RESUMO
Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.
Assuntos
Anticorpos/genética , Formação de Anticorpos/genética , Células Clonais/imunologia , Genômica , Animais , Anticorpos/imunologia , Células CHO , Efeitos da Posição Cromossômica/genética , Cricetulus , Dosagem de Genes/genética , Dosagem de Genes/imunologia , Genoma/genética , Humanos , Plasmídeos/genética , TransgenesRESUMO
Position effect variegation (PEV) is a perturbation of genes expression resulting from the changes in their chromatin organization due to the abnormal juxtaposition with heterochromatin. The exact molecular mechanisms of PEV remain enigmatic in spite of the long history of PEV studies. Here, we developed a genetic model consisting of PEV-inducing chromosome rearrangement and a reporter gene under control of the UAS regulatory element. Expression of the reporter gene could be regulated by adjustment of the GAL4 transactivator activity. Two UAS-based systems of expression control were tested - with thermosensitive GAL4 repressor GAL80ts and GAL4-based artificial transactivator GeneSwitch. Both systems were able to regulate the expression of the UAS-controlled reporter gene over a wide range, but GAL80ts repressed the reporter gene more efficiently. Measurements of the heterochromatin-mediated repression of the reporter gene in the GAL4+GAL80ts system point to the existence of a threshold level of expression, above which no PEV is observed.
Assuntos
Efeitos da Posição Cromossômica/genética , Drosophila/genética , Heterocromatina/genética , Animais , Heterocromatina/metabolismo , Modelos GenéticosRESUMO
Eukaryotic genomes are broadly divided between gene-rich euchromatin and the highly repetitive heterochromatin domain, which is enriched for proteins critical for genome stability and transcriptional silencing. This study shows that Drosophila KDM4A (dKDM4A), previously characterized as a euchromatic histone H3 K36 demethylase and transcriptional regulator, predominantly localizes to heterochromatin and regulates heterochromatin position-effect variegation (PEV), organization of repetitive DNAs, and DNA repair. We demonstrate that dKDM4A demethylase activity is dispensable for PEV. In contrast, dKDM4A enzymatic activity is required to relocate heterochromatic double-strand breaks outside the domain, as well as for organismal survival when DNA repair is compromised. Finally, DNA damage triggers dKDM4A-dependent changes in the levels of H3K56me3, suggesting that dKDM4A demethylates this heterochromatic mark to facilitate repair. We conclude that dKDM4A, in addition to its previously characterized role in euchromatin, utilizes both enzymatic and structural mechanisms to regulate heterochromatin organization and functions.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Heterocromatina/metabolismo , Histona Desmetilases/metabolismo , Animais , Biocatálise , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Efeitos da Posição Cromossômica/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Fertilidade/genética , Regulação da Expressão Gênica , Inativação Gênica , Histonas/metabolismo , Lisina/metabolismo , Metilação , Mutação/genética , Domínios Proteicos , Transcrição GênicaRESUMO
The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome.
Assuntos
Efeitos da Posição Cromossômica/genética , Proteínas Cromossômicas não Histona/genética , Replicação do DNA/genética , Epigênese Genética/genética , Proteínas de Schizosaccharomyces pombe/genética , Animais , Caenorhabditis elegans/genética , Centrômero/genética , Cromatina/efeitos dos fármacos , Cromatina/genética , DNA de Cadeia Simples/efeitos dos fármacos , Drosophila melanogaster/genética , Epigênese Genética/efeitos dos fármacos , Heterocromatina/efeitos dos fármacos , Heterocromatina/genética , Histonas/genética , Hidroxiureia/farmacologia , Nucleossomos/genética , Schizosaccharomyces/genéticaRESUMO
Interpretation of variants of uncertain significance, especially chromosomal rearrangements in non-coding regions of the human genome, remains one of the biggest challenges in modern molecular diagnosis. To improve our understanding and interpretation of such variants, we used high-resolution three-dimensional chromosomal structural data and transcriptional regulatory information to predict position effects and their association with pathogenic phenotypes in 17 subjects with apparently balanced chromosomal abnormalities. We found that the rearrangements predict disruption of long-range chromatin interactions between several enhancers and genes whose annotated clinical features are strongly associated with the subjects' phenotypes. We confirm gene-expression changes for a couple of candidate genes to exemplify the utility of our analysis of position effect. These results highlight the important interplay between chromosomal structure and disease and demonstrate the need to utilize chromatin conformational data for the prediction of position effects in the clinical interpretation of non-coding chromosomal rearrangements.
