bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epigenetic regulating enzyme activity modification in natural cinnamon extract treated MCF‐7 breast cancer cells
Authors: Hall Seth , Hill Matthew , Almehmadi Seham , Aulthouse Amy , Kinder David , Stockert Amy 
Published: June 2021

As research continues to hone in on different tumorigenesis mechanisms, epigenetic dysregulation continues to show promise as a major contributing factor in cancer pathology. There are a variety of mechanisms involved in epigenetic regulation, but it can occur most prominently through direct DNA methylation and demethylation by DNA methyl‐transferase (DNMT) enzymes. Dysregulation of this epigenetic modifying mechanism from exogenous stimuli can lead to alteration of DNA transcription and protein expression within cells causing mutations to develop that result in increased cell proliferation and metastasis. Dietary polyphenols are known exogenous factors that influence epigenetic change. In particular, cinnamon is a natural product that contains many of these natural polyphenols, and it has been shown to affect protein expression in MCF‐7 breast cancer cells. With our current research, we have shown that cinnamon causes changes in activity of DNMT enzymes in MCF‐7 breast cancer cells. Results from a real‐time PCR assay for DNMT have shown modified levels of expression and activity with both enzymes in vitro. It remains to be determined whether these changes promote or inhibit tumorigenesis effects within cells, but do demonstrate that natural polyphenols found within cinnamon can cause epigenetic modification in cells. Future research will determine the resultant changes in protein expression and proliferative or anti‐proliferative nature of the effects that could arise from these epigenetic modifications.Support or Funding InformationDepartments of Pharmaceutical and Biomedical Sciences and Biological and Allied Health Sciences, Ohio Northern UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of 5‐Azacytidine on the Long‐term Stability of HLA Class I Upregulation
Authors: Hansen Adam , O'Donnell Robert W 
Published: June 2021

The epigenetic modifiers, such as DNA methylation inhibitors and histone deacetylase inhibitors, 5‐Azacytidine (5‐AzaC) and Vorinostat, have been shown to increase human leukocyte antigen (HLA) expression. The expression of HLA is required for a T‐cell response to detect tumor cells. A lack of HLA expression allows tumor cells to escape immune detection. It has been previously shown that 5‐AzaC is able to upregulate HLA expression in the cell line MDA‐MB‐435. However, the long‐term stability of increased HLA expression, following a transient absence of 5‐AzaC, is not known. Two flasks of MDA‐MB‐435 cells, one being continually treated twice a week with 5‐AzaC at 0.1 μg/mL, and the other, an untreated control flask are being propagated for long periods of time. Periodically the cells are harvested and incubated with control antibodies or the experimental antibodies, Anti‐HLA‐ABC and analyzed via flow cytometry. Our experiments will help determine if the length of 5‐AzaC exposure modifies HLA expression in MDA‐MB‐435 cells. Experimentation on the role of 5‐AzaC is also planned to optimize both the overall upregulation of HLA expression, and the long‐term expression following the removal of 5‐AzaC. Understanding the long‐term stability of HLA upregulation may prove to be beneficial and applicable to cancer immunotherapy, an expanding field of improved cancer treatments.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of DNA Methylation Regulates Guanylyl Cyclase/Natriuretic Peptide Receptor‐A Gene Expression
Authors: Kumar Prerna 
Published: June 2021

Cardiac hormone atrial natriuretic peptide binds to guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and produces second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. The molecular and epigenetic mechanisms that contribute to the activation of Npr1 (coding for GC‐A/NPRA) gene expression are not well understood. The present study was aimed at understanding the mechanisms involved in DNA methylation inhibitor, 5‐Azacytidine (5‐aza)‐mediated Npr1 (coding for GC‐A/NPRA) gene transcription. The studies were carried out in rat thoracic aortic vascular smooth muscle cells (RTASMC), cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and treated with 5‐aza for 24 h. The MethPrimer search result showed the presence of three cytosine‐phosphate‐guanine (CpG) islands on the Npr1 full length promoter, namely island 1 (−886 to −752, 134 bp), island 2 (−310 to −58, 152 bp), and island 3 (−514 to +310, 464 bp) from the transcription start site. 5‐azacytidine significantly induced mRNA levels of Npr1 gene by 4‐fold and NPRA protein expression by 3‐fold in RTASMCs in a dose‐dependent manner. Treatment of cells with 5‐aza significantly (p<0.05) attenuated DNA methyltransferase 1 (DNMT1) protein expression in a dose‐dependent manner. However, there was no noticeable change in the DNMT3a and DNMT3b protein levels. There was a significant reduction in the DNMT activity in 5‐aza‐treated RTASMCs compared with vehicle–treated cells as quantified by EpiQuik DNMT activity assay. The present findings provide a novel regulatory mechanism for Npr1 gene transcription, an important player in the control of hypertension and cardiovascular homeostasis.Support or Funding InformationThis work was supported by an Institutional Development Award (IDeA) from the NIGMS and a partial support from Tulane Carol Lavin Bernick grant award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancer regulation by H3K4 methyltransferases MLL3/MLL4
Authors: Ge Kai 
Published: June 2021

