scholarly journals A Novel Negative Fe-Deficiency-Responsive Element and a TGGCA-Type-Like FeRE Control the Expression ofFTR1inChlamydomonas reinhardtii

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Xiaowen Fei ◽  
Mats Eriksson ◽  
Yajun Li ◽  
Xiaodong Deng
Keyword(s):  
Iron Deficiency ◽  
Chlamydomonas Reinhardtii ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Fe Deficiency ◽  
Start Site ◽  
Response Elements ◽  
Responsive Element ◽  
Necessary And Sufficient

We have reported three Fe-deficiency-responsive elements (FEREs),FOX1, ATX1,andFEA1, all of which are positive regulatory elements in response to iron deficiency inChlamydomonas reinhardtii. Here we describeFTR1, another iron regulated gene and mutational analysis of its promoter. Our results reveal that the FeREs ofFTR1distinguish itself from other iron response elements by containing bothnegativeandpositiveregulatory regions. InFTR1, the−291/−236 region from the transcriptional start site is necessary and sufficient for Fe-deficiency-inducible expression. This region contains two positive FeREs with a TGGCA-like core sequence: the FtrFeRE1 (ATGCAGGCT) at−287/−279 and the FtrFeRE2 (AAGCGATTGCCAGAGCGC) at−253/−236. Furthermore, we identified a novel FERE, FtrFeRE3 (AGTAACTGTTAAGCC) localized at−319/−292, which negatively influences the expression ofFTR1.

scholarly journals Identification and transcriptional regulation of the mir-218-1 alternative promoter

2020 ◽  
Author(s):  
Brenna A. Rheinheimer ◽  
Lukas Vrba ◽  
Bernard W Futscher ◽  
Ronald L Heimark
Keyword(s):  
Transcriptional Regulation ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Chromatin Immunoprecipitation ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Alternative Promoter ◽  
Ductal Adenocarcinoma ◽  
Start Site ◽  
Public Datasets ◽  
Intron 4

AbstractBackgroundmiRNAs are small, endogenous non-coding RNAs approximately 22 nucleotides in length that account for approximately 1% of the genome and play key regulatory roles in multiple signaling pathways. mir-218-1 is an intronic miRNA located within intron 15 of the SLIT2 gene. Public datasets showed enrichment of H3K4me3 within intron 4 of the SLIT2 gene. Therefore, we sought to determine the genomic location and transcriptional regulatory elements of the mir-218-1 candidate alternative promoter in pancreatic ductal adenocarcinoma.MethodsExpression of mir-218 was evaluated in a panel of pancreatic ductal adenocarcinoma cell lines. The mir-218-1 candidate alternative promoter was characterized by chromatin immunoprecipitation, Sequenom, and luciferase assays. Transcriptional regulation of the mir-218-1 candidate alternative promoter was assessed using chromatin immunoprecipitation and an inhibitor to NF-kB.ResultsWe found that expression of mir-218-1 does not correlate with SLIT2 expression and that mir-218-1 has a novel transcriptional start site separate from the SLIT2 promoter. This novel transcriptional start site showed transcriptional activity and was regulated by NF-kB.Conclusionsmir-218-1 is transcribed from an independent and novel transcriptional start site located within intron 4 of the SLIT2 gene in pancreatic ductal adenocarcinoma. Additionally, mir-218-1 expression is regulated by Nf-kB at this alternative transcriptional start site in pancreatic cancer.

scholarly journals Characterization of the promoter of human adipocyte hormone-sensitive lipase

1997 ◽  
Vol 328 (2) ◽  
pp. 453-461 ◽  
Author(s):  
Jacques GROBER ◽  
Henrik LAURELL ◽  
Régis BLAISE ◽  
Béatrice FABRY ◽  
Stéphane SCHAAK ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Rnase H ◽  
Hormone Sensitive Lipase ◽  
Start Codon ◽  
Start Site ◽  
Rnase Protection ◽  
Human Adipocyte ◽  
Hormone Sensitive

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5ʹ untranslated region (5ʹ-UTR). A single 5ʹ-UTR of approx. 70 nt was detected in RNase H mapping experiments. Two 5ʹ-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase protection experiments, with probes derived from the two products, showed that human adipocyte HSL mRNA contains only the 70 nt product. Primer extension analysis mapped the transcriptional start site 74 nt upstream of the start codon. In HT29, a human cell line expressing HSL, the presence of the short or the long 5ʹ-UTR is mutually exclusive. The short and long 5ʹ-UTR exons were located 1.5 and approx. 13 kb respectively upstream of the first coding exon. Various portions of the 5ʹ-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.

scholarly journals Transcriptional activation of vesicular monoamine transporter 2 in the pre-B cell line Ea3.123

1999 ◽  
Vol 337 (2) ◽  
pp. 193-199 ◽  
Author(s):  
Fiona WATSON ◽  
Damian G. DEAVALL ◽  
Janet A. MACRO ◽  
Rachel KIERNAN ◽  
Rod DIMALINE
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Vesicular Monoamine Transporter ◽  
Start Site ◽  
Monoamine Transporter ◽  
Vesicular Monoamine Transporter 2

Uptake and storage of monoamines in secretory granules is accomplished by vesicular monoamine transporters, and it is likely that vesicular monoamine transporter 2 (VMAT2) is important for histamine transport in vivo. In the present study we have used the pre-B-cell line Ea3.123 to investigate the mechanisms involved in the transcriptional activation of the VMAT2 gene. In Ea3.123 cells, VMAT2 mRNA abundance was increased following mobilization of intracellular calcium, and this increased mRNA expression was paralleled by changes in l-histidine decarboxylase mRNA, suggesting that VMAT2 may be responsible for sequestration of histamine into secretory vesicles in this cell line. We cloned the 5´-flanking region of the VMAT2 gene and determined its transcriptional start site by primer extension of rat VMAT2 mRNA. There was no TATA or TATA-like sequence upstream of this region; instead there were GC-rich elements, Ca2+/cAMP-response-element- and SP1-binding motifs. Approx. 900 bp upstream of the transcriptional start site was a purine–pyrimidine repeat sequence that may form a Z-DNA structure. A series of 5´-deletional VMAT2-promoter segments cloned upstream of a luciferase reporter were capable of driving transcription and indicated the presence of multiple regulatory elements, while stimulation with ionomycin or PMA resulted in an increased level of the transcriptional activity of the 5´-promoter segments studied.

scholarly journals Putative cis-regulatory elements predict iron deficiency responses in Arabidopsis roots

2019 ◽  
Author(s):  
Birte Schwarz ◽  
Christina B. Azodi ◽  
Shin-Han Shiu ◽  
Petra Bauer
Keyword(s):  
Transcription Factor ◽  
Iron Deficiency ◽  
Large Scale ◽  
Computational Models ◽  
Regulatory Elements ◽  
Transport Systems ◽  
Fe Deficiency ◽  
Grass Species ◽  
Bhlh Transcription Factor ◽  
Fe Uptake

AbstractIron (Fe) is a key cofactor in many cellular redox processes, including respiration and photosynthesis. Plant Fe deficiency (-Fe) activates a complex regulatory network which coordinates root Fe uptake and distribution to sink tissues, while avoiding over-accumulation of Fe and other metals to toxic levels. In Arabidopsis (Arabidopsis thaliana), FIT (FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR), a bHLH transcription factor (TF), is required for up-regulation of root Fe acquisition genes. However, other root and shoot -Fe-induced genes involved in Fe allocation and signaling are FIT-independent. The cis-regulatory code, i.e. the cis-regulatory elements (CREs) and their combinations that regulate plant -Fe-responses, remains largely elusive. Using Arabidopsis genome and transcriptome data, we identified over 100 putative CREs (pCREs) that were predictive of -Fe-induced up-regulation of genes in root tissue. We used large-scale in vitro TF binding data, association with FIT-dependent or FIT-independent co-expression clusters, positional bias, and evolutionary conservation to assess pCRE properties and possible functions. In addition to bHLH and MYB TFs, also B3, NAC, bZIP, and TCP TFs might be important regulators for -Fe responses. Our approach uncovered IDE1 (Iron Deficiency-responsive Element 1), a -Fe response CRE in grass species, to be conserved in regulating genes for biosynthesis of Fe-chelating compounds also in Arabidopsis. Our findings provide a comprehensive source of cis-regulatory information for -Fe-responsive genes, that advances our mechanistic understanding and informs future efforts in engineering plants with more efficient Fe uptake or transport systems.One sentence summary>100 putative cis-regulatory elements robustly predict Arabidopsis root Fe deficiency-responses in computational models, and shed light on the mechanisms of transcriptional regulation.

