bluuebunny/crossref_metadata_2025 huggingface dataset:
First Year Medical Student Authorship of Clinical Cases
Authors: Bateman Robert C , Chastain Paul , Stephens Melissa
Published: November 2023

Getting the diagnosis right is a critical skill for a physician and learning this skill should start early in a medical student's training. To this end, students in their first year at William Carey University College of Osteopathic Medicine must write and orally present an original clinical case on an assigned topic as a part of their medical biochemistry course. At the first of the 2016 spring semester the class was randomly divided into groups of three and each group assigned a unique topic consistent with the content covered in the course. The group was then asked to write an original clinical presentation of a new patient along with a listing of four possible diagnoses. This case was distributed to the entire class without a final diagnosis and the class voted via clicker slide on the final diagnosis after the group presented their case and the four diagnostic possibilities. The group then explained/justified their differential, decided on the diagnosis, described treatment and prognosis, and briefly explained the underlying biochemical basis of the disease/disorder. Assessment of the written and oral components was via rubric and comprised 10% of the student grade for the semester. The final exam was derived completely from aspects of the student‐authored cases, bringing accountability to the class for their own work as well as those of their classmates. The average grade on the final was the highest exam average of the entire year, suggesting a high level of mastery of the thirty‐six clinical cases authored by the students.
Observations and practical tips on metal affinity chromatography and protein refolding techniques
Authors: Tovar Carmel N , Mullins John A , Odunuga Odutayo O
Published: November 2023

Immobilized metal affinity chromatography (IMAC) is a specific high‐capacity technique used in large‐scale purification of proteins. In spite of the robustness of the technique, several factors still affect column binding capacity, yield and purity of proteins during IMAC. Using (His)6‐tagged proteins with comparably high levels of expression, we investigated the effect of protein size, flow‐rate, number and position of (His)6 tag on protein yield using commercial Ni2+ IMAC columns. Results suggest that regardless of manipulation of flow rate, (His)6 tag placement and number, size of protein is the major factor affecting binding and yield during IMAC purification. Small and medium sized proteins (<50 kDa) bind more tightly to the column than bigger proteins, resulting in higher yield during purification. In another study, we employed a modified gradient gel filtration technique to successfully refold a spectrum of highly aggregation‐prone enzymatic proteins back to their native states during denaturing purification. Some of the proteins chosen are well‐known to precipitate significantly during refolding. However, using this technique, precipitation was either so minimal or non‐existent. Enzymatic assays and dynamic molecular mass determination confirmed that the proteins were correctly refolded and biologically active. This strategy provides a robust approach to refolding denatured proteins that can also be used simultaneously as buffer exchange and additional purification step.

Support or Funding Information

The Welch Foundation
Predictors of Success on the MCAT for Post‐Baccalaureate Pre‐Medicine Students
Authors: Dobrydneva Yuliya , Schwartz Lisa
Published: November 2023

Students who aspire to enter medical school have to take the MCAT (Medical College Admission Test). In 2015, the MCAT was redesigned, covering a new set of foundational concepts. In 2015, a 12‐month post‐baccalaureate pre‐medicine certificate program at the GW School of Medicine and Health Sciences was launched to address the changes to the new MCAT. Furthermore, the program prepares students for medical school coursework, like biochemistry, histology, physiology and pharmacology. We designed an integrated curriculum as a sequence of three 10‐week courses (Organic Chemistry I and Organic Chemistry II ‐ both with laboratory ‐ and Biochemistry I) to teach the material required for pre‐medical students. The objectives of these courses are closely aligned with MCAT objectives. The GW post‐bac pre‐med program is intended for career changers who typically majored in the social sciences or humanities, and thus have little or no science background (“scientifically naive”). Therefore, in our admissions process, we need to identify predictors of future success in heavily scientific courses for students who have minimal prior exposure to sciences. We've analyzed data of the first cohort of ten students to determine what predictors appeared to be positively or negatively associated with MCAT performance. Conclusions of this pilot study will be expanded to the second cohort of students (27) who are currently enrolled in the GW post‐bac pre‐med program and will take the MCAT exam in summer 2017.
Modeling A Protein Story (MAPS): A Project‐based Learning Program Connecting Gene and Protein Sequence and Structure‐function Relationships with Physical Models
Authors: Munzenmaier Diane H.
Published: November 2023

The MAPS program consists of content‐driven multimedia online modules focused on the genetics and protein structure/function/dysfunction relationships associated with a specific protein essential to grades 9–12 life sciences curricula. Teams of students, led by a teacher within a classroom, home school, or extracurricular setting, progress through hands‐on activities using models and materials such as gene and protein sequences, molecular, protein folding and protein structure models as well as use of 3D molecular visualization and design software. Following a progressive exploration of the featured protein, teams choose a “protein story” extension where they gain a deeper understanding of a related topic of interest. They then design and 3D print a physical model that helps them to tell their specific molecular story. Teams write abstracts, create posters describing their work and present their posters using their physical models at local, regional and national conferences.