Enhancers control cell type‐specific gene expression and are marked by H3K4me1. Active enhancers are further marked by H3K27ac. We previously identified CBP/p300 as major H3K27 acetyltransferases and MLL3/4 as major enhancer H3K4me1 methyltransferases (EMBO J 2011; eLife 2013). During differentiation of adipocytes, myocytes and ES cells, MLL3/4 co‐localizes with lineage‐determining TFs (LDTFs) on active enhancers and are required for enhancer activation, cell type‐specific gene expression, and cell differentiation (eLife 2013; PNAS 2016). Further, we show that while enhancer‐priming by MLL3/4 is dispensable for cell identity maintenance, it controls cell fate transition by orchestrating CBP/p300‐mediated enhancer activation. MLL4 protein, rather than MLL4‐mediated H3K4me1, controls p300 recruitment to enhancers, suggesting that MLL4 may regulate enhancer activation independent of its enzymatic activity (PNAS 2016; NAR 2017).Our recent findings identify Brd4 as an enhancer epigenomic reader that links active enhancers with cell identity gene induction in differentiation. Our data suggest a model of sequential actions of epigenomic regulators on enhancers: 1) pioneer TFs and LDTFs recruit MLL3/4 to prime enhancer regions and label them with H3K4me1; 2) MLL3/4 facilitate the binding of CBP/p300, which activate enhancers and label them with H3K27ac; 3) H3K27ac and acetylated TFs are recognized by Brd4, which recruits Mediator and RNA Pol II to establish enhancerpromoter interactions and activate cell type‐specific gene expression (Nat Comm, 2017).To investigate the role of MLL3/4‐mediated H3K4me1 in enhancer activation and cell differentiation, we have generated two mouse lines: one expressing H3.3K4M, a lysine‐4‐to‐methionine (K4M) mutation of histone H3.3 that inhibits H3K4 methylation, and the other carrying conditional double knockout of MLL3/4 enzymatic SET domain. Unexpectedly, expression of H3.3K4M, or deletion of the SET domain, in lineage‐specific precursor cells destabilizes MLL3/4, prevents enhancer activation, and impairs cell differentiation and tissue development (NAR 2018). We are using CRISPR to generate enzyme‐dead knockin mutants of MLL3/4 in mice. The phenotypes of these mice will be reported at the meeting.By immunoprecipitation followed by mass spectrometry analysis, we have found that endogenous MLL3/4 complex interacts with SWI/SNF chromatin remodeling complex in cells. The biological significance of such physical interactions will be reported at the meeting.Support or Funding InformationSupported by the Intramural Research Program of NIDDK, NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isothermal Calorimetric Measurements of Metal Ion Interaction with TAR RNA
Authors: Song Jessica , Grover Neena 
Published: June 2021

The trans‐activation response (TAR) element of HIV‐1 is essential for transcriptional elongation in HIV‐1. It consists of a stem‐loop containing a trinucleotide bulge (UCU) and is capped by a hexaloop sequence located in the 5′ UTR in HIV‐1. The trinucleotide bulge interacts with the arginine‐rich basic domain of the Tat protein to increase the rate of transcription by nearly 100‐fold. Thermodynamic studies in our laboratory have previously examined the effects of TAR RNA interactions with divalent metal ions to show that single nucleotide modifications can lead to differences in RNA‐ion interactions. Thermal denaturation studies show an increase in wild type construct stability in 10 mM Ca2+ or Mg2+ as compared to 1 M KCl. In this study, the thermodynamics of metal ion binding to the TAR RNA sequence is investigated by directly measuring the heat released using isothermal titration calorimetry (ITC). TAR RNA derived constructs were titrated with magnesium, calcium, and arginine containing buffers to measure the enthalpic contributions and the binding affinity of the ions to further understand different types of RNA‐metal ion interactions.Support or Funding InformationDepartment of Chemistry and BiochemistryColorado CollegeThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermodynamics of the Kink‐Turn Motif in RNA
Authors: McCaskill Darryl Cole , Grover Neena 
Published: June 2021

