The role of the epidermis enhancer element in positive and negative transcriptional regulation of ebony in Drosophila melanogaster

Abstract The spatiotemporal regulation of gene expression is essential to ensure robust phenotypic outcomes. Pigmentation patterns in Drosophila are determined by pigments biosynthesized in the developing epidermis and the cis-regulatory elements (CREs) of the genes involved in this process are well-characterized. Here we report that the known primary epidermal enhancer (priEE) is dispensable for the transcriptional activation of ebony (involved in light-colored pigment synthesis) in the developing epidermis of D. melanogaster. The evidence was obtained by introducing an approximately 1 kbp deletion at the priEE by genome editing. The effect of the priEE deletion on pigmentation and on the endogenous expression pattern of a mCherry-fused ebony allele was examined in the abdomen. The expression levels of the mCherry-fused ebony in the priEE-deleted strains were slightly higher than that of the control strain, indicating that the sequences outside the priEE have an ability to drive an expression of this gene in the epidermis. Interestingly, the priEE deletion resulted in a derepression of this gene in the dorsal midline of the abdominal tergites, where dark pigmentation is present in the wild-type individuals. This indicated that the priEE fragment contains a silencer. Furthermore, the endogenous expression pattern of ebony in the two additional strains with partially deleted priEE revealed that the silencer resides within a 351-bp fragment in the 5' portion of the priEE. These results demonstrated that deletion assays combined with reporter assays are highly effective in detecting the presence of positively and negatively regulating sequences within and outside the focal CREs.

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

10.1101/2021.03.25.436947 ◽

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.

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Tetracycline-controlled transgenic targeting from the SCL locus directs conditional expression to erythrocytes, megakaryocytes, granulocytes, and c-kit-expressing lineage-negative hematopoietic cells

Blood ◽

10.1182/blood-2005-12-012104 ◽

2006 ◽

Vol 108 (5) ◽

pp. 1533-1541 ◽

Cited By ~ 16

Author(s):

Ernesto Bockamp ◽

Cecilia Antunes ◽

Marko Maringer ◽

Rosario Heck ◽

Katrin Presser ◽

...

Keyword(s):

Bone Marrow ◽

Expression Pattern ◽

Bone Marrow Cells ◽

Regulatory Elements ◽

Hematopoietic Stem ◽

Tetracycline Transactivator ◽

Adult Mice ◽

Endogenous Expression ◽

Conditional Expression

The stem cell leukemia gene SCL, also known as TAL-1, encodes a basic helix-loop-helix transcription factor expressed in erythroid, myeloid, megakaryocytic, and hematopoietic stem cells. To be able to make use of the unique tissue-restricted and spatio-temporal expression pattern of the SCL gene, we have generated a knock-in mouse line containing the tTA-2S tetracycline transactivator under the control of SCL regulatory elements. Analysis of this mouse using different tetracycline-dependent reporter strains demonstrated that switchable transgene expression was restricted to erythrocytes, megakaryocytes, granulocytes, and, importantly, to the c-kit-expressing and lineage-negative cell fraction of the bone marrow. In addition, conditional transgene activation also was detected in a very minor population of endothelial cells and in the kidney. However, no activation of the reporter transgene was found in the brain of adult mice. These findings suggested that the expression of tetracycline-responsive reporter genes recapitulated the known endogenous expression pattern of SCL. Our data therefore demonstrate that exogenously inducible and reversible expression of selected transgenes in myeloid, megakaryocytic, erythroid, and c-kit-expressing lineage-negative bone marrow cells can be directed through SCL regulatory elements. The SCL knock-in mouse presented here represents a powerful tool for studying normal and malignant hematopoiesis in vivo.

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Neural lineage induction reveals multi-scale dynamics of 3D chromatin organization

10.1101/004168 ◽

2014 ◽

Author(s):

Aleksandra Pekowska ◽

Bernd Klaus ◽

Felix Alexander Klein ◽

Simon Anders ◽

Malgorzata Oles ◽

...

Keyword(s):

Gene Expression ◽

Regulation Of Gene Expression ◽

Embryonic Stem ◽

Neural Induction ◽

Interaction Network ◽

Regulatory Elements ◽

Chromatin Organization ◽

Spatial Proximity ◽

Enhancer Element ◽

Neural Lineage

Regulation of gene expression underlies cell identity. Chromatin structure and gene activity are linked at multiple levels, via positioning of genomic loci to transcriptionally permissive or repressive environments and by connecting cis-regulatory elements such as promoters and enhancers. However, the genome-wide dynamics of these processes during cell differentiation has not been characterized. Using tethered chromatin conformation capture (TCC) sequencing we determined global three-dimensional chromatin structures in mouse embryonic stem (ES) and neural stem (NS) cell derivatives. We found that changes in the propensity of genomic regions to form inter-chromosomal contacts are pervasive in neural induction and are associated with the regulation of gene expression. Moreover, we found a pronounced contribution of euchromatic domains to the intra-chromosomal interaction network of pluripotent cells, indicating the existence of an ES cell-specific mode of chromatin organization. Mapping of promoter-enhancer interactions in pluripotent and differentiated cells revealed that spatial proximity without enhancer element activity is a common architectural feature in cells undergoing early developmental changes. Activity-independent formation of higher-order contacts between cis-regulatory elements, predominant at complex loci, may thus provide an additional layer of transcriptional control.

