Cooperative activation of Xenopus rhodopsin transcription by paired-like transcription factors
© Reks et al.; licensee BioMed Central Ltd. 2014
Received: 23 March 2013
Accepted: 1 February 2014
Published: 6 February 2014
In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking.
We investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element.
We have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription.
In vitro studies have largely supported those in transgenic animals, namely that Nrl and Crx are the key regulators of rhodopsin transcription . Nrl stimulates transcription of the rhodopsin promoter via NRE in transfected non-retinal cells [15, 16]. Furthermore, mutagenesis of Nrl has revealed multiple domains required for rhodopsin promoter transactivation [17, 18]. Similarly, Crx alone stimulates transcription of the rhodopsin promoter in transfected non-retinal cells . Although it has a more promiscuous target, Crx binds to a core sequence, TAAT [7, 19]. Rax, paired-like homeobox transcription factors, bind to the Ret1 and BAT1 sites [8, 20, 21] and alone weakly activates the rhodopsin promoter [8, 22]. Crx and Nrl interact physically and functionally in vitro. Crx binds to Nrl through its homeodomain . The binding site on Nrl is not as well defined [17, 18]. Both Crx and Nrl interact with other transcription factors, such as TBP , Fiz1  and Qrx , as well as chromatin modifiers . However, the most intriguing feature of rhodopsin transcriptional activation in vitro is its cooperative (or synergistic) activation by Crx and Nrl [15, 23, 26, 27]. Together they transactivate the rhodopsin promoter an order of magnitude or more than the additive effect of the individual transcription factors. This has led to the hypothesis that in rods rhodopsin transcription is activated by the cooperative activity of Crx and Nrl.
Current models for transcriptional activation describe the complex interplay between distal and proximal promoter regulatory elements, transcription factors and the basal transcription machinery (for example [28–30]). The usual hypothesis postulates that expression levels are determined by accessibility of the cis-elements (regulated by chromatin environment ) and the availability of functionally active transcription factors . This leads to the localization of transcription factors on the promoter for a significant length of time and thus promotes interaction with other general transcription factors assembling on the promoter . Thus, cis-elements are thought to increase the local concentration of transcription factors and to promote protein-protein interactions. Therefore, to account for rhodopsin transcription levels, it is important to quantitatively examine the contribution of DNA binding site architecture to activation of the rhodopsin promoter by Crx and Nrl. Our aim was to characterize the arrangement of DNA binding motifs in the RPP, i.e.to determine the requirements for cooperative (synergistic) activation by Nrl-Crx. Employing an in vitro system coupled with mutational analysis of the RPP, we characterized the specific role of the paired-like cis-elements in activation of the Xenopus rhodopsin promoter by Crx and its Xenopus homolog, Otx5, Rax and a novel Xenopus Rax family member, Rax2b [20, 33].
Rhodopsin proximal promoter activity in transfected HEK293 cells
Both cell lines exhibited robust transactivation of the XOP promoter by the combination of human Crx (hCrx) and Nrl (hNrl) or Xenopus homologs of Crx (Otx5) and Nrl (LNrl) (Figure 2B, ). However, the Xenopus homologs stimulated luciferase activity more strongly than their human counterparts. The higher levels of stimulation are in general agreement with our previous results, although the relative stimulation by transcription factors is somewhat higher here, mostly likely due to differences in experimental conditions.
Surprising, the activity in the presence of hNrl-hCrx was 2-fold higher in HEK293T cells compared to HEK293 cells, and contrary to the corresponding basal promoter activities (Figure 2A). It is typical to express activation by transcription factors as the relative activity, i.e. the ratio (or fold) of the activity in the presence and absence of transcription factors. For example, the activation of XOP by hNrl-hCrx is 100-fold in HEK293T compared to only 20-fold activation in HEK293 cells (Figure 2A). Thus, there is a 5-fold discrepancy in the estimated synergistic activation by hNrl-hCrx in HEK293T cells compared to HEK293 cells. This difference is a result of the higher non-specific activity of XOP as well as the lower maximal activation by hNrl-hCrx in HEK293 cells. For clarity and to facilitate comparisons between different experiments, we present all transcriptional activity data in relative light units (RLU).