The MAPS program builds deep content knowledge as well as higher level integration of content through an original and unique format based on methods that have been proven to be important for student learning. Each program year begins with foundational concepts to which higher level concepts are built upon throughout the course of the program. These concepts are explored using hands‐on activities within small groups of students, guided by a teacher. Teams are encouraged to wonder, ask questions, and construct hypotheses. At the end of each unit, videos are provided to assist in pulling together and reinforcing the concepts learned through the activities before they move on to the next section. This format utilizes and encourages proven effective educational strategies such as active learning, cooperative learning, and inquiry‐based learning. Once a basic level of understanding of the protein of interest is reached, teams then choose, research and develop their unique related molecular story, design and build a physical model that represents their story, and communicate their story to the public, developing necessary presentation skills. The ability for each team to choose an extension or “protein story” that they find particularly intriguing provides additional engagement and motivation and allows for more open‐ended discovery and learning.
Promoting rural student enthusiasm for STEM by establishing a model biotechnology company in their high school
Authors: Koci Matthew , Boller Bruce , Ali Rizwana , Orders Amy
Published: November 2023

The jobs of the 21st century require a higher level of skill, training, and practical competency in science than our high schools are currently able to provide students. This is especially true for students in rural and under‐served communities. To ensure the ability of these students to find stable and lucrative careers, we need to develop novel teaching approaches that provide better workforce development while attracting students to careers in science. This project seeks to address this issue by establishing a recombinant protein expression “mini‐biotech company” in the Bertie Early College High School, in Windsor, North Carolina. This mini‐biotech company serves as the platform to teach Essential and Common Core Standards of biology and chemistry. By going through the steps of initial process development, process scale‐up, production, and validation to ultimately produce recombinant proteins, the students get a better understanding of the concepts, technologies, inter‐relatedness of the different fields. Additionally, the students are producing proteins for active university research projects related to the industries in Bertie County. This helps the students gain a fuller appreciation for how science and biotechnology are related to the jobs in their community and better plot their own path to a science‐based career. Over the course of this project, the students developed mentoring networks with university faculty and scientists at various biotechnology companies. They have also attended local and national scientific meetings. Collectively the participants in this program gain practical work relevant experience leading to an understanding of biotechnology beyond most undergraduate students.

Support or Funding Information

This project is supported by a Student Science Enrichment Program grant from the Burroughs Welcome Fund
Design and Implementation of a Semester‐Long Course‐Based Research Experience in Biochemistry that is Suitable for Introductory and Upper Level Students
Authors: Shelby Shameka
Published: November 2023

Development of students' critical thinking skills and problem solving ability is paramount for many undergraduate science programs. As such, many programs attempt to provide students with individual research opportunities as a capstone experience. Previous studies have also shown that underclassmen greatly benefit from early exposure to research opportunities as seen by increased retention and student problem solving capacity. However, due to resource limitations, research opportunities for non‐senior students can be limited. To provide opportunities for a variety of students to gain exposure to research, a course‐based research experience was designed and conducted. Students utilized a bioinformatics approach to develop hypotheses concerning protein interactions with Mer Receptor Tyrosine Kinase, a protein that is necessary in maintaining the structural integrity of the retina. Students designed experiments to identify specific sites of interactions with SH2‐domain proteins. Techniques including site‐directed mutagenesis, PCR, affinity chromatography to purify recombinant protein, SDS‐PAGE, and in‐vitro pulldowns were utilized by students. The semester culminated in students testing their hypotheses and producing manuscripts. Evaluation of this course by students indicated increases in critical thinking skills, problem solving abilities, and expressed interest in pursuing further research opportunities. Underclassmen that participated in the course also benefitted from mentor‐mentee relationships developed with upperclassmen due to the collaborative nature of the course. The structure of the course also allows for further studies to be conducted based on novel findings and is highly adaptable to receptor tyrosine kinases found in other tissue types.
A POGIL Based Laboratory Manual for Undergraduate Biochemistry
Authors: Wright Amanda , Davis Bryan , Krzyslak Amanda
Published: November 2023

Current laboratory practices often involve students coming to lab having read a detailed protocol, performing the experiment, and leaving the lab rarely understanding the meaning behind the methods or the connection to research in general. They lack a conceptual understanding of the process and often do not understand how to manipulate data. Traditional methods do not provide students opportunities to think through the underlying scientific principles. Not only does this laboratory format not represent ‘real’ science, it leaves students unprepared for the often unpredictable and intimidating nature of experimental design. Research suggests that inquiry‐based pedagogical techniques can help promote student engagement and improve educational outcomes. Here, we present portions of a newly developed laboratory manual with which students will participate in the building of experimental protocols, implementing the experiments, and processing the data. Through this process, students will develop an understanding of scientific principles and data analysis, and think creatively in regards to experimental design. These practices have also been shown to increase student engagement in the laboratory. Current laboratory manuals, do not emphasize the building of these essential skills for anyone pursuing further education or a career in biochemistry, molecular biology, or related fields. We hope to bring these high impact learning practices to the biochemistry laboratory providing inquiry‐based experiments to better develop and engage our students in the biochemistry lab. In order to train the next generation of scientists, we must allow students to explore the scientific world in a safe, engaging, and meaningful way. It is our hope that the lab manual will be a pioneering advancement in the way educators approach undergraduate laboratories. This manual will be of great utility at both large institutions where access to a research environment is limited to a small population of the students and small institutions where resources are limited.
Ammonium Sulfate Selectively Extracts Invertase Activity from a Mixture of Precipitated Yeast Proteins
Authors: Cook Rachel M , Gontkovic Anna J , Lawrence Jim , Timerman Anthony
Published: November 2023

