Presentation Order:
Alyssa
Hannah
Shah
Keaton
Time: biweekly, Wednesday 9:30-11:50
Writing 9:30-10:00---5-10 min/person
Research Presentation: 30 minutes talk+discussion
HL
Fibroblastic reticular cells direct the initiation of T cell responses via CD44
Viruses have evolved mechanisms to evade host immunity, and identifying the pathways they target is essential for understanding antiviral defense and improving therapy. Although CD8 T cell responses are critical for viral control, the stromal mechanisms that organize these responses remain poorly understood. Using mutant murine CMV viruses, structural binding studies, and in vivo mouse infection models, the authors showed that murine cytomegalovirus suppresses antiviral immunity by targeting stromal cells in secondary lymphoid organs rather than immune cells directly. They found that the viral protein m11 binds CD44 on fibroblastic reticular cells, blocks its interaction with hyaluronic acid, and impairs dendritic cell migration and CD8 T cell priming. Deletion of the viral CD44-binding protein increased dendritic cell accumulation in the splenic white pulp, enhanced dendritic cell–T cell interactions, strengthened antiviral CD8 T cell responses, and reduced viral burden in vivo. Although these findings require further validation in human viral infections, this study identifies a previously unrecognized stroma-based immune checkpoint that viruses can exploit to evade adaptive immunity, opening new directions for targeting stromal pathways in antiviral therapies.
ST
Reduced cyclin D3 expression in erythroid cells protects against malaria
Malaria has acted as a strong evolutionary pressure, selecting genetic variants that modify red blood cell (RBC) biology and influence disease severity. Here, the authors investigated the variant rs112233623-T in an erythroid enhancer of the CCND3 gene, which encodes cyclin D3, a regulator of cell cycle and metabolism. Using genetic and population analyses, they showed that this variant is enriched in Sardinia and associated with altered RBC traits, consistent with reduced CCND3 activity. In human erythroblasts, they found using RT–qPCR and western blotting that rs112233623-T lowers CCND3 expression, and using flow cytometry that it slows G1–S cell cycle progression, leading to fewer but larger RBCs. Mechanistically, enhancer assays and binding studies revealed that the variant disrupts the activator SMAD3 and favors the repressor GATA1, reducing CCND3 expression, with pathway perturbation experiments confirming SMAD-dependent regulation. Functionally, malaria infection assays showed impaired Plasmodium falciparum growth in variant RBCs, which correlated with increased reactive oxygen species (ROS), similar to the protective mechanism seen in G6PD deficiency. Overall, the study identifies a regulatory variant linking erythroid cell cycle control and oxidative stress to malaria resistance, although the metabolic link between CCND3 and ROS remains to be defined.
AJM
The transition from monocyte to tissue-resident macrophage requires DHPS
Tissue-resident macrophages (RTMs) are crucial to tissue homeostasis and can be replenished by self-renewal or differentiated from monocytes. However, common mechanisms controlling both RTM maintenance and monocyte differentiation across various tissues have not been elucidated. Herein, the authors investigated LyzM-Cre mediated knockout mice of deoxyhypusin synthase (DHPS), an enzyme for spermidine-dependent hypusine modification of translation initiation factor eIF5, revealing loss of RTMs across tissues. Instead, these niche were enriched in monocytes and early differentiated monocytes-derived macrophages that were deficient in cell adhesion, phagocytosis, and recycling, leading to tissue disorder. While the authors did not elucidate an exact pathway for DHPS to impart its role, they provide a novel target that both could target RTM survival and modulate monocyte action in tissues, like tumors, prior to infiltration.