Assuntos
Efeitos da Posição Cromossômica/genética , Mapeamento Cromossômico , Cromossomos Humanos/genética , Rearranjo Gênico/genética , Predisposição Genética para Doença/genética , Genoma Humano/genética , Pontos de Quebra do Cromossomo , Regulação da Expressão Gênica/genética , Variação Genética/genética , Humanos , Hibridização in Situ Fluorescente , Cariótipo , Fenótipo , Translocação Genética/genéticaRESUMO
BACKGROUND: Array-comparative genomic hybridization (array-CGH) is a widely used technique to detect copy number variants (CNVs) associated with developmental delay/intellectual disability (DD/ID). AIMS: Identification of genomic disorders in DD/ID. MATERIALS AND METHODS: We performed a comprehensive array-CGH investigation of 1,015 consecutive cases with DD/ID and combined literature mining, genetic evidence, evolutionary constraint scores, and functional information in order to assess the pathogenicity of the CNVs. RESULTS: We identified non-benign CNVs in 29% of patients. Amongst the pathogenic variants (11%), detected with a yield consistent with the literature, we found rare genomic disorders and CNVs spanning known disease genes. We further identified and discussed 51 cases with likely pathogenic CNVs spanning novel candidate genes, including genes encoding synaptic components and/or proteins involved in corticogenesis. Additionally, we identified two deletions spanning potential Topological Associated Domain (TAD) boundaries probably affecting the regulatory landscape. DISCUSSION AND CONCLUSION: We show how phenotypic and genetic analyses of array-CGH data allow unraveling complex cases, identifying rare disease genes, and revealing unexpected position effects.
Assuntos
Variações do Número de Cópias de DNA/genética , Proteínas de Ligação a DNA/genética , Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Adolescente , Adulto , Criança , Pré-Escolar , Efeitos da Posição Cromossômica/genética , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Deficiências do Desenvolvimento/patologia , Feminino , Estudos de Associação Genética , Genômica , Humanos , Lactente , Deficiência Intelectual/patologia , Masculino , Linhagem , Fenótipo , Deleção de Sequência/genética , Adulto JovemRESUMO
Chromatin plays a critical role in faithful implementation of gene expression programs. Different post-translational modifications (PTMs) of histone proteins reflect the underlying state of gene activity, and many chromatin proteins write, erase, bind, or are repelled by, these histone marks. One such protein is UpSET, the Drosophila homolog of yeast Set3 and mammalian KMT2E (MLL5). Here, we show that UpSET is necessary for the proper balance between active and repressed states. Using CRISPR/Cas-9 editing, we generated S2 cells that are mutant for upSET We found that loss of UpSET is tolerated in S2 cells, but that heterochromatin is misregulated, as evidenced by a strong decrease in H3K9me2 levels assessed by bulk histone PTM quantification. To test whether this finding was consistent in the whole organism, we deleted the upSET coding sequence using CRISPR/Cas-9, which we found to be lethal in both sexes in flies. We were able to rescue this lethality using a tagged upSET transgene, and found that UpSET protein localizes to transcriptional start sites (TSS) of active genes throughout the genome. Misregulated heterochromatin is apparent by suppressed position effect variegation of the wm4 allele in heterozygous upSET-deleted flies. Using nascent-RNA sequencing in the upSET-mutant S2 lines, we show that this result applies to heterochromatin genes generally. Our findings support a critical role for UpSET in maintaining heterochromatin, perhaps by delimiting the active chromatin environment.
