scholarly journals Transvection in the Drosophila Abd-B Domain: Extensive Upstream Sequences Are Involved in Anchoring Distant cis-Regulatory Regions to the Promoter

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 1031-1050 ◽  
Author(s):  
László Sipos ◽  
József Mihály ◽  
François Karch ◽  
Paul Schedl ◽  
János Gausz ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Morphological Difference ◽  
Upstream Region ◽  
Homeotic Genes ◽  
Dependent Manner ◽  
Discrete Elements ◽  
Regulatory Regions ◽  
B Locus ◽  
Stage Of Development ◽  
Trans Regulation

Abstract The Abd-B gene, one of the three homeotic genes in the Drosophila bithorax complex (BX-C), is required for the proper identity of the fifth through the eighth abdominal segments (corresponding to parasegments 10–14) of the fruitfly. The morphological difference between these four segments is due to the differential expression of Abd-B, which is achieved by the action of the parasegment-specific cisregulatory regions infra-abdominal-5 (iab-5), -6, -7 and -8. The dominant gain-of-function mutation Frontabdominal-7 (Fab-7) removes a boundary separating two of these cis-regulatory regions, iab-6 and iab-7. As a consequence of the Fab-7 deletion, the parasegment 12- (PS12-) specific iab-7 is ectopically activated in PS11. This results in the transformation of the sixth abdominal segment (A6) into the seventh (A7) in Fab-7 flies. Here we report that point mutations of the Abd-B gene in trans suppress the Fab-7 phenotype in a pairing-dependent manner and thus represent a type of transvection. We show that the observed suppression is the result of trans-regulation of the defective Abd-B gene by the ectopically activated iab-7. Unlike previously demonstrated cases of trans-regulation in the Abd-B locus, trans-suppression of Fab-7 is sensitive to heterozygosity for chromosomal rearrangements that disturb homologous pairing at the nearby Ubx locus. However, in contrast to Ubx, the transvection we observed in the Abd-B locus is insensitive to the allelic status of zeste. Analysis of different deletion alleles of Abd-B that enhance trans-regulation suggests that an extensive upstream region, different from the sequences required for transcription initiation, mediates interactions between the iab cis-regulatory regions and the proximal Abd-B promoter. Moreover, we find that the amount of DNA deleted in the upstream region is roughly proportional to the strength of trans-interaction, suggesting that this region consists of numerous discrete elements that cooperate in tethering the iab regulatory domains to Abd-B. Possible implications of the tethering complex for the regulation of Abd-B are discussed. In addition, we present evidence that the tenacity of trans-interactions in the Abd-B gene may vary, depending upon the tissue and stage of development.

scholarly journals Cis and trans interactions between the iab regulatory regions and abdominal-A and abdominal-B in Drosophila melanogaster.

Genetics ◽  
1995 ◽  
Vol 139 (2) ◽  
pp. 835-848 ◽  
Author(s):  
J E Hendrickson ◽  
S Sakonju
Keyword(s):  
Drosophila Melanogaster ◽  
Chromosomal Rearrangements ◽  
Transcription Unit ◽  
Homeotic Genes ◽  
Regulatory Regions ◽  
In Trans ◽  
Trans Regulation ◽  
Cis And Trans ◽  
In Cis ◽  
Target Promoter

Abstract The infra-abdominal (iab) elements in the bithorax complex of Drosophila melanogaster regulate the transcription of the homeotic genes abdominal-A (abd-A) and Abdominal-B (Abd-B) in cis. Here we describe two unusual aspects of regulation by the iab elements, revealed by an analysis of an unexpected complementation between mutations in the Abd-B transcription unit and these regulatory regions. First, we find that iab-6 and iab-7 can regulate Abd-B in trans. This iab trans regulation is insensitive to chromosomal rearrangements that disrupt transvection effects at the nearby Ubx locus. In addition, we show that a transposed Abd-B transcription unit and promoter on the Y chromosome can be activated by iab elements located on the third chromosome. These results suggest that the iab regions can regulate their target promoter located at a distant site in the genome in a manner that is much less dependent on homologue pairing than other transvection effects. The iab regulatory regions may have a very strong affinity for the target promoter, allowing them to interact with each other despite the inhibitory effects of chromosomal rearrangements. Second, by generating abd-A mutations on rearrangement chromosomes that break in the iab-7 region, we show that these breaks induce the iab elements to switch their target promoter from Abd-B to abd-A. These two unusual aspects of iab regulation are related by the iab-7 breakpoint chromosomes that prevent iab elements from acting on Abd-B and allow them to act on abd-A. We propose that the iab-7 breaks prevent both iab trans regulation and target specificity by disrupting a mechanism that targets the iab regions to the Abd-B promoter.

