In recent years, functional genomics approaches combining genetic information with bulk RNA-sequencing data have identified the downstream expression effects of disease-associated genetic risk factors through so-called expression quantitative trait locus (eQTL) analysis. Single-cell RNA-sequencing creates enormous opportunities for mapping eQTLs across different cell types and in dynamic processes, many of which are obscured when using bulk methods. Rapid increase in throughput and reduction in cost per cell now allow this technology to be applied to large-scale population genetics studies. To fully leverage these emerging data resources, we have founded the single-cell eQTLGen consortium (sc-eQTLGen), aimed at pinpointing the cellular contexts in which disease-causing genetic variants affect gene expression. Here, we outline the goals, approach and potential utility of the sc-eQTLGen consortium. We also provide a set of study design considerations for future single-cell eQTL studies.

The perception that the inheritance of phenotypic traits operates solely through genetic means is slowly being eroded: epigenetic mechanisms have been shown to induce heritable changes in gene activity in plants^1,2 and metazoans^1,3. Inheritance of DNA methylation patterns provides a potential pathway for environmentally induced phenotypes to contribute to evolution of species and populations^1,2,3,4,5. However, in basal metazoans, it is unknown whether inheritance of CpG methylation patterns occurs across the genome (as in plants) or as rare exceptions (as in mammals)⁴. Here, we show that DNA methylation patterns in a reef-building coral are determined by genotype and developmental stage, as well as by parental environment. Transmission of CpG methylation from adults to their sperm and larvae demonstrates genome-wide inheritance. Variation in the hypermethylation of genes in adults and their sperm from distinct environments suggests intergenerational acclimatization to local temperature and salinity. Furthermore, genotype-independent adjustments of methylation levels in stress-related genes were strongly correlated with offspring survival rates under heat stress. These findings support a role of DNA methylation in the intergenerational inheritance of traits in corals, which could extend to enhancing their capacity to adapt to climate change.

The silencing of tumor suppressor genes (TSGs) by aberrant DNA methylation occurs frequently in acute myeloid leukemia (AML). This epigenetic alteration can be reversed by 5-aza-2’-deoxcytidine (decitabine, 5-AZA-CdR). Although 5-AZA-CdR can induce complete remissions in patients with AML, most patients relapse. The effectiveness of this therapy may be limited by the inability of 5-AZA-CdR to reactivate all TSGs due to their silencing by other epigenetic mechanisms such as histone methylation or chromatin compaction. EZH2, a subunit of the polycomb repressive complex 2, catalyzes the methylation of histone H3 lysine 27 (H3K27) to H3K27me3. 3-Deazaneplanocin-A (DZNep), an inhibitor of methionine metabolism, can reactivate genes silenced by H3K27me3 by its inhibition of EZH2. In a previous report, we observed that 5-AZA-CdR, in combination with DZNep, shows synergistic antineoplastic action against AML cells. Gene silencing due to chromatin compaction is attributable to the action of histone deacetylases (HDAC). This mechanism of epigenetic gene silencing can be reversed by HDAC inhibitors such as trichostatin-A (TSA). Silent TSGs that cannot be reactivated by 5-AZA-CdR or DZNep have the potential to be reactivated by TSA. This provides a rationale for the use of HDAC inhibitors in combination with 5-AZA-CdR and DZNep to treat AML.

Fluoride ions are highly reactive, and their incorporation in forming dental enamel at low concentrations promotes mineralization. In contrast, excessive fluoride intake causes dental fluorosis, visually recognizable enamel defects that can increase the risk of caries. To investigate the molecular bases of dental fluorosis, we analyzed the effects of fluoride exposure in enamel cells to assess its impact on Ca²⁺ signaling. Primary enamel cells and an enamel cell line (LS8) exposed to fluoride showed decreased internal Ca²⁺ stores and store-operated Ca²⁺ entry (SOCE). RNA-sequencing analysis revealed changes in gene expression suggestive of endoplasmic reticulum (ER) stress in fluoride-treated LS8 cells. Fluoride exposure did not alter Ca²⁺ homeostasis or increase the expression of ER stress–associated genes in HEK-293 cells. In enamel cells, fluoride exposure affected the functioning of the ER-localized Ca²⁺ channel IP₃R and the activity of the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump during Ca²⁺ refilling of the ER. Fluoride negatively affected mitochondrial respiration, elicited mitochondrial membrane depolarization, and disrupted mitochondrial morphology. Together, these data provide a potential mechanism underlying dental fluorosis.

Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca ²⁺ release–activated Ca ²⁺ (CRAC) channels and store-operated Ca ²⁺ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated Stim1/2 ^K14cre mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in Stim1/2 ^K14cre mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in Stim1/2 -deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of Stim1/2 ^K14cre mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel.