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FARE 2021 Special interest Group (SIG) Awards

FARE2021 Scientific Interest Group (SIG) Awards

SIG Awards were introduced to the Fellows Award for Research Excellence (FARE) Competition in 2018 to further recognize and merit the work of FARE awardees, and to provide an opportunity for them to showcase their research to the wider scientific community. The SIG Award involves the selection of a winning abstract from the FARE Competition by participating SIGs that they deem to be of high scientific merit. These awardees are then invited to present their work at one of the SIG meetings.

 

Participating SIGs:

 

SIG Scholar Awards:

 

FARE2021 SIG Awardees

Antibody Interest Group

Lawrence Wang

Mr. Lawrence Wang (NIAID-VRC)

A Potent Human Monoclonal Antibody Prevents Malaria By Binding A Subdominant Epitope on the P. falciparum Circumsporozoite Protein and Neutralizing Sporozoites in the Skin and Liver

Passive immunization with human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) is a promising approach to prevent malaria. PfCSP is the most abundant SPZ surface protein and is essential for parasite motility and infection of hepatocytes. PfCSP has three domains: an N-terminus, a central region composed of repeating tetrapeptides (1 NPDP, 4 NVDP, and 38 NANP repeats in the 3D7 reference strain), and a C-terminus. Nearly all neutralizing mAbs isolated to date bind the immunodominant NANP major repeats. Recently, the isolation of highly potent human mAbs against the unique tetrapeptide, NPDP, at the junction of the N-terminus and repeat region identified this "junctional epitope" as a new site of neutralization. This discovery has led to ongoing efforts to identify new, potent mAbs against other subdominant epitopes of PfCSP. Here, we report the first neutralizing human mAb (called L9) that preferentially binds the subdominant NVDP minor repeats, followed by the NANP major repeats, of PfCSP. L9 IgG bound to recombinant PfCSP with high affinity and valency in two distinct steps by isothermal titration calorimetry: the first step involved ~2 IgG (affinity = 22 nM) and the second step involved ~9 IgG (affinity = 47 nM). Genomic analyses indicate that the NVDP repeats recognized by L9 are conserved in 100% of worldwide field isolates, suggesting that L9 would react broadly with circulating strains. L9 was more protective against mosquito bite challenge with transgenic P. berghei SPZ expressing PfCSP in mice than six recently published neutralizing human mAbs that preferentially bind NPDP and NANP repeats. Furthermore, L9 mediated sterile protection against intravenously challenged Pf SPZ in chimeric mice with humanized livers (FRG-huHep) at serum concentrations as low as 4 µg/mL. Intravital imaging demonstrated that L9 mediated protection by multiple mechanisms at various physiological sites. In the skin, L9 limited SPZ motility and invasion of dermal capillaries. In the liver, L9 prevented SPZ egress from sinusoids and subsequent traversal and infection of hepatocytes. Furthermore, L9 was directly cytotoxic to SPZ in vitro and in the skin and liver. These data highlight the NVDP minor repeats as a new site of vulnerability on PfCSP; furthermore, clinical development of L9 has been initiated based on its potency in pre-clinical models.

 

Bioinformatics Scientific Interest Group

Kun Wang

Dr. Kun Wang (NCI-CCR)

Research Bio: Dr. Wang is a postdoctoral fellow from the Cancer Data Science Lab (CDSL) in NCI. He was trained as a computational biologist at the University of Maryland, College Park, USA. His current research work focuses on developing deconvolution tools to dissect tumor mixture for tumor microenvironment characterization using omics data and building predictive models for cancer immunotherapy. In addition, his research interests also include synthetic lethality-based cancer therapy, computational tumor diagnosis and single cell analysis.

 

Decode Chromatin Interest Group

Giulia Riparini

Dr. Giulia Riparini (NIAMS)

Polycomb Ezh2 Orchestrates a Crosstalk between Skeletal Muscle Stem Cells and Mesenchymal Stromal Cells During Muscle Regeneration