The purpose of this work was to improve the first step in a sequence of Chemistry 365 (Biochemistry) lab exercises in which students purify the enzyme invertase from a yeast extract that contains many other different proteins. The essence of the first step in this protein purification exercise is to precipitate many different proteins using aqueous solutions of ethanol and then selectively extract soluble proteins from the pellet in a buffer composed of 10 mM sodium phosphate at pH 7.0. Our results show that the addition of ammonium sulfate to the extraction buffer greatly reduces the total mass of proteins extracted from the pellet with a minimal loss of extracted invertase activity. SDS‐PAGE analysis of the final fraction prepared by this modified method reveals a sample with a fewer number of contaminating protein bands than that observed in a commercial sample of “pure‐invertase” purchased from Sigma Chemical Company.
Genetic Engineering of Human Cells Using Lentivirus in a High School Laboratory Course
Authors: Safranek Conrad , Ross Emily , Rosenberg Martin , Cole Austin , Chandra Atharv , Pemberton Luke , Zarrinnegar Hanna , Alonzo Amanda , De Luke , Sweeney Robert
Published: November 2023

Human cell culture, lentiviral genetic engineering, and fluorescence microscopy within a BSL‐2 laboratory provide for an unparalleled learning opportunity in molecular biology. Here we report course content that includes regulatory requirements needed to establish a BSL‐2 high school laboratory and provides detailed written and video protocols, adapted to a high school schedule, for visibly definitive positive controls in genetic engineering. This course can be fully implemented in one semester. Mechanistic descriptions and lab protocols for the following genetic engineering techniques are included in this work: lentiviral production and transduction with a GFP transgene (permanent gene delivery), with sgRNA for GFP and CAS9 payload (CRISPR; permanent gene disruption), with a GFP shRNA (permanent RNA interference), and transfection with GFP siRNA (transient RNA interference). Positive controls serve as a critical foundation for teaching fundamentals of molecular biology as well as for novel research. Students extract deeper meaning from visual representations of biological concepts, which makes GFP a particularly impactful tool in practicing positive controls. Also, students working under BSL‐2 lab conditions solidify lab safety skills that are fundamental to molecular biology. To our knowledge, there has never been published such a comprehensive guide to implementation of a BSL‐2 laboratory with associated genetic engineering protocols for visible positive controls. This information will be helpful to other secondary schools considering such programs as well as undergraduate molecular biology programs looking to incorporate these principles and techniques. All of the course content and protocols is presented and freely available at www.nuevaresearch.org.

Support or Funding Information

This work was supported by The Nueva School.
Establishing a Zebrafish Laboratory Exercise to be Used in a Course‐Based Undergraduate Research Experience (CURE) in Introductory Biology
Authors: Bruton Laura , Gruber Alex , Kalo Kadijah , Morrissette Jeffrey
Published: November 2023

Zebrafish (Danio rerio) are biological model organisms as they are a predictive model for human diseases and development. They are also an excellent teaching tool. At Lawrence Technological University, we are establishing a population of zebrafish of all ages for the purpose of using them in a course‐based undergraduate research experience in a first‐year biology course. We have purchased a stand‐alone, portable, zebrafish “E‐Rack” from Aquaneering (San Diego, CA), which is composed of eight 1.8 L and five 2.8 L tanks. Using this system, we have been able to maintain stable water quality that has allowed us to successfully house, mate, and rear zebrafish to adulthood. We use a single pair mating procedure to obtain fertilized eggs each week that can grow to adulthood in approximately 12 weeks. We are presently designing the laboratory procedures using larval zebrafish to introduce an authentic research experience into our freshman introductory biology laboratory course. One key advantage of larval zebrafish is their transparency which allows for easy viewing of the heart and blood vessels with a low‐power light microscope. In this CURE, students will perform a cardiotoxicity assay by first lightly anesthetizing one‐month‐old larval fish. Students will then measure and record fish heart rates under the microscope. Next, they will add a variety of compounds of their own choosing to test their potential cardiotoxic effects based on observed changes to heart rate and contractility. We will have students begin with known effectors of heart rate such as nicotine and caffeine, and then move on to testing their unknowns. At the end of the course, students will produce research posters which they will present at Lawrence Tech's Research Day conference and/or other local and national conferences. We will determine the effectiveness of this new teaching strategy using the established Classroom Undergraduate Research Experience (CURE) survey administered by Dr. David Lopatto at Grinnell University.

