypoxia. With rs7901656 on the FAS gene as a paradigm, we show how allele specific transcription factor complex assembly and disruption by a causal variant contributes to disease and phenotypic diversity. This finding provides the highly needed mechanistic insight into how the molecular etiology of regulatory SNPs can be understood in functional terms.
Department of Paediatric Pneumology, Neonatology and Allergology, Hannover Medical School, Hannover, Germany; Graduate School of Excellence, MD/PhD Programme Molecular Medicine Hannover Biomedical Research School, Hannover Biomedical Research School, Hannover Medical School, Hannover, Germany.
Chidiebere U
CU
Awah
Department of Paediatric Pneumology, Neonatology and Allergology, Hannover Medical School, Hannover, Germany.
Stephanie
S
Tamm
Department of Paediatric Pneumology, Neonatology and Allergology, Hannover Medical School, Hannover, Germany.
Silke
S
Hedtfeld
Institute for Human Genetics, Hannover Medical School, Hannover, Germany.
Doris
D
Steinemann
Department of Paediatric Pneumology, Neonatology and Allergology, Hannover Medical School, Hannover, Germany; Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Centre for Lung Research, Germany.
Burkhard
B
Tümmler
Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.
Georgios
G
Tsiavaliaris
Department of Paediatric Pneumology, Neonatology and Allergology, Hannover Medical School, Hannover, Germany; Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Centre for Lung Research, Germany. Electronic address: Mekus.Frauke@mh-hannover.de.
Frauke
F
Stanke
D000483:Alleles; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007700:Kinetics; D016328:NF-kappa B / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D019014:fas Receptor / Q000235:genetics*
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D019014:fas Receptor
Allele-specific TF binding; Cystic fibrosis; HIF1a; Higher order multimeric TF complexes; NF-KBp50; NF-KBp65; Non-coding SNP; TF-binding affinity