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Association of Ddx5/p68 protein with the upstream erythroid enhancer element (EHS1) of the Klf1 gene

10.1101/743435 ◽

2019 ◽

Author(s):

Xiaoyong Chen ◽

Felix Lohmann ◽

James J Bieker

Keyword(s):

Transcription Factor ◽

Expression Pattern ◽

Transcriptional Activation ◽

Affinity Purification ◽

Hypersensitive Site ◽

Enhancer Element ◽

Restricted Expression Pattern ◽

Dnase Hypersensitive Site ◽

Essential Transcription Factor ◽

Klf1 Gene

AbstractEKLF/KLF1 is an essential transcription factor that plays a global role in erythroid transcriptional activation. It’s own regulation is of interest, as it displays a highly restricted expression pattern, limited to erythroid cells and its progenitors. Here we use biochemical affinity purification to identify the Ddx5/p68 protein as a potential activator of KLF1 by virtue of its interaction with the erythroid-specific DNAse hypersensitive site (EHS1) upstream enhancer element. We postulate that its range of interactions with other proteins known to interact with this element render it part of the enhanseosome complex critical for optimal expression of KLF1.

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A revised model for promoter competition based on multi-way chromatin interactions at the α-globin locus

Nature Communications ◽

10.1038/s41467-019-13404-x ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 13

Author(s):

A. Marieke Oudelaar ◽

Caroline L. Harrold ◽

Lars L. P. Hanssen ◽

Jelena M. Telenius ◽

Douglas R. Higgs ◽

...

Keyword(s):

Transcriptional Activation ◽

Regulation Of Gene Expression ◽

Regulatory Elements ◽

Chromatin Domain ◽

Gene Promoters ◽

Multiple Promoters ◽

Chromatin Interactions ◽

Technological Advances ◽

Single Complex ◽

Membrane Bound

AbstractSpecific communication between gene promoters and enhancers is critical for accurate regulation of gene expression. However, it remains unclear how specific interactions between multiple regulatory elements contained within a single chromatin domain are coordinated. Recent technological advances which can detect multi-way chromatin interactions at single alleles can provide insights into how multiple regulatory elements cooperate or compete for transcriptional activation. Here, we use such an approach to investigate how interactions of the α-globin enhancers are distributed between multiple promoters in a mouse model in which the α-globin domain is extended to include several additional genes. Our data show that gene promoters do not form mutually exclusive interactions with enhancers, but all interact simultaneously in a single complex. These findings suggest that promoters do not structurally compete for interactions with enhancers, but form a regulatory hub structure, which is consistent with recent models of transcriptional activation occurring in non-membrane bound nuclear compartments.

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Dempster-Shafer Theory for the Prediction of Auxin-Response Elements (AuxREs) in Plant Genomes

BioMed Research International ◽

10.1155/2018/3837060 ◽

2018 ◽

Vol 2018 ◽

pp. 1-13 ◽

Cited By ~ 2

Author(s):

Nesrine Sghaier ◽

Rayda Ben Ayed ◽

Riadh Ben Marzoug ◽

Ahmed Rebai

Keyword(s):

Data Fusion ◽

Transcriptional Activation ◽

False Positive ◽

Fuzzy Theory ◽

Regulation Of Gene Expression ◽

Evidence Theory ◽

Regulatory Elements ◽

Auxin Response ◽

Shafer Theory ◽

Comparison Of The Results

Auxin is a major regulator of plant growth and development; its action involves transcriptional activation. The identification of Auxin-response element (AuxRE) is one of the most important issues to understand the Auxin regulation of gene expression. Over the past few years, a large number of motif identification tools have been developed. Despite these considerable efforts provided by computational biologists, building reliable models to predict regulatory elements has still been a difficult challenge. In this context, we propose in this work a data fusion approach for the prediction of AuxRE. Our method is based on the combined use of Dempster-Shafer evidence theory and fuzzy theory. To evaluate our model, we have scanning the DORNRÖSCHEN promoter by our model. All proven AuxRE present in the promoter has been detected. At the 0.9 threshold we have no false positive. The comparison of the results of our model and some previous motifs finding tools shows that our model can predict AuxRE more successfully than the other tools and produce less false positive. The comparison of the results before and after combination shows the importance of Dempster-Shafer combination in the decrease of false positive and to improve the reliability of prediction. For an overall evaluation we have chosen to present the performance of our approach in comparison with other methods. In fact, the results indicated that the data fusion method has the highest degree of sensitivity (Sn) and Positive Predictive Value (PPV).