scholarly journals Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements

2018 ◽  
Author(s):  
Verena Thormann ◽  
Laura V. Glaser ◽  
Maika C. Rothkegel ◽  
Marina Borschiwer ◽  
Melissa Bothe ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Glucocorticoid Receptor ◽  
Binding Site ◽  
Genome Engineering ◽  
Target Genes ◽  
Transcriptional Start Site ◽  
Chromatin Accessibility ◽  
Start Site ◽  
Response Elements ◽  
Genomic Response

The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used homology directed repair (HDR)-mediated genome editing to add a single GR binding site directly upstream of the transcriptional start site of four genes. To our surprise, we found that the addition of a single GR binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two out of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Further, by introducing GR binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.

Localization of transcriptional regulatory elements and nuclear factor binding sites in mouse ribosomal protein gene rpL32

1989 ◽  
Vol 9 (5) ◽  
pp. 2067-2074
Author(s):  
M L Atchison ◽  
O Meyuhas ◽  
R P Perry
Keyword(s):  
Ribosomal Protein ◽  
Dna Sequences ◽  
Binding Sites ◽  
Protein Gene ◽  
Transcriptional Start Site ◽  
Ribosomal Protein Gene ◽  
Regulatory Elements ◽  
Sequence Motif ◽  
Start Site ◽  
Nuclear Factors

The DNA sequences required for expression of the ribosomal protein gene rpL32 were identified by transient-expression assays of chimeric rpL32-chloramphenicol acetyltransferase genes. These studies showed that maximal rpL32 expression requires sequences in a 150- to 200-base-pair region spanning the transcriptional start site. Three discrete regions of importance were identified: one between positions -79 and -69 and two others located downstream of the transcriptional start site. Progressive 5' or 3' deletions caused stepwise decreases in expression, which suggested a complex interplay of redundant or compensatory elements. Gel mobility shift assays were used to identify trans-acting nuclear factors which bind to segments of the rpL32 promoter that are known to be important for transcription. Evidence for several distinct nuclear factors is presented. The binding sites for these factors were localized to the following regions: -79 to -69, -36 to -19, -19 to +11, +11 to +46 in exon I, and within the first 31 base pairs of intron 1. One of these factors may bind to multiple sites within the promoter region. Interestingly, the factor that binds to a sequence motif in the first exon also binds to similar motifs in a comparable region of the c-myc gene.

scholarly journals Enhanceosome Formation over the Beta Interferon Promoter Underlies a Remote-Control Mechanism Mediated by YY1 and YY2

2005 ◽  
Vol 25 (22) ◽  
pp. 10159-10170 ◽  
Author(s):  
Martin Klar ◽  
Juergen Bode
Keyword(s):  
Transcription Factor ◽  
Computational Analysis ◽  
Control Mechanism ◽  
Transcriptional Start Site ◽  
The Novel ◽  
Start Site ◽  
Dna Structures ◽  
Beta Interferon ◽  
Responsive Element ◽  
Upstream Control

ABSTRACT The expression of beta interferon genes from humans and mice is under the immediate control of a virus-responsive element (VRE) that terminates 110 bp upstream from the transcriptional start site. Whereas a wealth of information is available for the enhanceosome that is formed on the VRE upon the signals generated by viral infection, early observations indicating the existence of other far-upstream control elements have so far remained without a molecular fundament. Guided by a computational analysis of DNA structures, we could locate three as-yet-unknown transcription factor-binding regions at −0.5, −2, and −3 kb. Our present study delineates the interplay of factors YY1 and YY2 as it occurs at the sites at −3 kb and −2 kb (otherwise called HS1 and HS2), consistent with the idea that the novel factor YY2 antagonizes the negative actions exerted by YY1. Differences between the human and murine control regions will be described.

