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1.
Yi Chuan ; 43(9): 816-821, 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34702695

RESUMO

In interphase eukaryotic nuclei, chromatin is folded to form a higher-order topological structure. The spatial organization of such chromatin domain has an important impact on the regulation of gene expression. As a key architectural structural protein, CTCF (CCCTC-binding factor) plays an important role in the formation of chromatin three-dimensional chromatin structure. CTCF can also bind to many insulator elements in the genome and insulate enhancers from activating target genes via modulating remote chromatin interactions. A recent study by Dr. Chunliang Li and his team at St. Jude Children's Research Hospital in the United States showed that when CTCF was acutely degraded, significant changes were found in the three-dimensional structure of chromatin. The mechanism by which CTCF binding sites function as insulator elements was investigated by Prof. Qiang Wu's team at Institute of Systems Biomedicine and Shanghai Jiao Tong University in China and Prof. Bing Ren's team at Ludwig Institute for Cancer Research in the United States. Here we mainly review and discuss some of these latest progresses.


Assuntos
Genoma , Elementos Isolantes , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Criança , China , Cromatina/genética , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Elementos Isolantes/genética
2.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34531299

RESUMO

Habituation and sensitization (nonassociative learning) are among the most fundamental forms of learning and memory behavior present in organisms that enable adaptation and learning in dynamic environments. Emulating such features of intelligence found in nature in the solid state can serve as inspiration for algorithmic simulations in artificial neural networks and potential use in neuromorphic computing. Here, we demonstrate nonassociative learning with a prototypical Mott insulator, nickel oxide (NiO), under a variety of external stimuli at and above room temperature. Similar to biological species such as Aplysia, habituation and sensitization of NiO possess time-dependent plasticity relying on both strength and time interval between stimuli. A combination of experimental approaches and first-principles calculations reveals that such learning behavior of NiO results from dynamic modulation of its defect and electronic structure. An artificial neural network model inspired by such nonassociative learning is simulated to show advantages for an unsupervised clustering task in accuracy and reducing catastrophic interference, which could help mitigate the stability-plasticity dilemma. Mott insulators can therefore serve as building blocks to examine learning behavior noted in biology and inspire new learning algorithms for artificial intelligence.


Assuntos
Algoritmos , Aplysia/fisiologia , Inteligência Artificial , Elementos Isolantes , Redes Neurais de Computação , Níquel/química , Sinapses/fisiologia , Animais , Elétrons , Modelos Neurológicos , Plasticidade Neuronal
3.
Nat Commun ; 12(1): 4170, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234130

RESUMO

Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Animais Geneticamente Modificados , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Drosophila/genética , Técnicas de Silenciamento de Genes , Genoma de Inseto , Elementos Isolantes/genética , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , RNA-Seq , Proteínas Repressoras/genética , Fatores de Transcrição/genética
4.
Nat Genet ; 53(7): 1064-1074, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34002095

RESUMO

Insulators play a critical role in spatiotemporal gene regulation in animals. The evolutionarily conserved CCCTC-binding factor (CTCF) is required for insulator function in mammals, but not all of its binding sites act as insulators. Here we explore the sequence requirements of CTCF-mediated transcriptional insulation using a sensitive insulator reporter in mouse embryonic stem cells. We find that insulation potency depends on the number of CTCF-binding sites in tandem. Furthermore, CTCF-mediated insulation is dependent on upstream flanking sequences at its binding sites. CTCF-binding sites at topologically associating domain boundaries are more likely to function as insulators than those outside topologically associating domain boundaries, independently of binding strength. We demonstrate that insulators form local chromatin domain boundaries and weaken enhancer-promoter contacts. Taken together, our results provide genetic, molecular and structural evidence connecting chromatin topology to the action of insulators in the mammalian genome.


