bluuebunny/crossref_metadata_2025_split huggingface dataset:
Maternal Versus Lifelong High‐Fat Diet Differentially Alters Genome‐Wide DNA Methylation in Rat Liver
Authors: Moody Laura , Kriska Adam , Chen Hong , Pan Yuan‐Xiang 
Published: November 2023

Early life diet programs offspring such that discrepancy between intrauterine and postnatal nutrition will have negative consequences later in life. Specifically, exposure to high‐fat diet (HF) at critical developmental stages is potentially associated with metabolic syndromes; however, it is unclear which epigenetic mechanisms are responsible for such outcomes. We hypothesize that due to differences in DNA methylation, a mismatched perinatal diet and post‐weaning diet is more metabolically detrimental than a lifelong HF diet. In the present experiment, male Sprague‐Dawley rats were exposed to HF diet during gestation and lactation. At weaning, half the animals were kept on the HF diet (HF/HF) and half were switched to a control AIN‐93G diet (HF/C). Complementary Methylated DNA Immunoprecipitation coupled with high‐throughput sequencing (MeDIP‐seq) and Methylation‐sensitive Restriction Enzyme digestion followed by sequencing (MRE‐seq) were used to quantify DNA methylation in the liver. We identified 3,966 differentially methylated regions (DMRs) between HF/C and HF/HF groups. Of these DMRs, 35% were mapped to gene bodies while 6% fell within promoter or downstream regions. Of the differentially methylated genes, 21 were clustered in the type II diabetes mellitus and the adipocytokine signaling pathways, two metabolically relevant systems that are known to respond to HF intake and lead to insulin resistance. Quantitative PCR showed altered expression of many genes in these pathways, including carnitine palmitoyltransferase 1A (Cpt1a), tumor necrosis factor receptor (Tnfrsf1b), retinoid X receptor (Rxra and Rxrb), and acetyl‐CoA carboxylase (Acacb). The remaining 59% of DMRs were not associated with protein coding genes. Sixteen of these DMRs were mapped to non‐coding RNAs including 9 miRNAs, 9 snRNAs, 6 snoRNAs, 1 telomerase, and 1 RNase P. Interestingly, several genes in the type II diabetes mellitus and the adipocytokine signaling pathways were predicted targets of these differentially methylated miRNAs. Our results indicate that compared to a lifelong HF diet, exposure to mismatched early and late life diets induces DNA methylation that impairs insulin‐ and lipid‐related pathways. Furthermore, methylation and expression of miRNA may provide an additional level of epigenetic regulation that drives these metabolic outcomes.Support or Funding InformationUIUC Research Board grant #12192; Data Purchase Program from University of Illinois Library
Steroid Receptors can Activate Unique FoxA1 Binding Patterns through a Highly Dynamic Mechanism Associated with a Fast DNA Residence Time
Authors: Swinstead Erin E , Miranda Tina B , Paakinaho Ville , Baek Soongjoon , Hawkins Mary , Karpova Tatiana , Ball David , Mazza Davide , Lavis Luke D , Morisaki Tatsuya , Grøntved Lars , Presman Diego M , Hager Gordon L 
Published: November 2023

The transcription factor (TF) FoxA1 has been implicated in steroid receptor (SR) binding patterns serving as a pioneer factor; however, the effects of SRs on the function of FoxA1 have been controversial. Classically it has been proposed that FoxA1 has a slow residence time on DNA allowing chromatin to remain open permitting access of SRs to binding sites. Conversely, it has recently been identified that SRs can alter the binding landscape of each other facilitating selective access to the chromatin by a mechanism termed dynamic assisted loading. To determine if this phenomenon extends to FoxA1 we characterised the estrogen receptor (ER), glucocorticoid receptor (GR), and FoxA1 in three breast cancer cell lines. Genome‐wide analysis of ChIP and DNaseI hypersensitivity assays upon hormone treatment show that both SRs can recruit FoxA1 to specific binding sites associated with an increase in chromatin accessibility. This indicates chromatin reorganization upon activation of ER and GR and recruitment of FoxA1 binding through an assisted loading mechanism. Most importantly, we have established the DNA residence time of FoxA1 by single‐molecule tracking in living cell at a rate of ~8–10 seconds. This finding is supported by the lack of a detectable FoxA1 DNase footprint together indicating fast chromatin interactions. These results suggest that many TFs in a given cell have the potential to affect the binding landscape of other TFs, depending on the chromatin context. In addition, this study has shifted our classical understanding of pioneer factors in breast cancer, demonstrating that steroid receptors can alter the response of FoxA1 though highly dynamic and fast DNA interactions.Support or Funding InformationThis research was supported by grants from the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.
Characterization of the RNA‐binding Motif of Phosphoglycerate Kinase 1 – a Moonlighting Function for a Metabolic Enzyme
Authors: Walker Austin , Friend Kyle 
Published: November 2023