Fibrosis is the result of excessive extracellular matrix (ECM) deposition. Fibro-adipogenic progenitors (FAPs), a muscle interstitial mesenchymal cell population, are the main cellular source of matrix-producing myofibroblasts. Therefore, a proper regulation of FAP activity is crucial to avoid pathological fibrosis and tissue degeneration. Recently, high levels of transforming growth factor-B1 (TGF-B1) have been shown to activate FAPs and induce their differentiation into a fibrogenic lineage leading to excessive ECM deposition and fibrosis. Interestingly, we have found that deletion of the histone methyltransferase Ezh2, a master regulator of muscle stem cell (MuSC) transcriptional identity, prevents muscle regeneration and results in the release of inflammatory cytokines, such as TGF-B1. Therefore, we want to study whether Ezh2 regulates FAP cell fate during muscle regeneration. To explore this mechanism, we developed a conditional and Tamoxifen inducible knockout mouse model to ablate Ezh2 from MuSCs in adult mice. We used Ezh2 floxed mice crossed with transgenic animals expressing Cre recombinase under the control of the Pax7 promoter. The right tibialis anterior (TA) muscles of control and Pax7creEREzh2fl mice were injured with the snake venom toxin Notexin and muscles were then collected to evaluate the regenerative process. Seven days after injury, proliferation of MuSCs was severely impaired in the regenerating muscles of Ezh2 ablated mice compared to littermate controls and myofibers were smaller with a reduced cross-sectional area (CSA). These muscle defects were still detected 28 days after injury. Impairment in regeneration was concomitant with a significant increase in FAP numbers in Ezh2 ablated mice compared to control mice seven days after injury and FAP expansion remained detectable over the course of the examination (28 days post-injury). These data suggest the necessity of MuSC signaling on FAPs to ensure a proper regulation of these mesenchymal progenitors during muscle regeneration. Current experiments are aimed at elucidating the mechanisms by which MuSCs signal to FAPs to understand the molecular bases underlying their crosstalk. Fibrosis of skeletal muscle is a hallmark of muscular dystrophies, aging, and severe muscle injuries. Thus, a better understanding of the mechanisms underlying muscle fibrosis will help to advance our knowledge and develop innovative anti-fibrotic therapies to reverse fibrosis in such pathologic conditions.

 

Developmental Biology Scientific Interest Group

Shaohe Wang

Dr. Shaohe Wang (NIDCR)

Reconstituting Branching Morphogenesis by Engineering Cell Adhesion

During embryogenesis, many organs undergo branching morphogenesis to form a tree-like hierarchical structure. Stratified epithelia, such as in embryonic pancreas and salivary gland, branch by repeated clefting, where indentations at the surface extend inwards to split one epithelial bud into several. Clefting requires growth factor signaling and involves extensive dynamics of epithelial cells and the extracellular matrix. However, it remains unclear how numerous epithelial buds arise from the interplay of cell and matrix dynamics. Here, we used two-photon microscopy to perform long-term volumetric live imaging at high spatiotemporal resolution using transgenic mouse salivary glands expressing a green nuclear marker together with a red epithelial reporter or a red membrane marker. This imaging strategy has enabled us to follow individual cells within virtually the entire gland for several rounds of clefting. Through quantitative image analysis, we found that the surface epithelial cells form an integral layer with the basement membrane, which together expands uniformly and folds inward to drive clefting that leads to new bud formation. Interestingly, cell divisions of surface epithelial cells always produce one or two interior daughter cells, all of which eventually return to the surface to make a delayed contribution to the surface expansion. We propose that the robust surface return of interior daughter cells is driven by differential adhesion between low- and high-E-cadherin cells, whereas the surface layer integrity is maintained by preferential cell-matrix over cell-cell adhesions of low-E-cadherin cells at the surface. Importantly, we successfully reconstituted this mode of branching by substantially reducing E-cadherin expression and inducing basement membrane formation in 3D spheroid cultures of engineered cells that normally do not branch. Furthermore, we showed that the reconstituted branching requires integrin-mediated cell-matrix adhesions, and can be promoted by elevating the cell-matrix adhesion strength. Our results demonstrate a fundamental self-organization mechanism based on preferential cell-matrix adhesion greater than cell-cell adhesion that can explain how stratified epithelia undergo branching morphogenesis.

 

Inflammatory Disease Interest Group

Ai Ing Lim

Dr. Ai Ing Lim (NIAID)

Pre-birth Tissue-Specific Immune Programming

Environmental exposures, especially during the developmental window from conception to early life, can have long-term impacts on individual health. In particular, infections during pregnancy by severe, vertically transmittable pathogens can lead to numerous adverse outcomes for offspring. Unclear is to what extent transient, non-transmittable infections affect the well-being of offspring. To address this question, we used an attenuated strain of the gut pathogen Yersinia Pseudotuberculosis (YptbYopM) as a model. Tracking of YptbYopM after oral infection of timed-pregnant mice showed that infection by this pathogen is highly transient and incapable of vertical transmission to offspring. Strikingly, offspring of transiently infected dams harbored profound alterations in gut immunity, including substantially higher numbers of T helper 17 cells (Th17) in both the small and large intestine but not at other barrier tissues throughout adulthood. Cross-fostering and serum transfer experiments demonstrated that soluble factors from maternal circulation were sufficient to prenatally imprint offspring intestinal immunity. High-throughput screening revealed this in utero imprinting is mediated by the cytokine interleukin-6 (IL-6). Using mice with a cell-specific deletion in the IL-6 receptor, transient infection-induced maternal IL-6 was found to act directly on fetal intestinal epithelial cells and as a result epigenetically enhance their expression of factors (e.g. serum amyloid A) that are associated with epithelial-immune cells communication. Consequently, offspring delivered from transiently infected dams displayed enhanced Th17-mediated protective immunity against intestinal infections including, Salmonella typhimurium and Citrobacter rodentium. In contrast but in line with heightened activation of epithelial cells, offspring from transiently infected dams were more susceptible to experimentally induced intestinal inflammation. Our results thus propose transient maternal infections can prenatally imprint a defined IL-6-dependent program on intestinal epithelial-immune cell crosstalk that promote protective immunity but in defined settings exacerbates susceptibility to inflammatory disorders.