scholarly journals Characterization of regulatory elements in the 5'-flanking region of the rat GPIIb gene by studies in a primary rat marrow culture system

Blood ◽  
1994 ◽  
Vol 84 (10) ◽  
pp. 3385-3393 ◽  
Author(s):  
KL Block ◽  
K Ravid ◽  
QH Phung ◽  
M Poncz
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Regulatory Element ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Start Site ◽  
Specific Expression ◽  
Flanking Region ◽  
High Level ◽  
Marrow Cells

Abstract Glycoprotein (GP)IIb/IIIa, an integrin complex found on the surface of platelets, is a receptor for fibrinogen and other ligands, and is involved in platelet aggregation. Because GPIIb is specifically expressed in megakaryocytes, we have studied the 52-flanking region of the rat (r) GPIIb gene as a model of a megakaryocyte-specific gene. The studies presented here used a rat marrow expression system, which allows the study of primary cells undergoing terminal differentiation into megakaryocytes. The determination of megakaryocyte-specific expression of DNA constructs was possible by immunomagnetically separating megakaryocytes from total bone marrow cells. Transient expression constructs, containing varying lengths of the 52-flanking region from -39 to -912 bp, localized a regulatory element between -460 and -439 bp upstream of the transcriptional start site. This region contains a GATA consensus binding element between -457 and -454 (GATA454). Further constructs demonstrated that this GATA binding element was indeed essential for expression. A 25-bp substitution, covering the region -450 to -426 immediately downstream of the GATA454, demonstrated that this region was essential for full expression, which suggests that this region may interact with the GATA454 site in promoting high-level lineage-specific expression. To define regulatory elements between the GATA454 and the transcriptional start site further, we tested additional constructs derived from the original -912 construct; each of which contained the GATA454 but had different 50-bp deletions from -450 to the start site. Virtually all of these constructs continued to show high-level tissue-specific expression. The deleted -150 to -101 construct had twice the level of expression of the full-length wild-type construct; therefore, this region may contain a negative regulatory element. Comparison of our data with expression studies performed with the 52-region of the human GPIIb gene using HEL cells, a cell line with some megakaryocytic properties, demonstrates significant differences, which may reflect our use of primary rate bone marrow cells. In particular, our study points to the importance of the GATA454 for high levels of GPIIb expression in developing megakaryocytes.

scholarly journals Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences

2004 ◽  
Vol 32 (15) ◽  
pp. 4512-4523 ◽  
Author(s):  
Y. V. Sun ◽  
D. R. Boverhof ◽  
L. D. Burgoon ◽  
M. R. Fielden ◽  
T. R. Zacharewski
Keyword(s):  
Gene Expression ◽  
Transcriptional Start Site ◽  
Genomic Sequences ◽  
Mouse Tissue ◽  
Start Site ◽  
Orthologous Genes ◽  
Multiple Sequence ◽  
Response Elements

Abstract Comparative approaches were used to identify human, mouse and rat dioxin response elements (DREs) in genomic sequences unambiguously assigned to a nucleotide RefSeq accession number. A total of 13 bona fide DREs, all including the substitution intolerant core sequence (GCGTG) and adjacent variable sequences, were used to establish a position weight matrix and a matrix similarity (MS) score threshold to rank identified DREs. DREs with MS scores above the threshold were disproportionately distributed in close proximity to the transcription start site in all three species. Gene expression assays in hepatic mouse tissue confirmed the responsiveness of 192 genes possessing a putative DRE. Previously identified functional DREs in well-characterized AhR-regulated genes including Cyp1a1 and Cyp1b1 were corroborated. Putative DREs were identified in 48 out of 2437 human–mouse–rat orthologous genes between −1500 and the transcriptional start site, of which 19 of these genes possessed positionally conserved DREs as determined by multiple sequence alignment. Seven of these nineteen genes exhibited 2,3,7,8-tetrachlorodibenzo- p -dioxin-mediated regulation, although there were significant discrepancies between in vivo and in vitro results. Interestingly, of the mouse–rat orthologous genes with a DRE between −1500 and +1500, only 37% had an equivalent human ortholog. These results suggest that AhR-mediated gene expression may not be well conserved across species, which could have significant implications in human risk assessment.

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