RESUMO
Ensuring the public has a fundamental understanding of human-microbe interactions, immune responses, and vaccines is a critical challenge in the midst of a pandemic. These topics are commonly taught in undergraduate- and graduate-level microbiology and immunology courses; however, creating engaging methods of teaching these complex concepts to students of all ages is necessary to keep younger students interested when science seems hard. Building on the Tactile Teaching Tools with Guided Inquiry Learning (TTT-GIL) method we used to create an interactive lac operon molecular puzzle, we report here two TTT-GIL activities designed to engage diverse learners from middle schoolers to masters students in exploring molecular interactions within the immune system. By pairing physical models with structured activities built on the constructivist framework of Process-Oriented Guided Inquiry Learning (POGIL), TTT-GIL activities guide learners through their interaction with the model, using the Learning Cycle to facilitate construction of new concepts. Moreover, TTT-GIL activities are designed utilizing Universal Design for Learning (UDL) principles to include all learners through multiple means of engagement, representation, and action. The TTT-GIL activities reported here include a web-enhanced activity designed to teach concepts related to antibody-epitope binding and specificity to deaf and hard-of-hearing middle and high school students in a remote setting and a team-based activity that simulates the evolution of the Major Histocompatibility Complex (MHC) haplotype of a population exposed to pathogens. These activities incorporate TTT-GIL to engage learners in the exploration of fundamental immunology concepts and can be adapted for use with learners of different levels and educational backgrounds.
RESUMO
Activation of germline promoters is central to V(D)J recombinational accessibility, driving chromatin remodeling, nucleosome repositioning, and transcriptional read-through of associated DNA. We have previously shown that of the two TCRß locus (Tcrb) D segments, Dß1 is flanked by an upstream promoter that directs its transcription and recombinational accessibility. In contrast, transcription within the DJß2 segment cluster is initially restricted to the J segments and only redirected upstream of Dß2 after D-to-J joining. The repression of upstream promoter activity prior to Tcrb assembly correlates with evidence that suggests DJß2 recombination is less efficient than that of DJß1. Because inefficient DJß2 assembly offers the potential for V-to-DJß2 recombination to rescue frameshifted V-to-DJß1 joints, we wished to determine how Dß2 promoter activity is modulated upon Tcrb recombination. In this study, we show that repression of the otherwise transcriptionally primed 5'Dß2 promoter requires binding of upstream stimulatory factor (USF)-1 to a noncanonical E-box within the Dß2 12-recombination signal sequence spacer prior to Tcrb recombination. USF binding is lost from both rearranged and germline Dß2 sites in DNA-dependent protein kinase, catalytic subunit-competent thymocytes. Finally, genotoxic dsDNA breaks lead to rapid loss of USF binding and gain of transcriptionally primed 5'Dß2 promoter activity in a DNA-dependent protein kinase, catalytic subunit-dependent manner. Together, these data suggest a mechanism by which V(D)J recombination may feed back to regulate local Dß2 recombinational accessibility during thymocyte development.