Assuntos
Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Transcrição Genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/química , Elementos Facilitadores Genéticos , Humanos , Elementos Isolantes , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica
5.
PLoS Genet ; 17(4): e1009536, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33901190

RESUMO

Several distinct activities and functions have been described for chromatin insulators, which separate genes along chromosomes into functional units. Here, we describe a novel mechanism of functional separation whereby an insulator prevents gene repression. When the homie insulator is deleted from the end of a Drosophila even skipped (eve) locus, a flanking P-element promoter is activated in a partial eve pattern, causing expression driven by enhancers in the 3' region to be repressed. The mechanism involves transcriptional read-through from the flanking promoter. This conclusion is based on the following. Read-through driven by a heterologous enhancer is sufficient to repress, even when homie is in place. Furthermore, when the flanking promoter is turned around, repression is minimal. Transcriptional read-through that does not produce anti-sense RNA can still repress expression, ruling out RNAi as the mechanism in this case. Thus, transcriptional interference, caused by enhancer capture and read-through when the insulator is removed, represses eve promoter-driven expression. We also show that enhancer-promoter specificity and processivity of transcription can have decisive effects on the consequences of insulator removal. First, a core heat shock 70 promoter that is not activated well by eve enhancers did not cause read-through sufficient to repress the eve promoter. Second, these transcripts are less processive than those initiated at the P-promoter, measured by how far they extend through the eve locus, and so are less disruptive. These results highlight the importance of considering transcriptional read-through when assessing the effects of insulators on gene expression.


Assuntos
Proteínas de Drosophila/genética , Elementos Facilitadores Genéticos/genética , Proteínas de Homeodomínio/genética , Elementos Isolantes/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Animais , Cromatina/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica/genética , Proteínas de Choque Térmico HSP70/genética , RNA Antissenso/genética , Transcrição Genética
6.
Nucleic Acids Res ; 49(D1): D1094-D1101, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33095860

RESUMO

Most mutations in cancer genomes occur in the non-coding regions with unknown impact on tumor development. Although the increase in the number of cancer whole-genome sequences has revealed numerous putative non-coding cancer drivers, their information is dispersed across multiple studies making it difficult to understand their roles in tumorigenesis of different cancer types. We have developed CNCDatabase, Cornell Non-coding Cancer driver Database (https://cncdatabase.med.cornell.edu/) that contains detailed information about predicted non-coding drivers at gene promoters, 5' and 3' UTRs (untranslated regions), enhancers, CTCF insulators and non-coding RNAs. CNCDatabase documents 1111 protein-coding genes and 90 non-coding RNAs with reported drivers in their non-coding regions from 32 cancer types by computational predictions of positive selection using whole-genome sequences; differential gene expression in samples with and without mutations; or another set of experimental validations including luciferase reporter assays and genome editing. The database can be easily modified and scaled as lists of non-coding drivers are revised in the community with larger whole-genome sequencing studies, CRISPR screens and further experimental validations. Overall, CNCDatabase provides a helpful resource for researchers to explore the pathological role of non-coding alterations in human cancers.


Assuntos
Carcinogênese/genética , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Neoplasias/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Carcinogênese/metabolismo , Carcinogênese/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos Facilitadores Genéticos , Genes Reporter , Humanos , Elementos Isolantes , Luciferases/genética , Luciferases/metabolismo , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Fases de Leitura Aberta , Regiões Promotoras Genéticas , RNA não Traduzido/classificação , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Regiões não Traduzidas , Sequenciamento Completo do Genoma
7.
Chromosoma ; 129(3-4): 255-274, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33140220

RESUMO

Insulator proteins orchestrate the three-dimensional organization of the genome. Insulators function by facilitating communications between regulatory sequences and gene promoters, allowing accurate gene transcription regulation during embryo development and cell differentiation. However, the role of insulator proteins beyond genome organization and transcription regulation remains unclear. Suppressor of Hairy wing [Su(Hw)] is a Drosophila insulator protein that plays an important function in female oogenesis. Here we find that su(Hw) has an unsuspected role in genome stability during cell differentiation. We show that su(Hw) mutant developing egg chambers have poorly formed microtubule organization centers (MTOCs) in the germarium and display mislocalization of the anterior/posterior axis specification factor gurken in later oogenesis stages. Additionally, eggshells from partially rescued su(Hw) mutant female germline exhibit dorsoventral patterning defects. These phenotypes are very similar to phenotypes found in the important class of spindle mutants or in piRNA pathway mutants in Drosophila, in which defects generally result from the failure of germ cells to repair DNA damage. Similarities between mutations in su(Hw) and spindle and piRNA mutants are further supported by an excess of DNA damage in nurse cells, and because Gurken localization defects are partially rescued by mutations in the ATR (mei-41) and Chk1 (grapes) DNA damage response genes. Finally, we also show that su(Hw) mutants produce an elevated number of chromosome breaks in dividing neuroblasts from larval brains. Together, these findings suggest that Su(Hw) is necessary for the maintenance of genome integrity during Drosophila development, in both germline and dividing somatic cells.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Instabilidade Genômica , Elementos Isolantes , Fenótipo , Animais , Feminino , Genótipo , Oogênese/genética , Ovário/citologia , Ovário/metabolismo
8.
FASEB J ; 34(11): 14736-14749, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32924169

