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 Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Antagonistically Regulate Fission Yeast fbp1Transcription by Employing Different Modes of Action at Two Upstream Activation Sites

2000 ◽  
Vol 20 (17) ◽  
pp. 6426-6434 ◽  
Author(s):  
Lori A. Neely ◽  
Charles S. Hoffman
Keyword(s):  
Signal Transduction ◽  
Transcriptional Regulation ◽  
Protein Kinase ◽  
Mapk Pathway ◽  
Transcriptional Start Site ◽  
Mitogen Activated Protein Kinase ◽  
Signal Transduction Pathways ◽  
Start Site ◽  
Mitogen Activated Protein ◽  
Kinase A

ABSTRACT A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of theSchizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized twocis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation offbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulatefbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site.

scholarly journals Transcriptional Regulation ofFucosyltransferase 1Gene Expression in Colon Cancer Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Fumiko Taniuchi ◽  
Koji Higai ◽  
Tomomi Tanaka ◽  
Yutaro Azuma ◽  
Kojiro Matsumoto
Keyword(s):  
Gene Expression ◽  
Colon Cancer ◽  
Transcriptional Regulation ◽  
Binding Site ◽  
Transcriptional Start Site ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Luciferase Assay ◽  
Start Site ◽  
Lewis Y

Theα1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of H antigens, Lewis B, and Lewis Y. In this study, we clarified the transcriptional regulation of FUT1 in the DLD-1 colon cancer cell line, which has high expression of Lewis B and Lewis Y antigens, expresses theFUT1gene, and showsα1,2-fucosyltransferase (FUT) activity. 5′-rapid amplification of cDNA ends revealed a FUT1 transcriptional start site −10 nucleotides upstream of the site registered at NM_000148 in the DataBase of Human Transcription Start Sites (DBTSS). Using the dual luciferase assay,FUT1gene expression was shown to be regulated at the region −91 to −81 nt to the transcriptional start site, which contains the Elk-1 binding site. Site-directed mutagenesis of this region revealed the Elk-1 binding site to be essential for FUT1 transcription. Furthermore, transfection of the dominant negative Elk-1 gene, and the chromatin immunoprecipitation (CHIp) assay, supported Elk-1-dependent transcriptional regulation ofFUT1gene expression in DLD-1 cells. These results suggest that a defined region in the 5′-flanking region of FUT1 is critical for FUT1 transcription and that constitutive gene expression ofFUT1is regulated by Elk-1 in DLD-1 cells.

scholarly journals Transcriptional regulation of SLIT2 expression in pancreatic cancer cell lines

2020 ◽  
Author(s):  
Brenna A. Rheinheimer ◽  
Lukas Vrba ◽  
Bernard W Futscher ◽  
Ronald L Heimark
Keyword(s):  
Pancreatic Cancer ◽  
Transcriptional Regulation ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Cell Lines ◽  
Cancer Cell ◽  
Pancreatic Cancer Cell ◽  
Trichostatin A ◽  
Cancer Cell Lines ◽  
Ductal Adenocarcinoma ◽  
Liver Cancers

AbstractBackgroundSLIT2 has been shown to serve as a tumor suppressor in breast, lung, colon, and liver cancers. Additionally, expression of SLIT2 has been shown to be epigenetically regulated in prostate cancer. Therefore, we sought to determine transcriptional regulation of SLIT2 in pancreatic ductal adenocarcinoma.MethodsRNA expression of SLIT2, SLIT3, and ROBO1 was examined in a panel of pancreatic ductal adenocarcinoma cell lines while protein expression of ROBO1 and SLIT2 was examined in tumor tissue. Methylation of the SLIT2 promoter was determined using Sequenom while histone modifications were queried by chromatin immunoprecipitation. Reexpression of SLIT2 was tested by treatment with 5-aza-2’deoxycytidine and Trichostatin A.ResultsPancreatic cancer cell lines fall into three distinct groups based on SLIT2 and ROBO1 expression. The SLIT2 promoter is methylated in pancreatic ductal adenocarcinoma and SLIT2 expression is dependent on the level of methylation at specific CpG sites. Treatment with 5-aza-2’deoxycytidine (but not Trichostatin A) led to SLIT2 reexpression. The SLIT2 promoter is bivalent in pancreatic ductal adenocarcinoma and histone marks around the transcriptional start site are responsible for transcription.ConclusionsLoss of SLIT2 expression modulated by epigenetic silencing may play a role in pancreatic ductal adenocarcinoma progression.