 

Mary Lytle Rhim Young Investigator Award      

Supreet Agarwal

Dr. Supreet Agarwal (NCI-CCR)

Research Bio: Dr. Agarwal is a Research Fellow at the Center of Cancer Research, National Cancer Institute, NIH. His current research focuses on understanding mechanisms of drug resistance in advanced prostate cancer and performing preclinical trials using mouse models, patient-derived tumor xenografts, and tumor organoids as model systems. Dr. Agarwal has identified B7H3 as a potential therapeutic target in advanced metastatic prostate cancer and is currently testing an antibody-drug-conjugate in preclinical trials. He also has a strong background in bioinformatics and statistical analysis.

 

 

Matrix Biology Interest Group

Dinusha Rajapakse

Dr. Dinusha Rajapakse (NEI)

Amelotin, an enamel protein is expressed in retinal pigment epithelium (RPE) and associated with hydroxyapatite (HAP) deposit formation in dry age-related macular degeneration (AMD)

AMD is the main cause of irreversible vision loss in aging populations. There are no effective therapies for dry AMD which is the most common form of the disease. Early signs of AMD are drusen which are accumulations of extracellular lipids, proteins, and HAP minerals in the retina. HAP nodules are implicated in the progression of AMD. Drusen deposit growth is associated with precipitation of HAP and binding of proteins to HAP surface, but the proteins involved in the mineralization process is yet to be identified. We have previously reported serum-deprivation of RPE cells in culture mimics some features of AMD and using this model we identified amelotin (AMTN), a protein promoting hydroxyapatite mineralization in tooth enamel. HAP is also formed in our culture model and is blocked by siRNA inhibition of AMTN. Here using human donor eye tissue, we investigated AMTN expression in AMD. 6 normal and 22 AMD pairs of eyes from male and female donors, age range of 65-96 years were investigated. Eyes were genotyped for AMD-related polymorphism Y402H of complement factor H. Eyes were cut and sectioned through the macula for microscopy. Immunohistochemistry was performed to identify AMD eyes with lesions and categorize for types of drusen. In situ hybridization probes were used for detection of AMTN mRNA, anti-Amelotin antibody for AMTN protein and Bone-Tag 680RD to stain HAP. In situ hybridization showed that AMTN is expressed in RPE in eyes with dry AMD, particularly in soft drusen regions containing HAP nodules. AMTN was not found in hard drusen, normal RPE, or donor eyes diagnosed with wet AMD. Immunofluorescence labeling showed AMTN in RPE surrounding soft drusen and isolated drusen with large calcified nodules showed heterogeneous HAP staining, with intense staining of the crusts whereas AMTN was localized in a ring around the HAP structures. Eyes with Y/Y genotype did not contain soft drusen with HAP and had no AMTN signal. In contrast, donor eyes with H/H and H/Y genotypes, had soft drusen with HAP and AMTN. Our findings confirm that AMTN is expressed in diseased human RPE, specifically in dry AMD and may be a key protein in the organization of HAP mineralization in soft drusen implicated in clinical AMD progression. AMTN provides a new possibility for therapeutic intervention. Next, we will perform experiments to identify AMTN specific inhibitors by high throughput screening and test their effects on HAP mineralization on our human AMTN transgenic mice.

 

Metabolomics Scientific Interest Group

Yuhong Luo

Dr. Yuhong Luo (NCI-CCR)

Inhibition of protein arginine methyltransferase 5 ameliorates MYC-driven hepatocellular carcinoma

Liver cancer is one of the leading causes of cancer-related deaths worldwide, with incidence and death rates increasing by about 3% per year in western countries. Hepatocellular carcinoma (HCC) is the most common liver cancer, representing up to 90% of all primary hepatic malignancies. Current first-line treatment for HCC is limited to the kinase inhibitors sorafenib and lenvatinib, but resistance is a matter of course. Thus, there is an urgent need to develop new agents for HCC early detection and treatment. MYC, a basic helix-loop-helix (bHLH)-leucine zipper transcription factor with broad effects on various biological processes, is frequently dysregulated in human HCC. In the current study, HCC was induced in mice with hepatic-specific disruption of Myc (Myc-dHep) and control mice (Myc-floxed) by administration of diethylnitrosamine (DEN). Hepatic-specific Myc disruption suppressed HCC tumorigenesis. Dynamic LC/MS-based metabolomics analysis of urine samples from Myc-dHep and Myc-floxed mice revealed that dimethylarginine, especially symmetric dimethylarginine (SDMA), were increased in HCC in a MYC-dependent manner. We found that protein arginine methyltransferase 5 (Prmt5) was a novel direct target gene of Myc. Consistent with the mouse data, the level of SDMA was increased in the urine samples from HCC patients compared with healthy people. MYC and PRMT5 were both increased in human liver tumor specimens compared with adjacent non-tumor tissues. Administration of GSK3326595, a PRMT5 inhibitor in trials for the treatment of non-Hodgkin's lymphoma and solid tumors, to human MYC-overexpressing transgenic mice that spontaneously develop HCC, suppressed the growth of liver tumors as monitored by serial magnetic resonance imaging (MRI) scanning. Flowcytometry and immunofluorescence staining showed a surprising and unexpected increased lymphocyte infiltration in the GSK3326595-treated liver tumors compared with liver tumors from vehicle-treated mice, which may contribute to enhanced anti-tumor immunity and decreased tumor growth. Collectively, this study suggested that PRMT5, encoded by a MYC target gene, could be a new drug target for HCC treatment.

