scholarly journals The study of the regulatory region of the Drosophila melanogaster Notch gene by new methods of directed genome editing

2019 ◽  
Vol 23 (2) ◽  
pp. 199-202
Author(s):  
O. V. Andreyenkov ◽  
E. I. Volkova ◽  
N. G. Andreyenkova ◽  
S. A. Demakov
Keyword(s):  
Drosophila Melanogaster ◽  
Genome Editing ◽  
Regulatory Region ◽  
Polytene Chromosomes ◽  
Notch Pathway ◽  
Hereditary Diseases ◽  
Chromatin State ◽  
Open Chromatin ◽  
New Methods ◽  
Notch Gene

The Notch gene plays a key role in the development of organs and tissues of neuroectodermic origin, including the nervous system. In eukaryotic organisms, the Notch pathway is involved in cell fate determination. The Notch gene was first discovered in Drosophila melanogaster. In mammals, the family of Notch receptors includes four homologues. In humans, mutations in the Notch gene cause several hereditary diseases and carcinogenesis. Studies of the regulatory zone of the Notch gene in D. melanogaster have been conducted for several decades. We review their results and methods. The regulatory zone of the Notch gene is in the region of open chromatin state that corresponds to the 3C6/3C7 interband on the cytological map of polytene chromosomes of D. melanogaster salivary glands. The development of new methods for directed genome editing made it possible to create a system for introducing directed changes into the regulatory zone of the gene. Using the CRISPR/Cas9 system, we obtained a directed 4-kilobase deletion including the 5’-regulatory zone, promoter, and the first exon of the Notch gene and introduced the attP site into the first intron of the Notch gene. This approach enabled targeted changes of the sequence of the regulatory and promoter regions of the gene. Thus, it provided a new powerful tool for studies of Notch gene regulation and the organization of the open chromatin state.

scholarly journals A silencer repressing redundant enhancer activities revealed by deleting endogenouscis-regulatory element ofebonyinDrosophila melanogaster

2021 ◽  
Author(s):  
Noriyoshi Akiyama ◽  
Shoma Sato ◽  
Kentaro M. Tanaka ◽  
Takaomi Sakai ◽  
Aya Takahashi
Keyword(s):  
Drosophila Melanogaster ◽  
Genome Editing ◽  
Expression Pattern ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Biosynthesis Pathway ◽  
Melanin Biosynthesis ◽  
Pigment Synthesis ◽  
Endogenous Expression ◽  
Spatiotemporal Regulation

AbstractThe spatiotemporal regulation of gene expression is essential to ensure robust phenotypic outcomes. Pigmentation patterns inDrosophilaare formed by the deposition of different pigments synthesized in the developing epidermis and the role ofcis-regulatory elements (CREs) of melanin biosynthesis pathway-related genes is well-characterized. These CREs typically exhibit modular arrangement in the regulatory region of the gene with each enhancer regulating a specific spatiotemporal expression of the gene. However, recent studies have suggested that multiple enhancers of a number of developmental genes as well as those ofyellow(involved in dark pigment synthesis) exhibit redundant activities. Here we report the redundant enhancer activities in thecis-regulatory region of another gene in the melanin biosynthesis pathway,ebony, in the developing epidermis ofDrosophila melanogaster. The evidence was obtained by introducing an approximately 1 kbp deletion at the endogenous primary epidermis enhancer (priEE) by genome editing. The effect of the priEE deletion on pigmentation and on the endogenous expression pattern of amCherry-taggedebonyallele was examined in the thoracic and abdominal segments. The expression level ofebonyin the priEE-deleted strains was similar to that of the control strain, indicating the presence of redundant enhancer activities that drive the broad expression ofebonyin the developing epidermis. Additionally, the priEE fragment contained a silencer that suppressesebonyexpression in the dorsal midline of the abdominal tergites, which is necessary for the development of the subgenusSophophora-specific dark pigmentation patterns along the midline. The endogenous expression pattern ofebonyin the priEE-deleted strains and the reporter assay examining the autonomous activity of the priEE fragment indicated that the silencer is involved in repressing the activities of both proximal and distant enhancers. These results suggest that multiple silencers are dispensable in the regulatory system of a relatively stable taxonomic character. The prevalence of other redundant enhancers and silencers in the genome can be investigated using a similar approach.Author summaryGenes are expressed at the right timing and place to give rise to diverse phenotypes. The spatiotemporal regulation is usually achieved through the coordinated activities of transcription-activating and transcription-repressing proteins that bind to the DNA sequences called enhancers and silencers, respectively, located near the target gene. Most studies identified the locations of enhancers by examining the ability of the sequence fragments to regulate the expression of fused reporters. Various short enhancers have been identified using this approach. This study employed an alternative approach in which the previously identified enhancer that regulates expression ofebony(a gene involved in body color formation) was deleted in a fruitfly,Drosophila melanogaster, using the genome-editing technique. The knockout of this enhancer did not affect the transcription level of the gene to a large extent. This indicated the presence of transcription-activating elements with redundant functions outside the deleted enhancer. Additionally, the transcription ofebonyat the midline of the abdomen, which is repressed in the normal flies, were derepressed in the enhancer-deleted flies, which indicated that the deleted enhancer fragment contained a silencer that negatively regulates multiple enhancer activities in a spatially restricted manner.