RESUMO

CCCTC-binding factor (CTCF) sites interact with each other in the chromatin environment, establishing chromatin domains. Our previous study showed that interaction between CTCF sites is cell type-specific around the ß-globin locus and is dependent on erythroid-specific activator GATA-1. To find out molecular mechanisms of the cell type-specific interaction, we directly inhibited GATA-1 binding to the ß-globin enhancers by deleting its binding motifs and found that histone H3K27 acetylation (H3K27ac) was decreased at CTCF sites surrounding the ß-globin locus, even though CTCF binding itself was maintained at the sites. Forced H3K27ac by Trichostatin A treatment or CBP/p300 KD affected the interactions between CTCF sites around the ß-globin locus without changes in CTCF binding. Analysis of public ChIA-PET data revealed that H3K27ac is higher at CTCF sites forming short interactions than long interactions. GATA-1 was identified as a representative transcription factor that relates with genes present inside the short interactions in erythroid K562 cells. Depletion of GATA-1-reduced H3K27ac at CTCF sites near erythroid-specific enhancers. These results indicate that H3K27ac at CTCF sites is required for cell type-specific chromatin interactions between them. Tissue-specific activator GATA-1 appears to play a role in H3K27ac at CTCF sites in erythroid cells.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Células Eritroides/metabolismo , Fator de Transcrição GATA1/metabolismo , Código das Histonas , Elementos Isolantes , Acetilação , Cromatina/química , Elementos Facilitadores Genéticos , Fator de Transcrição GATA1/genética , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Células K562 , Ligação Proteica , Globinas beta/genética
9.
Genetics ; 216(3): 689-700, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32948625

RESUMO

Polycomb group (PcG) proteins are an important group of transcriptional repressors that act by modifying chromatin. PcG target genes are covered by the repressive chromatin mark H3K27me3. Polycomb repressive complex 2 (PRC2) is a multiprotein complex that is responsible for generating H3K27me3. In Drosophila, PRC2 is recruited by Polycomb Response Elements (PREs) and then trimethylates flanking nucleosomes, spreading the H3K27me3 mark over large regions of the genome, the "Polycomb domains." What defines the boundary of a Polycomb domain? There is experimental evidence that insulators, PolII, and active transcription can all form the boundaries of Polycomb domains. Here we divide the boundaries of larval Polycomb domains into six different categories. In one category, genes are transcribed toward the Polycomb domain, where active transcription is thought to stop the spreading of H3K27me3. In agreement with this, we show that introducing a transcriptional terminator into such a transcription unit causes an extension of the Polycomb domain. Additional data suggest that active transcription of a boundary gene may restrict the range of enhancer activity of a Polycomb-regulated gene.


Assuntos
Histonas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Animais , Drosophila melanogaster , Elementos Facilitadores Genéticos , Elementos Isolantes
10.
Plant Cell Rep ; 39(12): 1743-1753, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32959125

RESUMO

KEY MESSAGE: Genetic analysis identifies multiple, potential protein binding sites important for insulator function in Arabidopsis thaliana: Rap1 site in UASrpg, Su(Hw) site in UASrpg, and CTCF site in BEAD1c. Three non-plant insulators UASrpg, BEAD1c, and gypsy isolated from Ashbya gossypii, Homo sapiens and Drosophila melanogaster gypsy retrotransposon, respectively, demonstrate insulator function in transgenic Arabidopsis thaliana. Here, the hypothesis that DNA sequences functional in A. thaliana are the same as those in the original host as previously assumed, was tested. Genetic analyses of the cloned fragments in an enhancer blocking assay system was performed through deletions and mutations to identify more precisely which sequences within the cloned fragments function as insulators. Significant loss of insulator activity was observed when the UASrpg Rap1 binding site R2 was mutated but not R1. Cloned fragments containing BEAD1c are effective insulators in our assay system and the previously investigated gypsy insulator is non-functional. Further analyses identified potential Su(Hw) and CTCF sites within UASrpg, of which only the Su(Hw) site was functional. Thus, the activity of non-plant insulators in A. thaliana is context dependent. These results support the hypothesis that insulator function is conserved across kingdoms.


