bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of novel actin‐associated proteins at enteropathogenic Escherichia coli and Listeria monocytogenes actin‐rich structures
Authors: Chua Michael Dominic , Walker Brittany Dominique , Li Serina , Guttman Julian Andrew 
Published: June 2021

Enteropathogenic Escherichia coli (EPEC) and Listeria monocytogenes generate actin‐rich structures that are used for their colonization of host cells. EPEC remains extracellular, attaches to the host plasma membrane and secretes a variety of effector proteins that are used to control the host cells. The most dramatic morphological phenotype generated during these infections is the formation of actin‐rich protrusions called pedestals that are needed for E. coli‐based disease. L. monocytogenes enters its host cell and once in the cytosol it generates a bacterial protein (called ActA) that recruits actin polymerizing proteins to 1 pole of the microbe forming a comet tail that is used for movement within and amongst the host cells Previously, our lab conducted a mass spectrometry analysis of concentrated EPEC pedestals and over 90 novel proteins were identified. From this list, we selected a subset for confirmatory analysis and to determine whether their presence required the pre‐formation of the actin‐rich structures. These proteins included a calponin protein (CNN), dihydropyrimidinase‐like protein (CRMP4), nucleoside kinase (NK), mitogen‐activated protein pathway kinase (MK), and a ubiquitin‐conjugating enzyme (Ube2N). Because these novel proteins were observed in EPEC pedestals, we hypothesized that these five protein candidates are important for EPEC pedestals and L. monocytogenes comet tails. To study this, we first confirmed that these proteins were enriched in EPEC pedestals by immunolocalizing the proteins in EPEC‐infected cells. CNN, and NK were found throughout the full length of the pedestal while CRMP4, MK, and Ube2N were concentrated at the apical tip of EPEC pedestals. In L. monocytogenes‐infected cells, CNN and NK were found in actin clouds, comet tails, and listeriopods. CRMP4 immunolocalized only at invasion sites and listeriopods while MK was only present at listeriopods. To ensure that recruitment of these proteins were independent of bacterial attachment on the host cell and required formation of the actin‐rich structures, we infected cells with either a tir‐deficient EPEC mutant or an actA‐deficient L. monocytogenes mutant as both mutants cannot generate actin‐structures. None of the five proteins were recruited to the mutants suggesting that their presences are dependent on the formation of the actin‐rich structures. From these results, we have identified novel proteins that are important for the various stages of EPEC and L. monocytogenes infections. By identifying these proteins, we have begun to elucidate the complex web of proteins involved in the formation and maintenance of actin structures during these infections.Support or Funding InformationThis study was funded through NSERC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The highly conserved proteins Nucleolin and Sub1 play critical roles in regulating G4 DNA‐induced genome instability.
Authors: Kim Nayun 
Published: June 2021

