TY - JOUR
T1 - Functional characterization of QT interval associated SCN5A enhancer variants identify combined additive effects
AU - Gunamalai, Lavanya
AU - Singh, Parul
AU - Berg, Brian
AU - Shi, Leilei
AU - Sanchez, Ernesto
AU - Smith, Alexa
AU - Breton, Ghislain
AU - Bedford, Mark T.
AU - Balciunas, Darius
AU - Kapoor, Ashish
N1 - Publisher Copyright:
© 2024 The Author(s)
PY - 2025/1/9
Y1 - 2025/1/9
N2 - Several empirical and theoretical studies suggest the presence of multiple enhancers per gene that collectively regulate gene expression, and that common sequence variation impacting on the activities of these enhancers is a major source of inter-individual gene expression variability. However, for the vast majority of genes, enhancers and the underlying regulatory variation remains unknown. Even for the genes with well-characterized enhancers, the nature of the combined effects from multiple enhancers and their variants, when known, on gene expression regulation remains unexplored. Here, we have evaluated the combined effects from five SCN5A enhancers and their regulatory variants that are known to collectively correlate with SCN5A cardiac expression and underlie QT interval association in the general population. Using small deletions centered at the regulatory variants in episomal reporter assays in a mouse cardiomyocyte cell line, we demonstrate that the variants and their flanking sequences play critical role in individual enhancer activities, likely being a transcription factor (TF) binding site. By oligonucleotide-based pulldown assays on predicted TFs, we identify the TFs likely driving allele-specific enhancer activities. Using all 32 possible allelic synthetic constructs in reporter assays, representing the five bi-allelic enhancers, we demonstrate combined additive effects on overall enhancer activities. Using transient enhancer assays in zebrafish embryos we demonstrate that four elements act as enhancers in vivo. Together, these studies uncover the TFs driving the enhancer activities of QT interval associated SCN5A regulatory variants, reveal the additive effects from allelic combinations of these regulatory variants, and prove their potential to act as enhancers in vivo.
AB - Several empirical and theoretical studies suggest the presence of multiple enhancers per gene that collectively regulate gene expression, and that common sequence variation impacting on the activities of these enhancers is a major source of inter-individual gene expression variability. However, for the vast majority of genes, enhancers and the underlying regulatory variation remains unknown. Even for the genes with well-characterized enhancers, the nature of the combined effects from multiple enhancers and their variants, when known, on gene expression regulation remains unexplored. Here, we have evaluated the combined effects from five SCN5A enhancers and their regulatory variants that are known to collectively correlate with SCN5A cardiac expression and underlie QT interval association in the general population. Using small deletions centered at the regulatory variants in episomal reporter assays in a mouse cardiomyocyte cell line, we demonstrate that the variants and their flanking sequences play critical role in individual enhancer activities, likely being a transcription factor (TF) binding site. By oligonucleotide-based pulldown assays on predicted TFs, we identify the TFs likely driving allele-specific enhancer activities. Using all 32 possible allelic synthetic constructs in reporter assays, representing the five bi-allelic enhancers, we demonstrate combined additive effects on overall enhancer activities. Using transient enhancer assays in zebrafish embryos we demonstrate that four elements act as enhancers in vivo. Together, these studies uncover the TFs driving the enhancer activities of QT interval associated SCN5A regulatory variants, reveal the additive effects from allelic combinations of these regulatory variants, and prove their potential to act as enhancers in vivo.
KW - Additive
KW - Enhancer
KW - QT interval
KW - SCN5A
KW - Transcription factor
KW - Variant
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U2 - 10.1016/j.xhgg.2024.100358
DO - 10.1016/j.xhgg.2024.100358
M3 - Article
C2 - 39354714
AN - SCOPUS:85207787847
SN - 2666-2477
VL - 6
JO - Human Genetics and Genomics Advances
JF - Human Genetics and Genomics Advances
IS - 1
M1 - 100358
ER -