Faculty Bibliography

In mammals, the conserved telomere binding protein Rap1 serves a diverse set of nontelomeric functions, including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which Rap1 modulates gene expression. Using a separation-of-function allele, we show that Rap1 transcriptional regulation is largely independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci. Instead, Rap1 interacts with the TIP60/p400 complex and modulates its histone acetyltransferase activity. Notably, we show that deletion of Rap1 in mouse embryonic stem cells increases the fraction of two-cell-like cells. Specifically, Rap1 enhances the repressive activity of Tip60/p400 across a subset of two-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential up-regulation of genes proximal to MERVL elements in Rap1-deficient settings implicates these endogenous retroviral elements in the derepression of proximal genes. Altogether, our study reveals an unprecedented link between Rap1 and the TIP60/p400 complex in the regulation of pluripotency.

During animal embryogenesis, homeostasis and disease, tissues push and pull on their surroundings to move forward. Although the force-generating machinery is known, it is unknown how tissues exert physical stresses on their substrate to generate motion in vivo. Here, we identify the force transmission machinery, the substrate and the stresses that a tissue, the zebrafish posterior lateral line primordium, generates during its migration. We find that the primordium couples actin flow through integrins to the basement membrane for forward movement. Talin- and integrin-mediated coupling is required for efficient migration, and its loss is partially compensated for by increased actin flow. Using Embryogram, an approach to measure stresses in vivo, we show that the rear of the primordium exerts higher stresses than the front, which suggests that this tissue pushes itself forward with its back. This unexpected strategy probably also underlies the motion of other tissues in animals.

The gut microbiome shapes local and systemic immunity. The liver is presumed to be a protected sterile site. As such, a hepatic microbiome has not been examined. Here, we showed a liver microbiome in mice and humans that is distinct from the gut and is enriched in Proteobacteria. It undergoes dynamic alterations with age and is influenced by the environment and host physiology. Fecal microbial transfer experiments revealed that the liver microbiome is populated from the gut in a highly selective manner. Hepatic immunity is dependent on the microbiome, specifically Bacteroidetes species. Targeting Bacteroidetes with oral antibiotics reduced hepatic immune cells by ~90%, prevented APC maturation, and mitigated adaptive immunity. Mechanistically, our findings are consistent with presentation of Bacteroidetes-derived glycosphingolipids to NKT cells promoting CCL5 signaling, which drives hepatic leukocyte expansion and activation, among other possible host-microbe interactions. Collectively, we reveal a microbial - glycosphingolipid - NKT - CCL5 axis that underlies hepatic immunity.

Unlike other checkpoint inhibitors, our targeted immunotherapeutic localizes to any solid tumor and simultaneouslyshields an agent of immuno suppression while presenting a signal for immunostimulation. Phosphatidylserine (PS)exposure on the extracellular surface of living tumor cells and their vasculatures provides one avenue by which thetumor microenvironment promotes immunosuppression. Extracellular surface PS is inherent to a tumor and itsvasculature, even for inoperable tumors, and its expression cannot be mutated nor affected by acquired drugresistance. Annexin A5 (AnxA5) is a direct, high-affinity PS-binding protein that localizes to cells with PS exposed onthe outer plasma membrane. In our studies, we conjugated a proprietary modified AnxA5, lacking cellularinternalization, to TNFalpha (AnxA5 -TNFalpha) to convert the immunosuppresive environs of a murine 4T1 triplenegative breast cancer (TNBC) into an immunostimulated one. This strategy localized the immune response to the tumor and minimized side effects, as evidenced by a lack of toxicity for up to 7 days in non-tumor bearing Balb/cfemale mice given up to 1 mg/kg. Proper assembly and functionality of AnxA5 -TNFalpha was verified simultaneouslyby ellipsometry, an optical technique similar to plasmon resonance. Fully assembled constructs were tested forbinding to PS coated slides. The degree of light polarization is proportional to the amount of PS bound by the AnxA5complex. Samples could be further incubated with TNF receptors to verify TNFalpha activity. Based on dose escalationstudies in 4T1 tumor-bearing mice where the TNBC tumors were grown in the mammary fat pads, optimal dosages were determined for AnxA5 -TNFalpha (18 mug) and AnxA5 alone as a control (180 mug). These doses were furthertested in a 4T1 growth inhibition study. Tumor size was tracked by caliper in two groups of mice (n=5/group)receiving drug treatment on days 12, 14 and 16 and a repeated measures ANOVA was conducted onmeasurements taken before, during and post-treatment. While median tumor size did not differ between control and drug treatment groups during the pre-treatment interval (p=0.84), there was a significant difference post-treatment(p<0.001) with mice receiving AnxA5 -TNFalpha having much smaller TNBC tumors. Tumors from the study were embedded in paraffin, sectioned (5 mum) and the overall immune cell content determined by H&E staining. Once it was evident there was a greater quantity of immune cells in AnxA5 -TNFalpha treated tumors vs. controls, sections were stained with validated antibodies to identify and count the immunoactivated T-cells, NK-cells and macrophages. There was a 3X greater mean percentage of CD8 and CD4 T-cells in mice receiving drug vs. control(p=0.03) along with 2.5X and 5X increases in NK-cells and M1 immunoactive macrophages, respectively.
Conclusion(s): Our AnxA5 -TNFalpha inhibits the PS inhibitor while simultaneously activating TNF activators!

