Poster Abstracts

#2: Adams, Philip

RNA-RNA interactomes of Hfq and ProQ reveal overlapping and competing roles for RNA chaperones

Philip P. Adams, Sahar Melamed, Aixia Zhang, Hongen Zhang, and Gisela Storz

Bacteria encounter diverse stresses that they must overcome in order to survive. Small regulatory RNAs (sRNAs), which function by base-pairing with other RNAs, have emerged as critical components in bacterial stress responses. Thisbase pairing between sRNAs and their targets was shown to be facilitated bythe RNA chaperoneHfq in many bacteria. However, recent studies suggest that the FinO-domain protein ProQ also binds RNA and has a global regulatory function. To gain insight into the respective roles of Hfq and ProQ and to compare the regulatory networks associated with the two RNA chaperones, we took advantage of the recently-developed RIL-seq (RNA Interaction by Ligation and sequencing) approach. In this technique, an sRNA is ligated to its RNA-target when bound to a particular chaperone, allowing for downstream sequencing and identification of the ligated-RNAs. Using Escherichia coli, RIL-seq was used to examine the sRNA-RNA interactomes for both Hfq and ProQ. Curiously, while some sRNA-RNA interactions were unique for each chaperone, 30% of the ProQ-bound RNA-pairs were also found with Hfq. To better understand the biological roles of the observed overlapping interactions, the effect of sRNAs on their putative targets found on both Hfq and ProQ was examined. We demonstrated that a novel sRNA, termed RbsZ, acts as a regulatory RNA sponge by base-pairing with a well-characterized sRNA, RybB, to decrease its levels. Mutational studies on this interaction confirmed the direct binding between the RNA-pair. Surprisingly, this regulation was almost completely dependent on Hfq, but was blocked with overexpression of ProQ. Collectively, this work is the first to examine the interactome of multiple RNA chaperones and uncovers ProQ as a novel protein sponge, which fine-tunes RNA-mediated gene regulation. Our results indicate the interactions between chaperones, sRNAs, mRNAs and RNA sponges to combat stress responses are more complex than initially imagined.

#3: Amyx, Melissa

Racial/ethnic differences in the association between early gestation plasma cotinine and nicotine concentrations and neonatal anthropometric measures

#4: Anderson, Afrouz / Khaksari, Kosar / Kharw, Siddharth / Malpani, Ravi

Continuous monitoring of the placental oxygenation

Afrouz Anderson, Siddharth Khare, Kosar Khaksari, Ravi Malpani, Amir Gandjbakhche

#5: Aoyama, Jordan

Investigating competition between activities of a dual function RNA

#6: Appu, Abhilash / Sadhukhan, Tamal

Cln3-mutations underlying juvenile NCL cause drastically reduced levels of lysosomal Ppt1-protein and Ppt1-enzyme activity and may contribute to JNCL

Abhilash P. Appu, Maria B. Bagh, Tamal Sadhukhan, Avisek Mondal, Sydney Casey and Anil B. Mukherjee

#7: Ashton, Nicholas

DNA polymerase iota is regulated by USP7-mediated deubiquitination

#8: Bloyd, Michelle

Associations between the Cyclic AMP-Dependent Protein Kinase A Pathway Genes and Nonalcoholic Fatty Liver Disease in Children

#9: Belgrad, Jillian

Possible Involvement of Myelinating Glia in Hereditary Paralytic Disorder

#10: Bondage, Devanand / Bonnington, Katherine / Ellis, Nicole / Johnston, Kathryn / Lehman, Stephanie

Characterization of Legionella pneumophila virulence mechanisms

Devanand Bondage, Katherine Bonnington, Nicole Ellis, Kathryn Johnston, Mitch Lee, Stephanie Lehman

#11: Brooks, Samira

Linking kidney iron overload in African American renal cancer patients with the SLC40A1 (Ferroportin) Q248H mutation

#12: Chakraborty, Shreeta

Gene regulation during development and differentiation

#13: Dashtestani, Hadis / Harrison, Doug / Millerhagen, John

Investigation of the action-observation network using electroencephalogram (EEG) and functional near-infra-red spectroscopy (fNIRS)

Hadis Dashtestani, Doug Harrison, John Millerhagen

#14: De, Sandip / Gehred, Natalie

Mechanism of enhancer-promoter communication in a chromatin domain context

Sandip De, Natalie Gehred, Miki Fujioka, Victoria Blake, James B. Jaynes and Judith Kassis

Chromosomes are folded into topologically associating domains [TADs] that help pack long stretches of DNA inside the eukaryotic nucleus. TADs strongly favor intra-domain chromatin contacts, restricting enhancers’ activity within the domain. We set out to understand the mechanism of enhancer-promoter communication better with respect to a chromatin domain.

We are investigating the enhancer-promoter communication of the ‘invected [inv]-engrailed [en]’ TAD (~113kb) in Drosophila. Expression of inv & en are spatially and temporally repressed by Polycomb proteins and co-activated by at least 20 specific enhancers. Our results show that a transgene inserted outside the inv-en domain can be expressed like inv-en if contains specific sequences known as PREs [Polycomb response elements] and motifs within it to facilitate activation of the reporter genes by the en enhancers. We also found that transgenes present outside the inv-en chromatin domain are in competition with inv-en endogenous genes for the enhancers. Surprisingly, transgenes present within the inv-en domain share the enhancers equally with the endogenous genes. Finally we show that in Drosophila, transcriptionally active domains marked with H3K36me3, not the insulator/architechtural proteins binding sites, act as the boundary for Polycomb domains. Unexpectedly, the Polycomb domain can even spread through binding sites for insulator/architechtural proteins. Overall, we were able to understand the interesting behavior of a chromatin domain either transcriptionally active or repressed.

#15: Dhar, Beenish

Role of H19 associated miRNAs in embryonic development

#16: Drougat, Ludivine

Study of the impact of the PRKACA gene amplification in transgenic mice

Ludivine Drougat, Paraskevi Salpea, Annabel Berthon, Edra London, Andrea Gutierrez Maria, Fabio Rueda Faucz, Constantine Stratakis

#17: Eraso, Ariel

Long-Distance Relationships: A screen to characterize genes helping SOX2 and its enhancer stay together despite being so far apart

Ariel Eraso, Pedro Rocha, Shreeta Chakraborty

Across the genome, DNA looping facilitates promoter activation leading to transcription. Short and long distance looping allows for enhancers to come into close proximity with their target promoters with a great degree of specificity. Previous experiments have demonstrated that formation of these loops is mediated by Cohesin extrusion of DNA into smaller segments with borders delineated by CTCF binding. Sox2's interactions with its enhancer 150 kb away are thought to be maintained by such looping. The ability of the enhancer to maintain specificity to Sox2 is of special importance in early development as Sox2 is an essential gene in assuring proper embryonic development. Embryos lacking wild type Sox2 expression fail to develop a proper ICM in an embryonically fetal phenotype. This phenotype can be recapitulated if Sox2's distal enhancer is deleted in mouse embryonic stem cells that differentiate instead into tropho-ectoderm like cells that give rise to the placenta. Deletion of CTCF sites thought to aid Cohesin in establishing the looping mediating Sox2 enhancer-promoter communications does not recapitulate the enhancer deletion phenotype in mouse embryonic stem cells. In fact, there is only a marginal, insignificant decrease in Sox2 expression. From this we hypothesize there must be other unknown proteins and factors allowing for Sox2 and its distal enhancer to maintain communication. This project aims to identify the unknown factors that may be involved in maintenance of Sox2 enhancer-promoter interactions using a genome-wide CRISPR screen to knockout every gene in the mouse genome. Targeting that leads to interruption of the long-distance communication will then be further characterized to determine the involvement of those loci in long-distance enhancer-promoter communication.