 

Neurobiology Interest Group

Protein Trafficking and Organelle Dynamics Interest Group

Luis Bonet Ponce

Dr. Luis Bonet Ponce (NIA)

The Parkinson' s disease kinase LRRK2 promotes tubulation and vesicular sorting from membrane damaged lysosomes

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are a cause of familial Parkinson's disease (PD) and variants in the LRRK2 locus are associated with the sporadic form of the disease. LRRK2 pathogenic mutations are gain of function mutations that produce a toxic hyperactive protein. Nonetheless, the biological functions of LRRK2 remain incompletely understood. Additional human genetic data nominate the lysosome as a crucial organelle for PD, and although cells carrying LRRK2 pathogenic mutations display lysosomal defects, the specific role of LRRK2 in the lysosome remains unclear. Here, we observed that LRRK2 is recruited to lysosomes that have a partially ruptured membrane in mouse primary astrocytes. Our data suggest that LRRK2 recruitment to membrane damaged lysosomes is parallel and independent to lysophagy. The proximity labeling method APEX2, coupled with quantitative mass spectrometry, revealed the motor adaptor protein JIP4 as a LRRK2 interactor at the lysosomal membrane. Using confocal microscopy, combined with pharmacological and genetic tools, we observed that LRRK2 is able to recruit JIP4 to permeabilized lysosomes in a kinase-dependent manner. Indeed, the LRRK2 pathogenic mutation located in the kinase domain (G2019S) increases JIP4 recruitment to the lysosomal membrane. LRRK2 recruitment of JIP4 occurs through two small GTPases, RAB10 and RAB35. Both RAB proteins are phosphorylated by LRRK2 (in their Thr73 and ThrT72 sites respectively) at the lysosomal membrane which preserves the membrane localization of both RABs and allows them to recruit JIP4. Using super-resolution live cell imaging microscopy and focus ion beam electron microscopy (FIB-SEM), we observed that once at the lysosomal membrane, JIP4 promotes the formation of LAMP1-negative/LRRK2-negative lysosomal tubules that release membranous content from ruptured lysosomes. Released vesicular structures are able to interact with other healthy lysosomes. Thus, we described a new process that uses lysosomal tubulation to release vesicular structures from permeabilized lysosomes, being the first evidence of lysosomal sorting in mammalian cells. LRRK2 orchestrates this process that we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2) that, given the central role of the lysosome in PD, is likely to be disease relevant.

 

Proteomics Scientific Interest Group

Brajesh Singh

Dr. Brajesh Singh (NIAID)

An E3 ubiquitin ligase as a novel target for anti-malaria therapy

Malaria is one of the deadliest infectious diseases worldwide. Despite more than a century of efforts to eradicate malaria, the disease remains a major and growing threat to the public health due to multi drug resistance (MDR). Emergence of MDR signifying to discover novel targets/drugs to combat malaria parasites and one such targets which is currently most explored is “parasite’s ubiquitin system”. Protein ubiquitylation is an important post-translational regulation, which has been shown to be necessary for life cycle progression and survival of human malaria parasites- Plasmodium falciparum (Pf). E3 ubiquitin ligases (E3s), a key player in protein ubiquitination and proteasomal degradation, are novel potential drug targets. E3s are very diverse and are major determinants that provide specificity for the substrate/protein recognition to be ubiquitinated. The hypothesis behind this study is that targeting the E3 ligase functions of ubiquitin system would selectively kill the malaria parasite. We generated transgenic P. falciparum parasite lines with specific E3 ligases tagged with pSli-HA-glmS sequence that enables us to knockdown (KD) the expression of E3 ubiquitin ligase (E3-3). Our drug assay results evidently indicated that proper expression of this protein (E3-3) is essential for the growth of parasites and KD of this protein causes parasites vulnerable to antimalarial drugs. KD of an E3 ligase (E3-3) reduces ubiquitination of many parasite proteins. Changes in specific protein expression after E3-3 KD were identified using 2D gel electrophoresis and tandem mass spectrometry (MS-MS), many of the proteins match the known protein targets of artemisinin- a currently most potent drug to fight malaria. E3-3 KD renders parasites more sensitive to artemisinin derivatives and changes protein expression of P. falciparum multidrug resistant protein 1 (PfMDR1) and P. falciparum chloroquine resistance transporter (PfCRT). Therefore, the P. falciparum E3-3 ligase could play a significant role in parasite modulating the expression of key drug transporters and/or targets. Thus, E3 ligase could be potential drug target for treating malaria infections.