Sequences containing multiple guanine‐runs can form the four‐stranded G‐quadruplex (G4) DNA. The significance of G4 DNA in etiology of blood cancers is underscored by the manifest enrichment of G4 sequence motifs proximal to the leukemia/lymphoma‐associated translocation breakpoints. But how G4 DNA‐motifs instigate chromosome abnormalities leading to malignant transformation is yet to be resolved. We developed quantitative assays to measure and characterize multiple types of G4 DNA‐induced genome instability events in the context of the yeast genome. This system allowed us to demonstrate that the level and direction of transcription are important determinants of the stability of G4 sequences. Using this approach, we demonstrated that the topological change ensuing in the absence of Topoisomerase I (Top1) can severely endanger genome instability at actively transcribed G4 motifs. The G4‐associated instability in top1Δ yeasts was suppressed partially by the bacterial topA (ecTopA), which removes negative supercoils and not at all by the gyrases (ecGyrase), which removes positive supercoils or the RNase H1 overexpression, which removes RNA:DNA hybrids or R‐loops. Taking advantage of the G4 DNA‐conducive cellular environment in the top1Δ yeasts, we also demonstrated that, upon disruption of Sub1, a general transcription co‐activator, genome instability linked to co‐transcriptionally formed G4 DNA is significantly elevated and that the highly conserved DNA binding domain of Sub1 and its human homolog PC4 is sufficient in preventing genome instability at actively transcribed G4 motifs. Consistent with its role in suppressing G4‐mediated instability, Sub1 interacts specifically with co‐transcriptionally formed G4 DNA in vivo and yeast cells become highly sensitivity to G4‐stabilizing chemical ligands by the loss of Sub1. We demonstrated that Sub1 forms a complex with the G4‐resolving helicase Pif1 suggesting a possible mechanism by which Sub1 suppresses G4‐associated genome instability. Additionally, we discuss our most recent findings regarding the role of the highly conserved nucleolar protein Nucleolin (Nsr1 in yeast) in G4 DNA‐associated genome instability. Deletion of Nsr1, a known G4 DNA‐binding protein, surprisingly led to a significant decrease of G4‐induced recombination. We present a model where interaction with Nsr1 shift the equilibrium between B DNA and G4 DNA toward stable G4 DNA and thereby create strong genome‐destabilizing replication block.Support or Funding InformationWork in the NK lab was supported by grants from the Welch Foundation (AU1875) and the National Institutes of Health (GM116007).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NELF‐E and CDYL1: Two novel players for switching off transcription at DNA damage sites
Authors: Ayoub Nabieh , Awwad Samah W. , Abu‐Zhayia Enas R. , Ben‐Oz Bella M. , Guttmann‐Raviv Noga Guttmann‐Raviv 
Published: June 2021

Transcription activity is rapidly and transiently paused in response to DNA double‐strand breaks (DSBs) to eliminate production of abnormal transcripts and to avoid deleterious collisions between transcription and repair machineries. Little is known about the mechanisms that ensure transcription repression following DNA damage. Here we describe two novel DNA‐damage responsive proteins, regulated by PARP1 activity, that foster DSB‐induced transcription silencing and promote DSB repair. First, using I‐SceI and CRISPR‐Cas9 endonucleases, we showed that the negative elongation factor member E (NELF‐E), subunit of the NELF complex, is preferentially recruited to DSBs induced upstream transcriptionally active rather than inactive genes. The preferential recruitment of NELF‐E to DNA breakage sites is dependent on the presence of RNA polymerase II [1]. Second, we showed that the chromodomain Y‐like (CDYL1) protein is rapidly recruited to damaged euchromatic regions. In addition, CDYL1 promotes the recruitment of EZH2 methyltransferase, stimulates local increase of the repressive methyl mark H3K27me3 and promotes transcription silencing at DSB sites. Furthermore, the “traffic‐light reporter” system revealed that CDYL1 mainly promotes homology‐directed repair of DSBs. Consequently, CDYL1 knockout cells display synthetic lethality with the chemotherapeutic agent, cisplatin [2]. In the meeting, I will also discuss unpublished data implicating CDYL1 in regulating histone crotonylation at DNA damage sites. Altogether, our findings identify NELF‐E and CDYL1 as novel components that play an essential role in the cellular response to DNA damage.Support or Funding InformationThis work was supported by grants from the Israel Science Foundation (ISF) (grant no. 2021242), the Israel Cancer association (grant no. 2019404), the Binational Science Foundation (grant no. 2023065), the Israel Cancer Research Fund (ICRF) (grant no. 2021762), Volkswagen Foundation (grant no. 2020594). Enas R. Abu‐Zhaiya and Samah W. Awwad are supported by the Council for Higher Education 19 fellowship for outstanding minority MSc and PhD students, respectively. N. Ayoub is supported by the Neubauer Family Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expanding the role of FDO1 in yeast chromosomal biology
Authors: Seman Melissa , Hoggard Timothy , Fox Catherine 
Published: June 2021

