Journal: European Journal of Human Genetics
Article Title: An MTF1 binding site disrupted by a homozygous variant in the promoter of ATP7B likely causes Wilson Disease
doi: 10.1038/s41431-018-0221-4
Figure Lengend Snippet: Functional interrogation of chr13:g.52,586,149T>C. a Luciferase reporter assays in HepG2 cells were performed to quantify differences in transactivation by a 379 bp fragment of the ATP7B promoter with the chr13:g.52,586,149T (Ref) or chr13:g.52,586,149C (Alt) allele, in the presence (+) or absence (−) of MTF1 overexpression (OE). Both with and without MTF1 OE, the reference allele drove significantly higher expression compared to the alternate allele (comparison 1 and 2, respectively). Both alleles experienced dramatic increases in activity with MTF1 OE (comparisons 3 and 4), but the reference allele yielded a greater increase in expression than did the alternate allele in this context (comparison 5). Bars represent mean ± SD. Two-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. b ChIP-qPCR was performed to determine the extent of MTF1 binding at the SNV locus (ATP7B) compared to a computationally predicted negative control (Neg Ctrl, in an intron of WDPCP) and to a previously published [32] experimentally validated locus (Pos Ctrl, in the 5′ UTR of SELENOH). Enrichment of DNA immunoprecipitated by MTF1-specific vs. non-specific isotype-matched control antibodies was measured in technical triplicate by quantitative PCR analysis. Bars represent mean ± SD. One-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. c Proposed model for disease-causing mechanism of chr13:g.52,586,149T>C: (1) Excess intracellular accumulation of copper, Cu2+, increases (2) expression or nuclear translocation of MTF1 [12, 13]. In wildtype individuals, MTF1 then binds at chr13:g.52,586,149T (3) to recruit transcriptional machinery for upregulating ATP7B expression [34] and eliminating copper through serum ceruloplasmin and, ultimately, through the bile. We propose that the Wilson Disease (WD) patient’s homozygous single nucleotide promoter variant chr13:g.52,586,149T>C exhibits reduced affinity to MTF1. (4) The result is an insufficient ATP7B transcriptional response, consequent copper accumulation, and symptoms characteristic of Wilson Disease
Article Snippet: For experiments involving overexpression of MTF1, the human MTF1-FLAG expression plasmid (ABclonal, Woburn, MA, USA: HG15046-CF) was transfected (200 ng per well) 24 h prior to introducing the luciferase vectors.
Techniques: Functional Assay, Luciferase, Over Expression, Expressing, Activity Assay, Two Tailed Test, Binding Assay, Negative Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, One-tailed Test, Translocation Assay, Variant Assay
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![Genomic context of the candidate causative variant. a UCSC Genome Browser (hg19 minus strand) view of the region near the candidate causative variant positioned 676 bp upstream of the canonical ATP7B translation start site (chr13:g.52,586,149A>G, cyan highlight and asterisk). The variant lies in a 100-way vertebrate alignment conserved element and region of high cross-species sequence conservation as computed by PhastCons and PhyloP [30]. The chromatin surrounding chr13:g.52,586,149T>C is hypersensitive to DNaseI (and, therefore, open and accessible) in HepG2 cells. The sequence cloned into the luciferase reporter vector is indicated by the black bar. b Genotype verification for chr13:g.52,586,149T>C (red asterisk) by Sanger sequencing. Representative chromatograms show the indicated number (n=x/y) of amplification products containing the reference (T) or alternate (C) allele for each individual. Box indicates a predicted binding site for <t>MTF1.</t> c The candidate causative variant (bolded, pink) in the Wilson Disease (WD) patient disrupts a key base in the MTF1 position weight matrix. This exact base (boxed) is unaltered in all primates and in dozens of other mammals, even as distant as Tasmanian devil (Supplementary Fig. 1b) (color figure online).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4090/pmc06244090/pmc06244090__41431_2018_221_Fig1_HTML.jpg)
![Functional interrogation of chr13:g.52,586,149T>C. a Luciferase reporter assays in HepG2 cells were performed to quantify differences in transactivation by a 379 bp fragment of the ATP7B promoter with the chr13:g.52,586,149T (Ref) or chr13:g.52,586,149C (Alt) allele, in the presence (+) or absence (−) of MTF1 overexpression (OE). Both with and without MTF1 OE, the reference allele drove significantly higher expression compared to the alternate allele (comparison 1 and 2, respectively). Both alleles experienced dramatic increases in activity with MTF1 OE (comparisons 3 and 4), but the reference allele yielded a greater increase in expression than did the alternate allele in this context (comparison 5). Bars represent mean ± SD. Two-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. b ChIP-qPCR was performed to determine the extent of MTF1 binding at the SNV locus (ATP7B) compared to a computationally predicted negative control (Neg Ctrl, in an intron of WDPCP) and to a previously published [32] experimentally validated locus (Pos Ctrl, in the 5′ UTR of SELENOH). Enrichment of DNA immunoprecipitated by MTF1-specific vs. non-specific isotype-matched control antibodies was measured in technical triplicate by quantitative PCR analysis. Bars represent mean ± SD. One-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. c Proposed model for disease-causing mechanism of chr13:g.52,586,149T>C: (1) Excess intracellular accumulation of copper, Cu2+, increases (2) expression or nuclear translocation of MTF1 [12, 13]. In wildtype individuals, MTF1 then binds at chr13:g.52,586,149T (3) to recruit transcriptional machinery for upregulating ATP7B expression [34] and eliminating copper through serum ceruloplasmin and, ultimately, through the bile. We propose that the Wilson Disease (WD) patient’s homozygous single nucleotide promoter variant chr13:g.52,586,149T>C exhibits reduced affinity to MTF1. (4) The result is an insufficient ATP7B transcriptional response, consequent copper accumulation, and symptoms characteristic of Wilson Disease](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4090/pmc06244090/pmc06244090__41431_2018_221_Fig2_HTML.jpg)