#18: Erben, Larissa

Analysis of neurodevelopment and schizophrenia-relevant behaviors in ErbB4 mutant mice lacking the Cyt-1 isoform

#19: Fister, Alexandra

The miR-204/NFATC1 axis controls developmental lymphangiogenesis in zebrafish

#20: Frail, Sarah

How to Be the Cell Gata6 Wants You to Be: Genetic and Epigenetic Mechanisms Governing Early Mammalian Development

See abstract below.

#21: Friend, Lindsey

Sinuous, a member of the claudin family, facilitates synaptic targeting of specific subtypes of ionotropic glutamate receptors

#22: Gaikwad, Swati

Reprogramming of mRNA translation efficiencies by impaired ribosome recycling at stop codons in vivo

#23: Gober, Ryan

A Newly Identified Mutant helps to Characterize the Potential Function of Vascular-associated Meningeal Cells

#24: Gorrell, Laura / Omari, Shakib

Procollagen quality control in Osteoblasts

#25: Greenspan, Leah

Blood Vessel Regeneration after Injury

#26: Gulay, Suna

eIF4A and eIF4E interactions with distinct residues of the Ded1 N-terminus stimulate Ded1 function in translation initiation in vivo

#27: Gulyás, Gergő

Oxysterol-binding protein-related protein 3 (ORP3) is a PKC-regulated lipid transporter that works at contact sites between the plasma membrane and endoplasmic reticulum

#28: Gupta, Neha

Role of Pat1 in translational control

Neha Gupta, Jon R. Lorsch, Alan G. Hinnebusch

#29: Gutierrez Maria, Andrea

Uncovering unknown Aurora kinase signaling in adrenal tumor cell line, NCI-H295

#30: Hareendran, Sangeetha

Exosome-based Carboxypeptidase E confers metastasis and CPE-shRNA loaded exosomes as a therapeutic agent inhibits tumor growth

At The End Of The Brainbow: Dissecting Chandelier Cell Connectivity

#32: Ivanov, Ivaylo

Regulated Ribosome Queuing Mediates uORF and eIF2 Phosphorylation Control of CHOP and GADD34 mRNAs

#33: Jha, Abhishek

Superiority of ⁶⁸Ga-DOTATATE PET/CT to other functional imaging modalities in the detection of SDHA-related metastatic pheochromocytoma and paraganglioma—A comparative prospective study

Abhishek Jha, Divya Mamilla, Melissa K. Gonzales, Mayank Patel, Corina Millo, Clara C. Chen, Alexander Ling, Isabel Tena, Marianne Knue, Geraldine O'Sullivan Coyne, Alice Chen, Naris Nilubol, Frank I. Lin, Constantine A. Stratakis, Peter Herscovitch, David Taïeb, Ali Cahid Civelek, Jorge Carrasquillo, and Karel Pacak

Background and Purpose

Pheochromocytoma and/or paraganglioma (PPGL) in patients harboring a succinate dehydrogenasesubunit genes (SDHx) mutation are at a higher risk for metastases resulting in poor outcome compared to other hereditary PPGLs. ⁶⁸Ga-DOTA(0)-Tyr(3)-octreotate (⁶⁸Ga-DOTATATE) positron emission tomography-computed tomography (PET/CT) is superior to other functional imaging modalities in the detection of SDHB-related metastatic PPGL. However, its performance in SDHA-related PPGL is currently unknown. The purpose of this prospective study was to evaluate and compare the diagnostic performance of ⁶⁸Ga-DOTATATE PET/CT to ¹⁸F-fluoro-2-deoxy-D-glucose (¹⁸F-FDG) PET/CT and ¹⁸F-L-dihydroxyphenylalanine (¹⁸F-FDOPA) PET/CT in metastatic SDHA-related PPGL.

Methods

⁶⁸Ga-DOTATATE PET/CT was prospectively performed in 10 consecutive patients (females: males, 4:6; mean age, 43.5±18.1 years) with metastatic SDHA-related PPGLs. They also underwent ¹⁸F-FDG (n=10) and ¹⁸F-DOPA (n=6) PET/CT. The mean duration between ⁶⁸Ga-DOTATATE, ¹⁸F-FDG, and ¹⁸F-FDOPA was 6±4 days. Per patient and per lesion detection rates were compared for all 3 imaging modalities. Per patient and per lesion detection rates were compared for all 3 imaging modalities. A composite of all the functional imaging studiesas an imaging comparator served as a reference standard for calculation of detection rates. The McNemar test was used to compare detection rates between ⁶⁸Ga-DOTATATE PET/CT and the other imaging modalities. Two-sided p values <0.05 were considered significant.

Results

All patients were positive for PPGLs, demonstrating 373 lesions on the imaging comparator. ⁶⁸Ga-DOTATATE PET/CT demonstrated a per lesion detection rate of 318/373 [85.3%, 95% confidence interval (CI): 81.2-88.7%]. ¹⁸F-FDG and ¹⁸F-FDOPA PET/CT showed significantly lower per-lesion detection rates of 245/373 (65.7%, 95% CI: 60.6-70.5%; p<0.0001) and 48/159 (30.2%, 95% CI: 23.2-38.0%; p<0.0001) respectively. The per patient detection rates of ⁶⁸Ga-DOTATATE and ¹⁸F-FDG PET/CT were both 10/10 (100%, 95% CI: 69.2-100%), and that of ¹⁸F-FDOPA PET/CT was 5/6 (83.3%, 95% CI: 35.9-99.6).

Conclusions

⁶⁸Ga-DOTATATE PET/CT showed a significantly higher per lesion detection rate compared to ¹⁸F-FDG and ¹⁸F-DOPA PET/CTand may be the preferred functional imaging modality to evaluate metastatic SDHA-related PPGL. Furthermore, owing to the ⁶⁸Ga-DOTATATE avidity of PPGLs in these patients, cold somatostatin analog and/or peptide receptor radionuclide therapy can be considered as therapeutic options.