scholarly journals GAGA Factor and the TFIID Complex Collaborate in Generating an Open Chromatin Structure at the Drosophila melanogaster hsp26 Promoter

2002 ◽  
Vol 22 (17) ◽  
pp. 6148-6157 ◽  
Author(s):  
Boris A. Leibovitch ◽  
Quinn Lu ◽  
Lawrence R. Benjamin ◽  
Yingyun Liu ◽  
David S. Gilmour ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Chromatin Structure ◽  
Random Sequence ◽  
Regulatory Region ◽  
Open Chromatin ◽  
Gaga Factor ◽  
Factor Binding ◽  
Hypersensitive Sites ◽  
Tfiid Complex

ABSTRACT The upstream regulatory region of the Drosophila melanogaster hsp26 gene includes two DNase I-hypersensitive sites (DH sites) that encompass the critical heat shock elements. This chromatin structure is required for heat shock-inducible expression and depends on two (CT) n •(GA) n elements bound by GAGA factor. To determine whether GAGA factor alone is sufficient to drive formation of the DH sites, we have created flies with an hsp26/lacZ transgene wherein the entire DNA segment known to interact with the TFIID complex has been replaced by a random sequence. The replacement results in a loss of heat shock-inducible hsp26 expression and drastically diminishes nuclease accessibility in the chromatin of the regulatory region. Chromatin immunoprecipitation experiments show that the decrease in TFIID binding does not reduce GAGA factor binding. In contrast, the loss of GAGA factor binding resulting from (CT) n mutations decreases TFIID binding. These data suggest that both GAGA factor and TFIID are necessary for formation of the appropriate chromatin structure at the hsp26 promoter and predict a regulatory mechanism in which GAGA factor binding precedes and contributes to the recruitment of TFIID.

scholarly journals Scalloped wings Is the Lucilia cuprina Notch Homologue and a Candidate for the Modifier of Fitness and Asymmetry of Diazinon Resistance

Genetics ◽  
1996 ◽  
Vol 143 (3) ◽  
pp. 1321-1337 ◽  
Author(s):  
Andrew G Davies ◽  
Anne Y Game ◽  
Zhenzhong Chen ◽  
Tracey J Williams ◽  
Stephen Goodall ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Cell Adhesion ◽  
Molecular Level ◽  
Polytene Chromosomes ◽  
Resistance Mutation ◽  
Lucilia Cuprina ◽  
N Gene ◽  
Genomic Fragment ◽  
Australian Sheep ◽  
Notch Gene

Abstract The Scalloped wings (Scl) gene of the Australian sheep blowfly, Lucilia cuprina, is shown to be the homologue of the Drosophila melanogaster Notch gene by comparison at the DNA sequence and genetic levels. A L. cuprina genomic fragment, which shows strong identity with the Notch (N) gene at the molecular level, hybridizes to the location of the Scl gene on polytene chromosomes. The two genes are functionally homologous; the dominant and recessive Notch-like phenotypes produced by mutations in the Scl gene allow these alleles to be classed as N-like or Abruptex-like. The Scl gene is under investigation as a candidate for the fitness and asymmetry Modifier (M) of diazinon resistance. We show that M affects the penetrance of wing and bristle phenotypes associated with two Scl alleles in a manner consistent with the M being an allele of Scl. In addition, we report a phenotypic interaction between the diazinon-resistance mutation, Rop-1, and the same alleles of Scl. We propose that the product of Rop-1, an esterase, may be involved in cell adhesion in developmental processes involving the Scl gene product.

scholarly journals Chromosomal Distribution of Transposable Elements in Drosophila melanogaster Test of the Ectopic Recombination Model for Maintenance of Insertion Site Number