Assuntos
Cromatina/genética , Efeitos da Posição Cromossômica/genética , Proteínas de Drosophila/genética , Heterocromatina/genética , Proteínas Nucleares/genética , Animais , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Genoma de Inseto , Sequenciamento de Nucleotídeos em Larga Escala , Histona Desacetilases/genética , Histonas/genética , Humanos , Processamento de Proteína Pós-Traducional/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de SequênciaRESUMO
Nucleolus organizer regions (NORs) are chromosomal loci where hundreds of rRNA genes are clustered. Despite being nearly identical in sequence, specific rRNA genes are selected for silencing during development via choice mechanism(s) that remain unclear. In Arabidopsis thaliana, rRNA gene subtypes that are silenced during development were recently mapped to the NOR on chromosome 2, NOR2, whereas active rRNA genes map to NOR4, on chromosome 4. In a mutant line deficient for ATXR5 or ATXR6-dependent histone H3 lysine 27 (H3K27) monomethylation, we show that millions of base pairs of chromosome 4, including the telomere, TEL4N, and much of NOR4, have been converted to the corresponding sequences of chromosome 2. This genomic change places rRNA genes of NOR2, which are normally silenced, at the position on chromosome 4 where active rRNA genes are normally located. At their new location, NOR2-derived rRNA genes escape silencing, independent of the atxr mutations, indicating that selective rRNA gene silencing is chromosome 2-specific. The chromosome 2 position effect is not explained by the NOR2-associated telomere, TEL2N, which remains linked to the translocated NOR, implicating centromere-proximal sequences in silencing.
Assuntos
Arabidopsis/genética , Efeitos da Posição Cromossômica/genética , Cromossomos de Plantas/genética , Região Organizadora do Nucléolo/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Cruzamentos Genéticos , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas , Genótipo , Hibridização Genética , Modelos Genéticos , RNA Ribossômico/genética , Telômero/genéticaRESUMO
Enhancers are regulatory elements that promote gene expression in a spatio-temporal way and are involved in a wide range of developmental and disease processes. Both the identification and subsequent functional dissection of enhancers are key steps in understanding these processes. Several high-throughput approaches were recently developed for these purposes; however, in almost all cases enhancers are being tested outside their native chromatin context. Until recently, the analysis of enhancer activities at their native genomic locations was low throughput, laborious and time-consuming. Here, we discuss the potential of a powerful approach, TRIP, to study the functioning of enhancers in their native chromatin environments by introducing sensor constructs directly in the genome. TRIP allows for simultaneously analyzing the quantitative readout of numerous sensor constructs integrated at random locations in the genome. The high-throughput and flexible nature of TRIP opens up potential to study different aspects of enhancer biology at an unprecedented level.
Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos , Genômica , Efeitos da Posição Cromossômica/genética , Regulação da Expressão Gênica/genética , Genoma Humano , HumanosRESUMO
The influence of local chromatin context on gene expression can be explored by integrating a transcription reporter at different locations in the genome as a sensor. Here we provide a detailed protocol for analyzing thousands of reporters integrated in parallel (TRIP) at a genome-wide level. TRIP is based on tagging each reporter with a unique barcode, which is used for independent reporter expression analysis and integration site mapping. Compared with previous methods for studying position effects, TRIP offers a 100-1,000-fold higher throughput in a faster and less-labor-intensive manner. The entire experimental protocol takes â¼42 d to complete, with high-throughput sequencing and data analysis requiring an additional â¼11 d. TRIP was developed by using transcription reporters in mouse embryonic stem (mES) cells, but because of its flexibility the method can be used to probe the influence of chromatin context on a variety of molecular processes in any transfectable cell line.
