Untitled

Gene silencing in the study of transcriptional regulation:
the AβH-J-J locus
A. FINOTTI 1, G. BREVEGLIERI 2, S. GKRITZAPI 1, R. GAMBARI 1, G. FERIOTTO 1 The starting point to explain how the main biological
processes occur is the understanding of the mechanisms
Department of Biochemistry and Molecular Biology which control the gene expression regulation. One of the
University of Ferrara, Ferrara, Italy mechanisms of transcriptional regulation is operating
2Laboratory for the Development of Pharmacological at the promoter level. In this context, the identification
and characterization of transcription factors involved on
of Thalassemia Biotechnology Centre promoter binding and regulation appears to be very
University of Ferrara, Ferrara, Italy important. Silencing strategies are useful methods to
study the function of transcription factors (TFs) as pro-
moter regulators, and they include: 1) decoy and 2) RNA
interference (RNAi) approaches. This article describes
a successful application of these two methods in the
differentially expressed in various tissues.4 The tran- study of transcriptional regulation of human AβH-J-J
scriptional regulation of the AβH-J-J locus is expected locus. Transcription factor decoy (TFD) approach was
to be quite complex; it occurs at least at three levels: 1) employed in order to determine the role of Sp1-related
transcription directed by the P1 promoter, located proteins; RNAi approach for upstream stimulatory tran-
scription factor (USF) depletion was used to study the
upstream of exon 1; 2) transcription directed by the role of this factor on P1 promoter directed expression.
P2 promoter, located between exon 1 and exon 2; and3) alternative splicing. To understand the transcrip- Key words: Transcription - Sp transcription factors -
Upstream stimulatory factors - RNA interference.
tional regulation of the AβH-J-J locus, the two pro-moter sequences P1 and P2 were identified and func-tionally characterized by (a) RT-PCR Real Time and 5’ The AβH-J-J locus is a human genomic sequence, RACE of total mRNA from human adult normal tissues characterized by this research group,1-3 that gen- and from different cell lines; (b) in vitro and in vivo erates three functionally distinct proteins, which are transfections of deletion mutants of luciferase reporterconstructs; (c) gel-shift and super-shift; (d) mutagene- Fundings.—This work was supported in part by grants from FIRB, sis; (e) co-transfection studies using expression vec- Ministero Università e Ricerca Scientifica e Tecnologica (60% and 40%).
tors; (f) chromatin immunoprecipitations (ChIP).2, 3 R.G. is supported by grants from AIRC, C.I.B., by Fondazione Cassa diRisparmio di Padova e Rovigo, Cofin-2002, by UE Obiettivo 2. This In conclusion, the AβH-J-J locus appears to be a research is also supported by Regione Emilia-Romagna (Spinner Project) very useful system to identify transcription factors and by Associazione Veneta per la Lotta alla Talassemia, Rovigo (AVLT).
involved in the regulation of expression of genomic Received on 29 May, 2008.
Accepted for publication on 3 June, 2008.
regions coding for multiple proteins exhibiting dif-ferent biological functions. In this respect, the silenc-ing of the biological activity of transcription factors Address reprint requests to: R. Gambari, Department of Biochemistry appears to be an approach suitable to generate infor- and Molecular Biology, University of Ferrara, via Borsari 46, 44100 Ferrara,Italy. E-mail: gam@unife.it SILENCING STRATEGIES OF TRANSCRIPTIONALREGULATION Figure 1.—Decoy approach targeting Sp1 transcription factors on P1 promoter. A) Double stranded synthetic oligonucleotide carrying theSp1 consensus sequence binds nuclear factors from HepG2 cells. 100 fold molar excess of unlabeled competitor was used in order todemonstrate specific binding (lanes 2,3). Arrows indicate the specific complexes; B) effect of TF “decoy” ODNs mimicking Sp1 consensussequences or scrambled, unrelated oligonucleotide, on P1 promoter specific mRNA levels. The cDNA obtained from total RNA was subjectedto quantitative real time RT-PCR for P1 promoter specific transcripts. Relative quantification is expressed as normalized fold depletion andwas calculated by the “Ct” method using scrambled ODN (Scramblemer) as control sample (value=1). Statistical significance: *P<0.05.