Phosphoglycerate Kinase 1 (PGK1) is an enzyme that plays an integral role in glucose metabolism, catalyzing the reaction that converts 1,3‐bisphosphoglycerate into 3‐phosphoglycerate in glycolysis, and the reverse reaction in gluconeogenesis. In addition, PGK1 has been shown to have an RNA binding activity. This suggests that, in addition to its role in metabolism, PGK1 “moonlights” as a RNA‐regulatory enzyme. In particular, PGK1 was shown to down‐regulate the expression of the mRNAs which it bound. The goal of the current study is to identify the RNA sequence(s) that binds PGK1, using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. By identifying PGK1's consensus sequence, we will further elucidate the role of the enzyme in the cell beyond its metabolic capabilities, perhaps uncovering a new layer of regulation. We will then test how well PGK1 binds these RNA sequences via fluorescence polarization.Support or Funding InformationDr. G. Ashley Allen ‘65 Students Research Grant Fund, Washington and Lee University Chemistry and Biochemistry Department, Lenfest Grant
An Investigation of the Role of the NuA4 and Swr1 Complexes in Coordination of Transcription and RNA Splicing
Authors: Avitto Alexa N , Paripati Nikita , Moazamian Aron , Martinez Jordan , Moazamian Ryan , Pleiss Jeffrey , Kress Tracy 
Published: November 2023

RNA splicing is a critical step in gene expression in which non‐protein coding introns are removed from newly transcribed RNA. RNA splicing is carried out by the dynamic spliceosome and may occur directly after or concurrent with transcription, suggesting a close relationship between these processes. Indeed, there is mounting evidence that transcription and RNA splicing are coordinated, however the mechanistic details underlying this coordination are still lacking. The NuA4 protein complex is a histone acetylation transferase (HAT) with known functions in transcription. NuA4 works in conjunction with Swr1, an ATP‐dependent chromatin remodeling enzyme. Histone acetylation by NuA4 recruits Swr1, which subsequently exchanges H2A for the variant histone H2AZ to activate transcription. Together, these complexes regulate the transcription of ribosomal protein genes (RPGs), which comprise a substantial proportion of the genes that contain introns in Saccharomyces cerevisiae. Thus, NuA4 and Swr1 are perfectly positioned to link transcription and RNA splicing. We identified genetic interactions between genes encoding spliceosome proteins and genes encoding components of the NuA4 (eaf3Δ, eaf7Δ) and Swr1 complexes (vps72Δ and swr1Δ), as well as HTZ1, which encodes H2AZ. Splicing‐specific microarray analysis and reverse transcription‐PCR studies revealed splicing defects in swr1Δ, eaf7Δ, and htz1Δ. In addition, as predicted by our genetic studies, the splicing defects in strains harboring deletions in splicing proteins were exacerbated by deletion of EAF7, SWR1 or HTZ1. These data support a role for NuA4 and Swr1 in RNA splicing. We are currently using chromatin immunoprecipitation assays to test whether NuA4 and Swr1 increase splicing efficiency by recruiting splicing proteins during transcription. Our preliminary results indicate that splicing factor recruitment is altered in the absence of EAF7. Together, these data support a model in which the NuA4 and Swr1 chromatin modification complexes interact with the splicing machinery to coordinate transcription and splicing.Support or Funding InformationCottrell College Science Award from the Research Corporation for Science Advancement (#20186)
Histone H3K36 Methylation is Required for Efficient Pre‐mRNA Splicing in Saccharomyces cerevisiae
Authors: Flood Danielle M , Kennedy Margaret C , Sorenson Matthew , Ucles Stefanie , Jha Deepak K , Strahl Brian D , Stevens Scott W , Kress Tracy L 
Published: November 2023

