Identification of distal regulatory regions in the human αIIb gene locus necessary for consistent, high-level megakaryocyte expression

The αIIb/β3-integrin receptor is present at high levels only in megakaryocytes and platelets. Its presence on platelets is critical for hemostasis. The tissue-specific nature of this receptor's expression is secondary to the restricted expression of αIIb, and studies of the αIIb proximal promoter have served as a model of a megakaryocyte-specific promoter. We have examined the αIIb gene locus for distal regulatory elements. Sequence comparison between the human (h) and murine (m) αIIb loci revealed high levels of conservation at intergenic regions both 5′ and 3′ to the αIIb gene. Additionally, deoxyribonuclease (DNase) I sensitivity mapping defined tissue-specific hypersensitive (HS) sites that coincide, in part, with these conserved regions. Transgenic mice containing various lengths of the hαIIb gene locus, which included or excluded the various conserved/HS regions, demonstrated that the proximal promoter was sufficient for tissue specificity, but that a region 2.5 to 7.1 kb upstream of the hαIIb gene was necessary for consistent expression. Another region 2.2 to 7.4 kb downstream of the gene enhanced expression 1000-fold and led to levels of hαIIb mRNA that were about 30% of the native mαIIb mRNA level. These constructs also resulted in detectable hαIIb/mβ3 on the platelet surface. This work not only confirms the importance of the proximal promoter of the αIIb gene for tissue specificity, but also characterizes the distal organization of the αIIb gene locus and provides an initial localization of 2 important regulatory regions needed for the expression of the αIIb gene at high levels during megakaryopoiesis.

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Identification of cis-regulatory elements required for larval expression of the Drosophila melanogaster alcohol dehydrogenase gene.

Genetics ◽

10.1093/genetics/124.3.637 ◽

1990 ◽

Vol 124 (3) ◽

pp. 637-646 ◽

Cited By ~ 1

Author(s):

V Corbin ◽

T Maniatis

Keyword(s):

Drosophila Melanogaster ◽

Alcohol Dehydrogenase ◽

Dna Sequences ◽

Regulatory Elements ◽

Malpighian Tubules ◽

Proximal Promoter ◽

Start Site ◽

Wild Type ◽

Tissue Specific ◽

Adh Gene

Abstract The Alcohol dehydrogenase (Adh) genes of two distantly related species, Drosophila melanogaster and Drosophila mulleri, display similar, but not identical, patterns of tissue-specific expression in larvae and adults. The regulatory DNA sequences necessary for wild-type Adh expression in D. mulleri larvae were previously reported. In this paper we present an analysis of the DNA sequences necessary for wild-type Adh expression in D. melanogaster larvae. We show that transcription from the proximal promoter of the melanogaster Adh gene is regulated by a far upstream enhancer and two or more elements near the transcription start site. The enhancer is tissue specific and stimulates transcription to high levels in fat body and to lower levels in midgut and malpighian tubules whether linked to the proximal promoter or to a heterologous promoter. The enhancer activity localized to at least two discrete regions dispersed over more than 1.7 kb of DNA. Deletion of any one of these subregions reduces Adh transcription in all three larval tissues. Similarly, two regions immediately upstream of the proximal promoter start site are necessary for wild-type transcription levels in all three tissues. Thus, each of the identified regulatory elements is sufficient for low levels of Adh gene expression in all three larval tissues, but maximal levels of expression requires the entire set.

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Remus: A Web Application for Prioritization of Regulatory Regions and Variants in Monogenic Diseases

Frontiers in Genetics ◽

10.3389/fgene.2021.638960 ◽

2021 ◽

Vol 12 ◽

Author(s):

Paweł Sztromwasser ◽

Damian Skrzypczak ◽

Arkadiusz Michalak ◽

Wojciech Fendler

Keyword(s):