Taken together, the low basal transcription and the higher Nrl-Crx stimulation in HEK293T cells are better for quantifying transcriptional stimulation of rhodopsin promoters. If we assume that the same phenomenon underlies the transcriptional differences between the two cell lines in basal activity and maximal stimulation. Therefore, we can eliminate differences in transfection efficiencies, plasmid copy number or rate of plasmid degradation. More likely are mechanisms which alter the luciferase template for transcription. One possibility is a difference in extrachromosomal plasmid packaging leading to alterations in plasmid conformation or episomal structure. Alternatively, there could be differences in the plasmid subcellular localization that lead to differences in transcription efficiency. Finally, the cells could have differences in transcription factors that lead to recognition of cryptic promoters in the plasmid backbone. It is possible that plasmids are differentially occupied by transcription complexes that are not readily displaced by hNrl-hCrx. These could account for the higher basal and lower stimulated activity in the HEK293 cells compared to HEK293T.
Xenopus rhodopsin promoter 5′upstream sequence
The only region of extended conservation in tetrapod rhodopsin promoters is the RPP (Figure 1). Comparison of the 5′ upstream sequence (5′US) of Xenopus with other vertebrate rhodopsin promoters did not reveal any significant conserved regions (> ~ 10 bp). However, there are many core homeobox sites (ATTA, Additional file 1: Table S3), including several Crx binding sites, present in the 5′US that could contribute to transcriptional activation. Crx binding sites have also been identified in the 5′US of mouse rhodopsin by chromatin immunoprecipitation and chromosome confirmation capture [11, 19]. In Xenopus transgenic frogs both −5361 and −503 genomic fragments drive rod-specific reporter gene expression . To examine the potential modulation of transcriptional activity by regions upstream of the Xenopus RPP (xRPP, -145/-10, ), we compared two different 5′US sequences: (−5361/-146 and −503/-146) to the activity of the xRPP. We controlled for plasmid size by placing the rhodopsin structural gene upstream or downstream of the xRPP ((Figure 2D). Both 5′US sequences increased the synergistic stimulation of the xRPP by LNrl-Otx5 (Figure 2D). However, XOP(−5361/+41) stimulation was 10-fold while XOP(−503/+41) was 5-fold higher than that of the xRPP. Plasmid size does not account for this difference, since the activity of the -503UP and -503DWN plasmids, which are similar in size to the −5361 plasmid, was closer to the −503 plasmid than the −5361 plasmid (Figure 2D). The basal activity decreased 2.4-fold with increased 5′US length. We also found that while human Nrl-Crx can strongly stimulate synergistic activation, there is no difference in the level of activity between the two promoter lengths containing upstream sequence and the xRPP as compared to the Xenopus homologs (Figure 2B). Taken together, these results show that the RPP plays the principal quantitative role in determining transcriptional synergy by Nrl-Crx. Furthermore, the 5′US enhancement of synergistic activation appears to be specific to Xenopus transcription factors. Qualitatively, the relative activity of the promoters containing these 5′US sequences (−503, -5361) is similar in transgenic Xenopus ([3, 37] and M. Haeri, personal communication). However, position effects and copy number variation prevent a direct comparison between transgenics and in vitro experiments.
Stable integration of Xenopus rhodopsin promoter
Role of XOP cis-elements in Otx5 activation
Identification of a novel Rax family member
Using a yeast one hybrid screen with the Ret1 cis-element (GCCAATTAA) as bait and a Xenopus laevis adult retina cDNA library, we identified a novel gene, which we name Rax2b (Accession number: JN392465). Rax2b is a member of the Rax paired-like homeodomain transcription factor family and in sequence comparisons is the paralog of Rax2a  (89.9% amino acid). Rax2b was detected by RT-PCR in embryos at stage 35/36 (Additional file 4: Figure S3A). All Rax family genes were expressed in the adult retina with Rax1a the most highly expressed followed by Rax2b, Rax2a and Rax1b (Additional file 4: Figure S3B).
Co-activation of rhodopsin transcription by the Rax family
Role of xRPP cis-elements in Rax co-activation
Rax co-activates preferentially through the Ret1 site
A recent study quantifying bovine rhodopsin promoter activity in mouse retinal explants reported that the BAT1 site was not only essential (required) for promoter activation but was the most efficient site in driving activity . The Ret4 site appeared to be less important in transcriptional activation in this live mouse cell system. Substitution of the mouse Ret1 and Ret4 sites with a BAT1 consensus sequence motif increased activity by 10-fold. Replacing the Ret4 sequence with BAT1 consensus sequence increased activity approximately 4-fold. These differences suggest that there may be species dependent differences in opsin gene regulation, either from subtle sequence variation or transcription factor complexes functional across different cells.
