Jennifer J. Brady

Reprogramming towards pluripotency requires AID-dependent DNA demethylation

Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2–3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.

Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing

Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.

The Replication Cycle of Varicella-Zoster Virus: Analysis of the Kinetics of Viral Protein Expression, Genome Synthesis, and Virion Assembly at the Single-Cell Level

ABSTRACT Varicella-zoster virus (VZV) is a human alphaherpesvirus that is highly cell associated in cell culture. Because cell-free virus yields are too low to permit the synchronous infections needed for time-resolved analyses, information is lacking about the sequence of events during the VZV replication cycle. To address this challenge, we differentially labeled VZV-infected inoculum cells (input) and uninfected (output) cells with fluorescent cell dyes or endocytosed nanogold particles and evaluated newly infected cells by confocal immunofluorescence or electron microscopy (EM) at the single-cell level at defined intervals. We demonstrated the spatiotemporal expression of six major VZV proteins, ORF61, IE62, IE63, ORF29, ORF23, and gE, representing all putative kinetic classes, for the first time. Newly synthesized ORF61, as well as IE62, the major VZV transactivator, appeared within 1 h, and they were targeted to different subnuclear compartments. The formation of VZV DNA replication compartments started between 4 and 6 h, involved recruitment of ORF29 to putative IE62 prereplication sites, and resulted in large globular nuclear compartments where newly synthesized viral DNA accumulated. Although considered a late protein, gE accumulated in the Golgi compartment at as early as 4 h. ORF23 capsid protein was present at 9 h. The assembly of viral nucleocapsids and mature enveloped VZ virions was detected by 9 to 12 h by time-resolved EM. Although syncytium formation is a hallmark of VZV infection, infection of neighboring cells did not require cell-cell fusion; its occurrence from 9 h is likely to amplify VZV replication. Our results define the productive cycle of VZV infection in a single cell as occurring in 9 to 12 h.

Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells

Telomere extension has been proposed as a means to improve cell culture and tissue engineering and to treat disease. However, telomere extension by nonviral, nonintegrating methods remains inefficient. Here we report that delivery of modified mRNA encoding TERT to human fibroblasts and myoblasts increases telomerase activity transiently (24‐48 h) and rapidly extends telomeres, after which telomeres resume shortening. Three successive transfections over a 4 d period extended telomeres up to 0.9 kb in a cell type‐specific manner in fibroblasts and myoblasts and conferred an additional 28 ± 1.5 and 3.4 ± 0.4 population doublings (PDs), respectively. Proliferative capacity increased in a dose‐dependent manner. The second and third transfections had less effect on proliferative capacity than the first, revealing a refractory period. However, the refractory period was transient as a later fourth transfection increased fibroblast proliferative capacity by an additional 15.2 ± 1.1 PDs, similar to the first transfection. Overall, these treatments led to an increase in absolute cell number of more than 1012‐fold. Notably, unlike immortalized cells, all treated cell populations eventually stopped increasing in number and expressed senescence markers to the same extent as untreated cells. This rapid method of extending telomeres and increasing cell proliferative capacity without risk of insertional mutagenesis should have broad utility in disease modeling, drug screening, and regenerative medicine.—Ramunas, J., Yakubov, E., Brady, J. J., Corbel, S. Y., Holbrook, C., Brandt, M., Stein, J., Santiago, J. G., Cooke, J. P., Blau, H. M. Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells. FASEB J. 29, 1930‐1939 (2015). www.fasebj.org

A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells

Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation‐induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID‐knockout mouse fibroblasts was similar to that of wild‐type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72‐h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.—Bhutani, N., Decker, M. N., Brady, J. J., Bussat, R. T., Burns, D. M., Corbel, S. Y., Blau, H. M. A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells. FASEB J. 27, 1107–1113 (2013). www.fasebj.org

Design of Protease Activated Optical Contrast Agents That Exploit a Latent Lysosomotropic Effect for Use in Fluorescence-Guided Surgery

There is a need for new molecular-guided contrast agents to enhance surgical procedures such as tumor resection that require a high degree of precision. Cysteine cathepsins are highly up-regulated in a wide variety of cancers, both in tumor cells and in the tumor-supporting cells of the surrounding stroma. Therefore, tools that can be used to dynamically monitor their activity in vivo could be used as imaging contrast agents for intraoperative fluorescence image guided surgery (FGS). Although multiple classes of cathepsin-targeted substrate probes have been reported, most suffer from overall fast clearance from sites of protease activation, leading to reduced signal intensity and duration in vivo. Here we describe the design and synthesis of a series of near-infrared fluorogenic probes that exploit a latent cationic lysosomotropic effect (LLE) to promote cellular retention upon protease activation. These probes show tumor-specific retention, fast activation kinetics, and rapid systemic distribution. We demonstrate that they are suitable for detection of diverse cancer types including breast, colon and lung tumors. Most importantly, the agents are compatible with the existing, FDA approved, da Vinci surgical system for fluorescence guided tumor resection. Therefore, our data suggest that the probes reported here can be used with existing clinical instrumentation to detect tumors and potentially other types of inflammatory lesions to guide surgical decision making in real time.