Proper gene expression involves multiple steps, including RNA splicing where non‐protein coding regions of RNA are removed from the RNA transcript. RNA splicing is carried out by the large and dynamic spliceosome, which is comprised of small nucleoprotein complexes (snRNPs) that assemble on an RNA molecule in a precise order. Assembly of the spliceosome occurs co‐transcriptionally and RNA splicing is tightly coordinated with transcription to ensure precise and efficient gene expression. However, the mechanisms that underlie this coordination are poorly understood. In order to identify proteins that function to coordinate RNA splicing with transcription we carried out a genetic interaction study using Saccharomyces cerevisiae. We identified negative genetic interactions between genes encoding RNA splicing factors and the SET2 gene, a histone methyltransferase that methylates lysine36 on histone H3 (H3K36) to regulate transcription. Furthermore, we show that mutations that block H3K36 methylation also have negative genetic interactions with splicing factor genes, suggesting that H3K36 methylation is important for RNA splicing. Indeed, we have shown that deletion of SET2 or point mutation of H3K36 inhibits RNA splicing and exacerbates splicing defects in yeast strains harboring deletions of splicing factor genes. Using chromatin immunoprecipitation, we demonstrate that deletion of SET2 reduces the association of snRNPs with chromatin. Furthermore, we tested for reciprocal/reverse coordination between RNA splicing and transcription. We found that mutation of Prp28, a helicase protein that normally promotes RNA splicing, alters RNA polymerase II association with DNA. We are currently testing whether perturbation of RNA splicing alters H3K36 methylation. Thus, we provide evidence that H3K36 methylation is required for appropriate RNA splicing in yeast and suggest a model in which Set2 or H3K36 methylation help to recruit splicing factors to RNA during transcription.Support or Funding InformationCottrell College Science Award from the Research Corporation for Science Advancement (#20186) to T.L.KAmerican Cancer Society (RSG‐05‐137‐01‐GMC) to S.W.S National Institutes of Health (GM084246 to S.W.S and GM110058 to B.D.S) Cancer Prevention & Research Institute of Texas (RP101501) to M.R.SGateway to Graduate School funded by The National Science Foundation (NSF‐URM) to D.M.F
Genetic and Biochemical Studies on the Role of Essential Protein Dib1 in Pre‐messenger RNA splicing
Authors: Bowman Emily , Hernandez Cody , Schreib Christian , Lucas Amber , Maeder Corina 
Published: November 2023

Pre‐messenger RNA splicing is a process that involves the removal of non‐coding regions of RNA and the ligation of protein‐coding regions in order to become a mature messenger RNA. This process involves the sequential addition of small nuclear ribonucleic protein particles, or snRNPs, that come together to form a pre‐catalytic spliceosome. Once formed, the U1 and U4 snRNPs leave to create the catalytically active spliceosome. The timing and regulation of this step is critical for splicing which must be accurate to a single nucleotide precision in order to prevent drastic effects further downstream in the translation of mRNA into protein.A key figure in this process is Dib1, a small 17kDa essential protein. Dib1 has been shown to be necessary for cell viability and critical for splicing, although its role in splicing has yet to be characterized. Based on the recent cryo‐EM map of the U4/U6‐U5 triple snRNP complex, Dib1 appears to occupy a space in close proximity of the loop region of the U5 snRNA. This snRNA is expected to interact with the pre‐mRNA in the catalytically active spliceosome. Thus, in order for the spliceosome to be activated, Dib1 would need to depart from its location. To examine the role of Dib1 in splicing we have created multiple alleles of the dib1 gene in order to study their effects on cell viability in S. cerevisae. Using these strains, we are exploring mutant growth phenotypes and the effects on in vitro splicing. In addition, we are examining the effects of the mutations on the protein's stability.Currently we have identified two temperature sensitive mutants through our mutational analysis. Using in vitro splicing assays, both were found to have splicing defects, although their defects differed. In order to determine why the splicing phenotypes differed, we are characterizing the effects of the mutations on the structure of Dib1. The proteins were purified using affinity chromatography followed by anion exchange chromatography. CD analysis of the purified protein was then performed. We will present these findings and discuss the implications on pre‐mRNA splicing.
Investigating the Mechanism by which the Small RNA MtlS Regulates the Mannitol Protein Transporter at the Post‐Translational Level in Vibrio cholerae
Authors: Hansen Elisabeth E , Coyle Maxwell C , Liu Jane M 
Published: November 2023