Genetics ◽  
1996 ◽  
Vol 144 (1) ◽  
pp. 197-204
Author(s):  
Christine Hoogland ◽  
Christian Biémont
Keyword(s):  
Drosophila Melanogaster ◽  
Transposable Elements ◽  
Dna Content ◽  
Recombination Rate ◽  
Alternative Hypothesis ◽  
Insertion Site ◽  
Polytene Chromosomes ◽  
Negative Relationship ◽  
Site Occupancy ◽  
Site Number

Abstract Data of insertion site localization and site occupancy frequency of P, hobo, I, copia, mdg1, mdg3, 412, 297, and roo transposable elements (TEs) on the polytene chromosomes of Drosophila melanogaster were extracted from the literature. We show that TE insertion site number per chromosomal division was significantly correlated with the amount of DNA. The insertion site number weighted by DNA content was not correlated with recombination rate for all TEs except hobo, for which a positive correlation was detected. No global tendency emerged in the relationship between TE site occupancy frequency, weighted by DNA content, and recombination rate; a strong negative correlation was, however, found for the 3L arm. A possible dominant deleterious effect of chromosomal rearrangements due to recombination between TE insertions is thus not the main factor explaining the dynamics of TEs, since this hypothesis implies a negative relationship between recombination rate and both TE insertion site number and site occupancy frequency. The alternative hypothesis of selection against deleterious effects of insertional mutations is discussed.

scholarly journals Inferring time series chromatin states for promoter-enhancer pairs based on Hi-C data

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Henriette Miko ◽  
Yunjiang Qiu ◽  
Bjoern Gaertner ◽  
Maike Sander ◽  
Uwe Ohler
Keyword(s):  
Time Series ◽  
Histone Modifications ◽  
Gene Activation ◽  
Expectation Maximization Algorithm ◽  
Chromatin State ◽  
Open Chromatin ◽  
Multiple Time ◽  
Enhancer Activity ◽  
Chromatin States ◽  
Multiple Time Points

Abstract Background Co-localized combinations of histone modifications (“chromatin states”) have been shown to correlate with promoter and enhancer activity. Changes in chromatin states over multiple time points (“chromatin state trajectories”) have previously been analyzed at promoter and enhancers separately. With the advent of time series Hi-C data it is now possible to connect promoters and enhancers and to analyze chromatin state trajectories at promoter-enhancer pairs. Results We present TimelessFlex, a framework for investigating chromatin state trajectories at promoters and enhancers and at promoter-enhancer pairs based on Hi-C information. TimelessFlex extends our previous approach Timeless, a Bayesian network for clustering multiple histone modification data sets at promoter and enhancer feature regions. We utilize time series ATAC-seq data measuring open chromatin to define promoters and enhancer candidates. We developed an expectation-maximization algorithm to assign promoters and enhancers to each other based on Hi-C interactions and jointly cluster their feature regions into paired chromatin state trajectories. We find jointly clustered promoter-enhancer pairs showing the same activation patterns on both sides but with a stronger trend at the enhancer side. While the promoter side remains accessible across the time series, the enhancer side becomes dynamically more open towards the gene activation time point. Promoter cluster patterns show strong correlations with gene expression signals, whereas Hi-C signals get only slightly stronger towards activation. The code of the framework is available at https://github.com/henriettemiko/TimelessFlex. Conclusions TimelessFlex clusters time series histone modifications at promoter-enhancer pairs based on Hi-C and it can identify distinct chromatin states at promoter and enhancer feature regions and their changes over time.

scholarly journals Transvection at the End of the Truncated Chromosome in Drosophila melanogaster

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1375-1387
Author(s):  
Mikhail Savitsky ◽  
Tatyana Kahn ◽  
Ekaterina Pomerantseva ◽  
Pavel Georgiev
Keyword(s):  
Drosophila Melanogaster ◽  
Homologous Chromosome ◽  
Regulatory Region ◽  
Proximal Region ◽  
The Other ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Transcription Start ◽  
Promoter Proximal Region

Abstract The phenomenon of transvection is well known for the Drosophila yellow locus. Thus enhancers of a promoterless yellow locus in one homologous chromosome can activate the yellow promoter in the other chromosome where the enhancers are inactive or deleted. In this report, we examined the requirements for trans-activation of the yellow promoter at the end of the deficient chromosome. A number of truncated chromosomes ending in different areas of the yellow regulatory region were examined in combination with the promoterless y alleles. We found that trans-activation of the yellow promoter at the end of a deficient chromosome required ∼6 kb of an additional upstream sequence. The nature of upstream sequences affected the strength of transvection: addition of gypsy sequences induced stronger trans-activation than addition of HeT-A or yellow sequences. Only the promoter proximal region (within -158 bp of the yellow transcription start) was essential for trans-activation; i.e., transvection did not require extensive homology in the yellow upstream region. Finally, the yellow enhancers located on the two pairing chromosomes could cooperatively activate one yellow promoter.