Support or Funding Information

Lawrence Technological University Quest Program

Lawrence Technological University Natural Sciences Department
Mechanism of DNA Binding by Human DNA Ligase 1
Authors: Jurkiw Tom , O'Brien Pat
Published: November 2023

Human DNA Ligase 1 (hLIG1) is an essential enzyme in the cell that seals single‐strand DNA breaks, or nicks, that occur between adjacent Okazaki Fragments during DNA replication. Although the phosphoryl transfer steps of hLIG1 have been previously characterized, many questions remain about how hLIG1 is able find and recognize nick sites. New approaches are needed to observe the rapid and transient interactions. Here we have investigated the kinetic mechanism for DNA binding using fluorescence approaches. By using a nicked DNA reporter and endogenous tryptophan fluorescence, we are able to monitor the DNA‐dependent steps of the ligation mechanism. Under the conditions used the assay appears to report on initial interactions with the nicked DNA substrate and the product release step, both of which are previously uncharacterized steps. Future experiments will extend these kinetic and thermodynamic studies to understand what features of hLIG1 and the nicked DNA substrate allow for rapid and faithful ligation of DNA breaks.

Support or Funding Information

Research was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number T32GM008353 and the Rackham Graduate School and Biological Chemistry Department at the University of Michigan.
Determination of Escherichia coli Genes Important for DNA Repair Following Alkylation
Authors: Joshi Christopher , Aiken Ariel , Nash Emma , Wong Kelly , Carlson Alyssa , Leifer Becky , Muenter Mark , Beuning Penny
Published: November 2023

Alkylating agents, common in industrial manufacturing and chemotherapy, can damage DNA by adding alkyl groups to nucleotides. Alkylation can decrease the stability of the N‐glycosidic sugar‐base bond, making it more apt to break; alkylation can also interfere with base pairing. Either of these perturbations can lead to mutations if not remedied. DNA repair mechanisms, including direct base repair, base excision repair, and nucleotide excision repair, play important roles in correcting these lesions. Our research aims to better understand the mechanisms behind repair of alkylated DNA, first by confirming which genes are most critical. An assay was designed to screen bacterial strains harboring single deletions of more than 20 genes, selected based on prior known association to DNA repair. The survival of E. coli AB1157 strains, each with one of the 20 genes deleted, was measured upon exposure to one of two alkylating agents: styrene oxide (SO) or chloroacetaldehyde (CAA). Strains with dam deleted were sensitive to SO and CAA, suggesting that DNA adenine methylase, the protein encoded by dam, plays a critical role in DNA alkylation repair. Strains lacking recA, which plays multiple roles in DNA damage responses, were quite sensitive to both agents. Strains with mfd, mutH, or mutY deleted were not more sensitive to SO and CAA than the wild‐type strains. Next, the survival of double knockouts will be assayed, which will help determine if these genes act in the same or different DNA repair pathways.

Support or Funding Information

This work was supported by the American Cancer Society and the NU Office of the Provost.
Effects of H4K16 and Gene Mutation on Sensitivity to DNA Damaging Agents and Silencing
Authors: Enya Blessing , Young Tiffany , Kirchmaier Ann
Published: November 2023

The “Central Dogma” involves replication of DNA, transcription of mRNA, and translation of mRNA into protein. When a gene maintains its sequence, but portrays heritable differences in expression, these differences are often as a result of a process known as epigenetics. Epigenetic regulation of gene expression involves histone modification, chromatin structure, and other factors in all eukaryotes. Using budding yeast (Saccharomyces cerevisiae) as a model to study epigenetics, has provided a strong understanding of how histone modifications and chromatin structures are inherited and maintained throughout the cell cycle. S. cerevisiae has also served as a good model for analyzing genetic interactions. Identifying genetic interaction can be performed by observing differences in growth rate in the presence and absence of DNA damaging agents, of single and double mutants compared to Wild‐type. Mutations that occur in many genes responsible for replication, chromatin assembly, or histone modifications cause growth sensitivity in the presence of DNA damage. However, it is not known what role H4 K16 acetylation plays in responses to DNA damage and if this modification genetically interacts with chromatin assembly factor and histone acetyltransferase mutants. We hypothesized that H4K16 acetylation mutants would synthetically interact with defects in chromatin assembly factors and histone modifying‐enzymes in the presence of DNA damage.

Several genes such as ASF1, CAC1, RTT106, HIF1, etc., were mutated and combined with wild‐type histones or H4K16 acetylation mutants. These combinations were assessed in synthetic interaction analyses in yeast, in order to evaluate how this modification influences responses to DNA damage and silencing. Yeast were grown in Yeast Peptone Dextrose media in the absence or presence of different drugs or environmental conditions such as hydroxyurea, methyl methane sulfonate, zeocin, and ultraviolet exposure. Images were taken and compared to see how each drug and mutant combination affect yeast growth. Results show presence of growth defects in single and double mutants in the presence relative to the absence of different DNA damaging agents, highlighting synthetic interaction between the gene mutation and histone mutants in yeast. Silencing was determined based on colony color to indicate expression of HMR:ADE2 in the mutated strains. Red colonies indicate the silencing of ADE2 and loss of gene expression. White colonies indicate fully expressed ADE2 and enhanced gene expression. The results suggest that loss of HMR:ADE2 silencing means defects of silent chromatin formation, and combination of H3K56 and H4K16 acetylation mutants affect yeast growth under different DNA damaging conditions. Combining mutated genes with other altered histone modification, to compare growth defects and DNA damage sensitivity will be conducted to understand genetic pathways regulating DNA damage responses and epigenetic processes.
Determination of E. coli cellular factors that contribute to survival upon exposure to the alkylating agent benzyl bromide
Authors: Aiken Ariel , Bellou Sirine , Wong Kelly , Nash Emma , Kramer Caitlin , Leifer Becky , Muenter Mark , Beuning Penny
Published: November 2023