Cholera, caused by the gram‐negative, halophilic bacterial pathogen Vibrio cholerae, remains a world health problem, causing millionsof deaths each year. Throughout its life‐cycle, V. cholerae is able to adapt to avariety of environments, from aquatic reservoirs to the human small intestine. This remarkable adaptability is likely due in part to gene regulation by small regulatory RNAs. We are examining one such regulatory pathway by studying the proteolysis of MtlA, the mannitol‐specific phosphotransferase system transporter. In the absence of environmental mannitol, the small RNA MtlS down‐regulates mtlA expression at the translational and post‐translational level. However, the mechanism by which MtlS post‐translationally directs MtlA proteolysis is currently unknown. Our lab has shown that MtlS induction significantly affects the expression of various proteins, which we hypothesize may be involved in MtlA proteolysis. Specifically, proteins up‐regulated by MtlS induction may act as proteases or protease adaptors. Proteins down‐regulated by MtlS induction may normally act to stabilize MtlA, such that when they are suppressed, MtlA degradation can occur. Currently, we are studying the potential role of three proteins—VC1899, GroL2, and VC1872—in MtlA proteolysis. VC1899 and GroL2 are both up‐regulated by MtlS induction, while VC1872 is down‐regulated. We created strains that overexpress or have knocked out one of each of these proteins, and analyzed the affect that these proteins have on MtlA levels over time in mannitol(MtlS‐repressing) and mannitol‐free (MtlS‐inducing) conditions. Additionally, we created knockout strains containing an arabinose‐inducible plasmid bearing MtlS, as an alternative method to analyze MtlA levels over time upon MtlS induction. Preliminary results suggest that VC1899, an uncharacterized protein thought to be involved in nucleic acid binding, represses MtlA protein levels. Single‐knockout mutants of either GroL2, an ortholog to the essential chaperone GroEL, or VC1872, a nun characterized protein with homology to serine protein kinases, do not display significant phenotypes with regard to cell growth or MtlA proteolysis. However, we are in the process of making double knockout mutant strains in order to test whether GroL2 and VC1872 may work in concert. Ultimately, gaining a better understanding of the mechanism of MtlA proteolysis could help to illuminate other regulatory systems in V. cholerae and other pathogens.Support or Funding InformationNIH grant R15AI090606 to J.M.L.
Unraveling the Activation Mechanism of Cullin‐RING Ubiquitin Ligases using Quantitative Cross‐linking Mass Spectrometry
Authors: Yu Clinton , Mao Haibin , Novitsky Eric , Rychnovsky Scott D. , Zheng Ning , Huang Lan 
Published: November 2023

Cullin‐RING ubiquitin ligases (CRLs) represent a superfamily of heterogeneous multi‐protein E3 ubiquitin ligases comprised of a cullin and RING protein catalytic core that mediate the ubiquitination of protein substrates. The full enzymatic activity of CRLs requires cullin modification by Nedd8 (i.e. neddylation). This ubiquitin‐like modification promotes both E2 recruitment and subsequent ubiquitin transfer, thereby stimulating the E3 activity of CRL ligases. Thus, neddylation represents a critical post‐translational modification that regulates protein ubiquitination by these complexes. Despite extensive structural studies on CRLs, certain structural details of CRLs during protein ubiquitination remain elusive. It has been shown that deamidation of Gln40 of Nedd8 to glutamate (Q40E) effectively inhibits CRL E3 activities, directly interfering with cellular functions. However, the molecular mechanism by which Nedd8(Q40E) affects CRL function is unclear. Cross‐linking mass spectrometry (XL‐MS) is a powerful strategy that can be utilized to characterize dynamic and heterogeneous protein complexes that are difficult to study using conventional structural tools. Quantitative cross‐linking mass spectrometry (QXL‐MS) not only allows the determination of physical contacts between and within proteins at the amino acid resolution, but also enables quantitative assessment of conformational changes in protein complexes in various states. To elucidate the structural basis underlying the activation/inactivation of CRLs through neddylation, we have employed novel QXL‐MS strategies to compare cross‐link profiles of different types of full‐length human Cul1‐Rbx1 complexes, as well as their interactions with E2‐conjugating enzyme Cdc34. Thus far we have uncovered major Nedd8‐induced structural rearrangements of the CRL1 catalytic core that are not induced by Nedd8(Q40E) conjugation, suggesting that the subtle change of a single Nedd8 amino acid is sufficient to re‐orient the topology of the CRL catalytic core. Our results provide new insights into how neddylation regulates the conformation and activity of CRLs.Support or Funding InformationThis work is supported by NIH RO1GM074830 to L.H, and R01GM106003 to L.H. and S. R..
Decreased Aggregation of Carboxypeptidase O upon Incorporation into Lipid Rafts
Authors: Ezeribe Hazel O , Burke Linnea C , Barfi Gifty B , Lyons Peter J 
Published: November 2023

