bluuebunny/crossref_metadata_2025_split huggingface dataset:
Understanding the Efficacy of Inhibitor TRDL‐551 on Replication Protein A (RPA) Binding Affinity
Authors: Kaur Jasmeet , Balakrishnan Lata 
Published: November 2023

Replication protein A (RPA), a single stranded DNA (ssDNA) binding protein, prevents ssDNA from being a target of cellular nucleases, recombining with other complementary sequences, or folding‐back on itself to form hairpin structures. Each of these scenarios can cause high rates of genome instability. Due to the protective function of RPA it now used as a therapeutic target for cancer cells, wherein DNA replication and repair often occur at high frequencies. For the current study, we utilized a chemical inhibitor of RPA, TDRL‐551, which has been previously characterized as a potent inhibitor of RPA binding to substrate. In this study, we chose to further analyze the TDRL‐551 inhibitor by testing different binding conditions either by the unmodified or acetylated form of RPA. Results from our laboratory have shown that acetylated RPA binds with higher affinity to ssDNA compared to the unmodified form. We tested RPA (unmodified and acetylated) binding under two different conditions; (i) RPA was pre‐bound to substrate and then incubated with inhibitor or (ii) RPA was pre‐bound to inhibitor and then exposed to substrate. Our results showed that in the first case, there was no effect of exposure to inhibitor on both unmodified and acetylated RPA binding. However, when RPA was first incubated with the inhibitor, both the unmodified and acetylated RPA showed reduced binding affinity to the ssDNA substrate. In conclusion, our studies show that the length of the DNA substrate, the post‐translational status of RPA and the time of exposure of the inhibitor to the RPA all play a significant role in determining the potency of this inhibitor. Further studies are being done to ascertain the efficacy of this inhibitor before using it in translational research.Support or Funding InformationThis work was supported by a grant from the National Institutes of Health GM098328 (to LB)
Identifying the role of genomic uracil in folate deficiency
Authors: Chon James , Field Martha , Gondokusumo Jabez , Stover Patrick 
Published: November 2023

Folate deficiency is implicated in deleterious health outcomes including neural tube defects (NTDs), neurodegeneration and colon cancer. Folate is a cofactor for the de novo synthesis of thymidylate (dTMP), and folate deficiency increases DNA damage, alters histone methylation, and increases uracil misincorporation into the genome during DNA synthesis. Excision of uracil in DNA is shown to cause DNA damage, but paradoxically, knockdown of uracil excision enzyme uracil DNA glycosylase (UNG) increases rates of DNA damage. The biological significance of uracil incorporation into DNA in folate deficiency is unknown, but shows tissue‐specific differences. It is not known whether uracil in DNA has functional or regulatory consequences, but uracil incorporation in DNA interferes with transcription factor binding in vitro. Uracil levels in nuclear DNA are postulated to be governed by the cellular dTTP/dUTP ratio. Cellular dUTP levels are regulated by the enzyme dUTP diphosphatase (dUTPase), which cleaves two phosphate groups from dUTP, yielding dUMP, which can be used as the substrate for synthesis of dTMP. Cellular dTTP levels are determined by rates of de novo dTMP biosynthesis, which requires folate cofactors and functions as a multi‐enzyme complex in the nucleus. In this study, we sought to determine the contribution of these two activities to uracil accumulation in DNA.The multienzyme complex that synthesizes dTMP is comprised of serine hydroxymethyltransferase (SHMT1 and SHMT2α), thymidylate synthase (TYMS), dihydrofolate reductase (DHFR), and methylenetetrahydrofolate dehydrogenase (MTHFD1). Both SHMT and MTHFD1 synthesize 5,10‐methylenetetrahydrofolate, the cofactor required to synthesize dTMP. SHMT also acts as a scaffold for the multienzyme complex and is critical for recruitment of these proteins at sites of DNA replication and repair. CRISPR knockout of SHMT1 in HeLa cells leads to a 2‐fold increase in uracil in DNA, compared to control cells. Treatment of HeLa cells with dUTPase siRNA leads to a 2.5‐fold increase in uracil in DNA and a 9‐fold increase in gamma‐H2A.X, a marker of DNA damage.These data suggest that dUTPase prevents genomic uracil incorporation by its enzymatic activity, keeping dUTP concentrations low, and that SHMT1 prevents uracil incorporation by its scaffolding activity, indicating that the uracil incorporation into DNA is regulated beyond the nuclear dTTP/dUTP ratio.Support or Funding InformationNational Institutes of Health Grants DK58144 and HD059120
Neural Crest‐Placode Cell Interactions during Cranial Ganglia Assembly
Authors: Taneyhill Lisa A. 
Published: November 2023