 

RNA Interest Group

Agnes Karasik

Dr. Agnes Karasik (NIDDK)

RNase L mediated antiviral response triggers translation of the non-coding regions of messenger RNAs

Viral infections often trigger the activation of a ribonuclease, RNase L, which plays an important role in the innate immune response. During viral infection, oligo-adenylate synthase (OAS) produces a small molecule, 2’-5’-oligoadenylate (2-5A), which dimerizes and activates RNase L. This activated form of RNase L then cleaves single stranded regions of viral and host RNAs and this activity is thought to promote clearance of the virus and apoptosis. Widespread cleavage of messenger RNAs (mRNA) leads to broad changes in their abundance but it remains unknown how this reduced pool of mRNAs is translated. Ribosome profiling (Ribo-seq) is a powerful high-throughput method to globally assess the distribution of ribosomes on mRNAs. Thus, we performed Ribo-seq on untreated and 2-5A treated (RNase L activated) A549 lung carcinoma cells. Activation of RNase L was previously shown to trigger translation in the 3’ untranslated regions (UTRs) of mRNAs in in vitro assays. Therefore, we first assessed if ribosomes are also present in these regions when RNase L is activated in vivo. We found that RNase L activation leads to substantial accumulation of ribosomes in the 3’UTR, but not in RNase L KO cells, suggesting specificity for this pathway. In addition, we observed a relative increase in ribosomes in other non-coding regions of mRNAs, such as in the 5’ UTRs and in alternate reading frames within protein coding regions. We also verified that ribosomes were actively translating in non-coding regions of mRNA using in vitro biochemical assays and by computationally dissecting positions of ribosomes from Ribo-seq experiments. Analysis of published Ribo-seq data on viral infected cells showed that these unconventional translation events also occur during viral infections. Since translation of non-coding regions of mRNAs was dependent on the catalytic activity of RNase L, we favor a model where cleavage of mRNA by RNase L leads to the translation of mRNA fragments. While the function of the synthesized cryptic peptides is unknown, we propose that they could be presented by MHC I molecules on the cell surface. These MHC I-peptide complexes are potentially recognized by the immune system as “non-self” and could therefore enhance viral clearance. Production of cryptic peptides during viral infections is potentially a novel component of the innate immune response that could be exploited for therapeutics in the future.

 

Single-Cell Genomics Interest Group

Christopher Rhodes

Dr. Christopher Rhodes (NICHD)

Integration of single cell transcriptomes and chromosome accessibility to detect regulatory elements critical to interneuron development

GABAergic interneurons are a heterogenous cell population and their dysfunction is associated with numerous brain disorders. Interneurons are born in the ganglionic eminences (MGE, LGE and CGE), with distinct subtypes arising from different GE domains. Despite substantial evidence suggesting that initial interneuron subtypes are specified within these neurogenic niches, only a handful of fate-determining genes have been identified. One possible explanation is that progenitors contain epigenetic signatures directing cells towards a particular interneuron fate that are not yet apparent in the transcriptome. Ample evidence indicates that epigenetic mechanisms induce heritable changes in gene expression and are critical in mediating cell fate and differentiation in a variety of tissues. Yet we lack a comprehensive understanding of how epigenetic states influence interneuron cell fate in normal development and disease models. To explore the relationship between interneuron cell fate and epigenetic mechanisms in neural progenitors, we performed single cell RNA (scRNA-Seq) and single cell assay for transposase-accessible chromatin (scATAC-Seq) sequencing to determine the transcriptome and chromatin accessibility in the embryonic MGE, LGE, CGE and cortex. This allows us to screen for candidate enhancers and other non-coding regions regulating expression of specific interneuron subtypes. We detected distinct genes and corresponding chromatin profiles that are restricted to specific progenitor zones and interneuron lineages. By examining ATAC peaks in lineage-specific genes across cell types, we detected putative enhancers associated with specific GEs and potentially distinct interneuron subtypes. To expand on these initial observations, we generated a conditional knockout of Enhancer of zeste homolog 2 (Ezh2) in MGE progenitors. Ezh2 regulates histone methylation and acts as a transcriptional repressor; thus loss of Ezh2 likely disrupts chromatin architecture and gene expression. These Ezh2 cKO mice displayed a significant decrease in PV+ interneurons in multiple brain regions. We are currently performing scRNAseq and scATACseq on the MGE of Ezh2 cKO to assess how the transcriptome and chromatin landscape is altered in these mice. These insights increase our understanding of how epigenetic modifications regulate interneuron fate and could open new avenues for understanding how disease-associated genes or genomic loci could perturb interneuron fate and maturation.