#34: Xiao, Ashley

Carboxypeptidase E-Neurotrophic factor-α 1: Obliteration of the enzymatic activity uncovers its novel receptor-mediated hippocampal neuroprotective and cognitive functions in mice

#35: Khalique, Abdul

Uncovering how conserved tRNA isopentenyltransferases generate variable subsets of i6A37-modified tRNAs; and a system for mitochondrial targeting and screening of point mutations in the human population for potential pathogenicity

#36: Kouse, Andrew

Effect of the small RNA GlnZ on carbon and nitrogen metabolism in E. coli

Andrew B. Kouse, Lauren Walling, Gisela Storz

Regulatory small RNAs (sRNAs) were initially identified as being encoded within intergenic regions. However, recent studies have shown that many are derived from the 3′ UTRs of mRNAs. One such sRNA, denoted GlnZ, corresponds to the 3′ UTR of the Escherichia coli glutamine synthase gene glnA. RNA interaction by ligation and sequencing (RIL-seq) analysis of Hfq-dependent sRNA targets showed that potential targets of GlnZ include aceE, sucA and glnP which encode enzymes of glycolysis, the TCA cycle and a glutamine transporter, respectively. These potential targets along with the genomic location of glnZ suggest that GlnZ is involved in modulating carbon and nitrogen metabolism. Based on these observations, GlnZ levels were examined following growth in the carbon sources glucose and glycerol as well as the nitrogen sources ammonium and glutamine. Northern analysis revealed that while GlnZ levels were not affected by carbon source or ammonium concentration, they were significantly reduced when cells were grown in the presence of glutamine. This glutamine-dependent regulation was found to be independent of transcription from the native glnZ promoter but dependent on RNase III as GlnZ levels were unchanged in a Drnc mutant with glutamine as the sole nitrogen source. The effect of GlnZ on its targets was examined using beta-galactosidase assays as well as Western blot analysis. Overexpression of GlnZ led to a decrease in beta-galactosidase activity and protein levels suggesting GlnZ represses these predicted targets. Together our data indicate that GlnZ is destabilized in a glutamine- and RNase III-dependent manner and that GlnZ negatively regulates GlnP, AceE and SucA synthesis, linking carbon and nitrogen metabolism.

#37: Krishnakumar, Pritesh

The Migration of the Zebrafish Lateral Line

#38: Kulkarni, Shardul

Temperature-Dependent Regulation of Upstream Open Reading Frame Translation in S. Cerevisiae

Shardul Kulkarni, Fujun Zhou, Neelam Sen, Hongen Zhang, Alan Hinnebusch, Jon Lorsch

Translation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as ACG, UUG, etc. are also used as start sites at low levels in S. cerevisiae. Initiation from NCCs or AUGs in the 5′-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different start codons. We performed ribosome profiling, a technique that determines the position and occupancy of ribosomes (ribosome-footprint) on the mRNAs. We identified ~1350 uORFs starting with NCCs or AUGs in 5′-UTRs with a strong ribosomal footprint and significant evidence of translation. Intriguingly, translation of some, but not all, uORFs was differentially modulated by growth temperature, depending on the specific mRNA. The position of the uORF-start site and its nature were shown to influence this modulation. Most regulated uORFs starting with AUG behave similarly, with their translation repressed at low temperature and activated at higher temperature, whereas uORFs starting with near-cognate codons do not show coherent behavior but can be activated, repressed, or remain unchanged at lowered or elevated temperatures. In addition, we found temperature-regulated upstream initiation events that lead to formation of N-terminal extensions (NTEs) that are in-frame with the mORF. Interestingly, many of these NTEs are present on the proteins with differential cellular localizations. Overall, our data suggest that changes in growth temperature can affect the use of start codons and modulate specific gene expression at the level of translation in S. cerevisiae.

#39: Lee, Dongjin

Spatial heterogeneity and molecular dynamics of mouse telencephalic progenitors at single-cell resolution

#40: Lee, Mitchell

Host-mediated allosteric activation of a virulence-promoting kinase secreted by the bacterial pathogen Legionella pneumophila

Mitchell Lee, Pei-Chung Lee, Matthias Machner

Legionella pneumophila is a bacterial pathogen that can replicate in human alveolar macrophages and cause a life-threatening pneumonia called Legionnaires' disease. During infection, L. pneumophila deploys over 300 proteins, called effectors, into the host cell, where they manipulate host pathways and processes to establish a replication vacuole. Studying interactions between these effectors and host factors therefore increases our understanding of the infection strategies used by intracellular pathogens and provides foundations for the development of anti-microbial therapeutics for infection. In this study, we discovered and dissected the interaction between the L. pneumophila kinase effector LegK2 and the host small GTPase Rab32, which regulates phagosome maturation and trafficking to lysosome-related organelles. Using an in vitro kinase reconstitution assay, we found that Rab32 is a substrate of LegK2 and, unexpectedly, also an allosteric activator of LegK2 kinase activity. We then demonstrated that LegK2 specifically interacts with active, GTP-bound Rab32 and that this interaction is required for allosteric activation of LegK2. To identify host substrates of LegK2, we incubated pre-formed Rab32:LegK2 complexes with a high-density human protein microarray. Several host proteins involved in cytoskeleton rearrangement were phosphorylated by the Rab32:LegK2 complex, including the actin bundle-stabilizing and anchoring protein dematin (EBP49). These results suggest that L. pneumophila secretes LegK2 to exploit host Rab32 and dematin via phosphorylation. Our future work will investigate how LegK2 modulates vesicle trafficking and biogenesis of the replication vacuole in host cells through interaction with Rab32 and phosphorylation of dematin, as well as the consequences of this modulation for intracellular replication of L. pneumophila.

#41: Li, Feng

Identification of an integrase-independent pathway of retrotransposition

#42: Lotspeich-Cole, Leda

Ascorbate-sensing uORF Controls GDP-L-galactose phosphorylase (GGP) mRNA Translation

#43: Mahadevan, Vivek / Peltekian, Areg

Developmental NMDA receptor ablation in a subset of interneurons confers circuit-wide aberrant gene expression and schizophrenia-like impairments

Vivek Mahadevan, Ramesh Chittajallu, Areg Peltekian, Kenneth A. Pelkey, Caroline Esnault, Yajun Zhang, Maric Dragan, Xiaoqing Yuan, Steven Hunt, Daniel Abebe, Ryan Dale, Timothy J. Petros, Chris J. McBain

Medial ganglionic eminence (MGE)-derived forebrain GABAergic interneurons comprise several classes of interneurons namely, the parvalbumin, somatostatin, and neurogliaform/ivy cells that critically regulate cortical circuit maturation and refinement. NMDA-receptor (NMDAR) complexes within MGE interneurons mediate their development, physiology, and recruitment during network activity and plasticity. NMDARs provide a crucial source of calcium entry into neurons, and their signaling is a dominant convergence point for cell-intrinsic excitation-transcription (E-T) coupling. However, a comprehensive understanding of NMDAR-mediated E-T coupling in distinct interneuron classes is currently lacking, although interneuronal NMDAR-hypofunction is well known to underlie network and cognitive abnormalities in neurodevelopmental disorders, notably schizophrenia (SCZ).