The cranial sensory ganglia are created from two motile cell populations, neural crest cells and placode cell‐derived neurons. Defects in the migration and interaction of these cells can cause abnormalities in craniofacial development and in the formation of the sensory nervous system, leading to conditions such as cleft lip/palate, neuralgia, and peripheral neuropathies. Although prior studies have shown that neural crest cells and placode cell‐derived neurons intermingle and coalesce during cranial ganglia assembly, the mechanisms underlying these processes are still elusive. To this end, we are using the cranial ganglia in the developing chick embryo as a model to elucidate the molecules and signaling pathways that mediate these intercellular interactions. Using gene perturbation assays, we have delineated important roles for cellular adherens junction components in controlling cranial ganglia formation throughout multiple stages of gangliogenesis. Furthermore, we have identified a member of the Annexin protein super‐family that functions during the neurogenesis of cranial placode cells and possibly other non‐placodal cell types. These findings provide additional insight into the mechanisms that orchestrate the cellular movements essential for cranial gangliogenesis, and, collectively, will impact our understanding of the normal formation of, and diseases associated with, the sensory nervous system. Importantly, our results may be directly translatable to other tissues generated from heterotypic cell types and will therefore have broad implications for human health.Support or Funding InformationThis work is supported by NIH R01DE024217 to L.A.T.
Enhancer H3K4 methyltransferase MLL4 controls cell fate transition
Authors: Ge Kai 
Published: November 2023

Enhancers control cell type‐specific gene expression and direct cell fate transition. Enhancers are marked by H3K4me1/2. We have shown previously that MLL4 (KMT2D) is a major enhancer H3K4me1/2 methyltransferase with partial functional redundancy with MLL3 (KMT2C). However, the role of MLL4 in enhancer regulation and cell fate transition is poorly understood. Here we show in embryonic stem cells (ESCs), MLL4 associates with, but is dispensable for the maintenance of, active enhancers of ESC identity genes. As a result, MLL4 is dispensable for cell identity gene expression and self‐renewal in ESCs. In contrast, MLL4 is essential for activation of de novo enhancers and induction of cell identity genes during ESC differentiation. Similarly, MLL4 is dispensable for maintaining fibroblast cell identity but is essential for reprogramming into induced pluripotent stem cells. Together with our findings in adipocytes and naïve CD4+ T cells, these results indicate that whileMLL4 is dispensable for maintaining cell identity, it controls cell fatetransitions by orchestrating de novoenhancer activation.Support or Funding InformationSupported by the intramural research program of NIDDK, NIH to K.G.
Dissection of Gene Regulatory Networks that control retinal cell fate specification
Authors: Wang Sui , Cepko Connie 
Published: November 2023