Alkylating agents have the ability to add alkyl groups onto DNA at various positions. There are many of these agents present in our environment that can ultimately lead to damaged DNA and mutations. While some mutations are beneficial many of the ones caused by these chemicals can have detrimental effects. Benzyl bromide is a known alkylating agent and can be a byproduct of industrial processes and then released into the environment. The goal of my work is to identify genes responsible for cellular responses to DNA damage caused by benzyl bromide using a library of E. coli knockout strains. E. coli knockout strains are being created using bacteriophage‐mediated transduction and the identities confirmed by colony PCR and DNA sequencing. The genes deleted in the knockout strains are those that encode various proteins that play a role in some aspect of DNA repair. While carrying out these experiments the knockouts in comparison to the wild type E. coli are expected to have a lower survival rate. After optimizing the assay conditions, we are determining the survival of strains in our library of now more than 30 knockout strains. Insight to how these E. coli strains react when exposed to this alkylating agent will give us more information on how these genes affect DNA repair and allow further studies to be done. We will be able to determine which genes have a more significant role in comparison to others and focus on other causes and possibly ways to reduce its effects.

Support or Funding Information

Supported by American Cancer Society (RSG‐12‐161‐01‐DMC) and NIEHS ROUTES program.
Affinity Characterization of TTD Domain of UHRF2 Histone Reader Protein.
Authors: Petzold Timothy Samuel
Published: November 2023

One proposed method for anticancer drug development is to target epigenetic proteins, which regulate genome expression and are involved in various cancers. The ubiquitous reader protein UHRF1 (Ubiquitin Like With PHD And Ring Finger Domains) is identified as an oncogene and its binding affinity to histone H3 has been well‐determined via its PHD and TTD domains. The homologue, UHRF2, is noted to have properties of a tumor‐suppressor and oncogene, dependent on the cancer, but its affinity for H3 has not been assessed. In this study, we characterized the binding affinity of the TTD domain of UHRF2. The dissociation constant between H3K9me3 and TTD of UHRF2 was determined by fluorescence polarization assays. Interestingly, our study suggests that the linker portion between TTD and PHD inhibits binding of the TTD to H3. This linker region may provide a promising approach for regulating the activity of UHRF2.
GPS2 regulates mitochondrial biogenesis via mitochondria retrograde signaling and modulation of nuclear‐encoded mitochondrial genes core promoter accessibility
Authors: Cardamone Maria Dafne , Tanasa Bogdan , Cederquist Carly T , Huang Jiawen , Mahdaviani Kiana , Orofino Joseph L , Lentucci Claudia , Li Wenbo , Rosenfeld Michael Geoff , Liesa Marc , Perissi Valentina
Published: November 2023

A key step in the evolution of eukaryotic cells has been the acquisition of oxidative metabolism through the symbiotic incorporation of an aerobic prokaryote. As this primordial organism evolved into an intracellular organelle, now known as mitochondrion, most of its genetic information was transferred to the nuclear genome. While this implies that mitochondrial biogenesis, health and functionality, ultimately depend on the nucleus being informed of the metabolic needs of the cell and the status of existing mitochondria, much is still unknown about direct mitochondria‐to‐nucleus communication strategies, particularly in mammalian cells. Here, we have identified a key mediator of the mitochondria retrograde signaling pathway in the transcriptional cofactor G‐Protein Suppressor 2 (GPS2). Our data reveals that GPS2 translocates from the outer mitochondrial membrane to the nucleus in a regulated manner to promote the activation of the nuclear‐encoded mitochondrial gene program through the establishment of a chromatin environment permissive for RNA polymerase II (POL2)‐mediated transcriptional activation. We will present in vitro data characterizing GPS2 role in regulating mitochondrial biogenesis during adipocyte differentiation and dissecting the molecular mechanism of GPS2‐mediated transcriptional regulation of nuclear encoded mitochondrial genes in both human and murine cell lines, as well as discuss the in vivo physiological relevance of this newly described pathway in brown adipose tissue. Together, our findings shed new light on the strategies evolved in eukaryotic cells to maintain efficient mitochondria:nuclear inter‐organellar communication, with important implications for our understanding of the molecular mechanisms underlying the regulation of mitochondria biogenesis and metabolic flexibility in both homeostasis and disease.

