An internal promoter underlies the difference in disease severity between N- and C-terminal truncation mutations of Titin
- University of California, San Francisco, United States
- University of North Carolina at Chapel Hill, United States
- Brigham Young University, United States
- University of Zurich, United States
Abstract
Truncating mutations in the giant sarcomeric protein Titin result in dilated cardiomyopathy (DCM) and skeletal myopathy. The most severely affected DCM patients harbor Titin truncations in the C-terminal two-thirds of the protein, suggesting that mutation position might influence disease mechanism. Using CRISPR/Cas9 technology, we generated six zebrafish lines with Titin truncations in the N-terminal (Z-disk and I-band) and C-terminal (A-band) regions. Although all exons were constitutive, C-terminal mutations caused severe myopathy whereas N-terminal mutations demonstrated mild phenotypes. Surprisingly, neither mutation type acted as a dominant negative. Instead, we found a conserved internal promoter at the precise position where divergence in disease severity occurs, with the resulting protein product partially rescuing N-terminal truncations. In addition to its clinical implications, our work may shed light on a long-standing mystery regarding the architecture of the sarcomere.
Article and author information
Author details
Ethics
Animal experimentation: All zebrafish and mouse experimental work conformed to the 'Guide for the Care and Use of Laboratory Animals' published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Animal work was performed according to institutional guidelines with the full approval of the University of California Institutional Animal Care and Use Committee (protocols AN090013-03 and AN107039-01)
Human subjects: Human genetic studies were performed according to institutional guidelines and with the full approval of the University of California San Francisco Committee on Human Research (CHR#10-00207) and all studies performed were in keeping with the original informed consent forms. Informed consent and consent to publish was obtained from all participants.
Copyright
© 2015, Zou 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.
Metrics
-
- 3,956
- views
-
- 845
- downloads
-
- 90
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
An internal promoter underlies the difference in disease severity between N- and C-terminal truncation mutations of Titin
eLife 4:e09406.
https://doi.org/10.7554/eLife.09406
Further reading
-
- Cell Biology
- Genetics and Genomics
Progression through the G1 phase of the cell cycle is the most highly regulated step in cellular division. We employed a chemogenetic approach to discover novel cellular networks that regulate cell cycle progression. This approach uncovered functional clusters of genes that altered sensitivity of cells to inhibitors of the G1/S transition. Mutation of components of the Polycomb Repressor Complex 2 rescued proliferation inhibition caused by the CDK4/6 inhibitor palbociclib, but not to inhibitors of S phase or mitosis. In addition to its core catalytic subunits, mutation of the PRC2.1 accessory protein MTF2, but not the PRC2.2 protein JARID2, rendered cells resistant to palbociclib treatment. We found that PRC2.1 (MTF2), but not PRC2.2 (JARID2), was critical for promoting H3K27me3 deposition at CpG islands genome-wide and in promoters. This included the CpG islands in the promoter of the CDK4/6 cyclins CCND1 and CCND2, and loss of MTF2 lead to upregulation of both CCND1 and CCND2. Our results demonstrate a role for PRC2.1, but not PRC2.2, in antagonizing G1 progression in a diversity of cell linages, including chronic myeloid leukemia (CML), breast cancer, and immortalized cell lines.
-
- Evolutionary Biology
- Genetics and Genomics
Copy number variants (CNVs) are an important source of genetic variation underlying rapid adaptation and genome evolution. Whereas point mutation rates vary with genomic location and local DNA features, the role of genome architecture in the formation and evolutionary dynamics of CNVs is poorly understood. Previously, we found the GAP1 gene in Saccharomyces cerevisiae undergoes frequent amplification and selection in glutamine-limitation. The gene is flanked by two long terminal repeats (LTRs) and proximate to an origin of DNA replication (autonomously replicating sequence, ARS), which likely promote rapid GAP1 CNV formation. To test the role of these genomic elements on CNV-mediated adaptive evolution, we evolved engineered strains lacking either the adjacent LTRs, ARS, or all elements in glutamine-limited chemostats. Using a CNV reporter system and neural network simulation-based inference (nnSBI) we quantified the formation rate and fitness effect of CNVs for each strain. Removal of local DNA elements significantly impacts the fitness effect of GAP1 CNVs and the rate of adaptation. In 177 CNV lineages, across all four strains, between 26% and 80% of all GAP1 CNVs are mediated by Origin Dependent Inverted Repeat Amplification (ODIRA) which results from template switching between the leading and lagging strand during DNA synthesis. In the absence of the local ARS, distal ones mediate CNV formation via ODIRA. In the absence of local LTRs, homologous recombination can mediate gene amplification following de novo retrotransposon events. Our study reveals that template switching during DNA replication is a prevalent source of adaptive CNVs.