Assuntos
Cromatina/metabolismo , Efeitos da Posição Cromossômica/genética , Genes Reporter/genética , Animais , Células Cultivadas , Elementos de DNA Transponíveis , Vetores Genéticos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Polinucleotídeos/genéticaRESUMO
Genes in multicellular organisms are expressed as part of a developmental program that is largely dependent on self-perpetuating higher-order chromatin states. The mechanism of establishing and maintaining these epigenetic events is well studied in Drosophila. The first known example of an epigenetic effect was that of (PEV) in Drosophila, which has been shown to be due to gene silencing via heterochromatin formation. We are investigating a process similar to Position Effect Variegation (PEV) using a mini-w transgene, called Pci, inserted in the upstream regulatory region of ci. The mini-white+ transgene in Pci is expressed throughout the adult eye; however, when other P or KP elements are present, a variegated eye phenotype results indicating random w+ silencing during development. This P element dependent silencing (PDS) can be modified by the haplo-suppressors/triplo-enhancers, Su(var)205 and Su(var)3-7, indicating that these heterochromatic modifiers also act dose dependently in PDS. Here we use a spontaneous derivative mutation of Pci called PciE1 (E1) that variegates like PDS in the absence of P elements, presumably due to an adjacent gypsy element insertion, to screen for second-site modifier mutations that enhance variable silencing of white+ in E1. We isolated 7 mutations in CG8878, an essential gene, that enhance the E1 variegated phenotype. CG8878, a previously uncharacterized gene, potentially encodes a serine/threonine kinase whose closest Drosophila paralogue, ballchen (nhk-1), phosphorylates histones. These mutant alleles enhance both PDS at E1 and Position Effect Variegation (PEV) at w(m4), indicating a previously unknown common silencing mechanism between the two.
Assuntos
Efeitos da Posição Cromossômica/genética , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Inativação Gênica , Mutação/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Animais , Homólogo 5 da Proteína Cromobox , Elementos Facilitadores Genéticos/genética , Evolução Molecular , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Fenótipo , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Zebrafish transgenesis is increasingly popular owing to the optical transparency and external development of embryos, which provide a scalable vertebrate model for in vivo experimentation. The ability to express transgenes in a tightly controlled spatio-temporal pattern is an important prerequisite for exploitation of zebrafish in a wide range of biomedical applications. However, conventional transgenesis methods are plagued by position effects: the regulatory environment of genomic integration sites leads to variation of expression patterns of transgenes driven by engineered cis-regulatory modules. This limitation represents a bottleneck when studying the precise function of cis-regulatory modules and their subtle variants or when various effector proteins are to be expressed for labelling and manipulation of defined sets of cells. Here, we provide evidence for the efficient elimination of variability of position effects by developing a PhiC31 integrase-based targeting method. To detect targeted integration events, a simple phenotype scoring of colour change in the lens of larvae is used. We compared PhiC31-based integration and Tol2 transgenesis in the analysis of the activity of a novel conserved enhancer from the developmentally regulated neural-specific esrrga gene. Reporter expression was highly variable among independent lines generated with Tol2, whereas all lines generated with PhiC31 into a single integration site displayed nearly identical, enhancer-specific reporter expression in brain nuclei. Moreover, we demonstrate that a modified integrase system can also be used for the detection of enhancer activity in transient transgenesis. These results demonstrate the power of the PhiC31-based transgene integration for the annotation and fine analysis of transcriptional regulatory elements and it promises to be a generally desirable tool for a range of applications, which rely on highly reproducible patterns of transgene activity in zebrafish.
Assuntos
Efeitos da Posição Cromossômica/genética , Marcação de Genes , Mutagênese Insercional/genética , Transgenes/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Encéfalo/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter/genética , Loci Gênicos/genética , Genoma/genética , Integrases/metabolismo , Cristalino/metabolismo , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Xenopus laevis/genéticaRESUMO
The SF1 insulator was found to contain a polyadenylation signal, which corresponded to the functional polyadenylation signal in embryonic S2 cells and the transgenic lines of Drosophila and bi-directional promoter that functioned in S2 cells. The studies performed did not confirm the ability of the SF1 insulator to protect expression of reporter gene white from the chromosome position effect in transgenic lines.