Assuntos
Arabidopsis/genética , Elementos Isolantes/genética , Animais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Humanos , Plantas Geneticamente Modificadas , Retroelementos , Proteínas de Ligação a Telômeros/genética
11.
Cells ; 9(8)2020 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-32784937

RESUMO

Understanding the packaging of DNA into chromatin has become a crucial aspect in the study of gene regulatory mechanisms. Heterochromatin establishment and maintenance dynamics have emerged as some of the main features involved in genome stability, cellular development, and diseases. The most extensively studied heterochromatin protein is HP1a. This protein has two main domains, namely the chromoshadow and the chromodomain, separated by a hinge region. Over the years, several works have taken on the task of identifying HP1a partners using different strategies. In this review, we focus on describing these interactions and the possible complexes and subcomplexes associated with this critical protein. Characterization of these complexes will help us to clearly understand the implications of the interactions of HP1a in heterochromatin maintenance, heterochromatin dynamics, and heterochromatin's direct relationship to gene regulation and chromatin organization.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Eucromatina/metabolismo , Heterocromatina/metabolismo , Animais , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Elementos Isolantes , Filogenia , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas
12.
Genetics ; 215(4): 1003-1012, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32554599

RESUMO

Chromatin domain insulators are thought to help partition the genome into genetic units called topologically associating domains (TADs). In Drosophila, TADs are often separated by inter-TAD regions containing active housekeeping genes and associated insulator binding proteins. This raises the question of whether insulator binding proteins are involved primarily in chromosomal TAD architecture or gene activation, or if these two activities are linked. The Boundary Element-Associated Factor of 32 kDa (BEAF-32, or BEAF for short) is usually found in inter-TADs. BEAF was discovered based on binding to the scs' insulator, and is important for the insulator activity of scs' and other BEAF binding sites. There are divergent promoters in scs' with a BEAF binding site by each. Here, we dissect the scs' insulator to identify DNA sequences important for insulator and promoter activity, focusing on the half of scs' with a high affinity BEAF binding site. We find that the BEAF binding site is important for both insulator and promoter activity, as is another sequence we refer to as LS4. Aside from that, different sequences play roles in insulator and promoter activity. So while there is overlap and BEAF is important for both, insulator and promoter activity can be separated.


Assuntos
Cromatina/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Elementos Isolantes , Regiões Promotoras Genéticas , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Proteínas do Olho/metabolismo , Feminino
13.
Front Biosci (Landmark Ed) ; 25: 1828-1838, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32472759

RESUMO

Latest advancements in genomics involving individuals from different races and geographical locations has led to the identification of thousands of common as well as rare genetic variants and copy number variations (CNVs). These studies have surprisingly revealed that the majority of genetic variation is not present within the coding region but rather in the non-coding region of the genome, which is also termed as "Medical Genome". This short review describes how mutations/variations within; regulatory sequences, architectural proteins and transcriptional regulators give rise to the aberrant gene expression profiles that drives cellular transformations and malignancies.


Assuntos
Elementos Facilitadores Genéticos/genética , Genoma Humano/genética , Elementos Isolantes/genética , Mutação , Neoplasias/genética , Regiões Promotoras Genéticas/genética , Variações do Número de Cópias de DNA , Genômica/métodos , Humanos , Polimorfismo de Nucleotídeo Único
14.
Genome Biol Evol ; 12(8): 1378-1391, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32437518

RESUMO

Chromosomal inversions are among the primary drivers of genome structure evolution in a wide range of natural populations. Although there is an impressive array of theory and empirical analyses that have identified conditions under which inversions can be positively selected, comparatively little data are available on the fitness impacts of these genome structural rearrangements themselves. Because inversion breakpoints can disrupt functional elements and alter chromatin domains, the precise positioning of an inversion's breakpoints can strongly affect its fitness. Here, we compared the fine-scale distribution of low-frequency inversion breakpoints with those of high-frequency inversions and inversions that have gone to fixation between Drosophila species. We identified a number of differences among frequency classes that may influence inversion fitness. In particular, breakpoints that are proximal to insulator elements, generate large tandem duplications, and minimize impacts on gene coding spans which are more prevalent in high-frequency and fixed inversions than in rare inversions. The data suggest that natural selection acts to preserve both genes and larger cis-regulatory networks in the occurrence and spread of rearrangements. These factors may act to limit the availability of high-fitness arrangements when suppressed recombination is favorable.