 

Systems Biology Scientific Interest Group

Marwa Afifi

Dr. Marwa Afifi (NCI-CCR)

Single cells exhibit heterogeneous cell cycle behavior during irreversible commitment to cellular senescence

In response to genotoxic stress, cells can irreversibly exit the cell cycle and become senescent. Premalignant lesions such as skin nevi, glandular carcinoma in-situ, and oral dysplasias are rich in senescent cells suggesting that cellular senescence is the first defense mechanism against malignant transformation. Cells that escape senescence gain the ability to divide uncontrollably and invade the underling tissue to become an invasive cancer. While it is known that cellular senescence is regulated by the p53-p21 or p16-Rb pathways, the distinct time point at which cells commit to senescence remains unknown. One main challenge in the field is that DNA damage, the main stimulus used to induce senescence, is itself inherently irreversible; the DNA damage response remains active after the initial DNA damage. To overcome this problem, we induced senescence using a combination of cyclin-dependent kinase (CDK)4/6 and MEK inhibitors, which can be withdrawn to restore signaling. We used MCF10A, MCF7, U2OS cell lines and primary human lung fibroblasts to study the senescence commitment point. We created an automated quantification approach to generate a robust senescence score by combining parameters such as increased nuclear area and beta-galactosidase activity in the perturbed cells. By varying the duration of time cells were exposed to the treatment, we found that some cells within each cell type, irreversibly commit to senescence as early as 2 days after treatment. Other cells committed to senescence after as many as 4 days, suggesting a high degree of single-cell variability. We then used long-term time-lapse imaging and single-cell tracking to observe cellular behaviors prior to entry into senescence. We found that cells that commit early to senescence were arrested in the G1 or G2 phases after treatment. However, cells committing to senescence at later time points adopted a different behavior, where they initially arrested in the G1 phase for several hours, escaped into S phase, and finally re-arrested in the G2 phase. Furthermore, some cells managed to divide multiple times in the presence of the drugs before arresting in either G1 or G2 phase. Our data indicate there are multiple paths cells take to irreversibly exit the cell cycle and commit to senescence. We are interested in identifying the underlying mechanisms regulating this heterogeneity, which will further elucidate vulnerable paths bypassed during malignant transformation.

 

Transcription Scientific Interest Group

Joyce Thompson

Dr. Joyce Thompson (NICHD)

Rapid reshuffling of master transcription factors allows exit from plasticity to establish cell-fates

In the pre-implantation mammalian embryo three cell-fates are specified- trophectoderm (TE), epiblast (Epi) and primitive endoderm (PrE), which contribute to the placenta, embryo proper, and yolk-sac, respectively. Epi or PrE fates are determined by the interplay between two master transcription factors (TFs), NANOG and GATA6, and arise from the inner cell mass (ICM) where these two counteracting TFs are initially co-expressed. How NANOG and GATA6 coordinate to maintain plasticity in the ICM and counteract each other to specify distinct cell fates irreversibly is not well understood. Using a Gata6-driven in vitro model of PrE differentiation we define how GATA6 modulates epigenetic mechanisms to promote a PrE fate while suppressing the NANOG-driven Epi fate. We have mapped the changing binding patterns of key PrE-specific TFs (GATA6, GATA4, and SOX17) and Epi TFs (NANOG, SOX2) during differentiation, along with changes in chromatin landscape and transcription, to define how these TFs alter the epigenome and transcriptome. Within four hours of differentiation, we detect widespread changes in chromatin landscape directed by GATA6. PrE-specific genes are upregulated by the combined action of GATA6, GATA4, and SOX17, while Epi genes are downregulated, solely by GATA6, via direct eviction of NANOG and SOX2. Repressive histone marks (H3K9me3 and H3K27me3) play a limited role in achieving repression, and reduction in Epi-specific enhancer activity is the predominant mechanism. We detect very few changes in large-scale 3D-chromatin reorganization, which most often emanate from dramatic redistribution of active and repressive histone marks. Surprisingly, we observe enhanced recruitment of NANOG and SOX2 at GATA6 target sites, despite their progressive transcriptional silencing through differentiation. This could be a mechanism by which the Epi network resists differentiation cues, attempting to maintain plasticity. An alternate mechanism could be GATA6-mediated re-direction of Epi factors to activate PrE genes while reducing their relative levels at Epi genes leading to their repression, effectively promoting a PrE-fate over an Epi-fate. In summary, our study describes how the PrE fate is established and provides a possible mechanism to address the long-standing question of how two master TFs maintain and oppose plasticity in the early embryo.