Transcriptional regulation of human abraxas brother protein 1 expression by yin yang 1

2020 ◽  
Author(s):  
Pan Song ◽  
Jian Hong ◽  
Yuan Wang ◽  
Xuelian Yao ◽  
Yiqun Zhan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Start Codon ◽  
Mobility Shift ◽  
Cis Acting ◽  
Yin Yang 1 ◽  
Activating Transcription Factor 4 ◽  
Yin Yang

Abraxas brother protein 1 (ABRO1) is a subunit of the deubiquitinating enzyme BRCC36-containing isopeptidase complex and plays important roles in cellular responses to stress by interacting with its binding partners, such as ubiquitin-specific peptidase 7, p53, activating transcription factor 4, THAP-domain containing 5, and serine hydroxymethyltransferase. However, the transcriptional regulation of ABRO1 remains unexplored. In this study, we identified and characterized the core regulatory elements of the human ABRO1 gene and mapped them to the ABRO1 promoter region. Additionally, 5′ rapid amplification of cDNA ends revealed that the transcriptional start site (TSS) was located −13 bp upstream from the start codon. Reporter gene, chromatin immunoprecipitation, and electrophoretic mobility shift assays demonstrated that ABRO1 transcription was regulated through cis-acting elements located in the region −89 to −59 bp upstream of the ABRO1 TSS and that these elements were targeted by yin yang 1 transcription factor (YY1). Moreover, YY1 overexpression increased human ABRO1 mRNA and protein expression, and small-interfering RNA-mediated downregulation of YY1 attenuated ABRO1 expression. These results suggested that YY1 positively regulated human ABRO1 expression by binding to cis-acting elements located in the ABRO1 TSS.

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.

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 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 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 Cell-specific expression of the macrophage scavenger receptor gene is dependent on PU.1 and a composite AP-1/ets motif.

1994 ◽  
Vol 14 (7) ◽  
pp. 4408-4418 ◽  
Author(s):  
K S Moulton ◽  
K Semple ◽  
H Wu ◽  
C K Glass
Keyword(s):  
Gene Family ◽  
Binding Site ◽  
Transcriptional Control ◽  
Transcriptional Start Site ◽  
Receptor Gene ◽  
Regulatory Elements ◽  
Type I ◽  
Start Site ◽  
Macrophage Differentiation ◽  
Ets Domain

The type I and II scavenger receptors (SRs) are highly restricted to cells of monocyte origin and become maximally expressed during the process of monocyte-to-macrophage differentiation. In this report, we present evidence that SR genomic sequences from -245 to +46 bp relative to the major transcriptional start site were sufficient to confer preferential expression of a reporter gene to cells of monocyte and macrophage origin. This profile of expression resulted from the combinatorial actions of multiple positive and negative regulatory elements. Positive transcriptional control was primarily determined by two elements, located 181 and 46 bp upstream of the major transcriptional start site. Transcriptional control via the -181 element was mediated by PU.1/Spi-1, a macrophage and B-cell-specific transcription factor that is a member of the ets domain gene family. Intriguingly, the -181 element represented a relatively low-affinity binding site for Spi-B, a closely related member of the ets domain family that has been shown to bind with relatively high affinity to other PU.1/Spi-1 binding sites. These observations support the idea that PU.1/Spi-1 and Spi-B regulate overlapping but nonidentical sets of genes. The -46 element represented a composite binding site for a distinct set of ets domain proteins that were preferentially expressed in monocyte and macrophage cell lines and that formed ternary complexes with members of the AP-1 gene family. In concert, these observations suggest a model for how interactions between cell-specific and more generally expressed transcription factors function to dictate the appropriate temporal and cell-specific patterns of SR expression during the process of macrophage differentiation.

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