Non-consensus GLI binding sites in Hedgehog target gene regulation
© Winklmayr et al; licensee BioMed Central Ltd. 2010
Received: 12 August 2009
Accepted: 13 January 2010
Published: 13 January 2010
The GLI transcription factors, mediators of the hedgehog signal bind with high affinity to the consensus sequence GACCACCCA. The affinity of variant single substitutions in GLI binding sites has been measured systematically, but the affinities of the variant binding sites appears low compared to the frequency of occurrence of variant sites in known GLI target gene promoters.
We quantified transcriptional activation by GLI using PTCH1 promoter based luciferase reporters containing all single substitutions of the GLI consensus binding site. As expected variants with very low affinity did not activate the reporter. Many lower affinity binding sequences are, however, functional in the presence of moderate GLI concentration. Using two natural non-consensus GLI site promoters we showed that substitution of the variant sequences by consensus leads to comparable activity.
Variant GLI binding sites with relatively low affinity can within natural promoters lead to strong transcriptional activation. This may facilitate the identification of additional direct GLI target genes.
Sequence specific binding of transcription factors in response to diverse cellular input signals is a major determinant in the regulation of transcription. Binding sequences for many factors have been identified by experiment and/or by a wealth of prediction methods (reviewed in ). Consensus binding sites were classically determined by SELEX experiments and verified by EMSA while more recently affinity measurements by methods better suited to moderate to large scale experimentation like microarray binding experiments have been used . Experimentally determined affinities or frequencies for each base at every position of a binding site can be represented as position weight matrices or sequence logos, which can be used for prediction of new binding sites [3, 4]. It is well known that not all sequences, which a transcription factor strongly binds to in vitro will also be bound in an in vivo context . Global chromatin immunoprecipitation can identify the sequences bound by a transcription factor within the cellular context but does not indicate whether the binding site is functional, i.e. whether the presence of a given TF at this site affects expression of the target gene. For this, additional information usually derived from microarray data, sequencing or promoter studies is required [6, 7].
Relative binding affinity is a good indicator of transcriptional activation or repression in an artificial system as shown for example by Kang et al for the Zif268 DNA binding domain joined to repressor or activator domains . A detailed description of the quantitative relationship between affinity and activation potential in the cell is difficult since in vivo activation depends on the presence of co-factors, additional transcription factors and the epigenetic state of the chromatin. On the other hand, a single high affinity binding site in combination with a minimal promoter frequently does not produce strong target gene activation and reporter constructs therefore usually contain several repeats of consensus binding sites to enhance reporter activity. In the analysis of specific promoters attention is usually first focussed on consensus sites though the functionality of variant sites for many transcription factors has been shown in vivo and in reporter gene assays. The effect of variation in a single site on activation and specificity has extensively been investigated in E coli. Within specific mammalian promoters the influence of variant sites on transcriptional activation has not been explored systematically.
The three GLI transcription factors, mediators of the hedgehog signal, comprise a DNA binding domain of five zinc fingers, which are very highly homologous in the three GLIs. Two of the five fingers are responsible for all but one of the protein-DNA base contacts . The GLIs can function as activators and/or repressors and regulate target genes in a highly context specific way. The consensus binding sequence GACCACCCA was first determined by Kinzler et al  and many direct GLI target genes have been identified. Hallikas et al  determined the affinities of all single base substitutions in the GLI consensus binding sequence using a fusion of luciferase with the GLI-DNA binding domain in an in vitro assay. These data together with information on species conservation were used in the novel EEL prediction program to identify GLI regulated genes within the mouse and human genome. These predictions were successful in identifying new target genes though some known target genes were not represented in the original version. This emphasizes the need to characterise in more detail the relationship between affinity and functionality of GLI binding sites in functional assays.
We therefore set out to investigate the activity of all single site variants of the consensus GLI binding site in a luciferase assay. Frequently GLI transcriptional activity is measured in an artificial construct containing multiple copies of the consensus site. Here we use a construct based on the PTCH1 promoter, which is functional in many different cell types and should approximate a "normal" control of gene expression. Using relatively low GLI concentration to enhance specificity we found that a rather large number of variant GLI binding sites was able to activate transcription within the PTCH1 promoter. We then proceeded to turn variant binding sites into consensus within two unrelated natural promoters containing essential non-consensus GLI binding sites and found that activity was not significantly enhanced.
Results and Discussion
A PTCH1 reporter system to measure the functionality of variant GLI binding sites
We then tested the functionality of the luciferase reporter system by comparing the ability of GLI2act to activate the reporter constructs containing the linker with the consensus sequence (PTCH1_VAR_(cons)) to the unmodified PTCH1 promoter luciferase reporter construct (PTCH1_WT) (Figure 1B). All results presented here were obtained with GLI2act, which is a strong activator. When GLI1 was used comparable results were obtained though activity was lower (data not shown, CS unpublished). As shown in Figure 1B both wild type (PTCH1_WT) and modified PTCH1 promoter construct (PTCH1_VAR(Cons)) were strongly induced in response to GLI2act with only slightly lower activation for the modified PTCH1 promoter (Figure 1B). As expected, the inactive variant 6G7G (GACCAGG CA) (Figure 1B) in PTCH1_WT as well as in PTCH1_VAR resulted in strongly reduced reporter activity. No activation was observed with PTCH1_VAR, with no inserted sequence. Thus, the modified PTCH1 reporter system is functional and can be used to systematically measure the effect of variation in GLI binding sequence on GLI target gene activation.