Assuntos
Diferenciação Celular/genética , Diferenciação Celular/imunologia , DNA Intergênico/química , Rearranjo Gênico da Cadeia delta dos Receptores de Antígenos dos Linfócitos T/imunologia , Subpopulações de Linfócitos T/imunologia , Fatores Estimuladores Upstream/antagonistas & inibidores , Fatores Estimuladores Upstream/fisiologia , Regiões 5' não Traduzidas/genética , Regiões 5' não Traduzidas/imunologia , Regiões 5' não Traduzidas/efeitos da radiação , Animais , Sequência de Bases , Diferenciação Celular/efeitos da radiação , Linhagem Celular , Cobalto , DNA Intergênico/efeitos da radiação , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/efeitos da radiação , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transdução de Sinais/efeitos da radiação , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/efeitos da radiação , Fatores Estimuladores Upstream/genéticaRESUMO
Vertebrate development requires the formation of multiple cell types from a single genetic blueprint, an extraordinary feat that is guided by the dynamic and finely tuned reprogramming of gene expression. The sophisticated orchestration of gene expression programs is driven primarily by changes in the patterns of covalent chromatin modifications. These epigenetic changes are directed by cis elements, positioned across the genome, which provide docking sites for transcription factors and associated chromatin modifiers. Epigenetic changes impact all aspects of gene regulation, governing association with the machinery that drives transcription, replication, repair and recombination, a regulatory relationship that is dramatically illustrated in developing lymphocytes. The program of somatic rearrangements that assemble antigen receptor genes in precursor B and T cells has proven to be a fertile system for elucidating relationships between the genetic and epigenetic components of gene regulation. This chapter describes our current understanding of the cross-talk between key genetic elements and epigenetic programs during recombination of the Tcrb locus in developing T cells, how each contributes to the regulation of chromatin accessibility at individual DNA targets for recombination, and potential mechanisms that coordinate their actions.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/metabolismo , Animais , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Recombinação Genética , Linfócitos T/citologiaRESUMO
Iron is an essential element for the survival of living cells. However, excess iron is toxic, and its uptake is exquisitely regulated by the ferric uptake regulator, Fur. In Salmonella, the Salmonella pathogenicity island 1 (SPI-1) encodes a type three secretion system, which is required for invasion of host epithelial cells in the small intestine. A major activator of SPI-1 is HilA, which is encoded within SPI-1. One known regulator of hilA is Fur. The mechanism of hilA regulation by Fur is unknown. We report here that Fur is required for virulence in Salmonella enterica serovar Typhimurium and that Fur is required for the activation of hilA, as well as of other HilA-dependent genes, invF and sipC. The Fur-dependent regulation of hilA was independent of PhoP, a known repressor of hilA. Instead, the expression of the gene coding for the histone-like protein, hns, was significantly derepressed in the fur mutant. Indeed, the activation of hilA by Fur was dependent on 28 nucleotides located upstream of hns. Moreover, we used chromatin immunoprecipitation to show that Fur bound, in vivo, to the upstream region of hns in a metal-dependent fashion. Finally, deletion of fur in an hns mutant resulted in Fur-independent activation of hilA. In conclusion, Fur activates hilA by repressing the expression of hns.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Salmonella typhimurium/fisiologia , Transativadores/biossíntese , Fatores de Virulência/biossíntese , Animais , Proteínas de Bactérias/genética , Imunoprecipitação da Cromatina , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/biossíntese , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Genes Bacterianos , Ilhas Genômicas , Camundongos , Camundongos Endogâmicos C3H , Óperon , Ligação Proteica , Proteínas Repressoras/genética , Salmonelose Animal/microbiologia , Salmonella typhimurium/patogenicidade , Análise de Sobrevida , Fatores de Transcrição/biossíntese , VirulênciaRESUMO