Support or Funding Information

Pilot and Feasibility Award from Boston Nutrition and Obesity Research Center (BNORC) to M.D.C Joslin/Boston University (Pilot and Feasibility Award) and NIH/NIDDK (R01DK100422) to V.P.
New Method for Isolation of Native Yeast Nucleosomes ‐ Development and Application
Authors: Kuznietsov Viacheslav I , Fox Catherine A , Denu John M
Published: November 2023

Due to the ease of genetic manipulation, yeast S. cerevisiae represent an attractive and accessible epigenetic model. However, isolation of proteins and protein complexes from yeast cells has always been a challenging and time‐consuming task associated with low yield and extensive degradation. To expand the utility of yeast as a powerful epigenetic tool, we developed a novel protocol for native chromatin purification. The protocol addresses numerous known, and previously unreported problems. Our method allows isolation of native nucleosomes with preserved combinatorial post translational modifications in a time‐ and cost‐effective manner. This procedure is suitable for downstream LC/MS analysis as well as recently developed matrix assisted reader chromatin capture (MARCC) and native chromatin precipitation (N‐chip) investigations.
Metabolic Regulation of Gene Expression by Histone Lysine β‐hydroxybutyrylation
Authors: Zhang Di , Xie Zhongyu , Chung Dongjun , Tang Zhanyun , Huang He , Dai Lunzhi , Qi Shankang , Li Jingya , Colak Gozde , Chen Yue , Peng Chao , Ruan Haibin , Wang Danli , Jensen Lindy M. , Kwon Oh Kwang , Lee SangKyu , Pletcher Scott D. , Tan Minjia , Lombard David B. , White Kevin P. , Zhao Hongyu , Li Jia , Roeder Robert G. , Yang Xiaoyong , Zhao Yingming
Published: November 2023

®‐hydroxybutyrate has been used to treat epilepsy and plays a neuro‐protective role in models of neurodegenerative diseases. Ketogenic diets are under evaluation as adjunctive treatment for patients with brain tumors and other malignancies. These lines of evidence suggest a regulatory role for ketone bodies beyond serving as an energy source. However, the mechanisms underlying these physiological and pharmacological effects of ketone bodies remain largely unknown.

We had previously identified lysine ®‐hydroxybutyrylation (Kbhb) as a new type of histone modification. We showed that histone Kbhb is dramatically induced in livers from mice subjected to prolonged fasting or streptozotocin‐induced diabetic ketoacidosis. By ChIP‐seq and RNA‐seq analysis, we demonstrated that histone Kbhb is a mark enriched in active gene promoters, and that the increased H3K9bhb levels during starvation are associated with genes up‐regulated in starvation‐responsive metabolic pathways. We now have new data to show that p300 can catalyze histone Kbhb both in vitro and in cultured cell lines. Using in vitro transcription assay with recombinant chromatin, we demonstrate that p300‐catalyzed histone Kbhb can activate transcription directly.

All together, our study suggest histone Kbhb as a new epigenetic regulatory mark that couples metabolism to gene expression, offering a new avenue to study chromatin regulation and the diverse functions of ®‐hydroxybutyrate in the context of important human pathophysiological states, including diabetes, epilepsy, and neoplasia.

Support or Funding Information

The work was supported by: NIH DK089098, P01 DK057751, CT DPH 2014‐0139 and Ellison Medical Foundation to X.Y.; NIH DK71900 and the Starr Foundation Tri‐Institutional Stem Cell Initiative (2014‐021) to R.G.R.; NIH R01GM101171 and R21CA177925 to D.L.; NIH GM59507 to H. Z.; NIH R01AG030593 and R01AG023166 to S.D.P.; National Natural Science Foundation of China (81125023) and Shanghai Commission of Science and Technology (14431902800) to J.L and J.L.; National Basic Research Program of China (973 Program) (No. 2014CBA02004) and the Shanghai Municipal Science and Technology Commission (No. 15410723100) to M.T.
DISCOVERING SELECTIVITY IN BET BROMODOMAIN EPIGENETIC REGULATION
Authors: Olp Mike
Published: November 2023

Bromodomains are ~110 amino‐acid chromatin interaction modules that bind acetyl‐lysine residues on histones and other nuclear proteins, thereby controlling gene expression through directing assembly of transcriptional complexes on chromatin. The human genome encodes 61 bromodomains distributed among 46 bromodomain‐containing proteins. Members of the bromodomain extraterminal domain (BET) family (BRD2, BRD3, BRD4, and BRDT) contain two bromodomains (BD1 and BD2) at their N‐termini followed by an extra‐terminal domain at their C‐termini. BET bromodomain inhibitors are currently in phase I clinical trials for the treatment of various hematological malignancies and solid tumors. In addition, inhibition of BET protein‐mediated transcription is protective in various models of inflammatory disease including infection, atherosclerosis, lung fibrosis, autoimmune diseases and diabetes. Yet despite their widespread relevance in human disease, little is known about the basic protein‐protein interactions that allow BET bromodomains to exert transcriptional regulation. For instance, the non‐selective nature of current BET bromodomain inhibitors has made it difficult to distinguish between activities carried out by individual BET proteins. To address this issue, we take a covalent fragment‐based approach toward selectively targeting BRD4, the BET family member thought to be the relevant therapeutic target for most cancer and inflammatory applications. In addition to achieving pharmacological specificity, we characterize the binding selectivity of BET bromodomains toward recently discovered acyl‐lysine marks beyond acetylation such as formylation, propionylation, butyrylation, crotonylation, hydroxyisobutyrylation, malonylation, succinylation and glutarylation. We then begin to investigate ways that differential bromodomain binding to these non‐acetyl lysine acylations may complicate regulation of BET‐mediated transcription in the pathogenesis of diabetes.
Identification of Inhibitors of Human Histone Methyltransferases SUV39H1 and SUV39H2
Authors: Spencer Angie C , Jahan Asmat , Shaikh Zahid , Liu Kebin
Published: November 2023

