Internal initiation of reverse transcription in a Penelope-like retrotransposon.

Eukaryotic retroelements are generally divided into two classes: long terminal repeat (LTR) retrotransposons and non-LTR retrotransposons. A third class of eukaryotic retroelement, the Penelope-like elements (PLEs), has been well-characterized bioinformatically, but relatively little is known about the transposition mechanism of these elements. PLEs share some features with the R2 retrotransposon from Bombyx mori, which uses a target-primed reverse transcription (TPRT) mechanism, but their distinct phylogeny suggests PLEs may utilize a novel mechanism of mobilization. Using protein purified from E. coli, we report unique in vitro properties of a PLE from the green anole (Anolis carolinensis), revealing mechanistic aspects not shared by other retrotransposons. We found that reverse transcription is initiated at two adjacent sites within the transposon RNA that is not homologous to the cleaved DNA, a feature that is reflected in the genomic "tail" signature shared between and unique to PLEs. Our results for the first active PLE in vitro provide a starting point for understanding PLE mobilization and biology.

Multi-omic Analysis of Human B-cell Activation Reveals a Key Lysosomal BCAT1 Role in mTOR Hyperactivation by B-cell receptor and TLR9.

B-lymphocytes play major adaptive immune roles, producing antibody and driving T-cell responses. However, how immunometabolism networks support B-cell activation and differentiation in response to distinct receptor stimuli remains incompletely understood. To gain insights, we systematically investigated acute primary human B-cell transcriptional, translational and metabolomic responses to B-cell receptor (BCR), Toll-like receptor 9 (TLR9), CD40-ligand (CD40L), interleukin-4 (IL4) or combinations thereof. T-independent BCR/TLR9 co-stimulation, which drives malignant and autoimmune B-cell states, jointly induced PD-L1 plasma membrane expression, supported by NAD metabolism and oxidative phosphorylation. BCR/TLR9 also highly induced the transaminase BCAT1, which localized to lysosomal membranes to support branched chain amino acid synthesis and mTORC1 hyperactivation. BCAT1 inhibition blunted BCR/TLR9, but not CD40L/IL4-triggered B-cell proliferation, IL10 expression and BCR/TLR pathway-driven lymphoma xenograft outgrowth. These results provide a valuable resource, reveal receptor-mediated immunometabolism remodeling to support key B-cell phenotypes including PD-L1 checkpoint signaling, and identify BCAT1 as a novel B-cell therapeutic target.

Cell type-specific delivery by modular envelope design.

The delivery of genetic cargo remains one of the largest obstacles to the successful translation of experimental therapies, in large part due to the absence of targetable delivery vectors. Enveloped delivery modalities use viral envelope proteins, which determine tropism and induce membrane fusion. Here we develop DIRECTED (Delivery to Intended REcipient Cells Through Envelope Design), a modular platform that consists of separate fusion and targeting components. To achieve high modularity and programmable cell type specificity, we develop multiple strategies to recruit or immobilize antibodies on the viral envelope, including a chimeric antibody binding protein and a SNAP-tag enabling the use of antibodies or other proteins as targeting molecules. Moreover, we show that fusogens from multiple viral families are compatible with DIRECTED and that DIRECTED components can target multiple delivery chassis (e.g., lentivirus and MMLV gag) to specific cell types, including primary human T cells in PBMCs and whole blood.

Natural history of Ebola virus disease in rhesus monkeys shows viral variant emergence dynamics and tissue-specific host responses.

Ebola virus (EBOV) causes Ebola virus disease (EVD), marked by severe hemorrhagic fever; however, the mechanisms underlying the disease remain unclear. To assess the molecular basis of EVD across time, we performed RNA sequencing on 17 tissues from a natural history study of 21 rhesus monkeys, developing new methods to characterize host-pathogen dynamics. We identified alterations in host gene expression with previously unknown tissue-specific changes, including downregulation of genes related to tissue connectivity. EBOV was widely disseminated throughout the body; using a new, broadly applicable deconvolution method, we found that viral load correlated with increased monocyte presence. Patterns of viral variation between tissues differentiated primary infections from compartmentalized infections, and several variants impacted viral fitness in a EBOV/Kikwit minigenome system, suggesting that functionally significant variants can emerge during early infection. This comprehensive portrait of host-pathogen dynamics in EVD illuminates new features of pathogenesis and establishes resources to study other emerging pathogens.