Structure–function analysis of varicella-zoster virus glycoprotein H identifies domain-specific roles for fusion and skin tropism

Enveloped viruses require membrane fusion for cell entry and replication. For herpesviruses, this event is governed by the multiprotein core complex of conserved glycoproteins (g)B and gH/gL. The recent crystal structures of gH/gL from herpes simplex virus 2, pseudorabies virus, and Epstein–Barr virus revealed distinct domains that, surprisingly, do not resemble known viral fusogens. Varicella-zoster virus (VZV) causes chicken pox and shingles. VZV is an α-herpesvirus closely related to herpes simplex virus 2, enabling prediction of the VZV gH structure by homology modeling. We have defined specific roles for each gH domain in VZV replication and pathogenesis using structure-based site-directed mutagenesis of gH. The distal tip of domain (D)I was important for skin tropism, entry, and fusion. DII helices and a conserved disulfide bond were essential for gH structure and VZV replication. An essential 724CXXC727 motif was critical for DIII structural stability and membrane fusion. This assignment of domain-dependent mechanisms to VZV gH links elements of the glycoprotein structure to function in herpesvirus replication and virulence.

An immunoreceptor tyrosine-based inhibition motif in varicella-zoster virus glycoprotein B regulates cell fusion and skin pathogenesis

Herpesvirus entry functions of the conserved glycoproteins gB and gH–gL have been delineated, but their role in regulating cell–cell fusion is poorly understood. Varicella-zoster virus (VZV) infection provides a valuable model for investigating cell–cell fusion because of the importance of this process for pathogenesis in human skin and sensory ganglia. The present study identifies a canonical immunoreceptor tyrosine-based inhibition motif (ITIM) in the gB cytoplasmic domain (gBcyt) and demonstrates that the gBcyt is a tyrosine kinase substrate. Orbitrap mass spectrometry confirmed that Y881, central to the ITIM, is phosphorylated. To determine whether the gBcyt ITIM regulates gB/gH–gL–induced cell–cell fusion in vitro, tyrosine residues Y881 and Y920 in the gBcyt were substituted with phenylalanine separately or together. Recombinant viruses with these substitutions were generated to establish their effects on syncytia formation in replication in vitro and in the human skin xenograft model of VZV pathogenesis. The Y881F substitution caused significantly increased cell–cell fusion despite reduced cell-surface gB. Importantly, the Y881F or Y881/920F substitutions in VZV caused aggressive syncytia formation, reducing cell–cell spread. These in vitro effects of aggressive syncytia formation translated to severely impaired skin infection in vivo. In contrast, the Y920F substitution did not affect virus replication in vitro or in vivo. These observations suggest that gB modulates cell–cell fusion via an ITIM-mediated Y881 phosphorylation-dependent mechanism, supporting a unique concept that intracellular signaling through this gBcyt motif regulates VZV syncytia formation and is essential for skin pathogenesis.

Molecular definition of a metastatic lung cancer state reveals a targetable CD109-Janus kinase-Stat axis

Lung cancer is the leading cause of cancer deaths worldwide, with the majority of mortality resulting from metastatic spread. However, the molecular mechanism by which cancer cells acquire the ability to disseminate from primary tumors, seed distant organs, and grow into tissue-destructive metastases remains incompletely understood. We combined tumor barcoding in a mouse model of human lung adenocarcinoma with unbiased genomic approaches to identify a transcriptional program that confers metastatic ability and predicts patient survival. Small-scale in vivo screening identified several genes, including Cd109, that encode novel pro-metastatic factors. We uncovered signaling mediated by Janus kinases (Jaks) and the transcription factor Stat3 as a critical, pharmacologically targetable effector of CD109-driven lung cancer metastasis. In summary, by coupling the systematic genomic analysis of purified cancer cells in distinct malignant states from mouse models with extensive human validation, we uncovered several key regulators of metastatic ability, including an actionable pro-metastatic CD109–Jak–Stat3 axis.

Mapping the in vivo fitness landscape of lung adenocarcinoma tumor suppression in mice

The functional impact of most genomic alterations found in cancer, alone or in combination, remains largely unknown. Here we integrate tumor barcoding, CRISPR/Cas9-mediated genome editing and ultra-deep barcode sequencing to interrogate pairwise combinations of tumor suppressor alterations in autochthonous mouse models of human lung adenocarcinoma. We map the tumor suppressive effects of 31 common lung adenocarcinoma genotypes and identify a landscape of context dependence and differential effect strengths.In vivo analysis of pairwise combinations of tumor suppressor losses using a barcode-based assay in mice identifies unpredicted genetic interactions and shows that the effects of tumor suppressor alterations can be context-dependent.