The yeast transcription factor Forkhead 1 (FKH1) has multiple roles in chromosomal biology. Our lab recently discovered a protein‐protein interaction between Fkh1 and Fdo1 that contributes to mating type switching, one of multiple nuclear roles for Fkh1. Little information exists for roles of Fdo1 in yeast, and we were interested in whether Fdo1 has additional roles in chromosomal biology. To this end, we have begun to characterize the yeast cell cycle in a fdo1‐dependent manner. Using flow cytometry, we have uncovered connections between fdo1 and cell cycle progression as well as genome stability.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Role of the Human CST Complex in Regulating the DNA Damage Response
Authors: Stewart Jason A. , Ackerson Stephanie M. 
Published: June 2021

CST (CTC1‐STN1‐TEN1) is an RPA‐like, ssDNA binding protein that functions in telomere replication and genome‐wide DNA replication rescue. Previous studies have shown that depletion of STN1 or CST overexpression leads to decreased or increased new origin firing following replication stress, suggesting a role for CST in dormant origin activation. Additionally, depletion of CST subunits leads to increased anaphase bridges and micronuclei. Recently, a conditional CTC1 knockout line was created in HCT116 cells. Removal of CTC1 was found to cause decreased cell proliferation and elongation of the telomeric ssDNA G‐overhang, leading to RPA accumulation and telomeric DNA damage signaling. Interestingly, rescue of the telomeric damage did not restore normal cell growth, suggesting that other factors contribute to the growth defects in the absence of CTC1. Here, we investigate the consequences of CTC1 deletion on genome‐wide replication and activation of the DNA damage response. We find that CTC1 deletion lead to a delayed but abrupt activation of p53 and p21 leading to decreased DNA replication and a G2/M arrest. We hypothesized that this cell cycle arrest was caused by ATR activation from defects in telomere or genome‐wide DNA replication. Instead, our results suggest that ATR signaling is defective in the absence of CTC1 and p53 is activated in an ATR‐independent manner. Overall, our findings suggest a novel and unexpected role of CST in promoting checkpoint signaling to prevent genome instability.Support or Funding InformationThis work is supported by the National Institutes of Health grant R00 GM104409 and startup funds from the University of South Carolina.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histone Deacetylase Inhibition Leads to Dose‐Dependent Suppression of Oncogene‐Associated Super‐Enhancers
Authors: Wheeler Graycen , Sanchez Gilson , Liu Xuedong 
Published: June 2021

In cancer cells, decreased histone acetylation leads to gene expression patterns that drive oncogenic processes. Histone deacetylase (HDAC) inhibitors are therefore used as therapeutics to selectively kill cancer cells. The complex processes by which these chromatin‐remodeling agents affect gene expression and ultimately cell fate are still not entirely understood. The Liu group seeks to characterize the mechanisms of the HDAC inhibitor largazole by using epigenomic and transcriptomic studies to compare its effects on transformed and non‐transformed cells at different doses. From broad chromatin immunoprecipitation sequencing and RNA sequencing, we observed that largazole has a striking effect on super‐enhancer regions and the genes associated with them. Largazole decreases the accumulation of RNA polymerase II at super‐enhancer elements and limits the transcription of genes under the regulation of those super‐enhancers. Largazole also alters the expression of many genes by decreasing the histone acetylation of poised distal enhancers in a dose‐dependent manner. Many of these genes are transcriptionally upregulated after treatment, as is canonically associated with an increased histone acetylation profile, but some transcripts are repressed. These insights into the epigenetic reprogramming that drives oncogenic processes provide fundamental information about tumorigenesis and could potentially serve as a foundation for the development of further oncology therapies.Support or Funding InformationNational Institutes of Health [T32GM008759 to G.W.]; National Cancer Institute and National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health [CA107098, AR068254 to X.L.]; National Cancer Institute [CA107098S1 to G.S.];This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA Binding Kinetics of CTCF in vitro
Authors: Sanborn Adrian L. , Kornberg Roger D. 
Published: June 2021