Salmonella-based platform for efficient delivery of functional binding proteins to the cytosol.

Protein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wide-ranging applications for basic research and therapeutic approaches. However, whereas small chemical molecules efficiently reach intracellular targets, the delivery of macromolecules into the cytosol of cells remains a major challenge; thus cytosolic applications of protein-based reagents are rather limited. Some pathogenic bacteria have evolved a conserved type III secretion system (T3SS) which allows the delivery of effector proteins into eukaryotic cells. Here, we enhance the T3SS of an avirulent strain of Salmonella typhimurium to reproducibly deliver multiple classes of recombinant proteins into eukaryotic cells. The efficacy of the system is probed with both DARPins and monobodies to functionally inhibit the paradigmatic and largely undruggable RAS signaling pathway. Thus, we develop a bacterial secretion system for potent cytosolic delivery of therapeutic macromolecules.

Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions.

SOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. How temporal fluctuations in their expression levels bias lineage commitment is unknown. Here, we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cells towards both neuroectodermal and mesendodermal fates in undirected differentiation. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

The elusive role of mitotic bookmarking in transcriptional regulation: Insights from Sox2.

The ability of some transcription factors to remain bound to specific genes on condensed mitotic chromosomes has been suggested to play a role in their rapid transcriptional reactivation upon mitotic exit. We have recently shown that SOX2 and OCT4 remain associated to mitotic chromosomes, and that depletion of SOX2 at the mitosis-G1 (M-G1) transition impairs its ability to maintain pluripotency and drive neuroectodermal commitment. Here we report on the role of SOX2 at the M-G1 transition in regulating transcriptional activity of embryonic stem cells. Using single cell time-lapse analysis of reporter constructs for STAT3 and SOX2/OCT4 activity, we show that SOX2/OCT4 do not lead to more rapid transcriptional reactivation in G1 than STAT3, a transcription factor that is excluded from mitotic chromosomes. We also report that only few endogenous target genes show decreased pre-mRNA levels after mitotic exit or in other cell cycle phases in the absence of SOX2 at the M-G1 transition. This suggests that bookmarked SOX2 target genes are not differently regulated than non-bookmarked target genes, and we discuss an alternative hypothesis on how mitotic bookmarking by SOX2 and other sequence-specific transcription factors could be involved in transcriptional regulation.

A role for mitotic bookmarking of SOX2 in pluripotency and differentiation.

Mitotic bookmarking transcription factors remain bound to chromosomes during mitosis and were proposed to regulate phenotypic maintenance of stem and progenitor cells at the mitosis-to-G1 (M-G1) transition. However, mitotic bookmarking remains largely unexplored in most stem cell types, and its functional relevance for cell fate decisions remains unclear. Here we screened for mitotic chromosome binding within the pluripotency network of embryonic stem (ES) cells and show that SOX2 and OCT4 remain bound to mitotic chromatin through their respective DNA-binding domains. Dynamic characterization using photobleaching-based methods and single-molecule imaging revealed quantitatively similar specific DNA interactions, but different nonspecific DNA interactions, of SOX2 and OCT4 with mitotic chromatin. Using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) to assess the genome-wide distribution of SOX2 on mitotic chromatin, we demonstrate the bookmarking activity of SOX2 on a small set of genes. Finally, we investigated the function of SOX2 mitotic bookmarking in cell fate decisions and show that its absence at the M-G1 transition impairs pluripotency maintenance and abrogates its ability to induce neuroectodermal differentiation but does not affect reprogramming efficiency toward induced pluripotent stem cells. Our study demonstrates the mitotic bookmarking property of SOX2 and reveals its functional importance in pluripotency maintenance and ES cell differentiation.