scholarly journals Antagonising Chromatin Remodelling Activities in the Regulation of Mammalian Ribosomal Transcription

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 961
Author(s):  
Kanwal Tariq ◽  
Ann-Kristin Östlund Farrants
Keyword(s):  
Stress Responses ◽  
Ribosomal Gene ◽  
Chromatin Remodelling ◽  
Chromatin State ◽  
Rrna Gene ◽  
Open Chromatin ◽  
Histone Chaperones ◽  
Embryonic Cells ◽  
Cell Type ◽  
Chromatin States

Ribosomal transcription constitutes the major energy consuming process in cells and is regulated in response to proliferation, differentiation and metabolic conditions by several signalling pathways. These act on the transcription machinery but also on chromatin factors and ncRNA. The many ribosomal gene repeats are organised in a number of different chromatin states; active, poised, pseudosilent and repressed gene repeats. Some of these chromatin states are unique to the 47rRNA gene repeat and do not occur at other locations in the genome, such as the active state organised with the HMG protein UBF whereas other chromatin state are nucleosomal, harbouring both active and inactive histone marks. The number of repeats in a certain state varies on developmental stage and cell type; embryonic cells have more rRNA gene repeats organised in an open chromatin state, which is replaced by heterochromatin during differentiation, establishing different states depending on cell type. The 47S rRNA gene transcription is regulated in different ways depending on stimulus and chromatin state of individual gene repeats. This review will discuss the present knowledge about factors involved, such as chromatin remodelling factors NuRD, NoRC, CSB, B-WICH, histone modifying enzymes and histone chaperones, in altering gene expression and switching chromatin states in proliferation, differentiation, metabolic changes and stress responses.

Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes

Chromosoma ◽  
1981 ◽  
Vol 82 (2) ◽  
pp. 205-216 ◽  
Author(s):  
F. Scalenghe ◽  
E. Turco ◽  
J. E. Edström ◽  
V. Pirrotta ◽  
M. Melli
Keyword(s):  
Drosophila Melanogaster ◽  
Polytene Chromosomes ◽  
Specific Region

A cDNA from Drosophila melanogaster encodes a lamin C-like intermediate filament protein

1993 ◽  
Vol 104 (4) ◽  
pp. 1263-1272 ◽  
Author(s):  
C.A. Bossie ◽  
M.M. Sanders
Keyword(s):  
Drosophila Melanogaster ◽  
Intermediate Filament ◽  
Blot Analysis ◽  
Polytene Chromosomes ◽  
Intermediate Filament Protein ◽  
Chromosome 2 ◽  
Cross Hybridization ◽  
Intermediate Filament Proteins ◽  
Nuclear Lamins ◽  
Filament Protein

A novel intermediate filament cDNA, pG-IF, has been isolated from a Drosophila melanogaster embryonic expression library screened with a polyclonal antiserum produced against a 46 kDa cytoskeletal protein isolated from Kc cells. This 46 kDa protein is known to be immunologically related to vertebrate intermediate filament proteins. The screen resulted in the isolation of four different cDNA groups. Of these, one has been identified as the previously characterized Drosophila nuclear lamin cDNA, Dm0, and a second, pG-IF, demonstrates homology to Dm0 by cross hybridization on Southern blots. DNA sequence analysis reveals that pG-IF encodes a newly identified intermediate filament protein in Drosophila. Its nucleotide sequence is highly homologous to nuclear lamins with lower homology to cytoplasmic intermediate filament proteins. pG-IF predicts a protein of 621 amino acids with a predicted molecular mass of 69,855 daltons. In vitro transcription and translation of pG-IF yielded a protein with a SDS-PAGE estimated molecular weight of approximately 70 kDa. It contains sequence principles characteristic of class V intermediate filament proteins. Its near neutral pI (6.83) and the lack of a terminal CaaX motif suggests that it may represent a lamin C subtype in Drosophila. In situ hybridization to polytene chromosomes detects one band of hybridization on the right arm of chromosome 2 at or near 51A. This in conjunction with Southern blot analysis of various genomic digests suggests one or more closely placed genes while Northern blot analysis detects two messages in Kc cells.

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