An atrial fibrillation-associated regulatory region modulates cardiac Tbx5 levels and arrhythmia susceptibility
- Amsterdam University Medical Centers, Netherlands
- Yonsei University, Republic of Korea
Abstract
Heart development and rhythm control are highly Tbx5 dosage-sensitive. TBX5 haploinsufficiency causes congenital conduction disorders, whereas increased expression levels of TBX5 in human heart samples has been associated with atrial fibrillation (AF). We deleted the conserved mouse orthologues of two independent AF-associated genomic regions in the Tbx5 locus, one intronic (RE(int)) and one downstream (RE(down)) of Tbx5. In both lines we observed a modest (30%) increase of Tbx5 in the postnatal atria. To gain insight into the effects of slight dosage increase in vivo, we investigated the atrial transcriptional, epigenetic and electrophysiological properties of both lines. Increased atrial Tbx5 expression was associated with induction of genes involved in development, ion transport and conduction, with increased susceptibility to atrial arrhythmias, and increased action potential duration of atrial cardiomyocytes. We identified an AF-associated variant in the human RE(int) that increases its transcriptional activity. Expression of the AF-associated transcription factor Prrx1 was induced in Tbx5RE(int)KO cardiomyocytes. We found that some of the transcriptional and functional changes in the atria caused by increased Tbx5 expression were normalized when reducing cardiac Prrx1 expression in Tbx5RE(int)KO mice, indicating an interaction between these two AF genes. We conclude that modest increases in expression of dose-dependent transcription factors, caused by common regulatory variants, significantly impact on the cardiac gene regulatory network and disease susceptibility.
Data availability
Adult left atrial RNAseq and ATACseq have been deposited under GEO accession numbers GSE189342 and GSE189498.
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Tbx5 regulatory element controls atrial function and heart sizeNCBI Gene Expression Omnibus, GSE189342.
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Tbx5 regulatory element controls atrial function and heart sizeNCBI Gene Expression Omnibus, GSE189498.
Article and author information
Author details
Vincent M Christoffels
Funding
CVON project 2014-18 CONCOR-genes (Young Talent Program)
- Fernanda M Bosada
CVON project 2014-18 CONCOR-genes
- Vincent M Christoffels
Fondation Leducq (14CVD01)
- Vincent M Christoffels
Dutch Cardiovascular Alliance (OUTREACH)
- Vincent M Christoffels
ZonMw (TOP 91217061)
- Vincent M Christoffels
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Housing, husbandry, all animal care and experimental protocols were in accordance with guidelines310 from the Directive 2010/63/EU of the European Parliament and Dutch government. Protocols were311 approved by the Animal Experimental Committee of the Amsterdam University Medical Centers.312 Animal group sizes were determined based on previous experience.
Copyright
© 2023, Bosada et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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An atrial fibrillation-associated regulatory region modulates cardiac Tbx5 levels and arrhythmia susceptibility
eLife 12:e80317.
https://doi.org/10.7554/eLife.80317
Further reading
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- Chromosomes and Gene Expression
- Evolutionary Biology
Gene regulation is essential for life and controlled by regulatory DNA. Mutations can modify the activity of regulatory DNA, and also create new regulatory DNA, a process called regulatory emergence. Non-regulatory and regulatory DNA contain motifs to which transcription factors may bind. In prokaryotes, gene expression requires a stretch of DNA called a promoter, which contains two motifs called –10 and –35 boxes. However, these motifs may occur in both promoters and non-promoter DNA in multiple copies. They have been implicated in some studies to improve promoter activity, and in others to repress it. Here, we ask whether the presence of such motifs in different genetic sequences influences promoter evolution and emergence. To understand whether and how promoter motifs influence promoter emergence and evolution, we start from 50 ‘promoter islands’, DNA sequences enriched with –10 and –35 boxes. We mutagenize these starting ‘parent’ sequences, and measure gene expression driven by 240,000 of the resulting mutants. We find that the probability that mutations create an active promoter varies more than 200-fold, and is not correlated with the number of promoter motifs. For parent sequences without promoter activity, mutations created over 1500 new –10 and –35 boxes at unique positions in the library, but only ~0.3% of these resulted in de-novo promoter activity. Only ~13% of all –10 and –35 boxes contribute to de-novo promoter activity. For parent sequences with promoter activity, mutations created new –10 and –35 boxes in 11 specific positions that partially overlap with preexisting ones to modulate expression. We also find that –10 and –35 boxes do not repress promoter activity. Overall, our work demonstrates how promoter motifs influence promoter emergence and evolution. It has implications for predicting and understanding regulatory evolution, de novo genes, and phenotypic evolution.
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- Chromosomes and Gene Expression
- Developmental Biology
The male-specific lethal complex (MSL), which consists of five proteins and two non-coding roX RNAs, is involved in the transcriptional enhancement of X-linked genes to compensate for the sex chromosome monosomy in Drosophila XY males compared with XX females. The MSL1 and MSL2 proteins form the heterotetrameric core of the MSL complex and are critical for the specific recruitment of the complex to the high-affinity ‘entry’ sites (HAS) on the X chromosome. In this study, we demonstrated that the N-terminal region of MSL1 is critical for stability and functions of MSL1. Amino acid deletions and substitutions in the N-terminal region of MSL1 strongly affect both the interaction with roX2 RNA and the MSL complex binding to HAS on the X chromosome. In particular, substitution of the conserved N-terminal amino-acids 3–7 in MSL1 (MSL1GS) affects male viability similar to the inactivation of genes encoding roX RNAs. In addition, MSL1GS binds to promoters such as MSL1WT but does not co-bind with MSL2 and MSL3 to X chromosomal HAS. However, overexpression of MSL2 partially restores the dosage compensation. Thus, the interaction of MSL1 with roX RNA is critical for the efficient assembly of the MSL complex on HAS of the male X chromosome.