Proper telomere length is essential for indefinite self-renewal of embryonic stem (ES) cells and cancer cells. Telomerase-deficient late generation mouse ES cells and human ALT cancer cells are able to propagate for numerous passages, suggesting telomerase-independent mechanisms responding for telomere maintenance. However, the underlying mechanisms ensuring the telomere length maintenance are unclear. Here, using late generation telomerase KO (G4 Terc^-/-) ESCs as a model, we show that Zscan4, highly upregulated in G4 Terc^-/- ESCs, is responsible for the prolonged culture of these cells with stably short telomeres. Mechanistically, G4 Terc^-/- ESCs showed reduced levels of DNA methylation and H3K9me3 at Zscan4 promoter and subtelomeres, which relieved the expression of Zscan4. Similarly, human ZSCAN4 was also derepressed by reduced H3K9me3 at its promoter in ALT U2 OS cells, and depletion of ZSCAN4 significantly shortened telomeres. Our results define a similar conserved pathway contributing to the telomere maintenance in telomerase-deficient late generation mESCs and human ALT U2OS cancer cells.

Clonal expansion is a process that can drive pathogenesis in human diseases, with atherosclerosis being a prominent example. Despite advances in understanding the etiology of atherosclerosis, clonality studies of vascular cells remain in an early stage. Recently, several paradigm-shifting preclinical studies have identified clonal expansion of progenitor cells in the vasculature in response to atherosclerosis. This review provides an overview of cell clonality in atherosclerotic progression, focusing particularly on smooth muscle cells and macrophages. We discuss key findings from the latest research that give insight into the mechanisms by which clonal expansion of vascular cells contributes to disease pathology. The further probing of these mechanisms will provide innovative directions for future progress in the understanding and therapy of atherosclerosis and its associated cardiovascular diseases.

LetB is a tunnel-forming protein found in the cell envelope of some double-membraned bacteria, and is thought to be important for the transport of lipids between the inner and outer membranes. In Escherichia coli the LetB tunnel is formed from a stack of seven rings (Ring1 - Ring7), in which each ring is composed of a homo-hexameric assembly of MCE domains. The primary sequence of each MCE domain of the LetB protein is substantially divergent from the others, making each MCE ring unique in nature. The role of each MCE domain and how it contributes to the function of LetB is not well understood. Here we probed the importance of each MCE ring for the function of LetB, using a combination of bacterial growth assays and cryo-EM. Surprisingly, we find that ΔRing3 and ΔRing6 mutants, in which Ring3 and Ring6 have been deleted, confer increased resistance to membrane perturbing agents. Specific mutations in the pore-lining loops of Ring6 similarly confer increased resistance. A cryo-EM structure of the ΔRing6 mutant shows that despite the absence of Ring6, which leads to a shorter assembly, the overall architecture is maintained, highlighting the modular nature of MCE proteins. Previous work has shown that Ring6 is dynamic and in its closed state, may restrict the passage of substrate through the tunnel. Our work suggests that removal of Ring6 may relieve this restriction. The deletion of Ring6 combined with mutations in the pore-lining loops leads to a model for the tunnel gating mechanism of LetB. Together, these results provide insight into the functional roles of individual MCE domains and pore-lining loops in the LetB protein.

Mechanical overload of the vascular wall is a pathological hallmark of life-threatening abdominal aortic aneurysms (AAA). However, how this mechanical stress resonates at the unicellular level of vascular smooth muscle cells (VSMC) is undefined. Here we show defective mechano-phenotype signatures of VSMC in AAA measured with ultrasound tweezers-based micromechanical system and single-cell RNA sequencing technique. Theoretical modelling predicts that cytoskeleton alterations fuel cell membrane tension of VSMC, thereby modulating their mechanoallostatic responses which are validated by live micromechanical measurements. Mechanistically, VSMC gradually adopt a mechanically solid-like state by upregulating cytoskeleton crosslinker, α-actinin2, in the presence of AAA-promoting signal, Netrin-1, thereby directly powering the activity of mechanosensory ion channel Piezo1. Inhibition of Piezo1 prevents mice from developing AAA by alleviating pathological vascular remodeling. Our findings demonstrate that deviations of mechanosensation behaviors of VSMC is detrimental for AAA and identifies Piezo1 as a novel culprit of mechanically fatigued aorta in AAA.

Single-cell RNA sequencing (scRNA-seq) enables molecular characterization of complex biological tissues at high resolution. The requirement of single-cell extraction, however, makes it challenging for profiling tissues such as adipose tissue, for which collection of intact single adipocytes is complicated by their fragile nature. For such tissues, single-nucleus extraction is often much more efficient and therefore single-nucleus RNA sequencing (snRNA-seq) presents an alternative to scRNA-seq. However, nuclear transcripts represent only a fraction of the transcriptome in a single cell, with snRNA-seq marked with inherent transcript enrichment and detection biases. Therefore, snRNA-seq may be inadequate for mapping important transcriptional signatures in adipose tissue. In this study, we compare the transcriptomic landscape of single nuclei isolated from preadipocytes and mature adipocytes across human white and brown adipocyte lineages, with whole-cell transcriptome. We show that snRNA-seq is capable of identifying the broad cell types present in scRNA-seq at all states of adipogenesis. However, we also explore how and why the nuclear transcriptome is biased and limited, as well as how it can be advantageous. We robustly characterize the enrichment of nuclear-localized transcripts and adipogenic regulatory lncRNAs in snRNA-seq, while also providing a detailed understanding for the preferential detection of long genes upon using this technique. To remove such technical detection biases, we propose a normalization strategy for a more accurate comparison of nuclear and cellular data. Finally, we show successful integration of scRNA-seq and snRNA-seq data sets with existing bioinformatic tools. Overall, our results illustrate the applicability of snRNA-seq for the characterization of cellular diversity in the adipose tissue.