Faculty Bibliography

Bacterial infection often leads to suppression of mRNA translation, but hosts are nonetheless able to express immune response genes through as yet unknown mechanisms. Here, we use a Drosophila model to demonstrate that antimicrobial peptide (AMP) production during infection is paradoxically stimulated by the inhibitor of cap-dependent translation, 4E-BP (eIF4E-binding protein; encoded by the Thor gene). We found that 4E-BP is induced upon infection with pathogenic bacteria by the stress-response transcription factor ATF4 and its upstream kinase, GCN2. Loss of gcn2, atf4, or 4e-bp compromised immunity. While AMP transcription is unaffected in 4e-bp mutants, AMP protein levels are substantially reduced. The 5' UTRs of AMPs score positive in cap-independent translation assays, and this cap-independent activity is enhanced by 4E-BP. These results are corroborated in vivo using transgenic 5' UTR reporters. These observations indicate that ATF4-induced 4e-bp contributes to innate immunity by biasing mRNA translation toward cap-independent mechanisms, thus enhancing AMP synthesis.

Progress in whole-genome sequencing using short-read (e.g., <150 bp), next-generation sequencing technologies has reinvigorated interest in high-resolution physical mapping to fill technical gaps that are not well addressed by sequencing. Here, we report two technical advances in DNA nanotechnology and single-molecule genomics: (1) we describe a labeling technique (CRISPR-Cas9 nanoparticles) for high-speed AFM-based physical mapping of DNA and (2) the first successful demonstration of using DVD optics to image DNA molecules with high-speed AFM. As a proof of principle, we used this new "nanomapping" method to detect and map precisely BCL2-IGH translocations present in lymph node biopsies of follicular lymphoma patents. This HS-AFM "nanomapping" technique can be complementary to both sequencing and other physical mapping approaches.

Medical education is undergoing significant transformation. Many medical schools are moving away from the concept of seat time to competency-based education and introducing flexibility in the curriculum that allows individualization. In response to rising student debt and the anticipated physician shortage, 35% of US medical schools are considering the development of accelerated pathways. The roadmap described in this paper is grounded in the experiences of the Consortium of Accelerated Medical Pathway Programs (CAMPP) members in the development, implementation, and evaluation of one type of accelerated pathway: the three-year MD program. Strategies include developing a mission that guides curricular development - meeting regulatory requirements, attaining institutional buy-in and resources necessary to support the programs, including student assessment and mentoring - and program evaluation. Accelerated programs offer opportunities to innovate and integrate a mission benefitting students and the public. ABBREVIATIONS: CAMPP: Consortium of accelerated medical pathway programs; GME: Graduate medical education; LCME: Liaison committee on medical education; NRMP: National residency matching program; UME: Undergraduate medical education.

Squamous cell carcinomas (SCCs) are heterogeneous tumors sustained by tumor-propagating cancer cells (TPCs). SCCs frequently resist chemotherapy through still unknown mechanisms. Here, we combine H2B-GFP-based pulse-chasing with cell-surface markers to distinguish quiescent from proliferative TPCs within SCCs. We find that quiescent TPCs resist DNA damage and exhibit increased tumorigenic potential in response to chemotherapy, whereas proliferative TPCs undergo apoptosis. Quiescence is regulated by TGF-beta/SMAD signaling, which directly regulates cell-cycle gene transcription to control a reversible G1 cell-cycle arrest, independent of p21CIP function. Indeed, genetic or pharmacological TGF-beta inhibition increases the susceptibility of TPCs to chemotherapy because it prevents entry into a quiescent state. These findings provide direct evidence that TPCs can reversibly enter a quiescent, chemoresistant state and thereby underscore the need for combinatorial approaches to improve treatment of chemotherapy-resistant SCCs.

Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.

With recent advances in stem cell technology, it is becoming efficient to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, which can subsequently be used for myriad purposes, ranging from interrogating mechanisms of cardiovascular disease, developing novel cellular therapeutic approaches, as well as assessing the cardiac safety profile of compounds. However, the relative inability to acquire abundant pure and mature cardiomyocytes still hinders these applications. Recently, it was reported that glucose-depleted culture medium supplemented with lactate can facilitate purification of hPSC-derived cardiomyocytes. Here, we report that fatty acid as a lactate replacement has not only a similar purification effect but also improves the electrophysiological characteristics of hPSC-derived cardiomyocytes. Glucose-depleted culture medium supplemented with fatty acid and 3,3',5-Triiodo-l-thyronine (T3) was used during enrichment of hPSC-derived cardiomyocytes. Compared to untreated control cells, the treated cardiomyocytes exhibited enhanced action potential (AP) maximum upstroke velocity (as shown by a significant increase in dV/dtmax), action potential amplitude, as well as AP duration at 50% (APD50) and 90% (APD90) of repolarization. The treated cardiomyocytes displayed higher sensitivity to isoproterenol, more organized sarcomeric structures, and lower proliferative activity. Expression profiling showed that various ion channel and cardiac-specific genes were elevated as well. Our results suggest that the use of fatty acid and T3 can facilitate purification and maturation of hPSC-derived cardiomyocytes.

Mammalian polymerase theta (Poltheta) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Poltheta-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Poltheta-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Poltheta and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.