Role of desiccation in the termination of expression of genes for storage proteins

1994 ◽  
Vol 4 (2) ◽  
pp. 149-173 ◽  
Author(s):  
L. Jiang ◽  
A. R. Kermode
Keyword(s):  
Gene Expression ◽  
Storage Protein ◽  
Storage Proteins ◽  
Transient Gene Expression ◽  
Upstream Region ◽  
Regulatory Regions ◽  
Tobacco Seed ◽  
Gene Construct ◽  
Stage Of Development ◽  
Expression Of Genes

AbstractThe final stage of development of orthodox seeds is maturation drying, a process often accompanied by a dramatic decline in storage-protein synthesis. When desiccation is imposed prematurely at certain stages prior to the completion of development, a switch in synthetic events is elicited. events unique to development, such as synthesis of storage protein, are terminated, while syntheses associated with germination and growth are initiated. We investigated whether desiccation plays a key role in effecting a decline in expression of genes for storage proteins by acting directly upon the regulatory regions of these developmental genes. The desiccation responsiveness of the 5′ and 3′ regulatory regions of the gene for the pea storage protein vicilin was tested in transgenic tobacco seed. Chimaeric genes were introduced into tobacco, these genes consisted of the coding region of the reporter gene for β-glucuronidase (GUS) and 5′ and 3′ regions from the vicilin gene, or, as controls, the same regions derived from constitutively expressed genes, presumed to be desiccation-insensitive – those from the cauliflower mosaic virus (35S) and nopaline synthase genes. In transgenic seed expressing the gene constructs containing the vicilin 5′ upstream region, GUS activities declined dramatically after imbibition in mature seed and after rehydration of prematurely dried tobacco seed. In contrast, GUS activities increased after seed rehydration when the constitutive viral (35S) promoter replaced the vicilin 5′ upstream region. The 3′ downstream region of the gene construct did not significantly affect the patterns of changes in GUS activities after seed rehydration. In studies of transient gene expression in castor bean cotyledons, premature desiccation led to termination ofGUSgene expression only when the gene construct contained the vicilin 5′ upstream region. This region may respond directly to desiccation; alternatively, changes totrans-acting factors important for expression of genes for storage-proteins may occur as a result of drying.

scholarly journals A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

1993 ◽  
Vol 13 (3) ◽  
pp. 1610-1618 ◽  
Author(s):  
J E Slansky ◽  
Y Li ◽  
W G Kaelin ◽  
P J Farnham
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Phase Boundary ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Protein S ◽  
S Phase ◽  
Dependent Manner

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence

1993 ◽  
Vol 13 (7) ◽  
pp. 3841-3849
Author(s):  
B Zenzie-Gregory ◽  
A Khachi ◽  
I P Garraway ◽  
S T Smale
Keyword(s):  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Upstream Region ◽  
Promoter Strength ◽  
Recognition Sequence ◽  
Specific Recognition ◽  
Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

Posterior stripe expression of hunchback is driven from two promoters by a common enhancer element

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 3067-3077 ◽  
Author(s):  
J.S. Margolis ◽  
M.L. Borowsky ◽  
E. Steingrimsson ◽  
C.W. Shim ◽  
J.A. Lengyel ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Drosophila Embryo ◽  
Upstream Region ◽  
Enhancer Element ◽  
Gap Gene ◽  
Gap Genes ◽  
Two Phases ◽  
Necessary And Sufficient