Web Application ◽

Diagnostic Yield ◽

Mirna Gene ◽

Pathogenic Mutation ◽

Regulatory Elements ◽

Monogenic Disease ◽

Regulatory Regions ◽

Tissue Specific ◽

Pathogenic Variants ◽

Monogenic Diseases

BackgroundAnalysis of variants in distant regulatory elements could improve the current 25–50% yield of genetic testing for monogenic diseases. However, the vast size of the regulome, great number of variants, and the difficulty in predicting their phenotypic impact make searching for pathogenic variants in the regulatory genome challenging. New tools for the identification of regulatory variants based on their relevance to the phenotype are needed.MethodsWe used tissue-specific regulatory loci mapped by ENCODE and FANTOM, together with miRNA–gene interactions from miRTarBase and miRWalk, to develop Remus, a web application for the identification of tissue-specific regulatory regions. Remus searches for regulatory features linked to the known disease-associated genes and filters them using activity status in the target tissues relevant for the studied disorder. For user convenience, Remus provides a web interface and facilitates in-browser filtering of variant files suitable for sensitive patient data.ResultsTo evaluate our approach, we used a set of 146 regulatory mutations reported causative for 68 distinct monogenic disorders and a manually curated a list of tissues affected by these disorders. In 89.7% of cases, Remus identified the regulator containing the pathogenic mutation. The tissue-specific search limited the number of considered variants by 82.5% as compared to a tissue-agnostic search.ConclusionRemus facilitates the identification of regulatory regions potentially associated with a monogenic disease and can supplement classical analysis of coding variations with the aim of improving the diagnostic yield in whole-genome sequencing experiments.

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UNDERSTANDING DISTAL TRANSCRIPTIONAL REGULATION FROM SEQUENCE, EXPRESSION AND INTERACTOME PERSPECTIVES

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720010004756 ◽

2010 ◽

Vol 08 (02) ◽

pp. 219-246 ◽

Cited By ~ 1

Author(s):

ARVIND RAO ◽

DAVID J. STATES ◽

ALFRED O. HERO ◽

JAMES DOUGLAS ENGEL

Keyword(s):

Computational Biology ◽

De Novo ◽

Sequence Data ◽

Regulatory Elements ◽

Proximal Promoter ◽

Regulatory Sequences ◽

Sequence Motifs ◽

Specific Sequence ◽

Specific Expression ◽

Regulatory Regions

Gene regulation in eukaryotes involves a complex interplay between the proximal promoter and distal genomic elements (such as enhancers) which work in concert to drive precise spatio-temporal gene expression. The experimental localization and characterization of gene regulatory elements is a very complex and resource-intensive process. The computational identification of regulatory regions that confer spatiotemporally specific tissue-restricted expression of a gene is thus an important challenge for computational biology. One of the most popular strategies for enhancer localization from DNA sequence is the use of conservation-based prefiltering and more recently, the use of canonical (transcription factor motifs) or de novo tissue-specific sequence motifs. However, there is an ongoing effort in the computational biology community to further improve the fidelity of enhancer predictions from sequence data by integrating other, complementary genomic modalities. In this work, we propose a framework that complements existing methodologies for prospective enhancer identification. The methods in this work are derived from two key insights: (i) that chromatin modification signatures can discriminate proximal and distally located regulatory regions and (ii) the notion of promoter-enhancer cross-talk (as assayed in 3C/5C experiments) might have implications in the search for regulatory sequences that co-operate with the promoter to yield tissue-restricted, gene-specific expression.

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funMotifs: Tissue-specific transcription factor motifs

10.1101/683722 ◽

2019 ◽

Cited By ~ 1

Author(s):

Husen M. Umer ◽

Karolina Smolinska-Garbulowska ◽

Nour-al-dain Marzouka ◽

Zeeshan Khaliq ◽

Claes Wadelius ◽

...

Keyword(s):

Large Scale ◽

Tissue Specificity ◽

Functional Characterization ◽

Regulatory Elements ◽

Web Based ◽

Tissue Specific ◽

Functional Variants ◽

Genome Wide ◽

Reporter Assays

ABSTRACTTranscription factors (TF) regulate gene expression by binding to specific sequences known as motifs. A bottleneck in our knowledge of gene regulation is the lack of functional characterization of TF motifs, which is mainly due to the large number of predicted TF motifs, and tissue specificity of TF binding. We built a framework to identify tissue-specific functional motifs (funMotifs) across the genome based on thousands of annotation tracks obtained from large-scale genomics projects including ENCODE, RoadMap Epigenomics and FANTOM. The annotations were weighted using a logistic regression model trained on regulatory elements obtained from massively parallel reporter assays. Overall, genome-wide predicted motifs of 519 TFs were characterized across fifteen tissue types. funMotifs summarizes the weighted annotations into a functional activity score for each of the predicted motifs. funMotifs enabled us to measure tissue specificity of different TFs and to identify candidate functional variants in TF motifs from the 1000 genomes project, the GTEx project, the GWAS catalogue, and in 2,515 cancer samples from the Pan-cancer analysis of whole genome sequences (PCAWG) cohort. To enable researchers annotate genomic variants or regions of interest, we have implemented a command-line pipeline and a web-based interface that can publicly be accessed on: http://bioinf.icm.uu.se/funmotifs.

