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While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a Drosophila zinc finger transcription factor, we show here that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters. Gene promoters are very often poised for expression by paused RNA polymerase II. Here, Ramalingam et al., identify a mechanism for the de novo establishment of paused Pol II at promoters and study its effects on expression.

Premise of the Study Loss of photosynthesis is a common and often repeated trajectory in nearly all major groups of photosynthetic eukaryotes. One small subset of “apochloritic” diatoms in the genus Nitzschia have lost their ability to photosynthesize and require extracellular carbon for growth. Similar to other secondarily nonphotosynthetic taxa, apochloritic diatoms maintain colorless plastids with highly reduced plastid genomes. Although the narrow taxonomic breadth of apochloritic Nitzschia suggests a single loss of photosynthesis in their common ancestor, previous phylogenetic analyses suggested that photosynthesis was lost multiple times. Methods We analyzed genes from the nuclear, plastid, and mitochondrial genomes for a broad set of taxa to test whether photosynthesis was lost one or multiple times in Bacillariales. We also sequenced and characterized the plastid genome of a nonphotosynthetic Nitzschia species. Key Results Phylogenetic analyses showed that genes from all three genetic compartments either supported or failed to reject monophyly of apochloritic Nitzschia species, consistent with a single loss of photosynthesis in this group. The plastid genomes of two apochloritic Nitzschia are highly similar in all respects, indicating streamlining of the plastid genome before the split of these two species. Conclusions A better understanding of the phylogeny and ecology of apochloritic Nitzschia, together with emerging genomic resources, will help identify the factors that have driven and maintained the loss of photosynthesis in this group of diatoms. Finally, some habitats host diverse communities of co‐occurring nonphotosynthetic diatoms, reflecting resource abundance or resource partitioning in ecologically favorable habitats.

Modern agriculture’s reliance on chemical fertilizers has greatly increased crop yields but has also contributed to soil degradation, environmental pollution, and unsustainable resource use. Plant growth–promoting rhizobacteria offer a sustainable alternative, enhancing plant growth through mechanisms such as nitrogen fixation, phosphate solubilization, siderophore production, and phytohormone modulation, while also protecting plants against pathogens and abiotic stresses. However, soil microbial communities are highly diverse and structurally complex, making the isolation of individual beneficial strains challenging. Traditional approaches rely on selective media and labor-intensive phenotypic screening, which often capture only a small fraction of soil diversity.To overcome these limitations, we combined computational predictions with targeted microbial isolation to identify bacteria associated with sorghum growth under low- and full-nitrogen conditions. Using a large-scale field dataset, we integrated 16S rRNA amplicon sequencing, soil properties, and plant phenotype data to perform change-point analysis, revealing bacterial strains correlated with plant traits in the rhizosphere and endosphere. Targeted limiting-dilution culturing of selected field samples yielded multiple candidates, including two Pseudomonas strains, AOC87 and AOC36, predicted to have positive and negative associations with plant performance, respectively. Genome sequencing and functional assays confirmed these predictions: the “positive” strain possessed a complete indole-3-acetic acid (IAA) degradation pathway, while AOC36 carried a type III secretion system. These results demonstrate that computational prediction pipelines can refine the search for functionally relevant microbes, bridging culture-based methods with high-throughput microbiome analysis and enabling mechanistic investigation.Building on these findings, Chapter Four explores the complexities of studying and applying candidate growth-promoting strains, specifically Variovorax, to sorghum. This chapter shows that Variovorax strains exhibit both conserved and variable traits: IAA degradation is broadly conserved, supported by syntenic IAA-degradation operons, yet the kinetics of degradation differ quantitatively across strains. Despite this conserved metabolic capacity, effects on sorghum root elongation were highly variable and did not directly correlate with IAA degradation rates. Instead, variability likely arises from strain-specific factors such as root colonization efficiency, persistence, and interactions with host hormonal pathways, including auxin–ethylene crosstalk. Collectively, these results highlight that while IAA degradation is a core feature of Variovorax, its influence on plant growth is context-dependent and shaped by ecological and physiological interactions with the host. Overall, this thesis establishes a data-driven framework that integrates microbial community analysis, spatial modeling, and strain-level characterization, enabling the discovery and functional testing of field-relevant microbes that impact crop performance.

• Although most of the tens of thousands of diatom species are photoautotrophs, a small number of heterotrophic species no longer photosynthesize. We sequenced the genome of a nonphotosynthetic diatom, Nitzschia Nitz4, to determine how carbon metabolism was altered in the wake of this trophic shift. • Nitzschia Nitz4 has retained its plastid and plastid genome, but changes associated with the transition to heterotrophy were cellular-wide and included losses of photosynthesis-related genes from the nuclear and plastid genomes, elimination of isoprenoid biosynthesis in the plastid, and remodeling of mitochondrial glycolysis to maximize adenosine triphosphte (ATP) yield. The genome contains a β-ketoadipate pathway that may allow Nitzschia Nitz4 to metabolize lignin-derived compounds. • Diatom plastids lack an oxidative pentose phosphate pathway (oPPP), leaving photosynthesis as the primary source of NADPH to support essential biosynthetic pathways in the plastid and, by extension, limiting available sources of NADPH in nonphotosynthetic plastids. • The genome revealed similarities between nonphotosynthetic diatoms and apicomplexan parasites for provisioning NADPH in their plastids and highlighted the ancestral absence of a plastid oPPP as a potentially important constraint on loss of photosynthesis, a hypothesis supported by the higher frequency of transitions to parasitism or heterotrophy in lineages that have a plastid oPPP.