Here, we conditionally ablated the requisite GRIN1 subunit of NMDARs in the MGE-derived interneurons throughout development and examined its impact on cell-type specific gene expression, and behavioral abnormalities. Combined single-cell RNA sequencing and MGE-specific translating RNA sequencing analyses revealed a dramatic reduction in the expression of the neuron-specific inducible-transcription factor NPAS4 and its target BTG2 in the GRIN1-ablated cortical interneurons. Moreover, we observed a robust increase in the expressions of synapse regulators NTNG1, NORBIN, and KCNH2 in GRIN1-ablated hippocampal interneurons. Surprisingly, we observed non- cell-autonomous gene expression changes in pyramidal neurons and non-neurons, indirectly as a result of GRIN1-ablated MGE-interneuron impairment. In particular, we observed a robust increase in the pyramidal-neuron expressed immediate early genes, NPTX2, BDNF, and ARC, indicating aberrant pyramidal neuron hyperactivity; and a marked decrease in the microglia-expressed CX3CR1, IGF2. Importantly, several aberrantly expressed genes we identified are established human schizophrenia risk genes. Moreover, these animals exhibit novelty-induced hyperlocomotion, social anxiety and motor abnormalities reminiscent of SCZ-like impairments. Together, our study establishes that developmental NMDAR dysregulation initiated in a subset of GABAergic interneurons promotes circuit-wide gene expression abnormalities resembling those associated with SCZ etiology.

#44: Mahgoub, Mohamed

Dual histone reader ZCWPW1 links PRDM9-induced histone methylation marks to meiotic double strand break machinery

Mohamed Mahgoub, Florencia Pratto, Kevin Brick, Todd Macfarlan

The alignment and exchange of chromosome material during crossover formation in meiosis is the cornerstone for germline production in the sexual life cycle. Crossovers result from genetic recombination during DNA repair of programmed double strand breaks (DSBs) in one of the homologues. In most mammals, DSBs do not occur at random but at hotspots that are determined by the rapidly evolving DNA binding zinc finger array of Prdm9 (PR domain zinc finger protein 9). PRDM9 catalyzes trimethylation of lysine 4 and lysine 36 of Histone 3 (H3K4me3 and H3K36me3 respectively) in nearby nucleosomes, which is essential for DSB formation at hotspots, but how these histone marks contribute to the recruitment of the DSB machinery is unknown. Here we identify Zinc Finger CW-Type and PWWP Domain Containing 1 (Zcwpw1) as a likely obligate cofactor for PRDM9 induced DSBs. Zcwpw1 is tightly co-expressed with Prdm9 during meiosis and contains two highly conserved domains: the zf-CW domain that binds to the H3K4me3 mark and the PWWP domain that recognizes the H3K36me3 mark. Independently, Prdm9 and Zcwpw1 share a striking co-evolutionary history with co-emergence in jawless fish and co-disappearance in amphibians, birds, and some fish species. We demonstrate that Zcwpw1 knock-out mice are azoospermic and have small testes due to arrest at the pachytene stage, with spermatocytes displaying incomplete DSB repair and synapsis. We hypothesize a model in which Prdm9 tags chromatin with dual histone marks that are recognized by Zcwpw1 that ultimately tethers DNA loops to the chromosomal axis for subsequent DSB formation and repair.

#45: Malloy, Cole

Pin1 isomerization of Kv4.2 regulates neuronal excitability in the mouse hippocampus

#46: Melamed, Sahar

Regulatory RNAs manipulate ribosomal components and transcription factors to affect flagella synthesis

Sahar Melamed, Joshua Mills, Aixia Zhang, Michal Jarnik, Gisela Storz

While flagella synthesis is extremely energy intensive and is under extensive transcriptional regulation, less is known about post-transcriptional regulation of flagella synthesis. Small RNAs (sRNAs) are widely utilized as post-transcriptional regulators; many base-pairing with mRNAs affecting their stability and/or translation. Yet, the only known target of the sRNAs in the flagella regulon is the flhDC mRNA encoding the transcription factor at the top of the transcription regulatory network. Therefore, we searched for additional sRNAs that might regulate the flagella regulon.

Here we report on four Escherichia coli sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor, Sigma 28, encoded by fliA. These sRNAs originate from the UTR regions of mRNAs, including three genes belonging to the flagella regulon. Interestingly, MotR and FliX modulate the synthesis of ribosomal proteins, which are required for the mass production of the flagella proteins. UhpU affects flagella synthesis by repressing regulators of the flagellar regulon. Consistent with these roles, overexpression of each of the sRNAs leads to a unique phenotype in terms of flagella length and number. The phenotypes for these sRNAs exemplify the diverse impact that sRNAs can have on complex regulatory networks.

#47: Mishra, Saurabh

Separate functions of the transcription termination activity of the NTD and the RNA cleavage activity of the CTD, of the RNA polymerase III subunit, Rpc11

#48: Mondal, Avisek

Palmitoyl-protein thioesterases-1 deficiency dysregulates lysosomal Ca++ homeostasis due to reduced level of IP3R1 and contributes to pathogenesis of a neurodegenerative lysosomal storage disease

#49: Noeker, Jacob

How Loss of Imprinting Leads to a Broken Heart: Endothelial to mesenchymal transition as a mechanism for cardiovascular anomalies associated with BWS

#50: O'Connell, Constandina

Characterization of Autophagy Control Identifies a Novel Selective Anti-Cancer Therapy

#51: Patel, Tusharkumar

The Role of Adiponectin in Melanocortin 3 Receptor (MC3R)-Insufficiency Obesity Phenotypes

Tushar P. Patel, Joo Yun Jun, Adrian M. Wong, Jordan A. Levine, Noah J. Levi, Robin B. Roberson, Jack A. Yanovski

The MC3R affects energy homeostasis through its role on central nervous system appetitive pathways but also appears to act on peripheral metabolic processes. We previously reported that knock-in mice replacing the murine Mc3r with hypoactive human double mutant (C17A + G241A) MC3R (MC3R^hDM/hDM)had increased feeding efficiency, body weight and fat mass, along with surprisingly increased serum adiponectin compared to wild type human MC3R knock-in mice (MC3R^hWT/hWT).We cross-bred MC3R^hWT/hWT and MC3R^hDM/hDM mice with adiponectin knockout mice to generate MC3R^hWT/hWT+Adipo^-/- and MC3R^hDM/hDM+Adipo^-/- mice. Body composition was measured using DEXA. WAT was tested for adipogenesis, lipogenesis, and lipolysis to understand the role of adiponectin in the adipocyte expansion and obesity phenotype of MC3R insufficiency. The high adiponectin of MC3R^hDM/hDM became undetectable in MC3R^hDM/hDM+Adipo^-/-. Fat mass, bone measures and feeding efficiency partially normalized in MC3R^hDM/hDM+Adipo^-/- towards those of MC3R^hWT/hWT+Adipo^-/-. In adipose tissue, compared to MC3R^hDM/hDM, MC3R^hDM/hDM+Adipo^-/- had reduced protein expression of ACC-1 and FASN (de-novo lipogenesis) and increased expression of multiple enzymes involved in lipolysis: pHSL, MAGL and ATGL. During differentiation, adipose-derived stem cells from MC3R^hDM/hDM+Adipo^-/- also accumulated significantly less neutral lipid and had lower mRNA expression of adipogenesis and lipogenesis markers compared to MC3R^hDM/hDM cells. These data suggest adiponectin signaling interacts with MC3R signaling to affect lipid turnover and nutrient partitioning. The MC3R genotype x adiponectin effects on lipolysis and lipogenesis suggest that there is crosstalk between the adiponectin and melanocortin 3 signaling pathways in the regulation of fat mass in adipose tissue. The hyperadiponectinemia observed in MC3R^hDM/hDM is an important contributor to its obese phenotype.

