Publications

The autoimmune disease systemic lupus erythematosus has a complex environmental and multifactorial genetic basis. Genome-wide association studies have recently identified numerous disease-associated polymorphisms, but it remains unclear in which cells and during which step of pathogenesis specific polymorphisms interact to cause disease. Using a mouse model in which the same activating mutation (CD45E613R) causes distinct genetic background-dependent disease phenotypes, we performed a screen for genetic modifiers of autoreactivity between anti-nuclear Ab (ANA)-resistant CD45E613R.B6 and ANA-permissive CD45E613R.BALB/c mice. Within a novel autoreactivity-associated locus on chromosome 9, we identify a putative modifier, TLR9. Validating a role for TLR9 in modifying autoreactivity in the context of the CD45E613R mutation, manipulation of TLR9 gene dosage eliminates ANA in CD45E613R.BALB/c mice, but confoundingly permits ANA in CD45E613R.B6 mice. We demonstrate that sensitivity to ANA is modulated by strength of TLR9 signal, because stronger TLR9(B6) signals, but not weaker TLR9(BALB/c) signals, negatively regulate CD45E613R B cell development during competitive reconstitution at the central tolerance checkpoint. Our results identify a novel autoreactivity-associated locus and validate Tlr9 as a candidate gene within the locus. We further demonstrate a novel role for TLR9 signal strength in central tolerance, providing insight into the interplay of disease-associated polymorphisms at a discrete step of systemic lupus erythematosus pathogenesis.

The mechanisms by which transcription factor haploinsufficiency alters the epigenetic and transcriptional landscape in human cells to cause disease are unknown. Here, we utilized human induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) to show that heterozygous nonsense mutations in NOTCH1 that cause aortic valve calcification disrupt the epigenetic architecture, resulting in derepression of latent pro-osteogenic and -inflammatory gene networks. Hemodynamic shear stress, which protects valves from calcification in vivo, activated anti-osteogenic and anti-inflammatory networks in NOTCH1(+/+), but not NOTCH1(+/-), iPSC-derived ECs. NOTCH1 haploinsufficiency altered H3K27ac at NOTCH1-bound enhancers, dysregulating downstream transcription of more than 1,000 genes involved in osteogenesis, inflammation, and oxidative stress. Computational predictions of the disrupted NOTCH1-dependent gene network revealed regulatory nodes that, when modulated, restored the network toward the NOTCH1(+/+) state. Our results highlight how alterations in transcription factor dosage affect gene networks leading to human disease and reveal nodes for potential therapeutic intervention.

Resistance to combined antiretroviral therapy (cART) in HIV-1-infected individuals is typically due to nonsynonymous mutations that change the protein sequence; however, the selection of synonymous or 'silent' mutations in the HIV-1 genome with cART has been reported. These silent K65K and K66K mutations in the HIV-1 reverse transcriptase (RT) occur in over 35% of drug-experienced individuals and are highly associated with the thymidine analog mutations D67N and K70R, which confer decreased susceptibility to most nucleoside and nucleotide RT inhibitors. However, the basis for selection of these silent mutations under selective drug pressure is unknown. Using Illumina next-generation sequencing, we demonstrate that the D67N/K70R substitutions in HIV-1 RT increase indel frequency by 100-fold at RT codons 65-67, consequently impairing viral fitness. Introduction of either K65K or K66K into HIV-1 containing D67N/K70R reversed the error-prone DNA synthesis at codons 65-67 in RT and improved viral replication fitness, but did not impact RT inhibitor drug susceptibility. These data provide new mechanistic insights into the role of silent mutations selected during antiretroviral therapy and have broader implications for the relevance of silent mutations in the evolution and fitness of RNA viruses.