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A modified pod specific promoter for high level heterologous expression of genes in legumes

Legume Research - An International Journal ◽

10.18805/lr-4073 ◽

2019 ◽

Author(s):

Aravind Kumar Konda ◽

Pallavi Singh ◽

Khela Ram Soren ◽

Narendra Pratap Singh

Keyword(s):

Gene Expression ◽

Regulatory Elements ◽

Specific Gene ◽

Specific Expression ◽

Tissue Specific ◽

Cis Acting ◽

Inducible Promoters ◽

Expression Of Genes ◽

Enhanced Expression ◽

High Level

Promoters are cis-acting regulatory elements that are usually present upstream to the coding sequences and determine the gene expression. Deployment of tissue specific and inducible promoters are constantly increasing for development of successful and stable multiple transgenic plants. To this end, as a strategy for enhanced expression of cis or transgenes, promoter engineering of the native msg promoter from soya bean has been carried out for executing pod specific expression of genes. Cis regulatory elements such as 5’UTR and poly (A) tract have been incorporated for imparting mRNA stability and translational enhancement to generate the modified 1.285 Kb pod specific promoter. Further to attain transcriptional enhancement the modified promoter has been cloned to generate Bi-directional Duplex Promoters (BDDP). The engineered msg promoter gene constructs can be deployed for high level tissue specific gene expression of cis/trans genes along with chosen terminator in chickpea. soybean and other legumes as well.

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A comparative analysis of chromatin accessibility in cattle, pig, and mouse tissues

10.1101/2020.08.13.249870 ◽

2020 ◽

Author(s):

Michelle M Halstead ◽

Colin Kern ◽

Perot Saelao ◽

Ying Wang ◽

Ganrea Chanthavixay ◽

...

Keyword(s):

Agricultural Research ◽

Rapid Evolution ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Model Organisms ◽

Individual Species ◽

Open Chromatin ◽

Tissue Specific ◽

Mouse Tissues ◽

Intergenic Regions

AbstractBackgroundAlthough considerable progress has been made towards annotating the noncoding portion of the human and mouse genomes, regulatory elements in other species, such as livestock, remain poorly characterized. This lack of functional annotation poses a substantial roadblock to agricultural research and diminishes the value of these species as model organisms. As active regulatory elements are typically characterized by chromatin accessibility, we implemented the Assay for Transposase Accessible Chromatin (ATAC-seq) to annotate and characterize regulatory elements in pigs and cattle, given a set of eight adult tissues.ResultsOverall, 306,304 and 273,594 active regulatory elements were identified in pig and cattle, respectively. 71,478 porcine and 47,454 bovine regulatory elements were highly tissue-specific and were correspondingly enriched for binding motifs of known tissue-specific transcription factors. However, in every tissue the most prevalent accessible motif corresponded to the insulator CTCF, suggesting pervasive involvement in 3-D chromatin organization. Taking advantage of a similar dataset in mouse, open chromatin in pig, cattle, and mice were compared, revealing that the conservation of regulatory elements, in terms of sequence identity and accessibility, was consistent with evolutionary distance; whereas pig and cattle shared about 20% of accessible sites, mice and ungulates only had about 10% of accessible sites in common. Furthermore, conservation of accessibility was more prevalent at promoters than at intergenic regions.ConclusionsThe lack of conserved accessibility at distal elements is consistent with rapid evolution of enhancers, and further emphasizes the need to annotate regulatory elements in individual species, rather than inferring elements based on homology. This atlas of chromatin accessibility in cattle and pig constitutes a substantial step towards annotating livestock genomes and dissecting the regulatory link between genome and phenome.

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A comparative analysis of chromatin accessibility in cattle, pig, and mouse tissues

BMC Genomics ◽

10.1186/s12864-020-07078-9 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Michelle M. Halstead ◽

Colin Kern ◽

Perot Saelao ◽

Ying Wang ◽

Ganrea Chanthavixay ◽

...

Keyword(s):

Agricultural Research ◽

Rapid Evolution ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Model Organisms ◽