#52: Pathak, Rakesh

LEDGF/p75 functions during transcription to alter splicing and termination

Zelia Ferreira Worman, Kory Johnson, Arianna Lechsinska, Rakesh Pathak, Henry L. Levin

HIV-1 insertion sites determined in cultured cells demonstrate the host chromatin-binding factor LEDGF/P75 interacts with integrase and directs integration to actively transcribed genes. Previously, we found LEDGF/p75 is responsible for an integration bias that favors transcription units with high numbers of introns. We also found that LEDGF/p75 interacts with a number of mRNA splicing factors. These data and work from other labs suggest LEDGF/p75 may play a role in mRNA splicing. However, early studies with fractionated extracts indicated LEDGF/p75 is a key transcription factor that has strong promoter co-activator activity with a variety of activators including TBP and TFIIA. To enhance our understanding of how insertion sites are selected, we examined the role of LEDGF/p75 in genome-wide expression levels, splice choices, splice fidelity, and transcription termination. We used deep coverage RNAseq of HEK293T cells with bi-allelic deletions of the PSIP1 gene, which encodes LEDGF/p75, and generated high resolution de novo maps of transcript isoforms. Our analysis identified a total of 733 genes with differentially expressed transcripts (>1.5 fold change) between HEK293T cells (+/+) and p75 K.O. (-/-). To determine which of these changes in expression are likely a result of LEDGF/p75 association we used integration densities as a proxy for which genes are bound by LEDGF/p75. We found 254 (35%) of differentially expressed genes were among the 4,000 genes that had the highest density of integration. This represents a strongly significant overlap relative to the genome with a p<9.6e^-30 as determined by hypergeometric distance. We examined the role of LEDGF/p75 in promoter activity and splicing of the 254 differentially regulated genes associated with LEDGF/p75 by comparing transcripts that have the same transcription start sites. If LEDGF/p75 contributes to promoter activity, we expect the amount of RNAs with the same start of transcription to be concordantly altered in the p75 K.O whereas a role in splicing would cause discordant changes. Because the 254 regulated genes often had multiple transcripts, we could organize the mRNAs into 131 groups based on having the same start site of transcription. Comparing p75 (+/+) to LEDGF/p75 K.O. cells, 95% of the groups exhibited discordant behavior where the amount of at least one mRNA was unchanged and at least one other is significantly increased or reduced in the p75 K.O. (-/-) cells. Importantly, one common distinguishing feature of many altered mRNAs was their site of termination was different from others in the group that were unaffected by the p75 K.O. Taken together, our results indicate that LEDGF/p75 functions during transcription elongation to alter splicing and termination but has little impact on promoter activity.

#53: Pemberton, Joshua

Membrane phospholipid composition directly influences mitochondrial dynamics.

Pemberton J.G., Kim Y.J., Korzeniowski M.K., Sengupta N., and T. Balla

The coordinated fusion and fission of mitochondria is critical for maintaining the functional integrity of this important organelle and several prominent human diseases are associated with defective mitochondrial dynamics; including abnormalities during the progression of cancers, diabetes, as well as severe neurodegenerative disorders. Although a growing number of proteins have been identified as regulators of mitochondrial fusion and fission, the direct role of membrane lipids during these processes has not been assessed experimentally. To address these shortcomings, we devised a protein-engineering platform to acutely remodel phospholipids within the outer mitochondrial membrane (OMM) of intact cells. Specifically, after multiple rounds of iterative mutagenesis, we were able to generate a significantly modified phospholipase C from bacteria (bPLC) that is capable of hydrolyzing the headgroup from an essential structural lipid, phosphatidylinositol (PI), to leave behind only the membrane-embedded diacylglycerol (DG) backbone. Spatial restriction of bPLC activity was achieved using a chemically-inducible protein dimerization system that allowed for the local conversion of PI to DG selectively within the cytosolic leaflet of the OMM. Interestingly, acute hydrolysis of PI in the OMM and the subsequent accumulation of DG was associated with the rapid and uniform fragmentation of the mitochondria. As important controls, recruitment of a catalytically-inactive variant of the bPLC failed to induce mitochondrial fragmentation and the dramatic structural effects induced by the active bPLC also did not alter the OMM potential or lumenal Ca²⁺ concentration; strongly suggesting that the changes observed in the mitochondrial morphology are not due to gross insults to the integrity of the OMM. Taken together, these results provide the first direct evidence that acute changes to the lipid composition of the OMM significantly influences the structure of the mitochondrial network and highlight the need to better understand how the spatial control of OMM lipid metabolism is impacted during mitochondrial-associated diseases.

#54: Piccus, Zoe

Using single knock-in approaches in mice to understand juvenile ALS

#55: Pillay, Laura

Characterizing the function of RHOA signaling in regulating vascular integrity and development

Laura M. Pillay, Matthew G. Butler, Keith A. Barnes, Joseph J. Yano, Andrew E. Davis, Daniel A. Castranova, Aniket V. Gore, Matthew R. Swift, James R. Iben, and Brant M. Weinstein

#56: Pitsava, Georgia

Exome sequencing identifies genes involved in cell adhesion and migration in bladder exstrophy cases

#57: Plevock Haase, Karen

Identification of Novel Aging Genes in Drosophila melanogaster using RNAi and Overexpression Screens

#58: Radoc, Jeannie

Preconception Antidepressant Use and Pregnancy Outcomes: A Secondary Analysis of EaGER Trail

Jeannie G. Radoc, Kerry Flannagan, Sunni L. Mumford, Lindsey Sjaarda, Neil Perkins, Josh Freeman, Victoria Andriessen, Robert M. Silver, Enrique F. Schisterman

Objective: In the past decade, antidepressant use has quadrupled in the United States, with reports of use in 1 out of 7 reproductive aged women. Many women are reluctant to take antidepressants while trying to conceive, despite limited data on the impact of preconception use on pregnancy outcomes. Our aim was to examine the association between preconception antidepressant use and fecundability, live birth, and pregnancy loss.