HL
Bidirectional CRISPR screens decode a GLIS3-dependent fibrotic cell circuit
Chronic inflammatory bowel disease (IBD) progression to fibrosis remains poorly understood at the level of stromal cell regulation. Integrated single-cell and spatial transcriptomic profiling of human IBD tissue identified an expanded inflammation-associated fibroblast (IAF) population enriched for profibrotic and immune-interacting gene programs. Bidirectional genome-wide CRISPR knockout and activation screens in fibroblasts systematically defined regulators of this state, converging on the transcription factor GLIS3 as a central upstream driver. Elevated GLIS3 program activity correlated with greater disease severity in patient biopsies. Fibroblast-specific deletion of Glis3 in mouse colitis models attenuated inflammation-associated fibrosis, establishing functional causality. While reliance on preclinical models leaves translational efficacy and safety in humans unresolved, these findings map a GLIS3-dependent stromal circuit linking inflammation to fibrosis and nominate a potential therapeutic target.
ST
CFAP20 salvages arrested RNAPII from the path of co-directional replisomes
Cells prevent harmful collisions between transcription and DNA replication, particularly at promoter-proximal regions where RNA polymerase II (RNAPII) frequently pauses and forms R-loops. While most previous work focused on head-on collisions, the mechanism that protects co-directional replication forks from paused RNAPII near promoters remained unclear. Using genome-wide mapping, CRISPR-based genetic screens, and DNA fiber assays, the authors identify CFAP20 as a nuclear factor that suppresses Mediator-dependent R-loops specifically at co-directional promoters. Loss of CFAP20 leads to promoter-specific R-loop accumulation, replication fork acceleration, reduced origin firing, and single-stranded DNA gap formation, which can be rescued by inactivating the Mediator kinase module. The major strength of the study is its demonstration that local transcriptional stress can lead to global replication defects, although the mechanism by which CFAP20 promotes RNAPII elongation remains unclear. Future studies should define the direct molecular interaction between CFAP20 and the transcription machinery and determine whether CFAP20 dysfunction contributes to genome instability in vivo.
AJM
Ligand-specific activation trajectories dictate GPCR signalling in cells
G-protein-coupled receptors (GPCRs) are crucial to cell communication and are considered the most important target for drug development. While biophysical analyses of purified GPCRs have elucidated unique conformational states, there are no studies in living cells that identify such dynamics, nor the impact of these states in signaling intensity and duration. Herein, the authors develop a novel biosensor which utilizes a non-canonical amino acid with a small fluorescent label incorporated into various regions of the extracellular domain of the GPCR M2R that emit measurable differences in fluorescence to ligand binding. Coupled with testing agonists of varying affinity and G-proteins of different activity, the authors revealed two distinct GPCR-G protein complexes of unique extracellular conformation that are dependent on ligand and G-protein binding. In sum, strong agonists favor stabilizing complexes of high activation of G-proteins, whereas weaker affinity agonists stabilize a low-efficacy complex; these complexes occur for the same ligand-receptor pair across time for distinct G-proteins. Overall, the authors demonstrate the utility of their GPCR biosensor in ligand-receptor dynamics to understand unique G-protein signaling cascades, which will aid drug advancements towards greater specificity/less off target effects.
KK
A direct role for a mitochondrial targeting sequence in signalling stress
HL
Synergy between regulatory elements can render cohesin dispensable for distal enhancer function
Long-range enhancers are thought to need cohesin-mediated DNA loops to reach promoters, yet many genes remain active when cohesin is removed. The authors rapidly depleted cohesin in mouse stem cells and used RNA-seq, high-resolution 3D genome mapping, and targeted deletions of promoter-proximal elements to test enhancer dependence. Surprisingly, loss of loops caused only modest transcriptional changes, and many distal enhancers still functioned. Mechanistically, promoter-proximal elements synergize with distal enhancers to drive transcription without physical contact. This shows gene activation depends on the combinatorial logic of regulatory elements, not solely on chromosome folding, explaining why structural disruptions often have limited effects.