Individual Species ◽

Open Chromatin ◽

Tissue Specific ◽

Mouse Tissues ◽

Intergenic Regions

Abstract Background Although considerable progress has been made towards annotating the noncoding portion of the human and mouse genomes, regulatory elements in other species, such as livestock, remain poorly characterized. This lack of functional annotation poses a substantial roadblock to agricultural research and diminishes the value of these species as model organisms. As active regulatory elements are typically characterized by chromatin accessibility, we implemented the Assay for Transposase Accessible Chromatin (ATAC-seq) to annotate and characterize regulatory elements in pigs and cattle, given a set of eight adult tissues. Results Overall, 306,304 and 273,594 active regulatory elements were identified in pig and cattle, respectively. 71,478 porcine and 47,454 bovine regulatory elements were highly tissue-specific and were correspondingly enriched for binding motifs of known tissue-specific transcription factors. However, in every tissue the most prevalent accessible motif corresponded to the insulator CTCF, suggesting pervasive involvement in 3-D chromatin organization. Taking advantage of a similar dataset in mouse, open chromatin in pig, cattle, and mice were compared, revealing that the conservation of regulatory elements, in terms of sequence identity and accessibility, was consistent with evolutionary distance; whereas pig and cattle shared about 20% of accessible sites, mice and ungulates only had about 10% of accessible sites in common. Furthermore, conservation of accessibility was more prevalent at promoters than at intergenic regions. Conclusions The lack of conserved accessibility at distal elements is consistent with rapid evolution of enhancers, and further emphasizes the need to annotate regulatory elements in individual species, rather than inferring elements based on homology. This atlas of chromatin accessibility in cattle and pig constitutes a substantial step towards annotating livestock genomes and dissecting the regulatory link between genome and phenome.

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Elements responsible for hormonal control and tissue specificity of L-type pyruvate kinase gene expression in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.4852 ◽

1992 ◽

Vol 12 (11) ◽

pp. 4852-4861 ◽

Cited By ~ 31

Author(s):

M H Cuif ◽

M Cognet ◽

D Boquet ◽

G Tremp ◽

A Kahn ◽

...

Keyword(s):

Gene Expression ◽

Small Intestine ◽

Pyruvate Kinase ◽

Tissue Specificity ◽

Proximal Region ◽

Hormonal Control ◽

Proximal Promoter ◽

Kinase Gene ◽

Tissue Specific

L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway specifically expressed in the liver and, to a lesser degree, in the small intestine and kidney. One important characteristic of L-PK gene expression is its strong activation by glucose and insulin and its complete inhibition by fasting or glucagon treatment. Having previously established that the entire rat L-PK gene plus 3.2 kbp of 5'-flanking region functions in mice in a tissue-specific and hormonally regulated manner, various deletions of these 3.2 kbp of 5'-flanking regions were tested in transgenic animals to map the cis-acting elements involved in transcriptional gene regulation. Our experiments indicate that the proximal region between -183 and +11 confers tissue specificity and contains all the information necessary for dietary and hormonal control of L-PK gene expression in vivo. We found, however, that the transcriptional activity generated by this proximal promoter fragment can be modulated by distal sequences in a tissue-specific manner. (i) Sequences between bp -183 and -392 seem to play a dual role in the liver and small intestine; they induce L-PK expression in the liver but repress it in the small intestine. (ii) Sequences from bp -392 up to -1170 do not seem to have any additional effect on promoter activity. (iii) Between bp -1170 and -2080, we found a putative extinguisher whose transcriptional inhibitory effect is much more marked in the small intestine than in the liver. (iv) Finally, between bp -2080 and -3200, we identified an activating sequence required for full expression of the gene in the liver.

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LINE retrotransposons characterize mammalian tissue-specific and evolutionarily dynamic regulatory regions

Genome Biology ◽

10.1186/s13059-021-02260-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Maša Roller ◽

Ericca Stamper ◽

Diego Villar ◽

Osagie Izuogu ◽

Fergal Martin ◽

...

Keyword(s):

Tissue Specificity ◽

Single Species ◽

Evolutionary Stability ◽

Regulatory Evolution ◽

Regulatory Regions ◽

Tissue Specific ◽

Long Interspersed Nuclear Elements ◽

Genomic Regions ◽

Regulatory Landscape ◽

Specific Line

Abstract Background To investigate the mechanisms driving regulatory evolution across tissues, we experimentally mapped promoters, enhancers, and gene expression in the liver, brain, muscle, and testis from ten diverse mammals. Results The regulatory landscape around genes included both tissue-shared and tissue-specific regulatory regions, where tissue-specific promoters and enhancers evolved most rapidly. Genomic regions switching between promoters and enhancers were more common across species, and less common across tissues within a single species. Long Interspersed Nuclear Elements (LINEs) played recurrent evolutionary roles: LINE L1s were associated with tissue-specific regulatory regions, whereas more ancient LINE L2s were associated with tissue-shared regulatory regions and with those switching between promoter and enhancer signatures across species. Conclusions Our analyses of the tissue-specificity and evolutionary stability among promoters and enhancers reveal how specific LINE families have helped shape the dynamic mammalian regulome.

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