The kink‐turn is a helix‐internal loop‐helix with a three‐nucleotide bulge loop containing a canonical stem (Watson‐Crick base pairing) on one side and non‐canonical stem containing two or three G•A sheared base pairs on the other side. The sheared base pairs in the non‐canonical stem give the construct the flexibility to adopt a 120° kink between the two stems. The kink‐turn is expected to form in the presence of metal‐ions to neutralize the charges on phosphates that are in close proximity. RNA constructs were designed to mimic the Haloarcula Marismortui KT7 from the 28S ribosome. Thermodynamic properties of constructs were tested in a 1 M KCl salt buffer and a 10 mM magnesium buffer at either pH 5.5 or 7. The wild‐type construct shows a −2.36 kcal/mol gain in stability in the magnesium buffer compared to the 1 M KCl buffer at pH 7. Direct measurements of the heat released upon ion‐binding to RNA is being measured using ITC to determine the enthalpy and binding constants of RNA‐magnesium interactions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Tissue‐Specific RNA Exosome Cofactors as an Approach to Define Disease Mechanism
Authors: de Amorim Julia L , Leung Sara , van Hoof Ambro , Corbett Anita H 
Published: June 2021

The RNA exosome, a 10‐subunit complex that mediates both RNA processing and degradation, plays a critical role in defining cellular expression profiles. This complex is ubiquitously expressed, essential, and critical for fundamental cellular functions, such as ribosomal RNA processing. Recent studies have linked mutations in genes encoding multiple subunits of the complex to tissue‐specific human disease. For example, mutations in the human EXOSC3 gene, coding for a subunit, cause Pontocerebellar Hypoplasia type 1b (PCH1b), a disease characterized by atrophy of the pons and cerebellum. The missense mutations encode single amino acid changes in conserved regions of the EXOSC3 protein. How these amino acid substitutions confer tissue‐specific phenotypes is not known. One possible mechanism underlying the distinct disease phenotypes could be a decrease in the interaction of the RNA exosome complex with cofactors that confer specificity for RNA targets. However, most studies that identify and characterize RNA exosome cofactors have been carried out in budding yeast and thus, do not provide insight into whether tissue‐specific cofactors could exist. Our studies use immunoprecipitation from neuronal cell culture (N2A) and mouse tissues to define RNA exosome cofactors. Biochemical experiments that employ cultured N2A cells identified RNA exosome interacting proteins that are enriched in both the nucleus and cytoplasm. Preliminary results from this analysis identify an association between the RNA exosome and a large complex of tRNA ligase enzymes, which could link defects in tRNA maturation to disease pathology. We are extending these studies to explore the possibility of tissue‐specific cofactors by immunoprecipitating EXOSC3 and analyzing co‐purifying proteins from the cerebellum (affected in disease) and the cortex (unaffected) in mouse brain. Finally, using CRISPR/Cas9 genome editing, we are testing whether the amino acid changes that occur in disease alter interactions with both known RNA exosome cofactors and interactors identified in our co‐immunoprecipitation studies. These studies will provide insight into both the functional consequences of amino acid substitutions in the RNA exosome that cause disease and the role of cofactors in conferring RNA target specificity.Support or Funding InformationR01GM130147This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of DED1 Suppressors Using Genomic Sequencing
Authors: Piciw Jenny , Winters Aidan , Drewes‐Tartarotti Tatiana , Bowman John , Whitworth Gregg , Hilliker Angie 
Published: June 2021