Assuntos
Efeitos da Posição Cromossômica/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Elementos Isolantes/genética , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/biossíntese , Proteínas de Drosophila/biossíntese , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Fatores de Processamento de RNA , Fatores de Transcrição/biossíntese , Transcrição GênicaRESUMO
A persistent question in epigenetics is how heterochromatin is targeted for assembly at specific domains, and how that chromatin state is faithfully transmitted. Stable heterochromatin is necessary to silence transposable elements (TEs) and maintain genome integrity. Both the RNAi system and heterochromatin components HP1 (Swi6) and H3K9me2/3 are required for initial establishment of heterochromatin structures in S. pombe. Here we utilize both loss of function alleles and the newly developed Drosophila melanogaster transgenic shRNA lines to deplete proteins of interest at specific development stages to dissect their roles in heterochromatin assembly in early zygotes and in maintenance of the silencing chromatin state during development. Using reporters subject to Position Effect Variegation (PEV), we find that depletion of key proteins in the early embryo can lead to loss of silencing assayed at adult stages. The piRNA component Piwi is required in the early embryo for reporter silencing in non-gonadal somatic cells, but knock-down during larval stages has no impact. This implies that Piwi is involved in targeting HP1a when heterochromatin is established at the late blastoderm stage and possibly also during embryogenesis, but that the silent chromatin state created is transmitted through cell division independent of the piRNA system. In contrast, heterochromatin structural protein HP1a is required for both initial heterochromatin assembly and the following mitotic inheritance. HP1a profiles in piwi mutant animals confirm that Piwi depletion leads to decreased HP1a levels in pericentric heterochromatin, particularly in TEs. The results suggest that the major role of the piRNA system in assembly of heterochromatin in non-gonadal somatic cells occurs in the early embryo during heterochromatin formation, and further demonstrate that failure of heterochromatin formation in the early embryo impacts the phenotype of the adult.
Assuntos
Proteínas Argonautas/genética , Efeitos da Posição Cromossômica/genética , Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Epigênese Genética , Interferência de RNA , Alelos , Animais , Animais Geneticamente Modificados , Proteínas Argonautas/metabolismo , Blastoderma/crescimento & desenvolvimento , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrião não Mamífero , Inativação Gênica , Heterocromatina/genética , Histona Desmetilases/metabolismoRESUMO
Position-effect variegation (PEV) results when a gene normally in euchromatin is juxtaposed with heterochromatin by rearrangement or transposition. When heterochromatin packaging spreads across the heterochromatin/euchromatin border, it causes transcriptional silencing in a stochastic pattern. PEV is intensely studied in Drosophila using the white gene. Screens for dominant mutations that suppress or enhance white variegation have identified many conserved epigenetic factors, including the histone H3 lysine 9 methyltransferase SU(VAR)3-9. Heterochromatin protein HP1a binds H3K9me2/3 and interacts with SU(VAR)3-9, creating a core memory system. Genetic, molecular, and biochemical analysis of PEV in Drosophila has contributed many key findings concerning establishment and maintenance of heterochromatin with concomitant gene silencing.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Efeitos da Posição Cromossômica/genética , Proteínas de Drosophila/genética , Drosophila/genética , Proteínas do Olho/genética , Rearranjo Gênico/genética , Inativação Gênica/fisiologia , Heterocromatina/genética , Modelos Genéticos , Animais , Efeitos da Posição Cromossômica/fisiologia , Drosophila/fisiologia , Rearranjo Gênico/fisiologia , Heterocromatina/fisiologia , Mutação/genéticaRESUMO
We have used a simple binomial model of stochastic transgene inactivation at the level of the chromosome or transgene, rather than the cellular level, for the analysis of two mouse transgenic lines that show variegated patterns of expression. This predicts the percentages of cells that express one, both or neither alleles of the transgene in homozygotes from the observed percentages of cells, which express the transgene in hemizygotes. It adequately explained the relationship between the numbers of cells expressing the transgene in hemizygous and homozygous mosaic 21OH/LacZ mouse adrenals and mosaic BLG/7 mouse mammary glands. The binomial model also predicted that a small proportion of cells in mosaic mammary glands of BLG/7 homozygotes would express both BLG/7 alleles but published data indicated that all cells expressing the transgene showed monoallelic expression. Although it didn't fit all of the BLG/7 data as precisely as a more complex model, which used several ad hoc assumptions to explain these results, the simple binomial model was able to explain the relationship in observed transgene expression frequencies between hemizygous and homozygous mosaic tissues for both 21OH/LacZ and BLG/7 mice. It may prove to be a useful general model for analysing other transgenic animals showing mosaic transgene expression.