Diatoms are prolific photosynthesizers responsible for some 20% of global primary production. In real terms, the oxygen in one of every five breaths traces back to photosynthesis by marine diatoms. Among the tens of thousands of diatom species, a small handful of colorless diatom species in the genus Nitzschia have lost photosynthesis altogether and rely exclusively on extracellular organic carbon for growth. I used DNA sequence data to reconstruct the phylogeny of this group, and found that nonphotosynthetic diatoms are monophyletic, indicating that photosynthesis was lost just one time over the course of some 200 million years of diatom evolution. Carbon metabolism in nonphotosynthetic diatoms, including the exact source of carbon used by these species, has not been fully characterized. We sequenced the nuclear genome of one species and used it to develop a comprehensive model of central carbon metabolism. Preliminary analysis of Nitzschia metabolism showed that it generally matches to the pattern of previously reported diatom metabolic networks. As well we found some hints regarding Nitzschia external carbon acquisition which possibly can help to explain its heterotrophic mode of life. Overall, this study has provided novel insights into the evolutionary origin and metabolism of non-photosynthetic diatoms, which are unique among diatoms in their ability to sustain their growth solely from extracellular carbon.

While enhancers are often regulated at the level of accessibility by pioneer factors, promoters tend to be constitutively accessible and poised for activation by paused Pol II — thus are often not considered as sites of developmental regulation. Here we show that the accessibility of promoters and the acquisition of paused Pol II can be subject to developmental regulation by pioneer factors. We show that Lola-I, a Drosophila zinc finger transcription factor, is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene expression. Lola-I mediates this function by binding to the edges of the promoter nucleosomes, which leads to their depletion, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers, providing further evidence that promoters and enhancers display unexpectedly similar characteristics.

Loss of photosynthesis is a common and often repeated trajectory in nearly all major groups of photosynthetic eukaryotes. One small subset of 'apochlorotic' diatoms in the genus Nitzschia have lost their ability to photosynthesize and require extracellular carbon for growth. Similar to other secondarily nonphotosynthetic taxa, apochloritic diatoms maintain colorless plastids with highly reduced plastid genomes. Although the narrow taxonomic breadth of apochloritic diatoms suggests a single loss of photosynthesis in the common ancestor of these species, previous phylogenetic analyses suggested that photosynthesis was lost multiple times. We sequenced additional phylogenetic markers from the nuclear and mitochondrial genomes for a larger set of taxa and found that the best trees for datasets representing all three genetic compartments provided low to moderate support for monophyly of apochlorotic Nitzschia, consistent with a single loss of photosynthesis in diatoms. We sequenced the plastid genome of one apochloritic species and found that it was highly similar in all respects to the plastid genome of another apochloritic Nitzschia species, indicating that streamlining of the plastid genome had completed prior to the split of these two species. Finally, it is increasingly clear that some locales host relatively large numbers apochloritic Nitzschia species that span the phylogenetic diversity of the group, indicating that these species co-exist because of resource abundance or resource partitioning in ecologically favorable habitats. A better understanding of the phylogeny and ecology of this group, together with emerging genomic resources, will help identify the factors that have driven and maintained the loss of photosynthesis in this group, a rare event in diatoms.

Chloroquine is an anti-malarial and immunosuppressant drug that has cationic amphipathic chemical properties. We performed genome-wide screens in human cells with chloroquine and several other widely used cationic amphipathic drugs (CADs) including the anti-depressants, sertraline (Zoloft) and fluoxetine (Prozac), the analgesic nortriptyline (Pamelor), the anti-arrhythmic amiodarone (Cordarone), and the anti-hypertensive verapamil (Calan) to characterize their molecular similarities and differences. Despite CADs having different disease indications but consistent with them sharing key chemical properties, we found CADs to have remarkably similar phenotypic profiles compared with non-CADs we and others have previously screened. The most significant genetic interaction for all CADs was the initiating step in sphingolipid biosynthesis catalyzed by serine palmitoyltransferase (SPT). A comparison of genome-wide screens performed with diverse pathogens from viruses, bacteria, plants, and parasites including Ebola, adeno-associated virus AAV2, HIV, Rotavirus, Influenza A, Zika virus, Picornavirus, Exotoxin A, Cholera toxin, Type III secretion system and Shiga toxin, Ricin toxin, and Toxoplasma gondii showed SPT as a top common host factor and 80% overlap overall in top hits specifically with CADs. Potential sphingolipid-mediated mechanisms for the host response- and virulence-modulating effects of CADs involve autophagy and SERPINE1/PAI-1 (plasminogen activator inhibitor-1). Chloroquine has recently shown potential as an anti-viral agent for the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease. Our study demonstrates that numerous readily available drugs molecularly function highly similar to chloroquine, which suggests they might be considered for further pre-clinical investigation in the context of SARS-CoV-2. More generally, our work suggests the diverse pathogen mitigating potential of drugs that inhibit host sphingolipid biosynthesis such as CADs. Competing Interest Statement The corresponding author, Timothy R Peterson, is the founder of Bio-I/O, a St. Louis-based biotech company specializing in drug target identification. Bio-I/O is the recipient of NIH/NIDDK funding (R42 DK121652), which is focused on different drugs than those studied herein, but is still in the space of drug target ID. Footnotes * https://github.com/tim-peterson/CADs-pathogens