Assuntos
Pontos de Quebra do Cromossomo , Inversão Cromossômica , Drosophila melanogaster/genética , Animais , Elementos Isolantes , Mutação , Seleção Genética
15.
Sci Adv ; 6(13): eaaz3152, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32232161

RESUMO

In mammals, a C2H2 zinc finger (C2H2) protein, CTCF, acts as the master regulator of chromosomal architecture and of the expression of Hox gene clusters. Like mammalian CTCF, the Drosophila homolog, dCTCF, localizes to boundaries in the bithorax complex (BX-C). Here, we have determined the minimal requirements for the assembly of a functional boundary by dCTCF and two other C2H2 zinc finger proteins, Pita and Su(Hw). Although binding sites for these proteins are essential for the insulator activity of BX-C boundaries, these binding sites alone are insufficient to create a functional boundary. dCTCF cannot effectively bind to a single recognition sequence in chromatin or generate a functional insulator without the help of additional proteins. In addition, for boundary elements in BX-C at least four binding sites for dCTCF or the presence of additional DNA binding factors is required to generate a functional insulator.


Assuntos
Fator de Ligação a CCCTC/genética , Dedos de Zinco CYS2-HIS2/genética , Proteínas de Drosophila/genética , Drosophila/genética , Drosophila/metabolismo , Elementos Isolantes , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Fenótipo
16.
Genome Biol ; 21(1): 75, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32293525

RESUMO

BACKGROUND: CTCF is a key insulator-binding protein, and mammalian genomes contain numerous CTCF sites, many of which are organized in tandem. RESULTS: Using CRISPR DNA-fragment editing, in conjunction with chromosome conformation capture, we find that CTCF sites, if located between enhancers and promoters in the protocadherin (Pcdh) and ß-globin clusters, function as an enhancer-blocking insulator by forming distinct directional chromatin loops, regardless whether enhancers contain CTCF sites or not. Moreover, computational simulation in silico and genetic deletions in vivo as well as dCas9 blocking in vitro revealed balanced promoter usage in cell populations and stochastic monoallelic expression in single cells by large arrays of tandem CTCF sites in the Pcdh and immunoglobulin heavy chain (Igh) clusters. Furthermore, CTCF insulators promote, counter-intuitively, long-range chromatin interactions with distal directional CTCF sites, consistent with the cohesin "loop extrusion" model. Finally, gene expression levels are negatively correlated with CTCF insulators located between enhancers and promoters on a genome-wide scale. Thus, single CTCF insulators ensure proper enhancer insulation and promoter activation while tandem CTCF topological insulators determine balanced spatial contacts and promoter choice. CONCLUSIONS: These findings have interesting implications on the role of topological chromatin insulators in 3D genome folding and developmental gene regulation.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Elementos Facilitadores Genéticos , Elementos Isolantes , Regiões Promotoras Genéticas , Sítios de Ligação , Caderinas/genética , Cromatina/química , Globinas beta/genética
17.
Cell Rep ; 30(10): 3218-3228.e5, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32160531

RESUMO

Drosophila Myb (Dm-Myb) encodes a protein that plays a key role in regulation of mitotic phase genes. Here, we further refine its role in the context of a developing tissue as a potentiator of gene expression required for proper RNA polymerase II (RNA Pol II) function and efficient H3K4 methylation at promoters. In contrast to its role in gene activation, Myb is also required for repression of many genes, although no specific mechanism for this role has been proposed. We now reveal a critical role for Myb in contributing to insulator function, in part by promoting binding of insulator proteins BEAF-32 and CP190 and stabilizing H3K27me3 Polycomb-group (PcG) domains. In the absence of Myb, H3K27me3 is markedly reduced throughout the genome, leading to H3K4me3 spreading and gene derepression. Finally, Myb is enriched at boundaries that demarcate chromatin environments, including chromatin loop anchors. These results reveal functions of Myb that extend beyond transcriptional regulation.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Elementos Isolantes/genética , Lisina/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas do Grupo Polycomb/química , Proteínas Proto-Oncogênicas c-myb/química , Proteínas Proto-Oncogênicas c-myb/metabolismo , Animais , Metilação , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , RNA Polimerase II/metabolismo , Sítio de Iniciação de Transcrição
18.
Genome Biol ; 21(1): 79, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32216817