BACKGROUND: Tetra-O-methyl nordihydroguaiaretic acid, also known as terameprocol (TMP), is a naturally occurring phenolic compound found in the resin of the creosote bush. We have shown previously that TMP will suppress production of certain inflammatory cytokines, chemokines and lipids from macrophages following stimulation with LPS or infection with H1N1 influenza virus. In this study our goal was to elucidate the mechanism underlying TMP-mediated suppression of cytokine and chemokine production. We focused our investigations on the response to LPS and the NF-κB protein RelA, a transcription factor whose activity is critical to LPS-responsiveness. METHODS: Reporter assays were performed with HEK293 cells overexpressing either TLR-3, -4, or -8 and a plasmid containing the luciferase gene under control of an NF-κB response element. Cells were then treated with LPS, poly(I:C), or resiquimod, and/or TMP, and lysates measured for luciferase activity.RAW 264.7 cells treated with LPS and/or TMP were used in ChIP and EMSA assays. For ChIP assays, chromatin was prepared and complexes precipitated with anti-NF-κB RelA Ab. Cross-links were reversed, DNA purified, and sequence abundance determined by Q-PCR. For EMSA assays, nuclear extracts were incubated with radiolabeled probes, analyzed by non-denaturing PAGE and visualized by autoradiography.RAW 264.7 cells treated with LPS and/or TMP were also used in fluorescence microscopy and western blot experiments. Translocation experiments were performed using a primary Ab to NF-κB RelA and a fluorescein-conjugated secondary Ab. Western blots were performed using Abs to IκB-α and phospho-IκB-α. Bands were visualized by chemiluminescence. RESULTS: In reporter assays with TLR-3, -4, and -8 over-expressing cells, TMP caused strong inhibition of NF-κB-dependent transcription.ChIP assays showed TMP caused virtually complete inhibition of RelA binding in vivo to promoters for the genes for TNF-α, MCP-1/CCL2, and RANTES/CCL5 although the LPS-dependent synthesis of IκB-α was not inhibited. EMSA assays did not reveal an effect of TMP on the binding of RelA to naked DNA templates in vitro.TMP did not inhibit the nuclear translocation of NF-κB RelA nor the phosphorylation of IκB-α. CONCLUSION: TMP acts indirectly as an inhibitor of NF-κB-dependent transcription by preventing RelA from binding the promoters of certain key cytokine and chemokine genes.
RESUMO
Developmental patterning of antigen receptor gene assembly in lymphocyte precursors correlates with decondensation of the chromatin surrounding individual gene segments. Ongoing V(D)J recombination is associated with hyperacetylation of histones H3 and H4 and the expression of sterile germline transcripts across the region of recombinational accessibility. Likewise, histone acetyltransferase and SWI/SNF chromatin remodeling complexes each appear to be required for recombination, and the PHD-finger of RAG-2 preferentially associates with recombination signal sequence (RSS) chromatin that contains H3 trimethylated on lysine 4. However, the regulatory mechanisms that direct chromatin alteration and rearrangement have proven elusive, due in large part to the interdependency of individual stages in gene activation, our limited understanding of functional significance of changes to the histone code, and the difficulty of modeling recombinational accessibility in existing experimental systems. Examining Tcrb assembly in developing thymocytes, we review the central roles of RSS elements and germline promoters as foci for epigenetic reorganization of recombinationally accessible gene segments in light of recent findings and persistent questions.
Assuntos
Rearranjo Gênico do Linfócito T , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Timo/enzimologia , VDJ Recombinases/metabolismo , Acetilação , Animais , Montagem e Desmontagem da Cromatina , Epigênese Genética , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , Timo/imunologia , Transcrição GênicaRESUMO
Campylobacter jejuni is an important human pathogen that causes millions of cases of food-borne enteritis each year. The C. jejuni respiratory chain is highly branched and contains at least four enzymes predicted to contain a metal binding pterin (MPT), with the metal being either molybdenum or tungsten. Also predicted are two separate transport systems, one for molybdenum encoded by modABC and a second for tungsten encoded by tupABC. Both transport systems were mutated and the activities of the four predicted MPT-containing enzymes were assayed in the presence of molybdenum and tungsten in wild-type and mod and tup backgrounds. Results indicate that mod is primarily a molybdenum transporter that can also transport tungsten, while tup is a tungsten-specific transporter. The MPT containing enzymes nitrate reductase, sulphite oxidase, and SN oxide reductase are strict molybdoenzymes while formate dehydrogenase prefers tungsten. A ModE-like protein regulates both transporters, repressing mod in the presence of both molybdenum and tungsten and tup only in the presence of tungsten. Like other ModE proteins, the C. jejuni ModE binds DNA through a helix-turn-helix DNA binding domain, but unlike other members of the ModE family it does not have a metal binding domain.
Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Molibdênio/metabolismo , Fatores de Transcrição/metabolismo , Tungstênio/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Campylobacter jejuni/genética , Transporte de Elétrons/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Formiato Desidrogenases/genética , Formiato Desidrogenases/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Molibdênio/química , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Nitrato Redutase (NAD(P)H)/metabolismo , Nitrato Redutases/química , Nitrato Redutases/genética , Nitrato Redutases/metabolismo , Filogenia , Engenharia de Proteínas , Pterinas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tungstênio/químicaRESUMO
V(D)J recombination involves the stepwise assembly of B and T cell receptor genes as lymphocytes progress through the early stages of development. While the mechanisms that restrict each step in recombination to its appropriate developmental stage are largely unknown, they share many of the components that regulate transcription. For example, enhancer-dependent modifications in histone acetylation and methylation are essential for both germline transcription and rearrangement of antigen receptor genes. Promoters positioned proximal to individual D and J gene segments in Tcra, Tcrb, Tcrd, IgH, and Igk also contribute to antigen receptor gene assembly, though their effects appear more localized than those of enhancers. Tcrb assembly initiates with D-to-J joining at each of the two D-J-C gene segment clusters in DN1/2 thymocytes. DJ joints are fused with Vbeta elements to complete Tcrb recombination in DN3 cells. We have previously shown that Dbeta2 is flanked by upstream and downstream promoters, with the 5' promoter being held inactive until D-to-J recombination deletes the NFkappaB-dependent 3' promoter. We now report that activity of the 5' promoter reflects a complex interplay among Runx1, GATA-3, and E47 transcription factors. In particular, while multiple E47 and Runx1 binding sites clustered near the Dbeta2 5'RS and overlapping inr elements define the core 5'PDbeta2, they act in concert with an array of upstream GATA-3 sites to overcome the inhibitory effects of a 110bp distal polypurine.polypyrimidine (R.Y) tract. The dependence of 5'PDbeta2 on E47 is consistent with the reported role of E proteins in post-DN1 thymocyte development and V-to-DJbeta recombination.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/imunologia , Fator de Transcrição GATA3/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Regiões Promotoras Genéticas , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Fatores de Transcrição TCF/imunologia , Animais , Sequência de Bases , Diferenciação Celular/imunologia , Linhagem Celular , Cromatina/imunologia , Cromatina/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Fator de Transcrição GATA3/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Transcrição TCF/metabolismo , Timo/imunologia , Timo/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Proteína Supressora de Tumor p53/metabolismo , Fatores Estimuladores Upstream/imunologia , Fatores Estimuladores Upstream/metabolismoRESUMO
We report a streamlined procedure to efficiently carry samples from chromatin to qPCR-compatible DNA in as little as 4 h. We use this streamlined ChIP to quantify histone H3 modifications at active (cad) and repressed (T early alpha) promoters in a Rag1-deficient pro-T cell line after 1-2 h IP. We further show that the protocol readily quantified histone modifications in chromatin from 10(4) Rag-deficient DN thymocytes. Taken together, these data outline a simple, cost-effective procedure for efficient ChIP analysis.
Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/isolamento & purificação , Histonas/metabolismo , Animais , Aspartato Carbamoiltransferase/genética , Aspartato Carbamoiltransferase/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Di-Hidro-Orotase/genética , Di-Hidro-Orotase/metabolismo , Técnicas de Silenciamento de Genes , Histonas/genética , Camundongos , Camundongos Knockout , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Linfócitos T/metabolismoRESUMO
Ag receptor genes are assembled through somatic rearrangements of V, D, and J gene segments. This process is directed in part by transcriptional enhancers and promoters positioned within each gene locus. Whereas enhancers coordinate reorganization of large chromatin stretches, promoters are predicted to facilitate the accessibility of proximal downstream gene segments. In TCR beta locus, rearrangement initiates at two D-J cassettes, each of which exhibits transcriptional activity coincident with DJ rearrangement in CD4/CD8 double-negative pro-T cells. Consistent with a model of promoter-facilitated recombination, assembly of the DJbeta1 cassette is dependent on a Dbeta1 promoter (PDbeta1) positioned immediately 5' of the D. Assembly of DJbeta2 proceeds independent from that of DJbeta1, albeit with less efficiency. To gain insight into the mechanisms that selectively alter D usage, we have defined transcriptional regulation at Dbeta2. We find that both DJbeta cassettes generate germline messages in murine CD44+CD25- double-negative 1 cells. However, transcription of unrearranged DJbeta2 initiates at multiple sites 400-550 bp downstream of the Dbeta2. Unexpectedly, loci from which germline promoter activity has been deleted by DJ rearrangement redirect transcription to sites immediately 5' of the new DJbeta2 joint. Our analyses suggest that 3'-PDbeta2 activity is largely controlled by NF-kappaB RelA, whereas 5'-PDbeta2 activity directs germline transcription of DJbeta2 joints from initiator elements 76 bp upstream of the Dbeta2 5' recombination signal sequence. The unique organization and timing of Dbeta2 promoter activity are consistent with a model in which promoter placement selectively regulates the rearrangement potential of Dbeta2 during TCR beta locus assembly.
Assuntos
Diferenciação Celular/genética , Diferenciação Celular/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Regiões Promotoras Genéticas/imunologia , Timo/imunologia , Timo/metabolismo , Transcrição Gênica/imunologia , Ativação Transcricional/imunologia , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Mutação em Linhagem Germinativa/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Timo/citologia , Timo/enzimologia , VDJ Recombinases/genéticaRESUMO
Mre11, Rad50, and Nbs1 function in a protein complex that is central to the metabolism of chromosome breaks. Null mutants of each are inviable. We demonstrate here that hypomorphic Rad50 mutant mice (Rad50(S/S) mice) exhibited growth defects and cancer predisposition. Rad50(S/S) mice died with complete bone marrow depletion as a result of progressive hematopoietic stem cell failure. Similar attrition occurred in spermatogenic cells. In both contexts, attrition was substantially mitigated by p53 deficiency, whereas the tumor latency of p53(-/-) and p53(+/-) animals was reduced by Rad50(S/S). Indices of genotoxic stress and chromosomal rearrangements were evident in Rad50(S/S) cultured cells, as well as in Rad50(S/S) and p53(-/-) Rad50(S/S) lymphomas, suggesting that the Rad50(S/S) phenotype was attributable to chromosomal instability. These outcomes were not associated with overt defects in the Mre11 complex's previously established double strand break repair and cell cycle checkpoint regulation functions. The data indicate that even subtle perturbation of Mre11 complex functions results in severe genotoxic stress, and that the complex is critically important for homeostasis of proliferative tissues.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Hematopoese/genética , Mutação , Neoplasias Experimentais/genética , Alelos , Animais , Sequência de Bases , Dano ao DNA , Reparo do DNA , Enzimas Reparadoras do DNA , DNA Complementar/genética , Feminino , Genes p53 , Hematopoese/fisiologia , Proteína Homóloga a MRE11 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Neoplasias Experimentais/fisiopatologia , Fenótipo , Recombinação Genética , Espermatogênese/genética , Espermatogênese/fisiologiaRESUMO
Antigen receptor gene assembly is regulated by transcriptional promoters and enhancers, which control the accessibility of gene segments to a lymphocyte-specific V(D)J recombinase. However, it remained unclear whether accessibility depends on the process of transcription itself or chromatin modifications that accompany transcription. By using T cell receptor beta substrates that integrate stably into nuclear chromatin, we show that promoter location, rather than germ-line transcription or histone acetylation, is a primary determinant of recombination efficiency. These spatial constraints on promoter positioning may reflect an RNA polymerase-independent mechanism to target adjacent gene segments for chromatin remodeling events that facilitate rearrangement.