 

Virology Interest Group: Kuan-Teh Jeang Scholar Award

Rebecca Broeckel

Dr. Rebecca Broeckel (NIAID)

A genome-wide CRISPRa screen reveals helicase with zinc finger 2 as a potent cellular inhibitor of Ebola virus

Ebola virus (EBOV) causes outbreaks of viral hemorrhagic fever in humans with a case fatality rate of 60%. EBOV is a major public health concern because of its extreme virulence, its capacity for human-to-human transmission, and the unpredictability of EBOV emergence. The host type I interferon (IFN) response is critical in controlling EBOV replication at the cellular level, but the specific host proteins that have direct antiviral activity against EBOV, termed restriction factors, are unknown. To identify new host restriction factors for EBOV, we employed a genome-wide CRISPR transcriptional activation (CRISPRa) screen in human liver cells. In this CRISPRa screen, endogenous gene expression is induced by a nuclease-dead Cas9, recruited transcriptional activators, and guide RNAs that target promoter regions (10 guides/gene, ~210,000 guides in total). To identify putative viral inhibitors, CRISPRa cells were infected with EBOV whose replication is cytopathic, and surviving cells were subjected to RNAseq. After selection with EBOV, approximately 300 genes were significantly enriched in surviving cells. The top antiviral gene candidate was helicase with zinc finger 2 (Helz2), which is a known IFN-stimulated gene. Helz2 proved to be a remarkably potent restriction factor for EBOV by blocking release of infectious virus by more than 1000-fold compared to the control. To identify the mechanism of Helz2 inhibition, viral RNA was measured over a single round of infection during Helz2 overexpression. Helz2 interfered with EBOV RNA replication, while virus binding and initial entry into cells were not affected. Immunofluorescence of Helz2 CRISPRa cells during infection showed that viral inclusion bodies, defined as the sites of viral RNA replication, were smaller and less developed in the presence of Helz2. Furthermore, Helz2 was shown to bind to the EBOV VP35 protein, a cofactor for the virus RNA polymerase and the major IFN antagonist in infected cells. Thus, Helz2 disrupts early viral RNA replication and may also function to increase the ability of virus-infected cells to sense infection and produce IFN. This work identifies a specific point of vulnerability of EBOV during RNA replication and thereby reveals a strategy to design antivirals that mimic Helz2 function. Furthermore, validation of additional ‘hits’ from this screen will expand our understanding of the antiviral gene repertoire of the cell against EBOV.

 

Woman Scientist Advisors (WSA) Scholar Award

Francesca Barone

Dr. Francesca Barone (NEI)

Outer retina reconstruction using a tissue engineered transplant

Degeneration of outer retina (photoreceptors/PRs and retinal pigment epithelium/RPE cells) is the most common cause of vision loss and blindness in diseases of the retina. Similar PR/RPE damage is seen in eye injuries (e.g. laser-induced RPE/PR damage in the civilian population or laser and blast injuries in a battlefield). Currently, there is no treatment for such degenerative eye conditions. Here, we have developed an induced pluripotent stem cell (iPSC) derived outer retina cell replacement therapy to directly replace degenerated PRs and RPE. This works leverages our recently approved Phase I trial to test the safety of iPSC-derived RPE-only patch in macular degeneration patients. The iPSC-RPE-patch is developed by growing a confluent monolayer of polarized RPE cells on a biodegradable poly (lactic-co-glycolic acid) (PLGA) scaffold. iPSC-derived PRs are grown on top of the RPE-patch to form a 3D outer retina tissue. This close proximity of RPE and PRs allows correct polarization and co-maturation of both cell types. To test in vivo functionality of this tissue, we developed a micro pulse laser induced RPE/PR damage pig model. A 532nm laser is specifically absorbed by RPE pigment, causing RPE and adjacent PRs to die of the laser heat. 48 hours post the laser injury, we transplanted a 4x2 mm PLGA-RPE-PR patch under the laser-damaged retina. Animals (N=4 pigs) were evaluated by live imaging of the eye including Optical Coherence Tomography (OCT) that allows us to monitor retina structure and Adaptive Optics (AO) that allows us to track individual PRs. Our results show that the PLGA/RPE/PRP patch was able to protect overlying lasered retina from dying. OCT shows improved retinal thickness and preserved structure over the lasered retinal area with the transplant as compared to lasered only area. AO analyses of the transplanted areas shows an increase of PR density compared to untreated areas. Lastly, histology confirmed the integration of human RPE and PR in the back of pig retina. RPE cells continue to express RPE-lineage markers like Microphthalmia-associated transcription factor (MITF), and photoreceptors express ARRESTIN (ARR3) and other PR markers. This work provides the first ever dual-RPE/PR transplants to treat degenerative eye diseases and civilian or battlefield eye injuries.

 

Marta Markiewicz Potoczny

Dr. Marta Markiewicz Potoczny (NCI-CCR)