Gene regulatory networks (GRNs) regulate critical events during development. In complex tissues, such as the mammalian central nervous system (CNS), networks likely provide the complex regulatory interactions needed to direct the specification of the many CNS cell types. To understand these GRNs, we started by dissecting a GRN that regulates a binary fate decision between two siblings in the murine retina, the rod photoreceptor and bipolar interneuron. A simple strategy was employed and developed to dissect the rod‐bipolar GRN. First, we identified enhancers of key transcription factors (TFs) that regulate the specification of rod and bipolar cells. Second, by analyzing enhancers, we uncovered upstream regulatory TFs that regulate these enhancers. Third, we established the relationships among different TFs, and mapped TFs into GRNs via performing gene perturbation assays. Specifically, the rod‐bipolar GRN centers on Blimp1, one of the TFs that regulates the rod versus bipolar cell fate decision. We identified a cis‐regulatory enhancer, B108, which mimics Blimp1 expression. Deletion of genomic B108 by CRISPR/Cas9 in vivo using electroporation abolished the function of Blimp1. Otx2 and RORβ were found to regulate Blimp1 expression via B108, and Blimp1 and Otx2 were shown to form a negative feedback loop that regulates the level of Otx2, which regulates the production of the correct ratio of rods and bipolar cells. Furthermore, based on this study, we developed novel enhancer screen assays and TF pull‐down assays to identify enhancers and regulatory TFs in a more profound and large‐scale fashion to facilitate GRN studies.Support or Funding InformationThis work was supported by HHMI (to C.L.C. and S.W.).
Regulation of Hepatic Mouse Major Urinary Protein Genes by the Zinc Finger and Hemeoboxes 2 and Alpha‐fetoprotein Regulator 2
Authors: Jiang Jieyun , Creasy Kate Townsend , Peterson Martha L , Spear Brett 
Published: November 2023

ObjectivesWe have previously found that AFP, H19, Glypican 3 (Gpc3) and Lipoprotein lipase (Lpl) are targets of Zinc Finger and Hemeoboxes (Zhx) 2 based on their elevated expression in the adult liver of BALB/cJ mice compared to other strains. Since mouse major urinary proteins (MUP) mRNA are expressed at lower levels in the adult liver of BALB/cJ mice compared to other mouse strains, we tested whether this trait is due to Zhx2.MethodsAt first, by using Real‐Time Polymerase Chain Reaction (PCR) techniques, we tested hepatic MUP mRNA levels in different mouse strains including BALB/c, BALB/cJ mice, BALB/cJ mice with Zhx2 liver transgene, and recently developed Zhx2 knock‐out mice. Next we performed liver regeneration and Afr2 analysis, C3H/HeJ (Afr2a) and C57BL/6 (Afr2b) mice (2–3 months old) were given intraperitoneal injections of 50 mL of mineral oil (MO) containing 10% (vol/vol) carbon tetrachloride (CCl4); control animals were given intraperitoneal injections of 50 mL of MO alone (22). Three days later, the animals were killed, and their livers were removed for RNA preparation and analysis. Lastly, we studied how Zhx2 regulates MUP using in‐vitro cell based assays.ResultsOur data indicate that MUP genes are target of Zhx2 regulation. In contrast to previous Zhx2 targets, which are elevated when Zhx2 levels are reduced, MUP expression decreases when Zhx2 levels are lower. AFP, H19, Gpc3 and Lpl are reactivated in regenerating liver and HCC. With all four genes, the level of induction is significantly less in C57BL/6 mice than in other mouse strains; this trait is governed by a single locus on mouse chromosome 2 called alpha‐fetoprotein regulator 2 (Afr2). The gene for the Afr2 phenotype has not yet been identified. Since all known Zhx2 targets are also regulated by Afr2, we tested whether MUP expression was influenced by this locus in regenerating liver. We found that MUP mRNA levels decreased in regenerating liver (in contrast to AFP, H19, Gpc3 and Lpl, which are increased in regenerating liver) and that this decrease is influenced by Afr2. Additionally, data from in vitro cell‐based assays demonstrated that Zhx2 could activate MUP24 promoter and increase MUP expression in AML cells.ConclusionTaken together, MUP is a new target of Zhx2 and it is positively regulated by Zhx2.
Regulatory Characteristics of Vibrio vulnificus gbpA Encoding a Mucin‐Binding Protein Essential for Pathogenesis
Authors: Choi Sang Ho , Jang Kyung Ku 
Published: November 2023