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Transcription Factor Action Orchestrates the Complex Expression Pattern of CRABS CLAW in Arabidopsis

Genes ◽

10.3390/genes12111663 ◽

2021 ◽

Vol 12 (11) ◽

pp. 1663

Author(s):

Thomas Gross ◽

Annette Becker

Keyword(s):

Transcription Factor ◽

Expression Pattern ◽

Transcriptional Activation ◽

Binding Sites ◽

Molecular Mechanisms ◽

Expression Patterns ◽

Regulation Of Gene Expression ◽

Transcript Abundance ◽

Dna Interaction ◽

Complex Expression

Angiosperm flowers are the most complex organs that plants generate, and in their center, the gynoecium forms, assuring sexual reproduction. Gynoecium development requires tight regulation of developmental regulators across time and tissues. How simple on and off regulation of gene expression is achieved in plants was described previously, but molecular mechanisms generating complex expression patterns remain unclear. We use the gynoecium developmental regulator CRABS CLAW (CRC) to study factors contributing to its sophisticated expression pattern. We combine in silico promoter analyses, global TF-DNA interaction screens, and mutant analyses. We find that miRNA action, DNA methylation, and chromatin remodeling do not contribute substantially to CRC regulation. However, 119 TFs, including SEP3, ETT, CAL, FUL, NGA2, and JAG bind to the CRC promoter in yeast. These TFs finetune transcript abundance as homodimers by transcriptional activation. Interestingly, temporal–spatial aspects of expression regulation may be under the control of redundantly acting genes and require higher order complex formation at TF binding sites. Our work shows that endogenous regulation of complex expression pattern requires orchestrated transcription factor action on several conserved promotor sites covering almost 4 kb in length. Our results highlight the utility of comprehensive regulators screens directly linking transcriptional regulators with their targets.

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In Silico Analysis of Osa-miR164 Gene Family in Rice (Oryza Sativa)

VNU Journal of Science Natural Sciences and Technology ◽

10.25073/2588-1140/vnunst.5304 ◽

2021 ◽

Vol 37 (3) ◽

Author(s):

Vuong Quang Tien ◽

Nguyen Huy Duong ◽

Dao Trong Nhan ◽

Phan Minh Vu ◽

Do Thi Phuc

Keyword(s):

Oryza Sativa ◽

Expression Pattern ◽

Target Genes ◽

Regulation Of Gene Expression ◽

In Silico Analysis ◽

Regulatory Elements ◽

Stress Conditions ◽

Gene Promoters ◽

Non Coding Rna ◽

Post Transcriptional Regulation

MicroRNA (miRNA) is a small non-coding RNA molecule containing about 22- 24 nucleotides, which functions in post-transcriptional regulation of gene expression. Previous reports have shown that miRNA plays an important role on the resistance ability of plants to adverse conditions. Rice (Oryza sativa) is a major food crop. Climate change makes the situation of salinity and drought in Vietnam worse, significantly affects rice cultivation area, leading to the decrease of the quantity and the quality of rice grains. In this research, we focused on miR164 family in rice. By using bioinformatics approach, we analyzed sequences of all osa-miR164 belonging to rice miR164 family, evaluated the expression profile of osa-miR164 under different stress conditions, predicted cis-regulatory elements on osa-miR164 gene promoters, and simultaneously predicted miR164-targeted genes and their expressions. The results showed the high conserve in mature osa-miR164 sequences but not in the precursor sequences, different expression pattern of osa-miR164 gene members under stress conditions and various cis-regulatory elements present in osa-miR164 gene promoters which may explain for diverse expression pattern of osa-miR164 genes. Some potential target genes of osa-miR164 were identified and their expressions under different stress conditions were analyzed.

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A revised model for promoter competition based on multi-way chromatin interactions

10.1101/612275 ◽

2019 ◽

Cited By ~ 1

Author(s):

A. Marieke Oudelaar ◽

Caroline L. Harrold ◽

Lars L. P. Hanssen ◽

Jelena M. Telenius ◽

Douglas R. Higgs ◽

...

Keyword(s):

Transcriptional Activation ◽

Regulation Of Gene Expression ◽

Regulatory Elements ◽

Chromatin Domain ◽

Gene Promoters ◽

Multiple Promoters ◽

Chromatin Interactions ◽

Super Enhancer ◽

Technological Advances ◽

Single Complex

AbstractSpecific communication between gene promoters and enhancers is critical for accurate regulation of gene expression. However, it remains unclear how specific interactions between multiple regulatory elements and genes contained within a single chromatin domain are coordinated. Recent technological advances allow for the investigation of multi-way chromatin interactions at single alleles in individual nuclei. This can provide insights into how multiple regulatory elements cooperate or compete for transcriptional activation. We have used these techniques in a mouse model in which the α-globin domain is extended to include several additional genes. This allows us to determine how the interactions of the α-globin super-enhancer are distributed between multiple promoters in a single domain. Our data show that gene promoters do not form mutually exclusive interactions with the super-enhancer, but all interact simultaneously in a single complex. These finding show that promoters within the same domain do not structurally compete for interactions with enhancers, but form a regulatory hub structure, consistent with the recent model of transcriptional activation in phase-separated nuclear condensates.

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