Impaired LRP6-TCF7L2 Activity Enhances Smooth Muscle Cell Plasticity and Causes Coronary Artery Disease

SummaryMutations in Wnt-signaling coreceptor LRP6 have been linked to coronary artery disease (CAD) by unknown mechanisms. Here, we show that reduced LRP6 activity in LRP6R611C mice promotes loss of vascular smooth muscle cell (VSMC) differentiation, leading to aortic medial hyperplasia. Carotid injury augmented these effects and led to partial to total vascular obstruction. LRP6R611C mice on high-fat diet displayed dramatic obstructive CAD and exhibited an accelerated atherosclerotic burden on LDLR knockout background. Mechanistically, impaired LRP6 activity leads to enhanced non-canonical Wnt signaling, culminating in diminished TCF7L2 and increased Sp1-dependent activation of PDGF signaling. Wnt3a administration to LRP6R611C mice improved LRP6 activity, led to TCF7L2-dependent VSMC differentiation, and rescued post-carotid-injury neointima formation. These findings demonstrate the critical role of intact Wnt signaling in the vessel wall, establish a causal link between impaired LRP6/TCF7L2 activities and arterial disease, and identify Wnt signaling as a therapeutic target against CAD

Evolutionary innovation in conserved regulatory elements across the mammalian tree of life

Transcriptional enhancers orchestrate cell type- and time point-specific gene expression programs. Evolution of enhancer sequences can alter target gene expression without causing detrimental misexpression in other contexts. It has long been thought that this modularity allows evolutionary changes in enhancers to escape pleiotropic constraints, which is especially important for evolutionary constrained developmental patterning genes. However, there is still little data supporting this hypothesis. Here we identified signatures of accelerated evolution in conserved enhancer elements across the mammalian phylogeny. We found that pleiotropic genes involved in gene regulatory and developmental processes were enriched for accelerated sequence evolution within their enhancer elements. These genes were associated with an excess number of enhancers compared to other genes, and due to this they exhibit a substantial degree of sequence acceleration over all their enhancers combined. We provide evidence that sequence acceleration is associated with turnover of regulatory function. We studied one acceleration event in depth and found that its sequence evolution led to the emergence of a new enhancer activity domain that may be involved in the evolution of digit reduction in hoofed mammals. Our results provide tangible evidence that enhancer evolution has been a frequent contributor to modifications involving constrained developmental signaling genes in mammals

Isradipine therapy in Cacna1dIle772Met/+ mice ameliorates primary aldosteronism and neurologic abnormalities

Somatic gain-of-function mutations in the L-type calcium channel CaV1.3 (CACNA1D gene) cause adrenal aldosterone-producing adenomas and micronodules. De novo germline mutations are found in a syndrome of primary aldosteronism, seizures, and neurologic abnormalities (PASNA) as well as in autism spectrum disorder. Using CRISPR/Cas9, we here generated mice with a Cacna1d gain-of-function mutation found in both adenomas and PASNA syndrome (Cacna1dIle772Met/+). These mice show reduced body weight and increased mortality from weaning to approximately 100 days of age. Male mice do not breed, likely due to neuromotor impairment, and the offspring of female mice die perinatally, likely due to lack of maternal care. Mice generated by in vitro fertilization showed elevated intracellular calcium in the aldosterone-producing zona glomerulosa, an elevated aldosterone/renin ratio, and persistently elevated serum aldosterone on a high-salt diet as signs of primary aldosteronism. Anesthesia with ketamine and xylazine induced tonic-clonic seizures. Neurologic abnormalities included hyperlocomotion, impaired performance in the rotarod test, impaired nest building, and slight changes in social behavior. Intracellular calcium in the zona glomerulosa, aldosterone levels, and rotarod performance responded to treatment with the calcium channel blocker isradipine, with implications for the therapy of patients with aldosterone-producing lesions and with PASNA syndrome

Y265C DNA Polymerase Beta Knockin Mice Survive Past Birth and Accumulate Base Excision Repair Intermediate Substrates

DNA is susceptible to damage by a wide variety of chemical agents that are generated either as byproducts of cellular metabolism or exposure to man-made and harmful environments. Therefore, to maintain genomic integrity, having reliable DNA repair systems is important. DNA polymeraseβis known to be a key player in the base excision repair pathway, and mice devoid of DNA polymerase beta do not live beyond a few hours after birth. In this study, we characterized mice harboring an impaired polβvariant. This Y265Cpolβ variant exhibits slow DNA polymerase activity but WT lyase activity and has been shown to be a mutator polymerase. Mice expressing Y265C polβ are born at normal Mendelian ratios. However, they are small, and 60% die within a few hours after birth. Slow proliferation and significantly increased levels of cell death are observed in many organs of the E14 homozygous embryos compared with WT littermates. Mouse embryo fibroblasts prepared from the Y265C polβ embryos proliferate at a rate slower than WT cells and exhibit a gap-filling deficiency during base ex-cision repair. As a result of this, chromosomal aberrations and single- and double-strand breaks are present at significantly higher levels in the homozygous mutant versus WT mouse embryo fibroblasts. This is study in mice is unique in that two enzymatic activities of polβ have been separated; the data clearly demonstrate that the DNA polymerase activity of polβ is essential for survival and genome stability

CpG island turnover events predict evolutionary changes in enhancer activity

Abstract Background Genetic changes that modify the function of transcriptional enhancers have been linked to the evolution of biological diversity across species. Multiple studies have focused on the role of nucleotide substitutions, transposition, and insertions and deletions in altering enhancer function. CpG islands (CGIs) have recently been shown to influence enhancer activity, and here we test how their turnover across species contributes to enhancer evolution. Results We integrate maps of CGIs and enhancer activity-associated histone modifications obtained from multiple tissues in nine mammalian species and find that CGI content in enhancers is strongly associated with increased histone modification levels. CGIs show widespread turnover across species and species-specific CGIs are strongly enriched for enhancers exhibiting species-specific activity across all tissues and species. Genes associated with enhancers with species-specific CGIs show concordant biases in their expression, supporting that CGI turnover contributes to gene regulatory innovation. Our results also implicate CGI turnover in the evolution of Human Gain Enhancers (HGEs), which show increased activity in human embryonic development and may have contributed to the evolution of uniquely human traits. Using a humanized mouse model, we show that a highly conserved HGE with a large CGI absent from the mouse ortholog shows increased activity at the human CGI in the humanized mouse diencephalon. Conclusions Collectively, our results point to CGI turnover as a mechanism driving gene regulatory changes potentially underlying trait evolution in mammals