Ded1, an RNA‐dependent ATPase, promotes translation initiation of many mRNAs in yeast. Ded1 is extremely important for the translation of mRNAs predicted to have substantial secondary structure (Sen et al., 2015). Ded1 interacts directly with eIF4A and eIF4G and this complex has increased helicase activity compared to either Ded1 or eIF4A alone (Hilliker et al., 2011; Gao et al., 2016). Consistent with an important role in translation, DDX3, the human homolog of Ded1, has been shown to be misregulated in several cancers and is hijacked by HIV to allow translation of highly structured HIV mRNAs (Valiente‐Echeverria et al., 2015; Xiang et al., 2016). Because it is critical to translation and influences other translation initiation factors, we are interested in how Ded1 itself is regulated and how that regulation impacts translation.We use genetic selections to identify regulators of Ded1 function using a well characterized ded1 mutant. A cold sensitive ded1 mutant (ded1‐dam1) lacks two regions important for Ded1's ability to promote translation; one of these regions promotes the interaction between Ded1 and eIF4G. Yeast containing this mutant as the sole copy of ded1 show a growth defect, a dramatic drop in global translation, and an accumulation of P‐bodies at the restrictive temperature (Hilliker et al., 2011). We have isolated 38 putative mutations of the cold‐sensitive growth conferred by ded1‐dam1 assembly mutant. These 38 ded1‐dam1 suppressor mutants were isolated through one of four mutagenesis processes, including extended growth, EMS mutagenesis, UV mutagenesis and spontaneous mutations.Classical ways of identifying suppressor mutations, including library production or screening, are labor intensive. To accelerate the identification of suppressors, mutations will be identified using high throughput sequencing. Of the 38 putative suppressors of ded1‐dam1, 28 putative suppressors were sequenced using Ilumina HiSeq 2500 technology. Putative suppressors were chosen for sequencing based on results of mitochondrial mutation screens and the strength of suppressor mutation, as determined by frogging of serial dilutions. We are currently developing a pipeline for processing the data and identifying the relevant SNPs within the genomic data. By identifying suppressors of this well‐defined ded1 mutant, we will identify new regulators of Ded1, an RNA helicase essential for translation initiation.Support or Funding InformationThe Thomas F. and Kate Miller Jeffress Memorial Trust Awards Program in Interdisciplinary Research; HHMI Grant to University of Richmond; Dr. Martha Carpenter Fellowship, University of RichmondThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deciphering the Mechanism of 14‐3‐3ζ‐Mediated Inhibition of Phosducin Binding to the G Protein Transducin Complex
Authors: Chen Yuwen , Gao Meng , Huang Yongqi 
Published: June 2021

Phosducin modulates phototransduction by sequestering Gtβγ subunit from Gtα subunit of the heterotrimeric G protein transducin complex during light adaptation. However, the interactions between phosducin and the Gtβγ subunit are inhibited through phosphorylation at S54 and S73 within the N‐terminal domain of phosducin. It has been found that phosphorylation at S54 and S73 of phosducin induces its binding to 14‐3‐3 proteins, which blocks interactions between phosducin and Gtβγ. In this work, molecular dynamics simulations and fluorescence resonance energy transfer (FRET) were performed to elucidate the mechanism of 14‐3‐3ζ‐mediated inhibition of phosducin binding to the Gtβγ complex. To simulate the association between 14‐3‐3ζ and phosducin and the dissociation of phosducin from the Gtβγ complex, coarse‐grained representation of proteins and structure‐based Gô‐model were applied. We investigated the influence of phosphorylation on the dynamics and stability of phosducin/Gtβγ complex and association of phosducin with 14‐3‐3ζ. Our simulations showed that phosphorylation of phosducin on S54 and S73 introduced strong electrostatic repulsion between S54 and S73, resulting in opening of a segment between S41 and E66. Consistently, FRET efficiency measurement showed that the distance between Q16 and Q57 increased after phosphorylation. Binding simulations of 14‐3‐3ζ with phosducin/Gtβγ complex in the presence or absence of S54/S73 phosphorylation showed that 14‐3‐3ζ interacted with the phosphorylated S54 motif first and then interacted with the phosphorylated S73 motif when phosducin was dissociated from the phosducin/Gtβγ complex. These results provide insights into the molecular mechanism of phosphorylation‐regulated and 14‐3‐3ζ‐mediated inhibition of Phosducin binding to the G protein transducin complex.Support or Funding InformationThis work was supported by National Natural Science Foundation of China (Grant number: 21603121) and Hubei University of Technology.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation States of PEA‐15 Control Binding Specificity and Regulate Cell Proliferation and Apoptosis
Authors: Crespo Sergio , Hassan Sherouk , Cabezas Andres , Wei Yufeng 
Published: June 2021