CTCF is a ubiquitously expressed transcription factor whose architectural role as an insulator is tied to its ability to form genomic loops and delimit boundaries of topologically associated domains (TADs). This capacity for binding of a single protein to affect distal regulation over megabases is both remarkable and poorly understood mechanistically. In current models, a cohesin‐associated complex expands a loop by tracking along DNA in both directions, while CTCF defines the loop size and position by halting this complex. However, a recent single molecule imaging study finds the DNA‐association lifetime of CTCF to be ten‐fold shorter than that of cohesin in vivo, suggesting that CTCF and cohesin act independently and dynamically (Hansen et al., 2017). This raises key questions about how CTCF forms and maintains domain boundaries and loops, and highlights the need for a careful study of CTCF‐DNA binding kinetics.Here, we purified human CTCF and characterized its DNA‐binding activity in vitro. Using mobility shift assays, we recapitulated the known high affinity of full length CTCF and the zinc finger domain for CTCF binding sites and its dependence on DNA methylation. We further used surface plasmon resonance to measure real time binding and unbinding kinetics of CTCF to methylated and unmethylated DNA targets. These experiments illuminate the role of DNA methylation in CTCF binding site selection, shed light on in vivo binding kinetics, and represent a first step towards the study of CTCF‐mediated looping in vitro.Support or Funding InformationThis research was supported by NIH Grants GM36659 and AI21144 (to R.D.K.) and a National Defense Science and Engineering Fellowship (to A.L.S.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐based design of Nucleosome Binding Peptides for controlling cell function
Authors: Teles Kaian Amorim , Santos Guilherme Martins 
Published: June 2021

Proteins that bind the nucleosome, the repetitive unit of chromatin, and the histone H4 tail are critical to establishing chromatin architecture and phenotypic outcomes. A myriad of histone modifications, which strongly induce changes in chromatin structure, have been associated with malignancies and other diseases, highlighting the crucial role of chromatin architectural changes in disease mechanisms. The nucleosome surface was hypothesised as a therapeutic target, however the development, design and function of exogenous nucleosome binding molecules remain unclear.Herein, we performed structure‐based calculations on the nucleosome surface interaction to four peptides (NBMs), which present different nucleosome binding sites, derived from Nucleosome Binding Proteins (NBPs) to analyse the nucleosome sensitivity. Further, we were able to generate a new peptide (GMIP) with two nucleosome binding sites. A series of in vitro studies were performed to verify the impact of all these peptides on the nucleosome, showing differential action on its stability. We also demonstrated that GMIP and a canonical peptide may induce chromatin condensation in vitro. Confocal microscopy using labeled GMIP showed that it penetrates, by a passive mechanism, the cell and binds to chromatin. Cell‐based assays (MTT and FACS) showed that NBMs tested here presented a strong inhibitory activity on tumor cells, but not on non‐tumoral cells, proliferation and viability. It was also shown that these peptides might induce chromatin condensation in vivo. Besides that, the fish embryo toxicity (FET) test showed specificity in actions of NBMs on the ability to induce tissue modifications.Finally, we correlated the structural impact of NBMs to cellular function, providing a unique map on the nucleosome for designing new molecules with important implications. Taking all of this into account, it is clear that the is right to consider the nucleosome surface as a potential therapeutic target to regulate a plethora of cellular disorders.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of Chimeric Histone Methyltransferase Complexes In Saccharomyces cerevisiae Generate Unique Phenotypes and Clarify the Roles of MLL1 and Set1 Complex Accessory Proteins
Authors: Chosed Renee J. , Klein David , Longan Emery , Baker Marian , Gogoli Sasha , Wang Jingtian , Alkoutami Sami 
Published: June 2021