Carboxypeptidase O (CPO) is a member of the M14 family of proteolytic enzymes. Many members of this family are secreted from cells and are involved in degradation of extracellular peptides; other carboxypeptidases remain within the secretory pathway where they are involved in the maturation of bioactive peptides. CPO is a GPI‐anchored carboxypeptidase that is expressed in epithelial cells of the small intestine. To further understand the function of CPO, we investigated its subcellular localization. CPO was found to be present in detergent‐insoluble complexes, suggesting a lipid raft localization and function. Immunofluorescence analysis of CPO in stably‐transfected MDCK cells, however, indicated a punctate distribution of aggregates having little co‐localization with lipid raft proteins. Incubation with cholesterol dramatically decreased this aggregation and resulted in the localization of CPO to puncta of uniform size (< 300nm diameter) and distribution. Similar results were obtained when cells were incubated with oleic acid, which also led to increased GPI anchoring and membrane association. We reasoned that increased membrane cholesterol might mediate conformational changes in CPO, via its GPI anchor, that result in decreased aggregation. We found that loss of the GPI anchor by deletion of the C‐terminal GPI signal peptide, but not the N‐terminal partial prodomain, resulted in greatly reduced expression of CPO in Sf9 cells, suggestive of a conformational and/or folding defect. A decrease in expression of this C‐terminally deleted CPO was also observed in HEK293T cells, although further analysis showed no alteration in protease susceptibility of CPO that might suggest large changes in conformation or folding. Our results suggest that incorporation of CPO into cholesterol‐rich lipid rafts leads to subtle changes in CPO conformation that prevent aggregation.Support or Funding InformationSupported by Faculty Research Grants to PJL from Andrews University
Brain Expressed X‐linked 3 (BEX3) is a High‐Order Oligomer with Partially Folded Regions
Authors: Cabral Katia , Raymundo Diana , Silva Viviane , Sampaio Laura , Hill Luis Fernando , Almeida Fabio , Cordeiro Yraima , Almeida Marcius 
Published: November 2023

The members of the family of Brain Expressed X‐linked (BEX) are mammal‐specific proteins involved in neurodegeneration, cell cycle and cancer. However, very limited data are available on the conformational state of any member of the BEX family. Recently, BEX3 has been predicted to be intrinsically disordered and also to represent an intracellular hub for cell signaling. In this study, we characterized the BEX3 structure using biophysical experimental data. The association of atomic force microscopy and small angle X‐ray scattering revealed that BEX3 forms a specific higher‐order oligomer that is consistent with a globular molecule. The partial proteinase K digestion has suggested that BEX3 structure contains a core which is proteolysis‐resistant around residues 55–120 with unfolded regions at the N‐ and C‐termini. Circular dichroism spectroscopy and solution nuclear magnetic resonance data revealed that BEX3 exists in solution as an oligomer with partially disordered segments at the N‐ and C‐termini and with a more compact helical structure in a central core. The self‐oligomerization of BEX3 has been previously reported in cell culture and is consistent with our in vitro data. We speculate whether the self‐association of BEX3 into a folded core offers a natural mechanism to protect it from degradation and possibly even to regulate its activity.Support or Funding InformationFAPERJ, CNPq, INBEB, CENABIO.
Hydrodynamic Radius Prediction of Intrinsically Disordered Proteins
Authors: Ricard Benjamin , Tomasso Maria , Whitten Steven 
Published: November 2023

Intrinsically disordered proteins are proteins that do not show a set three‐dimensional tertiary structure and are usually involved in signal‐transduction and transcription regulation. It is estimated that upwards of 33% of all eukaryotic proteins contain intrinsically disordered domains. It is known that hydrodynamic radius (Rh) of IDPs depends largely on their intrinsic propensity to conform into a polyproline‐II helix. A generalized understanding of the molecular organization of IDPs in response to perturbations is presented here. This model uses polyproline‐II propensities and net charge in order to simulate Rh and understand the structural properties of IDPs in terms of discrete changes in a system. Fundamental understanding of protein structures in IDPs may provide an avenue for novel drug design, diagnostics, and treatment.Support or Funding InformationResearch reported in this poster was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award R15GM115603.