Phosphoprotein enriched in astrocytes, 15 KDa (PEA‐15), is a small, cytoplasmic, death‐effector domain (DED) containing protein, found pervasively in mammals. It is ubiquitously expressed in different tissues, and has been known to be involved in several cellular pathways, including apoptosis and cell proliferation. This protein appeals to us and many other scientists because it is present in the cells in two different forms, phosphorylated PEA‐15 (at serine 104 and serine 116) and unphosphorylated PEA‐15, each interacting with different proteins at distinct biological pathways. PEA‐15 in its unphosphorylated state, interacts with ERK1/2 and prevents cell proliferation, while phosphorylated form interacts with Fas‐Associated Death Domain (FADD) protein and inhibits death receptor‐initiated apoptosis. Our initial NMR spectroscopic study showed that PEA‐15 undergoes conformational changes when it becomes phosphorylated at both serine residues, mimicked by serine to aspartic acid mutations (PEA‐15DD). The modulation of conformation switches the binding specificity of PEA‐15 from ERK1/2 to FADD, and changes its function from promoting cell death to cell survival. Based on our most recent results, we hypothesize that the DED conformation is allosterically controlled by the phosphorylation states of the C‐terminal tail, which in turn determines the binding specificity of the protein. Our ongoing research aims to broaden our understanding of the FADD/PEA‐15DD complex in vivo using NMR. In our phosphostasis model, we speculate that the balance between phosphorylated and unphosphorylated PEA‐15 is strictly regulated in different cell types and tissues to maintain the optimal cellular condition, and disruptions of the delicate balance could be detrimental to the cells, resulting in unregulated proliferation or elevated cell death.Support or Funding InformationU.S. Education Department Minority Science & Engineering Improvement Program Grant # P120A160084 NIH AREA Grant #CA179410USED Title III Part F, HSI‐STEM & Articulation Grant #P03C160155This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism of Competitive Binding Between HIF‐1α and CITED2 with TAZ1
Authors: Gao Meng , Huang Yongqi 
Published: June 2021

The cellular response to hypoxia is mediated by interactions between the α‐subunit of transcription factor HIF‐1 (HIF‐1α) and the TAZ1 domain of the transcriptional coactivators CBP and p300. CITED2 negatively regulates the transcriptional activity of HIF‐1. Structure studies have revealed that HIF‐1α and CITED2 bind to a partially overlapping surface of TAZ1 with similar affinity. Although the affinity of HIF‐1α and CITED2 for binding to TAZ1 is comparable, fluorescence and NMR experiments indicated that CITED2 efficiently displaced HIF‐1α from the HIF‐1α/TAZ1 complex. In this work, the binding mechanisms for HIF‐1α/TAZ1 and CITED2/TAZ1 interactions were studied by coarse‐grained molecular dynamics simulations. We found that the structure of HIF‐1α/TAZ1 complex was more dynamic than that of the CITED2/TAZ1 complex. Free energy analysis revealed that binding free energy barrier of the CITED2/TAZ1 complex was higher than that of HIF‐1α/TAZ1 complex, suggesting that the cooperativity of CITED2/TAZ1 interaction was stronger than that of HIF‐1α/TAZ1 interaction. Detailed analysis on the binding mechanism showed that CITED2 and HIF‐1α encountered different surfaces of TAZ1 to initiate the binding process. We further investigated the competitive binding between CITED2 and HIF‐1α with TAZ1 by carried out simulations using free CITED2 with HIF‐1α/TAZ1 complex, free HIF‐1α with CITED2/TAZ1 complex, and free CITED2, HIF‐1α and TAZ1. Consistent with previous NMR experiments, we observed formation of a ternary intermediate complex. Our results indicated that the dynamic interactions between HIF‐1α and TAZ1 allowed CITED2 efficiently displaced HIF‐1α from the HIF‐1α/TAZ1 complex.Support or Funding InformationThis work was supported by National Natural Science Foundation of China (Grant 21603121 to Y.H.) and Hubei University of Technology (M. G. and Y.H.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.