Genomic rearrangements involving the MLL1 gene are prevalent in human cancers, especially mixed lineage leukemia. This gene encodes an enzyme that methylates histone H3 on lysine 4 (H3K4) as a part of the MLL1 multi‐protein complex. The MLL1 complex regulates the expression of many genes via this epigenetic mark, including homeobox genes. Problematically, higher eukaryotes contain many functionally redundant complexes that complicate the study of MLL1 and associated cancers in a living system. Furthermore, due to the nature of the disease‐causing mutations, namely chromosomal rearrangements, MLL1 is not a promising drug target. However, several accessory proteins within the complex are required for catalytic activity, presenting possible drug targets themselves. Herein we present an in vivo system for the study of the MLL1 complex in Saccharomyces cerevisiae, making use of the homologous Set1/COMPASS complex. We genetically replaced COMPASS members from S. cerevisiae with their human homologs using antibiotic resistance cassettes. We then performed phenotypic characterization of chimeric COMPASS/MLL1 complexes assessing global H3K4 methylation status. Selected chimeric yeast‐human methyltransferase complexes conferred catalytic activity at varying degrees, while others did not confer methyltransferase activity. Notably, we observed H3K4 dimethylation levels comparable to wild type when human Ash2L replaced yeast Bre2 but reduced levels of H3K4 trimethylation with this same chimeric complex. Together, these data represent a proof of concept for simplifying the study of this clinically important protein complex in a tractable in vivo system, and also offer mechanistic insight into the functional role of a catalytically essential accessory protein within the MLL1 complex.Support or Funding InformationThis research was supported by grants from the National Center for Research Resources (5 P20 RR016461) and the National Institute of General Medical Sciences (8 P20 GM103499) from the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantitative analysis of lncRNA from human FF and FFPE brain speimens
Authors: Lv Yehui , Rao Yuliang , Dong Wenzhe 
Published: June 2021

Both Fresh frozen (FF) and formalin‐fixed paraffin‐embedded (FFPE) brain tissues are important resources for molecular medical study. However, long non‐coding RNA (lncRNA) analysis is restricted in FFPE samples due to its high‐level degradation. To compare the quality of lncRNA extracted from FF and FFPE brain specimens and to explore the possibility of lncRNA quantification in FFPE tissues, 10 paired of frozen and FFPE human brain tissues were collected from autopsies by our collaborating teams at the Institute of Criminal Scientific Technology in Shanghai (). We selected 2 endogenous control mRNA (ACTB and GAPDH) and 6 target lncRNA (MALAT1, H19, PTENP1, BC1, HAR1 and DD3) from literatures, and designed 2 short (<100 bp, non‐overlapping) and 1 long (about 200 bp) primers for each biomarker. After total RNA was extracted from all specimens, the expression level of each biomarker was analyzed by real‐time quantitative PCR (RT‐qPCR) and amplification efficiency (AE) was subsequently calculated. The degradation profiles of total RNA were evaluated by RNA integrity number (RIN), and RIN values of FFPE samples were significantly lower than FF samples (P<0.05). Following RT‐qPCR, short amplicon of all 6 RNA markers achieved optimal AE between 90% and 110% in FF samples, while AE of long amplicons differed remarkably with RNA type, and only 2 lncRNA (MALAT1 and BC1) and endogenous control mRNA reached the optimal efficiency. As for FFPE tissues, most short amplicons (except for HAR1) achieved the optimum efficiencies, however, AE of most long amplicons was overblown in FFPE samples, including ACTB and GAPDH. In present study, high concordance of RNA expression was also observed in 2 non‐overlapping short amplicons of both FF and FFPE samples. In conclusion, short amplicons of target lncRNA were amplified more efficiently than long amplicons and this study may provide an experimental basis for accurate lncRNA quantitative analysis with short amplicons in FFPE brain tissues.Support or Funding InformationAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethical permission was granted through the Science and Ethics Committee of Shanghai University of Medicine & Health Sciences. This study was funded by the Seed Fund of Shanghai University of Medicine & Health Sciences and Open Foundation of Shanghai Key Laboratory of Forensic Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.