AJM
Viral RNA blocks circularization to evade host codon usage control
Human cells have a bias for certain tRNA-codon pairs among synonymous codons, which results in differential translation rates across mRNAs. This fundamental mechanism, termed codon-usage bias, should constrain viral protein replication with discordant codon preference; however, nearly all viral genomes, including several common and pandemic-potential viruses for humans, such as influenza and SARS-CoV-2, have dissimilar codon usage preferences to us. Thus, the authors sought to determine the mechanism of viral codon-usage bias escape via careful plasmid construction to evaluate the impacts of codons, flanking untranslated regions (UTRs), and 3D structures on translation rate. Importantly, the authors revealed codon-usage bias is only relevant for UTR-mediated circularized mRNA. Meanwhile, viral 5’ UTRs enforce a linear structure during translation, which eliminates codon preference and escapes inhibition of protein elongation. This work lends support to several biological theories, including the closed-loop structure of canonical mRNA during translation and codon-usage regulation. Overall, this work has broad impacts on gene editing techniques and therapies. Future work can visualize linearization of viral genomes during translation as well as determine the spectrum of viruses utilizing this means of escape.
ST
AJM
Genetic elements promote retention of extrachromosomal DNA in cancer cells
Extra-chromosomal DNA (ecDNA) is a prevalent oncogene activator in diverse cancers, permitting heritable additive expression of oncogenes across cell generations. How ecDNA is retained has been well studied in the viral replication context, however, its mechanism in the human genome has not been established. Herein, the authors revealed a novel method, termed Retain-Seq, that sequences transduced plasmids with random human genome inserts to identify retention sequences that allow ecDNA retention across several rounds of cell division. The authors identify diverse retention sequences with a common thread of transcription-activating regions, including transcriptional start sites, promoters, and enhancer. Importantly, these regions are enriched in CpG sites. Given CpG methylation is crucial for promoter activity, the authors tested the impact of differential methylation status of ecDNA’s CpGs on retention, finding decreased ecDNA retention and subsequent cell proliferation and viability with increased CpG methylation. Overall, the authors elucidate an important biological means of oncogene activation that could pose a novel target for therapeutic intervention against cancer, and could be further applied to improve targeted personalized gene therapies in chronic disease.
KK
Atomically accurate de novo design of antibodies with RFdiffusion
Antibodies are critical to immune defense and as a novel therapeutic class; however, de novo development of antibodies to target specific epitopes requires costly and time-consuming in vivo screening. To address this challenge, the authors of this study created a computational framework to design antibodies against clinically relevant epitopes. A set of antibody designs generated with a modified version of their RFdiffusion structure prediction model, yielded several candidate antibodies that bound to the target, though not at the desired affinity. The authors then combined their model with an additional step to mutate the candidate designs in silico, eventually reaching the desired affinity. Although the current implementation of this approach requires more optimization and validation than is practical for most potential end users, it represents a promising innovation toward de novo antibody design.
HL
Secretome translation shaped by lysosomes and lunapark-marked ER junctions
The spatial organization that drives the translation of mRNAs encoding secretory and membrane proteins at the endoplasmic reticulum (ER), and their coordination with other organelles such as lysosomes, remains poorly understood. Using real-time single-molecule imaging, Choi et al. define the spatial relationship between secretome mRNA translation, ER junctions, and lysosomal proximity. They demonstrate that secretome mRNAs associate with the ER during active translation but are more diffuse when untranslated, and that translating mRNAs preferentially localize to ER three-way junctions termed “translation hotspots.” These hotspots are enriched for the ER-shaping protein Lunapark and frequently positioned near lysosomes. Functionally, efficient secretome mRNA translation depends on both Lunapark and intact lysosomal activity, with lysosomal metabolic state modulating local translation such that amino-acid starvation enhances, while lysosomal deacidification suppresses, translation near junctions. Together, these findings identify a novel mechanism in which Lunapark-marked ER junctions and lysosomes spatially coordinate the translation of secretory and membrane protein mRNAs, reframing ER architecture as an active regulator of translational control rather than a passive scaffold.
ST
Neoadjuvant immunotherapy in mismatch-repair-proficient colon cancers