Methods: Within the EAGeR trial, women aged 18-40 years old (n=1212) were screened for urinary concentrations of fluoxetine, sertraline, escitalopram, citalopram, and tricyclic antidepressants at up to 2 preconception time points and asked to report any antidepressant use for up to 12 months prior to conception. Women were followed for up to 6 months while attempting pregnancy. Cox proportional hazard regression was used to calculate fecundability odds ratios (FOR), and log-binomial regression to estimate risk ratios (RR) of live birth and pregnancy loss adjusting for age, body mass index, education level, employment, smoking frequency, alcohol use frequency, screened or self-reported marijuana use, and screened or self-reported opioid use.

Results: 183 (15%) women screened positive for antidepressant use during the preconception period, of which 113 also self-reported use. 235 women (19.1%) either screened positive or self-reported use. Women who either screened positive for or self-reported preconception antidepressant use had lower fecundability compared to those who did not use antidepressants (FOR 0.80; 95% CI 0.64, 1.00). No associations were observed between antidepressant use and live birth (RR 0.91; 95% CI 0.78, 1.05) or pregnancy loss (RR 1.04; 95% CI 0.76, 1.40).

Conclusions: Women who screened positive for or self-reported antidepressant use during preconception had reduced fecundability, though no associations were observed with live birth or pregnancy loss. Future work is needed to understand how different types and doses of antidepressants may negatively impact fecundability.

#59: Raina, Medha

Glucose and Low pH-Induced IsrB RNA Modulates Carbon Metabolism as Both a Base Pairing RNA and by Encoding a 28-Amino Acid Protein.

#60: Rajendran, Megha

Role of VDAC in α-Synuclein Induced Mitochondrial Dysfunction in Parkinson's Disease.

Perceived Stress and Salivary Cortisol Awakening Response in Youth

Sophie Ramirez, Lisa M. Shank, Manuela Jaramillo, Sarah G. Rubin, Deborah R. Altman, Elisabeth K. Davis, Sarah LeMay-Russell, Meghan Byrne, Sheila Brady, Miranda M. Broadney, Nichole R. Kelly, Marian Tanofsky-Kraff, Jack A. Yanovski

#62: Ranjan, Amitabh

The variant PAM2w motif of LARP4 promotes poly(A) RNA binding and contributes to a network of mRNA homeostasis

#63: Ray, Subhasis

Inhibitory control of sparse odor-coding in an insect mushroom body

#64: Regmi, Saroj

Nup96 has a key role in maintaining nuclear pore stability

#65: Rhodes, Christopher

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

Christopher Rhodes, Tim Petros

Mitotic progenitors in the ventricular/subventricular zones (VZ/SVZ) of the dorsal and ventral forebrain display similar cell cycle and neurogenic dynamics, yet each regiongenerates distinctneuronal subtypes. While the dorsal telencephalon gives rise to glutamatergic excitatory cells, GABAergic inhibitory interneuronsarise from three ganglionic eminences in the ventral telencephalon (MGE, LGE and CGE). GABAergic interneurons are an incredibly diverse population of cells thatperform critical roles in neural network function. Despite substantial evidence suggesting that initial interneuron subgroup fate is specified within the GEs, attempts to identify important fate determining genes in GE progenitors have largely failed. One possibility is that interneuron progenitors contain epigenetic signatures directing cells towards a particular interneuron fate that are not yet apparent in the transcriptome. For example, enhancers are critical to regulating gene expression, but we lack a comprehensive understanding of how such non-coding genomic regions influence cell fate. Transcription of a gene is highly dependent upon enhancers, which in turn are regulated by chromatin modifications.

To explore the relationship between enhancers and gene expression in distinct interneuron precursors, we utilize single cell assay transposase-accessible chromatin (scATAC-Seq) and single cell RNA (scRNA-Seq) sequencing to screen for candidate enhancers regulating expression of interneuron fate determining genes. We demonstrate that single cell sequencing approaches can detect distinct transcription and chromatin profiles restricted to specific progenitor zones (MGE, LGE, CGE or cortex). By examining ATAC peaks in lineage-specific genes across cell types, we were able to detect putative enhancers associated with interneuron fate determination. These insights increase our knowledge of fate determining mechanisms and could open new avenues for understanding how disease-associated genes could perturb interneuron fate and maturation.

#66: Rosario, Yvonne

A comparison of strategies for precise single-nucleotide edits in the zebrafish genome

Yvonne Rosario and Benjamin Feldman

#67: Sakhawala, Rima

Development of a NanoLuciferase Reporter System to Study C11 Function in Transcription Termination in S. pombe

#68: Schmidberger, LauraAnn

Roles of Autism-Linked Chromatin Remodelers in Neuronal Chromatin Organization

#69: Settas, Nikolaos

Kisspeptin and Kisspeptin receptor may be involved in the regulation of adrenocortical development and steroid hormone secretion.

#70: Sharma, Vinay

NF-α1/CPE neuroprotects against oxidative stress by activating ERK/BCL2 signaling via HTR1E receptor.

#71: Smith, Andrew

2-hydroxypropyl-B-cyclodextrin stabilizes swallow outcomes in Niemann-Pick Disease, type C Phase 0/I trial

#72: Shrestha, Deepika

Maternal BMI-increasing genetic risk score and fetal weights among diverse US ethnic groups

Deepika Shrestha, Stefanie Hinkle, Mohammad Rahman, Tsegaselassie Workalemahu, Fasil Tekola-Ayele

#73: Stover, Carissa

Investigating a Role for La Protein in an Innate Immune Signal Pathway via Shielding 5’-ppp-RNAs from Activating RIG-I

Carissa Stover, Sandy Mattijssen, Richard Maraia

#74: Subedi, Abhi

Unexpected diversity of raphe neurons in the zebrafish brain

Abhi Subedi, LauraAnn Schmidberger, Harold A. Burgess

Serotonin is an important neurotransmitter in the brain that has a role in regulation of anxiety and depression. The serotonergic neurons originate in the raphe nucleus is a highly conserved structure in the vertebrate brains, from the most primitive lamprey to human. Because of its highest concentration of serotonergic neurons in the brain and is also involved in modulating multiple behaviors including sleep, fear, stress, wakefulness, arousal, aggression, pain, and reward, there is a wide interest in this nucleus. Still, how one single region of the brain is involved in modulating these varieties of functions is still not clear. To answer this, we generated the individual neuronal projection map to see how and where individual neurons project to get an insight into the possible function of each individual neurons. Using transgenic driver (tph2:gal4) and a reporter (UAS:gal4FF:2A:mCherry) that silences effectively, we were able to image individual neurons of the raphe nucleus. Through high resolution confocal imaging of these neurons, we were able to generate the projection map and found that these neurons are highly diverse. The information gained from these experiments will help to understand the role of these neurons in regulating multiple behavior.