Approaches for gene silencing
TF is a positive modulator of the gene expression ofthe target gene. Therefore, the decoy approach is a Among the approaches available for “silencing” of powerful tool to modulate transcription of a target genes coding for transcription factors, the use of decoy gene and directly study the transcriptional control of oligonucleotides targeting proteins belonging to tran- scription factors (TFs) families, and the silencing RNA As examples of molecular targets for the decoy approach targeting mRNA sequences coding putative approach, proteins belonging to the NF-kB and Sp1 families are very interesting, since these transcriptionfactors are involved in the regulation of the expression Targeting transcription factors: the decoy approach of several genes relevant to human pathologies.14-16Thus, the development of experimental approaches The transcription factor decoy (TFD) strategy, that based on TF decoys to modulate gene transcription is based on displacement of the TFs to their consen- sus sequences present in the promoter of specificgenes, has been demonstrated to be very useful in modulating gene expression when directed to target FFECT OF A DECOY OLIGONUCLEOTIDE TARGETING SP1 TRAN- key TFs both in vitro and in vivo. SCRIPTION FACTORS ON P1-DIRECTED TRANSCRIPTION eral decoy oligodeoxynucleotides have been In order to verify the possible role of Sp1-related employed in order to study the modulation of the proteins in the transcription regulation of the AβH-J- expression of genes relevant to human diseases.8, 9 J locus the TFD approach was employed. First, the This approach is based on the intracellular delivery of specificity of protein-DNA interaction at Sp1 site was double stranded oligodeoxynucleotides able to com- verified using competitive electroforetic mobility shift pete for the binding of TF-related proteins to the dif- assay (EMSA). 32P-labeled double-stranded synthetic ferent consensus sequences in the promoter of specific oligonucleotide Sp1mer mimicking Sp1 transcription genes. Transcription is affected in a positive way if the factor binding site was incubated with HepG2 nuclear TF is a negative modulator and in a negative way if the extract in presence of 1) the same unlabeled Sp1mer SILENCING STRATEGIES OF TRANSCRIPTIONALREGULATION sequence, 2) unlabeled Scramblemer sequence (Figure1A, lanes 2,3), used as competitors. Then, this Sp1sequence was chosen as TF “decoy” ODNs, using thenon-related scramble sequence of the same length,Scramblemer, as control. For the decoy experiment,HepG2 cells were transfected with Lipofectamine 2 000and 2 μg/mL of each double stranded oligonucleotide.
Twenty-four hours later RNA extraction was per-formed and the transcripts relative to P1 promoter Figure 2.—RNA interference strategy for USF1 transcription factor activity were analyzed by using quantitative real-time silencing: effect on P1 promoter activity. pSingle-tTS-shRNA plasmid RT-PCR. The results obtained demonstrate that in cells vector (Clontech, Palo Alto, CA, USA) was used for cloning three treated with the Sp1 decoy a 64% reduction of tran- small hairpin double stranded DNA oligonucleotides targeting humanUSF1 RNA sequences or a scrambled DNA sequence. The recombi- scription directed by the P1 promoter was obtained.
nant vectors obtained were transiently transfected into HeLa cells.
In contrast, scrambled oligonucleotide exibited no Total RNA was extracted two days after transfection, and used for quan-titative Real Time RT-PCR for specific P1 promoter transcripts ampli- effects on transcription of the analyzed mRNA (Figure fication; ΔΔCt method was used to compare gene expression data and 1B). These results indicate a role of proteins belong- standard error of the mean was calculated. ing to the Sp1 superfamily in the regulation of theP1-promoter directed expression of the AβH-J-J locus.3 stimulatory factor (USF) transcription factors bindingTargeting mRNAs coding for transcription factors: the sites are also present (Finotti et al., manuscript in preparation). In order to study the role of proteinsbelonging to the USF family on the transcription of the Gene silencing by RNA interference (RNAi) is ush- human AβH-J-J locus directed by the P1 promoter, ering biological research into a new age, providing an silencing of the USF1 gene was performed using the extremely powerful tool for the analysis of loss-of- RNAi approach. A tetracycline-inducible plasmid function phenotypes in vertebrates where alternative expression vector was employed, engineered to approaches are very often arduous or even ineffec- express small hairpin RNAs (shRNA), which are tive.17, 18 RNAi is an evolutionarily conserved system processed in vivo into siRNA-like molecules capable mediated by, and targeted against, RNA. The RNAi of carrying out gene-specific silencing. The system is pathway is triggered by long double-stranded (ds)RNA designed so that expression of shRNA is induced that is cleaved by an endonuclease named Dicer to when doxycycline is added to the culture medium of produce short interfering (si)RNAs of 21 nt in length.
transfected cells. Three small hairpin double strand- These siRNAs can be incorporated into an RNAi effec- ed DNA oligos targeting three different human USF tor complex – the RNA-induced silencing complex – RNA sequences were designed and cloned into this where they direct degradation of RNA with sequence vector. A negative control scrambled shRNA, which homology to the siRNA.17 Because of the high effica- had no significant homology to human gene cy and specificity, the RNAi approach has become a sequences, was also designed to detect any non spe- required research tool to study transcriptional regu- cific effect. Two days after transient transfection of lation and gene functions in a wide variety of organ- recombinant expression vectors into HeLa cells, total isms.19-21 The aim of this approach in a transcription- RNA was extracted and used for quantitative Real al regulation study is the indirect inhibition of the Time RT-PCR, using primers targeting USF or P1 pro- binding of TFs to the consensus promoter sequence moter specific transcripts. The data demonstrated that obtained through the direct targeting and degrada- mRNA levels of P1 transcripts were significantly reduced when USF was silenced (Figure 2) referringscramble siRNA, which has no detectable effects on P1driven expression (data not reported). The particularly RNA INTERFERENCE APPROACH FOR USF TRANSCRIPTION FAC- evident depletion of P1 transcripts using USF1-C shRNA vector correlate with the major silencing effect In addition to the Sp1 sites in the P1 promoter of this recombinant construct on USF specific mRNAs region, E-box sequences homologous to upstream SILENCING STRATEGIES OF TRANSCRIPTIONALREGULATION Conclusions
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