Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis. Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster. Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

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Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster

10.1101/452649 ◽

2018 ◽

Author(s):

Victoria Jorgensen ◽

Jingxun Chen ◽

Helen Vander Wende ◽

Devon Harris ◽

Siu Wah Wong-Deyrup ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Alcohol Dehydrogenase ◽

Proximal Promoter ◽

Start Codon ◽

Transcriptional Interference ◽

Developmentally Regulated ◽

Larval Stages ◽

Nearby Region ◽

Distal Promoter ◽

Mrna Isoform

AbstractNeighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis. Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster. Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter does also result in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, we show that transcription from the distal promoter is sufficient to repress proximal transcription in larvae and that the degree of this repression depends on the degree of distal promoter activity. Finally, repression of the endogenous Adh proximal promoter is associated with the enrichment of histone 3 lysine 36 trimethylation (H3K36me3), a chromatin mark necessary for transcription-coupled gene repression in yeast. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.

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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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Evidence for an Integrated Gene Repression Mechanism Based on mRNA Isoform Toggling in Human Cells

10.1101/264721 ◽

2018 ◽

Author(s):

Ina Hollerer ◽

Juliet C. Barker ◽

Victoria Jorgensen ◽

Amy Tresenrider ◽

Claire Dugast-Darzacq ◽

...

Keyword(s):

Embryonic Stem ◽

Stem Cell Differentiation ◽

Open Reading Frames ◽

Proximal Promoter ◽

Embryonic Stem Cell Differentiation ◽

Transcriptional Interference ◽

Upstream Open Reading Frames ◽

Regulate Protein ◽

Distal Promoter ◽

Mrna Isoform

ABSTRACTWe recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference were used in an integrated manner to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that blocked translation of the ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2. We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs.

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The Alcohol Dehydrogenase Gene Is Nested in the outspread Locus of Drosophila melanogaster

Genetics ◽

10.1093/genetics/143.2.897 ◽

1996 ◽

Vol 143 (2) ◽

pp. 897-911 ◽

Cited By ~ 1

Author(s):

S McNabb ◽

S Greig ◽

T Davis

Keyword(s):

Drosophila Melanogaster ◽

Alcohol Dehydrogenase ◽

Pcr Amplification ◽

Transcription Unit ◽

Adjacent Gene ◽

Dehydrogenase Gene ◽

Chromosomal Breakpoints ◽

Splicing Patterns ◽

Somatic Musculature

Abstract This report describes the structure and expression of the outspread (osp) gene of Drosophila melanogaster. Previous work showed that chromosomal breakpoints associated with mutations of the osp locus map to both sides of the alcohol dehydrogenase gene (Adh), suggesting that Adh and the adjacent gene Adh' are nested in osp. We extended a chromosomal walk and mapped additional osp mutations to define the maximum molecular limit of osp as 119 kb. We identified a 6-kb transcript that hybridizes to osp region DNA and is altered or absent in osp mutants. Accumulation of this RNA peaks during embryonic and pupal periods. The osp cDNAs comprise two distinct classes based on alternative splicing patterns. The 5′ end of the longest cDNA was extended by PCR amplification. When hybridized to the osp walk, the 5′ extension verifies that Adh and Adh' are nested in osp and shows that osp has a transcription unit of ≥74 kb. In situ hybridization shows that osp is expressed both maternally and zygotically. In the ovary, osp is transcribed in nurse cells and localized in the oocyte. In embryos, expression is most abundant in the developing visceral and somatic musculature.

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GENETIC AND BIOCHEMICAL BASIS OF ENZYME ACTIVITY VARIATION IN NATURAL POPULATIONS. I. ALCOHOL DEHYDROGENASE IN DROSOPHILA MELANOGASTER

Genetics ◽

10.1093/genetics/89.2.371 ◽

1978 ◽

Vol 89 (2) ◽

pp. 371-388

Author(s):

John F McDonald ◽

Francisco J Ayala

Keyword(s):

Gene Regulation ◽

Drosophila Melanogaster ◽

Alcohol Dehydrogenase ◽

Natural Populations ◽

Difficult Problem ◽

Regulatory Genes ◽

Biochemical Basis ◽

Evolutionary Consequence ◽

The Third ◽

Adh Activity

ABSTRACT Recent studies by various authors suggest that variation in gene regulation may be common in nature, and might be of great evolutionary consequence; but the ascertainment of variation in gene regulation has proven to be a difficult problem. In this study, we explore this problem by measuring alcohol dehydrogenase (ADH) activity in Drosophila melanogaster strains homozygous for various combinations of given second and third chromosomes sampled from a natural population. The structural locus (Adh) coding for ADH is on the second chromosome. The results show that: (1) there are genes, other than Adh, that affect the levels of ADH activity; (2) at least some of these "regulatory" genes are located on the third chromosome, and thus are not adjacent to the Adh locus; (3) variation exists in natural populations for such regulatory genes; (4) the effect of these regulatory genes varies as they interact with different second chromosomes; (5) third chromosomes with high-activity genes are either partially or completely dominant over chromosomes with low-activity genes; (6) the effects of the regulatory genes are pervasive throughout development; and (7) the third chromosome genes regulate the levels of ADH activity by affecting the number of ADH molecules in the flies. The results are consistent with the view that the evolution of regulatory genes may play an important role in adaptation.

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Regulation of alcohol dehydrogenase in Drosophila melanogaster by dietary alcohol and carbohydrate

Insect Biochemistry ◽

10.1016/0020-1790(84)90034-9 ◽

1984 ◽

Vol 14 (2) ◽

pp. 231-242 ◽

Cited By ~ 61

Author(s):

Stephen W McKechnie ◽

Billy W Geer

Keyword(s):

Drosophila Melanogaster ◽

Alcohol Dehydrogenase

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The β1 Integrin Distal Promoter is Developmentally Regulated in Transgenic Mice

Cell Adhesion and Communication ◽

10.3109/15419069309097254 ◽

1993 ◽

Vol 1 (3) ◽

pp. 203-212 ◽

Cited By ~ 10

Author(s):

Emilio Hirsch ◽

Fiorella Balzac ◽

Cristina Pastore ◽

Guido Tarone ◽

Lorenzo Silengo ◽

...

Keyword(s):

Transgenic Mice ◽

Β1 Integrin ◽

Developmentally Regulated ◽

Distal Promoter

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Perturbation of chromatin architecture on ecdysterone induction of Drosophila melanogaster small heat shock protein genes.

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.332 ◽

1989 ◽

Vol 9 (1) ◽

pp. 332-335 ◽

Cited By ~ 7

Author(s):

S E Kelly ◽

I L Cartwright

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Heat Shock Protein ◽

Intergenic Region ◽

Small Heat Shock Protein ◽

Dnase I ◽

Developmentally Regulated ◽

Regulatory Interactions ◽

Chromatin Architecture ◽

Heat Shock Protein Genes

Alterations in the pattern of DNase I hypersensitivity were observed on ecdysterone-stimulated transcription of Drosophila melanogaster small heat shock protein genes. Perturbations were induced near hsp27 and hsp22, coupled with an extensive domain of chromatin unfolding in the intergenic region between hsp23 and the developmentally regulated gene 1. These regions represent candidates for ecdysterone regulatory interactions.

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