#75: Swearer, Avery

Cavefish: a model to study subcutaneous fat deposition

#76: Taimatsu, Kiyohito

A genetic screen for tissue specific epigenetic regulators

#77: Tanizaki, Yuta / Shibata, Yuki / Na, Wonho / Wang, Shouhong

Utilizing frog models to characterize the thyroid hormone-dependent postembryonic development of intestinal stem cells

Yuta Tanizaki, Yuki Shibata, Wonho Na, Shouhong Wang and Yun-Bo Shi

#78: Thompson, Joyce

Understanding the role of chromatin folding in shaping early embryonic development

#79: Tracy, Christopher

Investigating the transcriptional regulation and directionality of an enhancer at the Igf2/H19 locus

#80: Venero Galanternik, Marina

Studying the origin and function of novel vascular-associated cells in the zebrafish meninges

#81: Venkatesh, Sanan

Investigating Interneuron Epigenetics using Hi-C: Methodology and Analysis

#82: Ventresca, Christa

The Gene HDAC6 and its Effect on Drosophila Metabolism and Development

#83: Williamson, Nathan

Water diffusive mobility is almost entirely governed by lipid membranes

#84: Workalemahu, Tsegaselassie

Elevated maternal blood pressure during pregnancy alters placental DNA methylation

#85: Yano, Joseph

Characterizing the Role of Rhoa in Regulating Vascular Integrity

#86: Yau, Ka Chun

Dynamic control of the Ran-GTP gradient by Ran binding protein 1 (RanBP1) during mitosis

#87: Yazdankhah, Mojgan

Validation of Target Compounds for the Therapeutic Intervention of Smith-Lemli-Opitz Syndrome

#88: Young-Baird, Sara

Rescue of eIF2 mutant MEHMO syndrome phenotypes by ISRIB

#89: Temmen, Chelsie

Longitudinal Associations Between Maternal and Paternal Knowledge and Adolescent Alcohol and Marijuana Use

#90: Queralt Martin, Maria

Reduced affinity of mitochondrial VDAC3 for cytosolic proteins reveals a mechanism for isoform-specific physiology.

Voltage-dependent anion channels (VDACs) are the most abundant proteins in the mitochondrial outer membrane, where they are the primary cogs between the mitochondria and the cytosol, and have been implicated in numerous mitochondrial pathologies. Among the three known mammalian isoforms, VDAC3 is the least characterized and, in most cell types, the minimally expressed isoform. Yet, data in cell models demonstrate a distinctive function of VDAC3 and VDAC3-knockout mouse show a peculiar phenotype – male infertility – not observed with VDAC1. However, a high sequence similarity between VDAC1 and VDAC3 (~70%) is indicative of a similar pore-forming ability. Here we show that recombinant human VDAC3 forms highly conductive, stable, voltage-gated channels which are weakly anion-selective. These features are typical of VDAC1 and demonstrate that VDAC3 concurrently facilitates metabolite exchange. However, VDAC3 demonstrates unique properties when interacting with cytosolic proteins alpha-synuclein (α-syn) and tubulin. These two known potent regulators of VDAC1 induce similar characteristic (millisecond) blockages of VDAC3, but with hundred-fold reduced on-rates. Along with the facilitated translocation of α-syn through the channel in comparison with VDAC1, this suggests a distinct physiological role. The extra six cysteines of VDAC3 compared to VDAC1 do not influence basic channel function but modulate its interaction with α-syn. Thus, we conclude that interaction of VDAC3 with cytosolic proteins distinguishes it from VDAC1 and provides first mechanistic insights into the reported differences between these isoforms in cell and animal models.

Gur, Dvir

Structural color change and pattern formation in zebrafish

Dvir Gur

Animals including chameleons and certain species of fish, can dynamically tune their crystalline based structural colors, this color change is a prominent feature of many animals, and have important functions such as camouflage, kin recognition, and mate choice. The cellular machinery that facilities this remarkable phenomenon was yet to be discovered. Here, we have used zebrafish skin iridophores (specialized guanine crystals forming cells) as a model system to unravel the cellular basis of structural color change. Using a combination of super-resolution fluorescence microscopy, genetic manipulations, and cryogenic scanning electron microscopy, we have found that color change occurs due to tilting of the intracellular crystals and that the microtubules are the ones facilitating this process. While studying the color change mechanism we noted that only a subpopulation of iridophores is capable of color changing. This observation led to our discovery that there are two distinct types of iridophores in the zebrafish, which significantly differ in their ultrastructural organization, optical properties, transcriptome patterns, and their ability to change color upon stimulation.

Jain, Anshika

A new mutation that causes Multiple Mitochondrial Dysfunctional Syndrome (MMDS)

Anshika Jain, Nunziata Maio, Anamika Singh, Tracey A. Rouault

MMDS is a genetic disorder which results in fatal infantile encephalopathy in infants. This disease is caused by a mutation in NFU1 gene, which prevents NFU1 from transferring an essential cofactor, Fe-S cluster to several mitochondrial enzymes involved in the different stages of respiration (ex. SDHB and LAS). These defects in-turn cause pulmonary-hypertension and cardiomyopathy in patients.

An uncharacterized missense-mutation (p.Tyr231Cys) in NFU1 has been reported in SNP-database. We characterized the molecular significance of Y231 mutation and its role in MMDS. NFU1 has been previously identified as an Fe-S cluster carrier protein. We characterized the mechanism of its Fe-S cluster acquisition by establishing a direct interaction between NFU1 and Iron-Sulfur cluster scaffold protein (ISCU) via co-immunoprecipitation and yeast-two-hybrid assay. Interestingly, the interaction site on NFU1 was mapped to F230 and Y231 on NFU1 by alanine-scanning. Subsequently, to examine the effect of these two residues on the role of NFU1 as an Fe-S cluster carrier, we co-expressed WT-NFU1 or NFU1-FY mutant with ISCU in bacteria and purified holo-proteins (NFU1/mutant) in vivo anaerobically (to prevent the oxidation of Fe-S). Interestingly, the Fe-S cluster was detected only on the WT-protein. In vitro however, the apo form of both WT and mutant proteins could be successfully reconstituted in the presence of excess Fe and S. Furthermore, we observed a significant decrease in the protein levels of SDHB and LAS (the NFU1 target proteins) conferred by NFU1-FY mutant in the nfu1 knockout background cell lines. These results suggest that the FY-motif on NFU1 is important for a biological interaction and therefore, the cluster transfer between ISCU and NFU1, which in-turn provides for the downstream proteins. Furthermore, we used mass-spectrometry on NFU1 co-purified proteins from HeLa cells and identified new Fe-S proteins targeted by NFU1. Our study shows a new motif on NFU1 which causes MMDS and delineates the mechanism of Fe-S cluster acquisition by NFU1 and its downstream recipient proteins.