BackgroundAdhesion to mucus layer is a prerequisite for pathogenesis of enteropathogens.ResultsGbpA is a mucin‐binding protein essential for pathogenesis of Vibrio vulnificus, and IscR, CRP, and SmcR cooperatively regulate gbpA in a growth‐phase dependent manner.ConclusionCooperation of IscR, CRP, and SmcR precisely tunes gbpA expression during pathogenesis.SignificanceThis study elucidated the regulatory mechanism of gbpA transcription for the first time.Binding to mucin is the initial step for enteropathogens to establish pathogenesis. An open reading frame, gbpA, of Vibrio vulnificus was identified and characterized in this study. Compared to the wild type, the gbpA mutant was impaired in binding to mucin‐agar and the mucin‐secreting HT29‐MTX cells, and the impaired mucin binding was restored by the purified GbpA provided exogenously. The gbpA mutant was defective in lethality and intestinal colonization in a mouse model, indicating that GbpA is a mucin‐binding protein and essential for pathogenesis of V. vulnificus. The gbpA transcription was growth‐phase dependent, reaching a maximum during the exponential phase. The Fe‐S cluster regulator (IscR) and the cyclic AMP receptor protein (CRP) coactivated whereas SmcR, a LuxR homologue, repressed gbpA. The cellular levels of IscR, CRP, and SmcR were not significantly affected by one another, indicating that the regulator proteins function cooperatively to regulate gbpA rather than sequentially in a regulatory cascade. The regulatory proteins directly bind to the upstream of the gbpA promoter PgbpA. DNase I protection assays, together with the deletion analyses of PgbpA, demonstrated that IscR binds to two specific sequences centered at −164.5 and −106, and CRP and SmcR bind specifically to the sequences centered at −68 and −45, respectively. Furthermore, gbpA was induced by exposure to H2O2, and the induction appeared to be mediated by elevated intracellular levels of IscR. Consequently, the combined results indicated that IscR, CRP, and SmcR cooperate for precise regulation of gbpA during the V. vulnificus pathogenesis.Support or Funding InformationThis work was supported by Mid‐career Researcher Program Grants 2015R1A2A1A13001654 through National Research Foundation funded by Ministry of Science, ICT, and Future planning; the R&D Convergence Center Support Program of the Ministry of Agriculture, Food and Rural Affairs; and a grant (14162MFDS972) from Ministry of Food and Drug Safety, Republic of Korea.
Structural basis for histone H2B deubiquitination by the SAGA DUB module
Authors: Morgan Michael Thomas , Haj‐Yahya Mahmood , Ringel Alison E. , Bandi Prasanthi , Brik Ashraf , Wolberger Cynthia 
Published: November 2023

Monoubiquitinated histone H2B plays multiple roles in transcription activation. H2B is deubiquitinated by the Spt‐Ada‐Gcn5 acetyltransferase (SAGA) coactivator, which contains a four‐protein subcomplex known as the deubiquitinating (DUB) module. The crystal structure of the Ubp8/Sgf11/Sus1/Sgf73 DUB module bound to a ubiquitinated nucleosome reveals that the DUB module primarily contacts H2A/H2B, with an arginine cluster on the Sgf11 zinc finger domain docking on the conserved H2A/H2B acidic patch. The Ubp8 catalytic domain mediates additional contacts with H2B as well as with the conjugated ubiquitin. We find that the DUB module deubiquitinates H2B both in the context of the nucleosome and in H2A/H2B dimers complexed with the histone chaperone, FACT, suggesting that SAGA could target H2B at multiple stages of nucleosome disassembly and reassembly during transcription.Support or Funding InformationSupported by grant GM‐095822 from the National Institute of General Medical Sciences (C.W.) and by a Ruth L. Kirschstein National Research Service Award (M.M.). A. Brik is a Neubauer Professor. GM/CA @ APS has been funded by the National Cancer Institute (Y1‐CO‐1020) and the National Institute of General Medical Sciences (Y1‐GM‐1104).
Hematopoietic Cell Stress Response Gene Transcriptional Regulation By TFII‐I and E2F Family
Authors: Aryan Mahmoud 
Published: November 2023