The gap gene hunchback (hb) is required for the formation and segmentation of two regions of the Drosophila embryo, a broad anterior domain and a narrow posterior domain. Accumulation of hb transcript in the posterior of the embryo occurs in two phases, an initial cap covering the terminal 15% of the embryo followed by a stripe at the anterior edge of this region. By in situ hybridization with transcript-specific probes, we show that the cap is composed only of mRNA from the distal transcription initiation site (P1), while the later posterior stripe is composed of mRNA from both the distal and proximal (P2) transcription initiation sites. Using a series of genomic rescue constructs and promoter-lacZ fusion genes, we define a 1.4 kb fragment of the hb upstream region that is both necessary and sufficient for posterior expression. Sequences within this fragment mediate regulation by the terminal gap genes tailless (tll) and a huckebein, which direct the formation of the posterior hb stripe. We show that the tll protein binds in vitro to specific sites within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of gene expression by tll. We propose a model in which the anterior border of the posterior hb stripe is determined by tll concentration in a manner analogous to the activation of anterior hb expression by bicoid.

Discrete Polycomb-binding sites in each parasegmental domain of the bithorax complex

Development ◽  
1995 ◽  
Vol 121 (6) ◽  
pp. 1681-1689 ◽  
Author(s):  
A. Chiang ◽  
M.B. O'Connor ◽  
R. Paro ◽  
J. Simon ◽  
W. Bender
Keyword(s):  
Binding Sites ◽  
Polytene Chromosomes ◽  
Homeotic Genes ◽  
Bithorax Complex ◽  
Lacz Reporter Gene ◽  
Regulatory Regions ◽  
Cooperative Action ◽  
Polycomb Protein ◽  
Regulatory Dna ◽  
Segmental Expression

The Polycomb protein of Drosophila melanogaster maintains the segmental expression limits of the homeotic genes in the bithorax complex. Polycomb-binding sites within the bithorax complex were mapped by immunostaining of salivary gland polytene chromosomes. Polycomb bound to four DNA fragments, one in each of four successive parasegmental regulatory regions. These fragments correspond exactly to the ones that can maintain segmentally limited expression of a lacZ reporter gene. Thus, Polycomb acts directly on discrete multiple sites in bithorax regulatory DNA. Constructs combining fragments from different regulatory regions demonstrate that Polycomb-dependent maintenance elements can act on multiple pattern initiation elements, and that maintenance elements can work together. The cooperative action of maintenance elements may motivate the linear order of the bithorax complex.

scholarly journals Functional reconstruction of trans regulation of the Ultrabithorax promoter by the products of two antagonistic genes, trithorax and Polycomb.

1995 ◽  
Vol 15 (12) ◽  
pp. 6601-6612 ◽  
Author(s):  
Y L Chang ◽  
B O King ◽  
M O'Connor ◽  
A Mazo ◽  
D H Huang
Keyword(s):  
Expression Patterns ◽  
Proximal Promoter ◽  
Homeotic Genes ◽  
Promoter Activation ◽  
Range Interaction ◽  
Cis Acting ◽  
Functional Reconstruction ◽  
Long Range Interaction ◽  
Trans Regulation ◽  
Zinc Binding Domain

Maintenance of the "on-off" state of Drosophila homeotic genes in Antennapedia and bithorax complexes requires activities of the trithorax and Polycomb groups of genes. To identify cis-acting sequences for functional reconstruction of regulation by both trithorax and Polycomb, we examined the expression patterns of several Ubx-lacZ transgenes that carry upstream fragments corresponding to a region of approximately 50 kb. A 14.5-kb fragment from the postbithorax/bithoraxoid region of Ultrabithorax exhibited proper regulation by both trithorax and Polycomb in the embryonic central nervous system. Using a Drosophila haploid cell line for transient expression, we found that trithorax or Polycomb can function independently through this upstream fragment to activate or repress the Ultrabithorax promoter, respectively. Studies of deletion mutants of trithorax and Polycomb demonstrated that trithorax-dependent activation requires the central zinc-binding domain, while Polycomb-dependent repression requires the intact chromodomain. In addition, trithorax-dependent activity can be abrogated by increasing the amount of Polycomb, suggesting a competitive interaction between the products of trithorax and Polycomb. Deletion analysis of this fragment demonstrated that a 440-bp fragment contains response elements for both trithorax and Polycomb. Furthermore, we showed that the integrity of the proximal promoter region is essential for trithorax-dependent activation, implicating a long-range interaction for promoter activation.

scholarly journals Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region.