TRF2-independent telomere protection in pluripotent stem cells

Telomeres, the nucleoprotein structures at the ends of eukaryotic linear chromosomes, consist of repetitive G/C-rich DNA sequences and DNA binding proteins. Telomere homeostasis is critical for genome stability and cell survival with telomere length deregulation associated with increased cancer risk and premature aging. When telomeres become critically short or telomere binding proteins are defective, cells recognize dysfunctional telomeres as sites of DNA damage responses resulting in aberrant DNA repair leading to genomic instability. In mammals, depletion of TRF2, an essential telomere binding factor, results in massive levels of end-to-end fusions in all cell types tested, including neurons, fibroblasts and various cancer cell lines. Surprisingly, we found that TRF2 is dispensable for the survival of pluripotent mouse embryonic stem cells (ESCs). TRF2-depleted ESCs do not exhibit telomere fusions and can be expanded indefinitely, but lose viability when differentiated, suggesting the existence of a developmental switch that controls the mechanism of chromosome end-protection. To understand how telomere protection is maintained in TRF2-depleted ESCs we performed a genome-wide CRISPR/Cas9 knockout screen aimed at identifying genes that are synthetically lethal with TRF2. Based on this screen we identified approximately 30 genes which are essential for the proliferation of TRF2-depleted ESCs. We confirmed that two of these genes, the telomere-associated protein POT1b and the chromatin remodeling factor BRD2, are required for telomere protection in TRF2-null ESCs. Co-depletion of POT1b or BRD2 with TRF2 restores a canonical DNA damage response at telomeres resulting in frequent telomere fusions and cell death. Furthermore, we performed the RNAseq analysis and identified genes differentially expressed between TRF2-depleted and wild type ESCs. Unexpectedly, we found that TRF2 depletion in ESCs activates a totipotent-like 2-cell stage transcriptional program including high levels of ZSCAN4. We show that the upregulation of ZSCAN4 is both required and sufficient to establish TRF2-independent telomere protection. In summary, we found that ESCs have a unique response to telomere dysfunction elicited by TRF2 depletion. Our study provides novel insights into the field of telomere and stem cell biology and will contribute to understanding the nature of cancer cells maintaining telomeres by telomerase-independent mechanisms.

 

Victoria Avanzato

Ms. Victoria Avanzato (NIAID)

A structural basis for neutralization of Nipah virus via antibody-mediated targeting of the fusion glycoprotein

Nipah virus is a highly pathogenic paramyxovirus capable of causing severe neurologic and respiratory disease in humans. Bat populations act as the natural reservoir of Nipah virus, from which near annual spillover events into humans result in outbreaks with fatality rates reaching 90%. Currently, there are no licensed vaccines or therapeutics against Nipah virus, which, combined with its severe pathology, has led the WHO to classify Nipah as a priority pathogen, underscoring the urgent need for research and development of countermeasures. The Nipah virion surface is decorated with two glycoproteins, termed NiV-G and NiV-F, which allow the virus to attach to and enter host cells, respectively. As the sole proteinaceous antigens on the virus surface, NiV-G and NiV-F are exposed and accessible for host immune recognition and thus make attractive vaccine targets. Indeed, previous studies have shown that generation of neutralizing antibodies against these glycoproteins is critical to control Nipah virus disease in animal models. In order to identify immunologically vulnerable target sites on the virus surface and to elucidate the molecular basis for antibody-mediated neutralization of Nipah virus, we sought to define the epitopes of monoclonal antibodies against NiV-F using X-ray crystallography and cryo-electron microscopy. We determined the structures of NiV-F in complex with the Fab fragments of two neutralizing antibodies, mAb66 and mAb92. The crystal structure reveals that Fab66 binds an epitope at the most membrane distal apex region of NiV-F. The interaction is light chain dominated and insertion of the CDR3 loop into a shallow depression on the NiV-F surface allows several key tyrosine residues on Fab66 to form stabilizing contacts with NiV-F. Characterization of mAb92 using cryo-electron microscopy reveals an overlapping but distinct epitope also located at the membrane distal region of NiV-F, supporting the hypothesis that this apical region is accessible for immune targeting. Importantly, these membrane distal epitopes are highly conserved among Nipah virus isolates. Experiments to determine whether prophylactic treatment with mAb92 offers protection from a lethal Nipah challenge in hamster models are ongoing to evaluate the in vivo relevance of these epitopes. Combined, this work reveals the membrane distal region of NiV-F as a site of vulnerability on the Nipah virus surface and may inform the design of improved targeted vaccines in the future.

 

FARE2021 SIG Runners-up

Antibody Interest Group          

  • Ms. Victoria Avanzato (NIAID)
  • Dr. Maja Buszko (NIAID)
  • Dr. Sebastian Temme (NCI-CCR)
  • Dr. Chris Gonelli (NIAID-VRC)

Decode Chromatin Interest Group       

  • Dr. Arun Kumar Ganesan (NCI-CCR)
  • Dr. Joyce Thompson (NICHD)
  • Dr. Marta Markiewicz Potoczny (NCI-CCR)

Inflammatory Disease Interest Group  

  • Dr. Panagiotis Mastorakos (NINDS)

Matrix Biology Interest Group  

  • Dr. Shaohe Wang (NIDCR)
  • Dr. Giulia Riparini (NIAMS)

Neurobiology Interest Group   

  • Dr. Miranda Marvel (NICHD)
  • Dr. Cole Malloy (NICHD)

Protein Trafficking and Organelle Dynamics Interest Group      

  • Dr. Joshua Pemberton (NICHD)
  • Dr. Alexander Gasparski (NCI-CCR)

Systems Biology Scientific Interest Group         

  • Dr. Kun Wang (NCI-CCR)

Transcription Scientific Interest Group 

  • Dr. Xiyuan Zhang (NCI-CCR)