The effect of GLI binding site variants on PTCH1 promoter activation
Functional non-consensus GLI binding sites in Hh target gene promoters
Non-consensus GLI binding sites in GLI target gene promoters
To further explore the influence of binding affinity on transcriptional activation in a natural promoter context other than PTCH1 we chose the JUN and GLI1 promoters, both containing functional non-consensus GLI binding sites, for further analysis
Recent observations show that lower affinity binding sites for transcription factors can be identified by global ChIP  and occur quite frequently. Large scale affinity measurements as described in  showed that a large selection of transcription factors recognises many variations of the primary motifs and that even secondary motifs exist, which may possibly affect changes in transcriptional specificity. A visible influence of low affinity sites on gene expression in yeast has been described pointing to their potential relevance for modulating gene expression . Vokes et al  identified a number of GLI promoters/enhancers, which behave in a tissue specific way and are influenced by nuclear GLI concentration. In a more global study, groups of sites with high and lower affinities to REST repressor were shown to cluster into groups responsible for activation of target genes expressed commonly, specifically or uniquely in different cell lines . These data imply an important role for lower affinity sites in the context dependent control of transcription and point to the need for more detailed investigation of their function.
The results presented here specifically focus on the activation potential of binding sites of the GLI transcription factors, the mediators of the hedgehog signal. We measured activation in a standardised luciferase assay in the context of the PTCH1 promoter testing all single site mutations of the GLI consensus binding sequence. A rather large number of substitutions was shown to be active, which is consistent with the existence of many known GLI target gene promoters containing variant sites with lower binding affinity. Taking into account the contribution of a larger subset of binding sites with significant affinity the results presented in this study are likely to be helpful in the prediction and experimental validation of more direct GLI target genes.
Sequences of oligonucleotides used for cloning, site directed mutagenesis and ChIP
GCCGGACCT GG CA GTATTTGCTGC
CAAATACTG CC AGGTC CGGCTCGC
GGTTGCCTACCTG CC TGGTC TCTCT
AAGTAGAGAGACCA GG CA GGTAGGC
CACACACTGGGTTGCCTACCTGGGTG A TC TCTCTACTTTGGTGAGC
GCTCACCAAAGTAGAGAGA T CACCCA GGTAGGCAACCCAGTGTGTG
CACACACTGGGTTGCCTACCT T GGTGGTC TCTCTACTTTGGTGAGC
GCTCACCAAAGTAGAGAGACCACC A A GGTAGGCAACCCAGTGTGTG
GTTTGCGCTTCTCGT G GGTGGTC CGGGCTTGCGGCCCGGCGG
CCGCCGGGCCGCAAGCCCGGACCACC C A CGAGAAGCGCAAAC
CTTCGGAGTGTTCTCAACGTGGG T GG T C GACTCTCGGGAGACCGC
GCGGTCTCCCGAGAGTCG A CC A CCCA CGTTGAGAACACTCCGAAG
PTCH BS2 ChIP
Cell culture, transfection and luciferase reporter assays
HaCaT cells and GLI2actHaCaT  were cultured in Dulbecco's modified Eagle medium (high glucose, PAA, Pasching, Austria) with 10% fetal calf serum (PAA, Pasching, Austria) supplemented with streptomycin/penicillin (Pen/Strep100x stock solution, PAA, Pasching, Austria) at 37°C, 5% CO2. Cells were grown to 80% confluence in 24-well plates and transfected in triplicate with the pGL3 luciferase reporter plasmids, the GLI2act expression (80 ng/transaction sample) construct  or pcDNA4/TO as negative control using Superfect Transfection reagent (Quiagen Inc., Valencia, CA). LacZ expression plasmid (400 ng/transfection sample) was used for normalization of transfection efficiency. Cells were harvested 48 h after transfection, and luciferase activity measured with a LucyII luminometer (Anthos Labtec, Cambridge, UK) using Luciferase Assay Substrate (Promega, Madison, USA).
ChIP from GLI2actHaCaT was done as described in . Antibodies used were: polyclonal goat-anti-GLI2 (GLI2-N20) (Santa Cruz Biotechnology) for specific precipitation and species matched normal IgGs (Santa Cruz Biotechnology) for unspecific control. PCR primer sequences are listed in Table 2.
Electrophoretic Mobility Shift Assay
Transcription Start Site
Systematic Evolution of Ligands by Exponential Enrichment
Relative Light Unit.
We thank Stefan Wegenkittl (University of Applied Sciences, Salzburg) for advice with the statistical evaluation of the results. This work was supported by the Austrian Genome Project GENAU "Ultra-sensitive Proteomics and Genomics II" to AMF and FA, FWF Project 16518-B14 to FA, and the University of Salzburg priority program "Biosciences and Health".
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