Massively parallel synthetic promoter assays reveal the in vivo effects of binding site variants

Gene promoters typically contain multiple transcription factor binding sites (TFBSs), which may vary in affinity for their cognate transcription factors (TFs). One major challenge in studying cis-regulation is to understand how TFBS variants affect gene expression. We studied the in vivo effects of TFBS variants on cis-regulation using synthetic promoters coupled with a thermodynamic model of TF binding. We measured expression driven by each promoter with RNA-seq of transcribed sequence barcodes. This allowed reporter genes to be highly multiplexed and increased our statistical power to detect the effects of TFBS variants. We analyzed the effects of TFBS variants using a thermodynamic framework that models both TF-DNA interactions and TF-TF interactions. We found that this system accurately estimates the in vivo relative affinities of TFBSs and predicts unexpected interactions between several TFBSs. Our results reveal that binding site variants can have complex effects on gene expression due to differences in TFBS affinity for cognate TFs and differences in TFBS specificity for noncognate TFs

Seasonal distribution and drivers of surface fine particulate matter and organic aerosol over the Indo-Gangetic Plain

The Indo-Gangetic Plain (IGP) is home to 9 % of the global population and is responsible for a large fraction of agricultural crop production in Pakistan, India, and Bangladesh. Levels of fine particulate matter (mean diameter &lt;2.5 µm, PM2.5) across the IGP often exceed human health recommendations, making cities across the IGP among the most polluted in the world. Seasonal changes in the physical environment over the IGP are dominated by the large-scale south Asian monsoon system that dictates the timing of agricultural planting and harvesting. We use the WRF-Chem model to study the seasonal anthropogenic, pyrogenic, and biogenic influences on fine particulate matter and its constituent organic aerosol (OA) over the IGP that straddles Pakistan, India, and Bangladesh during 2017–2018. We find that surface air quality during pre-monsoon (March–May) and monsoon (June–September) seasons is better than during post-monsoon (October–December) and winter (January–February) seasons, but all seasonal mean values of PM2.5 still exceed the recommended levels, so that air pollution is a year-round problem. Anthropogenic emissions influence the magnitude and distribution of PM2.5 and OA throughout the year, especially over urban sites, while pyrogenic emissions result in localised contributions over the central and upper parts of IGP in all non-monsoonal seasons, with the highest impact during post-monsoon seasons that correspond to the post-harvest season in the agricultural calendar. Biogenic emissions play an important role in the magnitude and distribution of PM2.5 and OA during the monsoon season, and they show a substantial contribution to secondary OA (SOA), particularly over the lower IGP. We find that the OA contribution to PM2.5 is significant in all four seasons (17 %–30 %), with primary OA generally representing the larger fractional contribution. We find that the volatility distribution of SOA is driven mainly by the mean total OA loading and the washout of aerosols and gas-phase aerosol precursors that result in SOA being less volatile during the pre-monsoon and monsoon season than during the post-monsoon and winter seasons.</p

Microbiotas from Humans with Inflammatory Bowel Disease Alter the Balance of Gut Th17 and RORγt+ Regulatory T Cells and Exacerbate Colitis in Mice

Microbiota are thought to influence the development and progression of inflammatory bowel disease (IBD), but determining generalizable effects of microbiota on IBD etiology requires larger-scale functional analyses. We colonized germ-free mice with intestinal microbiotas from 30 healthy and IBD donors and determined the homeostatic intestinal T&nbsp;cell response to each microbiota. Compared to microbiotas from healthy donors, transfer of IBD microbiotas into germ-free mice increased numbers of intestinal Th17 cells and Th2 cells and decreased numbers of RORγt+ Treg cells. Colonization with IBD microbiotas exacerbated disease in a model where colitis is induced upon transfer of naive T&nbsp;cells into Rag1-/- mice. The proportions of Th17 and RORγt+ Treg cells induced by each microbiota were predictive of human disease status and accounted for disease severity in the Rag1-/- colitis model. Thus, an impact on intestinal Th17 and RORγt+ Treg cell compartments emerges as a unifying feature of IBD microbiotas, suggesting a general mechanism for microbial contribution to IBD pathogenesis

Rapid Synthesis Of Defined Eukaryotic Promoter Libraries

Current gene synthesis methods allow the generation of long segments of dsDNA. We show that these techniques can be used to create synthetic regulatory elements and describe a method for the creation of completely defined, synthetic variants of the PHO5 promoter from the budding yeast Saccharomyces cerevisae. 128 promoters were assembled by high-temperature ligation, cloned into plasmids by isothermal assembly, maintained in E. coli, and consequently transformed into yeast by homologous recombination. Synthesis errors occurred at frequencies comparable to, or lower than those achieved with current gene synthesis methods. The promoter synthesis method reported here is robust, fast, and readily accessible. Synthetically engineered promoter libraries will be useful tools for dissecting the intricacies of promoter input-output functions, and may serve as tunable components for synthetic genetic networks