TFII‐I is a ubiquitously expressed, multifunctional transcription factor that has been shown to activate and repress transcription. TFII‐I is relatively large and contains a DNA binding domain, a nuclear localization domain, and protein‐protein interaction domains, including 6 R‐repeats that resemble helix‐loop‐helix motifs and a leucine zipper. Genome wide analysis of TFII‐I in human erythroleukemia K562 cells through biotinylation and ChIP sequencing revealed TFII‐I interacting with DNA segments near binding sites for E2F transcription factors. Co‐immunoprecipitation experiments provided data suggesting that E2F1, E2F4, and E2F6 interact with TFII‐I. The E2F family of transcription factors has been shown to regulate G1/S transition stages during the cell cycle. E2F1 works as a transcriptional activator, thereby promoting cell cycle progression, whereas E2F4 and E2F6 are transcriptional repressors and block cell cycle progression. TFII‐I has also been shown to be involved in cell cycle regulation. Chromatin immunoprecipitation (ChIP) experiments revealed binding of TFII‐I and E2F transcription factors at regulatory DNA elements in the Activating Transcription Factor 3 (ATF3)‐gene locus. ATF3 is a transcription factor that binds to the cAMP response element and is a critical component of the cellular stress response. Expression of ATF3 is induced in response to amino acid starvation and/or endoplasmic reticulum stress. Results from the Encode database suggests that transcription factors E2F4 and E2F6 co‐localize with RNA polymerase II at the ATF3 promoter just upstream of a TFII‐I binding site. By treating K562 cells with histidinol and thapsigargin to induce amino acid starvation or endoplasmic reticulum stress, respectively, the upregulation of ATF3 correlated with differential E2F binding patterns in the promoter region. Moreover, TFII‐I knockdown prompted reduced interactions of E2F4 and E2F6 at the ATF3 gene locus. These findings direct us to conclude that the associations between E2F4/6 and TFII‐I may have intricate roles in the configuration of the ATF3 gene locus thereby regulating mechanisms of cellular stress response.Support or Funding InformationMahmoud Aryan, Alex X. Fan, PhD1, Mir Hossain, MS1, and Jörg Bungert, PhD1 1Department of Biochemistry and Molecular Biology, Center for Epigenetics, Powell Gene Therapy Center NIDDK: RO1DK083389, RO1D0K52356
O‐GlcNAc Transferase and O‐GlcNAcase Interact with Mi2β at the Aγ‐Globin Promoter
Authors: Zhang Zhen , Costa Flávia C. , Tan Ee Phie , Bushue Nathan , Costello Catherine E. , McComb Mark E. , Whelan Stephen A. , Peterson Kenneth R. , Slawson Chad 
Published: November 2023