RESUMO

Non-coding variants have been shown to be related to disease by alteration of 3D genome structures. We propose a deep learning method, DeepMILO, to predict the effects of variants on CTCF/cohesin-mediated insulator loops. Application of DeepMILO on variants from whole-genome sequences of 1834 patients of twelve cancer types revealed 672 insulator loops disrupted in at least 10% of patients. Our results show mutations at loop anchors are associated with upregulation of the cancer driver genes BCL2 and MYC in malignant lymphoma thus pointing to a possible new mechanism for their dysregulation via alteration of insulator loops.


Assuntos
Cromatina/química , Aprendizado Profundo , Elementos Isolantes , Neoplasias/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Mutação , Sequenciamento Completo do Genoma
19.
FEBS Open Bio ; 10(4): 644-656, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32087050

RESUMO

Synthetic biology circuits are often constructed with multiple gene expression units assembled in close proximity, and they can be used to perform complex functions in embryonic stem cells (ESCs). However, mutual interference between transcriptional units has not been well studied in mouse ESCs. To assess the efficiency of insulators at suppressing promoter interference in mouse ESCs, we used an evaluation scheme in which a tunable tetracycline response element promoter is connected to a constant Nanog promoter. The chicken hypersensitive site 4 (cHS4) insulator, widely used both for enhancer blocking and for barrier insulation in vitro and in vivo, was positioned between the two expression units for assessment. By inserting the cassette into various loci of the mouse ESC genome with PiggyBac transposon, we were able to quantitatively examine the protective effect of cHS4 by gradually increasing the transcriptional activity of the tetracycline response element promoter with doxycycline and then measuring the transcriptional activity of the Nanog promoter. Our results indicate that the cHS4 insulator has minimal insulating effects on promoter interference in mouse ESCs. Further studies show that the cHS4 insulation effect may be promoter specific and related to interaction with CCCTC-binding factor-mediated loop formation. In addition, we also compared DNA transposition and transgene expression with or without the cHS4 insulator using well-established ESC reporters. The results indicate that cHS4 has no apparent effects on DNA transposition and transgene expression levels, but exerts modest protective effects on long-term transgene silencing.


Assuntos
Expressão Gênica , Elementos Isolantes/genética , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas/genética , Transgenes , Animais , Células Cultivadas , Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Inativação Gênica , Genes Reporter , Vetores Genéticos , Camundongos , Proteína Homeobox Nanog/genética , Plasmídeos/genética , Biologia Sintética/métodos , Transfecção
20.
Transgenic Res ; 29(2): 171-186, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31919795

RESUMO

The expression of recombinant proteins in plants is a valuable alternative to bioreactors using mammalian cell systems. Ease of scaling, and their inability to host human pathogens, enhance the use of plants to generate complex therapeutic products such as monoclonal antibodies. However, stably transformed plants expressing antibodies normally have a poor accumulation of these proteins that probably arise from the negative positional effects of their flanking chromatin. The induction of boundaries between the transgenes and the surrounding DNA using matrix attachment regions (MAR) and insulator elements may minimize these effects. With the PHB-01 antibody as a model, we demonstrated that the insertion of DNA elements, the TM2 (MAR) and M4 insulator, flanking the transcriptional cassettes that encode the light and heavy chains of the PHB-01 antibody, increased the protein accumulation that remained stable in the first plant progeny. The M4 insulator had a stronger effect than the TM2, with over a twofold increase compared to the standard construction. This effect was probably associated with an enhancer-promoter interference. Moreover, transgenic plants harboring two transcriptional units encoding for the PHB-01 heavy chain combined with both TM2 and M4 elements enhanced the accumulation of the antibody. In summary, the M4 combined with a double transcriptional unit of the heavy chain may be a suitable strategy for potentiating PHB-01 production in tobacco plants.


Assuntos
Anticorpos/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Elementos Isolantes , Regiões de Interação com a Matriz/genética , Proteínas Recombinantes/metabolismo , Tabaco/genética , Transgenes/genética , Anticorpos/genética , Regulação da Expressão Gênica de Plantas , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Tabaco/crescimento & desenvolvimento
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