Metastasis of cancer represents a significant challenge in the fight against the disease. In human colorectal cancer (CRC) if the cancer has metastasized to the liver, the 5‐year survival rates drops to 10% making it the third leading cause of cancer‐related deaths in the United States. Hence, it is imperative to elucidate the mechanisms that characterize tumor progression in CRC. One such cell‐intrinsic pathway which induces programmed cell death, or apoptosis, is ligand cell surface death receptors, including Fas protein. In normal human colon tissues, Fas is constitutively expressed, but in primary colorectal cancer, Fas expression is often diminished and complete loss of Fas expression is a hallmark of human metastatic CRC. This silencing of Fas expression is perhaps a mechanism by which human colorectal cancer cells evade apoptosis. The decrease in Fas expression levels is associated with hypermethylation of histone 3 lysine 9 catalyzed by histone methyltransferases (HMTases) such as SUV39H1 and SUV39H2. Because of the role of methylation in silencing Fas expression, HMTase inhibitors represent potential anti‐cancer agents. Verticillin A, a natural compound from mild mushroom, has been identified as an inhibitor of HMTase activity and its action increased Fas expression levels in human colon carcinoma cells. However, Verticillin A is a broad‐based HMTase inhibitor and has been shown to cause toxicity in mice. Our goal in this study is to identify specific SUV39H1 and SUV39H2 inhibitors that are less toxic than Verticillin A. To investigate this, human SUV39H1 and SUV39H2 were sub‐cloned into an expression vector, transformed into an E. coli expression strain and purified using affinity chromatography. Gel electrophoresis and Western blot analysis confirmed the presence of human SUV39H1 and SUV39H2. The enzymatic activity of the HMTases was confirmed using an in vitro assay utilizing S‐(methyl‐3H) adenosyl‐L‐methionine (3H‐SAM) and a histone 3 peptide. Potential small molecule inhibitors of SUV39H1 and SUV39H2 were identified using molecular docking and tested against the recombinant proteins. From over 100 compounds, five compounds were found to inhibit SUV39H1 activity and two were found to inhibit SUV39H2 activity.
Biological Function and Histone Recognition of Family IV Bromodomain‐Containing Proteins
Authors: Lloyd Jonathan T. , Poplawaski Amanda , Lubula Mulu Y. , Carlson Samuel , Gay Jamie , Glass Karen C.
Published: November 2023

Bromodomain proteins function as epigenetic readers that recognize acetylated histone tails to facilitate transcription of target genes. There are approximately 60 known human bromodomains, and these are split into 8 sub‐families based on structural conservation. The bromodomain‐containing proteins in family IV consist of 7 members (BRPF1, BRPF2, BRPF3, BRD7, BRD9, ATAD2a, and ATAD2b). The bromodomains of each of these proteins recognize and bind acetyllysine residues on histone tails protruding from the nucleosome. However, the histone marks recognized by each bromodomain protein, can be very different. Our research revealed that the BRPF1 subunit of the MOZ histone acetyltransferase (HAT) recognizes histone acetylated at H2AK5ac, H4K12ac, H3K14ac, H4K8ac and H4K5ac1. While the bromodomain of BRD7, a member of the SWI/SNF complex, was shown to preferentially recognize histones H3K9ac, H3K14ac, H4K8ac, H4K12ac and H4K16ac2. The bromodomains of BRPF2 and BRPF3 are very similar in amino acid sequence, and function as part of the HBO1 HAT complex, but there is limited data on which histone ligands they bind3. Similarly, there is little known about the histone targets of the BRD9 and ATAD2b bromodomain proteins. Interestingly, the ATAD2a bromodomain was recently shown to preferentially bind to H4K5acK12ac in newly synthesized histones following DNA replication4. ATAD2 is a co‐activator of the estrogen and androgen receptors, and has also been shown to stimulate MYC and E2F‐dependent cell proliferation. Additionally, ATAD2a is overexpressed in a range of cancers including lung, breast, and prostate cancer, as well as ovarian and hepatocellular carcinoma. However, despite the physiological importance of the family IV bromodomains, little is known about how they function at the molecular or atomic level. Here, we summarize our understanding of how family IV bromodomains recognize and select for acetyllysine marks by providing the molecular details of ligand binding, which will be critical for the development of new therapeutic interventions targeting these bromodomains.