Sickle Cell Disease (SCD), caused by a point mutation in the adult β‐globin gene, leads to chronic damage to multiple organs and stroke, as well as cardiovascular abnormalities and dysfunction. However, SCD patients are phenotypically normal if they carry compensatory mutations that result in continued expression of fetal γ‐globin. Thus, a logical clinical goal for treatment of SCD is to up‐regulate fetal γ‐globin synthesis. One mode of γ‐globin silencing occurs at the GATA binding sites located at −566 or −567 relative to the Aγ‐globin or Gγ‐globin transcription start site, respectively, and is mediated through the DNA binding moiety GATA‐1 (GATA Binding Protein 1) and its recruitment of co‐repressor partners, FOG‐1 (Friend of GATA‐1) and Mi2β (CHD4, Chromodomain Helicase DNA Binding Protein 4). Post‐translational modification of transcription factors can regulate their activity. One such modification is O‐GlcNAcylation, which is the attachment of a single N‐acetyl‐glucosamine moiety to serine or threonine residues on nuclear, cytoplasmic and mitochondrial proteins. O‐GlcNAc is added to proteins by O‐GlcNAc transferase (OGT) and removed by O‐GlcNAcase (OGA). Here, using ChIP (Chromatin Immunoprecipitation) assays, we demonstrate that both OGT and OGA interact with the Aγ‐globin promoter when Aγ‐globin is repressed in wild‐type murine CID (Chemical Inducer of Dimerization)‐dependent human β‐YAC (β‐globin locus yeast artificial chromosome) bone marrow cells (BMCs). In addition, Mi2β and OGT are recruited to the −566 Aγ‐globin GATA silencer site in mouse embryonic day E18 fetal liver when Aγ‐globin is repressed. Furthermore, we demonstrate that OGT and OGA interact with Mi2β and FOG‐1, and Mi2β and FOG‐1 are O‐GlcNAcylated using co‐immunoprecipitation experiments. In order to confirm Mi2β O‐GlcNAcylation, we mapped one O‐GlcNAc site at S85 by mass spectrometry. Our data suggested that OGT and OGA can regulate GATA‐1/FOG‐1/Mi2β repressor complex activity at the Aγ‐globin promoter.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (NIGMS) of the National Institutes for Health under grant P20 GM12345, National Institute of Diabetes and Digestive and Kidney Diseases R01 DK100595 to C. Slawson and K. Peterson, and by National Heart, Lung, and Blood Institute contract HHSN268201000031C and NIGMS grant P41 GM104603 to C. E. Costello.
The dynamics of TAL effector – DNA interaction reveals optimal number of targeted nucleotides for maximum binding specificity
Authors: Rinaldi Fabio Cupri , Wilkins Katherine Elizabeth , Bogdanove Adam Joseph 
Published: November 2023

Transcriptional activator like effectors (TALEs) of plant pathogenic bacteria in the genus Xanthomonas have received special attention from the research community in recent years for their customizability as DNA targeting proteins. TALEs have evolved to bind DNA sequences in the promoters of host genes and upregulate expression. The DNA binding domain of TALEs is unique, comprising a series of modular repeats typically 34aa long that are essentially distinct only at positions 12 and 13, referred to as the RVD (repeat variable di‐residue). Individual base‐specific contacts by residue 13 across the repeats determine overall target specificity, with residue 12 playing a stabilizing role. Binding affinity and specificity of individual RVD‐nucleotide associations are increasingly well studied. However, there are still key questions concerning how TALEs can be designed to target DNA sequences with overall high binding affinity and specificity and how TALE target and off‐target sequences can be better predicted. Especially, the relationship between number of repeats and binding affinity has not yet been addressed quantitatively. For instance, in nature the average size of the TALE DNA binding domain is 17.5 repeats. Intuitively, one might assume that longer TALEs will have increased binding affinity and will be less likely to hit off‐target sites. We set out to test this assumption using biochemical methods to systematically study the relationship between the number of repeats of a set of designed TALEs and their respective binding affinities for the target DNA. We found that the binding affinity of TALEs in relation to their length saturates at approximately the average size of TALEs observed in nature. This suggests that increasing the number of repeats does not necessarily increase binding specificity and in fact has the opposite effect. Mathematically modeling the relationship supported this conclusion. Using the model improved the accuracy of TAL effector target prediction, providing empirical evidence in support of the model. This better understanding of the dynamics of TALE‐DNA interactions will benefit not only applications in biotechnology but also molecular characterization of the host‐pathogen interaction in the many important plant diseases caused by Xanthomonas spp.Support or Funding InformationBayer CropScience (BCS)National Science Foundation (NSF)National Institutes of Health (NIH)