1987 ◽  
Vol 7 (6) ◽  
pp. 2046-2051 ◽  
Author(s):  
A D Garcia ◽  
A M O'Connell ◽  
S J Sharp
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Upstream Region ◽  
Nucleotide Position ◽  
Wild Type ◽  
5S Rna ◽  
Cell Extracts

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

scholarly journals Genomic Insights into the Different Layers of Gene Regulation in Yeast

2011 ◽  
Vol 2011 ◽  
pp. 1-12
Author(s):  
José E. Pérez-Ortín ◽  
Daniel A. Medina ◽  
Antonio Jordán-Pla
Keyword(s):  
Gene Regulation ◽  
Transcription Initiation ◽  
Model Organism ◽  
New Methods ◽  
Control Gene Expression ◽  
Gene Sets ◽  
Initiation Step ◽  
Technical Features ◽  
Trans Regulation ◽  
Different Levels

The model organism Saccharomyces cerevisiae has allowed the development of new functional genomics techniques devoted to the study of transcription in all its stages. With these techniques, it has been possible to find interesting new mechanisms to control gene expression that act at different levels and for different gene sets apart from the known cis-trans regulation in the transcription initiation step. Here we discuss a method developed in our laboratory, Genomic Run-On, and other new methods that have recently appeared with distinct technical features. A comparison between the datasets generated by them provides interesting genomic insights into the different layers of gene regulation in eukaryotes.

Differential Requirements for Survivin in Hematopoietic Cell Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1257-1257
Author(s):  
Yanfei Xu ◽  
Sandeep Gurbuxani ◽  
Ganesan Keerthivasan ◽  
Amittha Wickrema ◽  
John D. Crispino
Keyword(s):  
Cell Cycle ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Caspase 3 ◽  
Terminal Differentiation ◽  
Dependent Manner ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Stage Of Development ◽  
Primary Mouse

Abstract The development of the complete repertoire of blood cells from a common progenitor, the hematopoietic stem cell, is a tightly controlled process that is regulated, in part, by the activity of lineage specific transcription factors. Despite our knowledge of these factors, the mechanisms that regulate the formation and growth of distinct, but closely related lineages, such as erythroid cells and megakaryocytes, remain largely uncharacterized. Here we show that Survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express Survivin throughout their maturation, up to the terminal stage of differentiation (orthochromatic), even after the cells exit the cell cycle. This is surprising because Survivin is generally expressed in a cell cycle dependent manner and not thought to be expressed in terminally differentiated cells. In contrast, purified murine megakaryocytes express nearly 5-fold lower levels of Survivin mRNA compared to erythroid cells. To investigate whether Survivin is involved in the differentiation and/or survival of hematopoietic progenitors, we infected primary mouse bone marrow cells with retroviruses harboring either the human Survivin cDNA or a mouse Survivin shRNA, and then induced erythroid and megakaryocyte differentiation in both liquid culture and colony-forming assays. These studies revealed that overexpression of Survivin promoted the terminal differentiation of red blood cells, while its reduction, by RNA interference, inhibited their differentiation. In contrast, downregulation of Survivin facilitated the expansion of megakaryocytes, and its overexpression antagonized megakaryocyte formation. In addition, consistent with a role for survivin in erythropoiesis, downregulation of Survivin expression in MEL cells led to a block in terminal differentiation. Finally, since caspase activity is known to be required for erythroid maturation, we investigated whether survivin associated with cleaved caspase-3 in erythroid cells. Immunofluorescence revealed that Survivin and cleaved caspase-3 co-localized to discrete foci within the cytoplasm of erythroid cells at the orthochromatic stage of development. Based on these findings, we hypothesize that Survivin cooperates with cleaved caspase-3 in terminal maturation of red blood cells. Together, our findings demonstrate that Survivin plays multiple, distinct roles in hematopoiesis.

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