Support or Funding Information

Supported by a National Institutes of Health grant, NIGMS 1R15GM104865, and an ACPHS scholarship of discovery grant.
Class I‐Specific HDAC Inhibitor Stimulates the Expression of Npr1 in Haplotype Mice by Enhanced Histone Acetylation at Different Lysine Residues
Authors: Kumar Prerna , Gogulamudi Venkateshwara R. , Nguyen Christian , Pandey Kailash N.
Published: November 2023

Guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) signaling activates natriuresis/diuresis, vasodilatation, and inhibits the cell proliferation and fibrosis. The objective of the present study was to gain insight into the mechanisms of Npr1 (coding for GC‐A/NPRA) expression and receptor function involving epigenetic regulatory mechanisms activated by class I histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103). We utilized 20‐ to 22‐weeks old male Npr1 gene‐knockout haplotype (1‐copy; Npr1+/−), wild‐type (2‐copy; Npr1+/+) and gene‐duplicated heterozygous (3‐copy; Npr1++/+) mice. The mice were injected intraperitonealy with MGCD0103 (5 mg/kg) at the alternate days for 2‐weeks. The Western blot analysis showed that MGCD0103 significantly increased the renal NPRA mRNA and protein levels in all mice groups compared with vehicle‐treated controls. There was marked increase in renal cGMP levels in MGCD0103‐treated mice groups. The Npr1 haplotype mice exhibited increased HDAC activity and decreased acetylated histone levels; however, 3‐copy mice showed lower HDAC activity compared with 2‐copy mice. Treatment with MGCD0103 significantly attenuated HDAC activity by almost 50% in Npr1 haplotype mice. A substantial global increase in acetylation levels of histone 3 at different lysine positions (H3‐K9/14ac, H3‐K18ac, and H3‐K27ac) and histone 4 lysine 8 (H4‐K8ac) was observed in the kidneys of MGCD0103‐treated mice. The present results suggest that MGCD0103 epigenetically regulates NPRA expression in vivo via HDAC inhibition and histone modifications, which might have important implications in the renal function in disease states.

Support or Funding Information

This work was supported by NIH grants (HL057531 and HL062147) and by an Institutional Development Award (IDeA) from the NIGMS.
Regulation of Histone Methylation via Methionine Metabolism
Authors: Haws Spencer A. , Denu John M.
Published: November 2023

S‐adenosylmethionine (SAM) is the sole methyl‐donor for methyltransferase reactions in the cell. However, given SAM's inherent instability and high energetic cost of synthesis, the mechanism by which methyltransferases acquire this metabolite poses an interesting problem as uptake via diffusion appears inefficient. Recent data suggest that SAM may be directly channeled from its synthetase to histone methyltransferases (HMTs) in response to glucose‐derived serine metabolism as well as oxidative stress. We have investigated the influence of one‐carbon metabolism on nuclear SAM utilization, using methionine restriction to mimic methyl‐donor metabolite depletion in HCT116 cells. When HCT116 cells are subjected to methionine restriction, we identify significant alterations in one‐carbon metabolism, including a complete loss of free intracellular methionine after 5 minutes and a 5‐fold reduction in SAM concentrations after 1 hour. To determine how these fluctuations in one‐carbon metabolism influence histone post‐translational modifications (PTMs), we used mass spectrometry to identify the global histone PTM profiles of HCT116 cells subjected to a methionine restriction time course. While we observed an overall decrease in histone methylation throughout the time course, we also detected a preservation of mono‐methyl PTMs on H3K9 and H3K27. Knowing that the mammalian SAM synthetase MAT2A is responsive to methionine availability, we next analyzed the histone PTM profiles of MAT2A knockdown cells via mass spectrometry. In agreement with the methionine restriction results, we observed maintenance of mono‐methyl PTMs on H3K9 and H3K27 while global histone methylation levels decreased. These results suggest that MAT2A may be preferentially providing SAM to H3K9 and H3K27 mono‐methyl HMTs during one‐carbon metabolite depletion in an attempt to preserve crucial epigenetic memory until environmental conditions improve.

Support or Funding Information

This project was supported by NIH GM059789‐15/P250V A.
An Analysis of Cycling DOF Factor‐Like Genes and Their Expression in Physcomitrella patens
Authors: Pang Jonathan C. , Hicks Karen A.
Published: November 2023

A great deal of research has been done on the subject of how flowering plants use changes in photoperiod and temperature to regulate reproductive development. However, it is unclear whether these molecular pathways are conserved in other major clades of the plant evolutionary tree. Here, we explore the molecular pathway for seasonal regulation of reproductive development in the bryophyte species Physcomitrella patens. Nonvascular plants, including bryophytes, diverged from vascular plants, a group that includes flowering plants, approximately 450 million years ago. Identifying genes in a bryophyte species that are similar in structure and function to genes responsible for seasonal regulation in flowering plants, would indicate that seasonal regulation arose in a common ancestor over 450 million years ago. Alternatively, it could be that seasonal regulation evolved independently in nonvascular plants. One way we explore the evolution of seasonal regulation is by investigating genes in Physcomitrella patens that appear to be related to Cycling DOF Factor (CDF), a gene involved in seasonal regulation in flowering plants. CDFs belong to the DOF (DNA‐binding One Zinc Finger) family of transcription factors. In flowering plants, CDFs inhibit the expression of genes that promote reproductive development. We seek to determine whether similar genes perform the same function in Physcomitrella patens. Working from RNA‐seq data collected from Physcomitrella patens, we identified CDF‐like genes that appear to be differentially expressed when exposed to a photoperiod and temperature that induce reproduction. Here, we present our methods and the results of our investigation of the expression of these CDF‐like genes using qRT‐PCR. Our findings on the expression of CDF‐like genes provide insight into whether regulatory pathways found in flowering plants are conserved in other plant lineages.