Assuntos
Efeitos da Posição Cromossômica/genética , Hemizigoto , Homozigoto , Esteroide 21-Hidroxilase/biossíntese , Córtex Suprarrenal/metabolismo , Alelos , Animais , Feminino , Expressão Gênica , Óperon Lac , Masculino , Camundongos , Camundongos Transgênicos , Esteroide 21-Hidroxilase/genéticaRESUMO
Eu-heterochromatic rearrangements transfer genes into the heterochromatin and cause their variegated inactivation (PEV). Genes affected by PEV often demonstrate association with heterochromatic nuclear compartment (a distinct area composed of heterochromatin sequences like satellite DNA and enriched in specific chromatin proteins e.g. HP1). Here, we investigate the nuclear localization and the expression levels of the genes subjected to PEV caused by chromosome inversion, In(2)A4. We demonstrate that the degree of PEV-caused gene inactivation depends on a developmental stage, and the maximum of repression corresponds to the gene expression activation period. In the case of In(2)A4 rearrangement we detect the dragging of affected euchromatic region into heterochromatic nuclear compartment and the increase in HP1 occupancy in this region. We developed a protocol of simultaneous RNA-DNA-protein staining to demonstrate firstly in a single cell a strong correlation between transcriptional activity of affected gene and its distance from chromosome 2 satellite DNA.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Efeitos da Posição Cromossômica/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Heterocromatina/genética , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Inversão Cromossômica/genética , DNA Satélite/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Eucromatina/genética , Regulação da Expressão Gênica , Inativação GênicaAssuntos
Proteína de Ligação a CREB/metabolismo , Diferenciação Celular/efeitos dos fármacos , Efeitos da Posição Cromossômica/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/genética , Receptor beta de Estrogênio/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Glucocorticoides/biossíntese , Heterocromatina/metabolismo , Proteínas de Homeodomínio , Modelos Genéticos , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Hipófise/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores de Esteroides/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional/genética , Vitamina K/farmacologia , Proteínas Wnt/metabolismo , Animais , Feminino , Humanos , MasculinoRESUMO
Classic "position-effect" experiments repositioned genes near telomeres to demonstrate that the epigenetic landscape can dramatically alter gene expression. Here, we show that systematic gene knockout collections provide an exceptional resource for interrogating position effects, not only near telomeres but at every genetic locus. Because a single reporter gene replaces each deleted gene, interrogating this reporter provides a sensitive probe into different chromatin environments while controlling for genetic context. Using this approach, we find that, whereas systematic replacement of yeast genes with the kanMX marker does not perturb the chromatin landscape, chromatin differences associated with gene position account for 35% of kanMX activity. We observe distinct chromatin influences, including a Set2/Rpd3-mediated antagonistic interaction between histone H3 lysine 36 trimethylation and the Rap1 transcriptional activation site in kanMX. This interaction explains why some yeast genes have been resistant to deletion and allows successful generation of these deletion strains through the use of a modified transformation procedure. These findings demonstrate that chromatin regulation is not governed by a uniform "histone code" but by specific interactions between chromatin and genetic factors.
Assuntos
Efeitos da Posição Cromossômica/genética , Epigênese Genética , Ordem dos Genes/genética , Saccharomyces cerevisiae/genética , Acetilação , Cromatina/metabolismo , Cromossomos Fúngicos/genética , Diploide , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Biblioteca Gênica , Genes Fúngicos/genética , Marcadores Genéticos , Heterozigoto , Histonas/metabolismo , Lisina/metabolismo , Metilação , Mutagênese Insercional/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genéticaRESUMO
A neDNA fragment that protects a transgene from position effect variegation when flanking it was tested for insulator properties. The fragment did not act as an insulator. A similarity of neDNA and various barrier elements was examined, and Drosophila melanogaster and Mus musculus chromosome DNA regions homologous to neDNA were analyzed. Additional conserved sites and DNA duplex destabilization sites were found in the neDNA sequence, and DNA conformational specifics were assumed for the chromosomal neighborhood of neDNA sites of the D. melanogaster and M. musculus genomes.