Mandal, Amrita

Retrograde transport is required for mitochondrial health and neuronal activity

Amrita Mandal, Katherine Pinter, Katie S. Kindt and Catherine M. Drerup

Neurons are some of the largest cells in the body. In addition to their size, these cells also have to develop polarized processes, maintain an electrical gradient, and sustain high levels of metabolic demand for an organism to sense and respond to its environment. To form and maintain their complex architecture, neurons need a large population of healthy mitochondria positioned appropriately throughout the cell. To populate and maintain mitochondria in long axonal processes in particular, neurons utilize axonal transport. Consequently, abnormalities in axonal mitochondrial transport are associated with many neurodegenerative diseases. Anterograde (axon terminal directed) transport of mitochondria is accomplished by Kinesin motors and is important for axon regeneration and growth. The exact role of retrograde (cell body directed) transport is not yet clear though we know it requires the cytoplasmic dynein molecular motor. We sought to understand the function of retrograde mitochondrial transport in mature axons. First, using photoconversion of mitochondria in vivo, we addressed the frequency of retrograde mitochondrial movement in long axons of the zebrafish posterior lateral line. We found retrograde transport occurs frequently and is required for redistribution of mitochondria throughout the neuron. Inhibition of retrograde mitochondrial movement leads to a significant decrease in cell body mitochondrial load and accumulation of this organelle in axon terminals. Using an array of fluorescent reporters, we subsequently assayed mitochondrial health and function in vivo with retrograde transport disruption. Inhibition of retrograde mitochondrial transport leads to a homeostatic imbalance of the oxidation/redox system, loss of membrane potential and reduced calcium buffering capacity in neurons. Finally, we assayed lateral line afferent responses to synaptic activity and found that mitochondrial imbalance in axons leads to impaired circuit activity. Altogether our data suggests retrograde transport is important to maintain a healthy pool of mitochondria in neurons which is critical for neuronal circuit activity.

Bellotti, Adriano

Proposed intermittent ion channel trafficking presents speed-stability tradeoff around transport bottlenecks

Adriano Bellotti, Dax Hoffman, Tim O'Leary

Neurons are excitable cells that contain processes that can extend hundreds or thousands of microns away from the cell body. This unique morphology presents several logistical obstacles regarding the function of the cell as a cohesive unit. One such challenge is the transport and regulation of ion channel concentrations throughout the dendritic arborization of pyramidal cells. Compounding the issue, Kv4.2 is a voltage-gated ion channel that is endogenously expressed with a particularly demanding profile showing increasing density with distance from the soma. Previous modeling studies confirm that such cargo demand profiles create transport bottlenecks and result in critical speed-precision tradeoffs.

We propose that ion channels such as Kv4.2 are trafficked intermittently in discrete on/off states to alleviate transport bottlenecks. This hypothesis is based on 100+ hours of dendrite recording following transfection of Kv4.2-GFP. Dendritic transport was observed in only 30% of total recording time, and dendritic activity appears to follow a dichotomous random process with puncta appearing in clustered bursts. Interestingly, axonal trafficking is present during 90% of total recording time, and puncta transport in axons appear to be continuous with no distinct off-state. Inferential statistical analysis of the dataset is performed to determine which stochastic process best describes the underlying intermittent phenomenon. Prospective models include the inhomogenous Poisson point process, the telegraph process, and combinations of these processes.

The observed intermittent phenomenon has been replicated in silico to assess its feasibility in relieving transport bottlenecks and improving system stability. Initial open-loop simulations reveal a speed-stability tradeoff governed by the frequency of the intermittent phenomenon. In closed-loop simulations, linear controllers with increased gains result in instability, whereas an on/off nonlinear switch controller can be amplified without instability. Complex analysis of poles and zeros is required to better understand the nonlinear system.

Frail, Sarah

How to Be the Cell Gata6 Wants You to Be: Genetic and Epigenetic Mechanisms Governing Early Mammalian Development

Sarah Frail, Joyce Thompson, Pedro Rocha

Most molecular factors involved in cell fate specification in the early embryo have a wide range of functions both in development and in adult tissues. Aberrant expression of these factors can lead to developmental malformations and to diseases like cancer. Despite the crucial role of these early developmental cell-fate decisions to our understanding of gene expression and disease, the mechanism by which the molecular factors involved dictate transcriptional programs is not completely understood. We are investigating the initiation of the second cell fate decision, during which cells of the inner cell mass of the blastocyst specify into primitive endoderm (PrE) or epiblast cell types—which will go on to become the fetus and the yolk sac, respectively. We are dissecting how Gata6, a transcription factor that drives PrE differentiation, initiates changes in chromatin status and transcription to achieve lineage specification. Using a new genome-wide sequencing technique called CUT&RUN, which allows for high-resolution mapping of protein-DNA interactions with as few as 100 cells, we will identify changes in transcription factor binding patterns and activation of tissue-specific regulatory elements over time both in vitro—using a differentiation inducible stem cell line—and in vivo—using maturing blastocysts. So far, we have characterized the genome-wide binding pattern of Gata6, Sox2, enhancer mark H3K27ac, and the insulator protein CTCF, allowing us to identify uniquely activated regulatory regions and the co-localization of key transcription factors. Next, we will characterize binding of these factors in vivo in mouse blastocysts. Establishing a genome-wide understanding of these early developmental cell fate decisions both in vitro and in vivo will help build a more complete picture of how cells establish and alter the pluripotency network to form a functioning complex organism and how disruption of this network can contribute to disease.

Sarah is also presenting a poster, #20, during the afternoon session.

Orr, Mona

Regulation of the Escherichia coli multidrug efflux pump AcrB by the small protein AcrZ

Arthur Neuberger, Catherine Newman, Mona Wu Orr, Pin-Chia Hsu, Firdaus Samsudin, Leana M. Ramos, Mekdes Debela, Syma Khalid, Gisela Storz, Ben F. Luisi, Dijun Du

Multidrug resistant bacteria are a global health problem. Resistance can be caused by increased expression and/or activity of efflux pumps, including the RND-family pumps that are overexpressed in some multidrug resistant clinical isolates. The E. coli RND-family pump AcrB was known to bind small protein AcrZ, which is required for resistance to some AcrB antibiotic substrates. However, how AcrZ recognizes and binds AcrB to affect activity remains unknown. To address these questions, AcrB alone and the AcrBZ complex were reconstituted in native E. coli lipid nanodiscs and the structures solved by cryo-EM. These structures revealed that AcrZ causes changes in AcrB at the site of interaction and in the distally located drug binding pocket and access channels. The changes were only observed in the structures derived from cryo-EM in lipid nanodiscs, suggesting that the local lipid environment is important for AcrB function. Cardiolipin is a modulator of other membrane proteins, and molecular dynamics simulations predicted cardiolipin enrichment around the AcrBZ complex. The most pronounced structural changes in AcrB were seen in the presence of both AcrZ and cardiolipin. To examine the effects of cardiolipin and AcrZ on AcrB, the corresponding genes were deleted, resulting in an additive effect on chloramphenicol sensitivity. Together, these results suggest that AcrZ and cardiolipin cause allosteric changes in AcrB to affect drug transport. Mutational analyses to identify the AcrZ determinants that affect AcrB interaction and activity showed that only proline 16 in AcrZ is required. Proline is a helix breaker that confers a kink to α-helices. Interestingly, restoring the proline further down the helix and increasing flexibility with glycine substitutions rescued resistance. Together, these studies help explain how AcrZ regulates AcrB. Further elucidation of this mechanism will be useful in informing drug design experiments for inhibitors of multidrug efflux pumps.