Rax is a co-activator of rhodopsin
We identified a novel retinal homeobox transcription factor, Rax2b, which is a paralog of Rax1a . Rax2 belongs to the aristaless-related paired-like homeobox gene family, which includes Rax1a and Rax1b. In promoter activity assays employing mutant promoter constructs, we demonstrated that while the Ret1 cis-element is not utilized by Otx5 for cooperative activation by Nrl-Otx5, it is utilized by Rax to enhance the level of synergistic activation. Furthermore, Rax activates, selectively and preferentially, through Ret1 and cannot substitute for Otx5 as a co-activator with Nrl of synergistic activation. These results suggest that the Ret1 site is used by Rax to fine tune synergistic activation by Nrl-Otx5. Based upon our results, we suggest that Rax provides the cell with an additional means of increasing rhodopsin expression levels. An unidentified repressor might also modulate transcription through the Ret1 site to inhibit transcription. Rax has been shown to bind to Ret1, BAT1 and Ret4 motifs in in vitro binding assays and to Ret1 by ChIP [8, 20, 21, 41]. Previously published studies have also reported that Rax, alone, can weakly activate rhodopsin transcription and in combination with Nrl-Crx, can enhance synergistic activation [8, 20, 41]. The Rax family (Rax1a and b, Rax2a and b), which is found in most vertebrate species, has been implicated in eye formation, playing a critical role in cell-fate determination and development [33, 41, 42]. Rax1 and Rax2 continue to be expressed in the adult retina. However, the functional role of Rax in the mature retina is not clear. Qrx, a paired-like homeodomain TF, appears to be the mammalian ortholog to Rax2 . It is expressed in both the outer and inner nuclear layers of the retina. In transient tranfection reporter activity assays, it activated rhodopsin expression through the Ret1 site, was a weak activator alone but enhanced Nrl-Crx synergy. Interestingly, mice do not appear to carry the Rax2 gene. However, the regulatory machinery for Rax2 seems to be intact, since transgenic mice strongly expressed an EGFP-tagged Qrx promoter fragment in photoreceptors.
Role of rhodopsin 5′ US
The transcriptional activity assays performed on different lengths of the Xenopus rhodopsin promoter provide additional insights into promoter activation. While the RPP was responsible for the cooperative synergistic activation by Nrl, Crx and Rax, the 5′ upstream promoter sequence (5′ US) distal to the RPP contributed to the activation as well. The level of activity was dependent on the length of the promoter as well as the species of TFs used to activate. This observation was reinforced by the promoter activity assay data in which all three paired-like cis-elements were mutated (m6), resulting in a dramatic but not complete loss of activation. These results suggest that there are regulatory elements upstream of the Xenopus RPP that enhance transcription and are species-specific. This seems plausible since a distal regulatory element, the rhodopsin enhancer element (RER), which activates transcription, has been described for the mammalian promoter and is required for maximal activation of expression in vivo[4, 5]. Although we could not identify any conserved sequence element similar to the mammalian RER, there are multiple core ATTA/CTTA sites present in the 5′US, including core Ret1 (CAATTA), BAT1 (GGATTA), Ret4 (GCTTA) and NRE (TGCTGA) motifs which could potentially bind Crx, Rax, other paired-like TFs or bZIP TFs, such as Nrl, which might contribute to the increased activity.
In the present study, we characterized regulatory regions of the Xenopus rhodopsin promoter and their contribution to transcriptional activation. The RPP is solely responsible for synergistic activation, however, the 5′US contributes to overall transcriptional activity. We defined quantitatively the individual contributions of Xenopus rhodopsin promoter cis-elements to synergistic activation of transcription by the paired like transcription factors, Rax and Crx/Otx5. We showed specificity of the interaction between the transcription factors and cis-elements. Rax preferentially binds to the Ret1 cis-element and augments synergy, while Otx5/Crx binds to both the BAT1 and Ret4 cis-elements, contributing equally to synergistic activation. These studies characterizing the rhodopsin promoter architecture will advance our understanding of the role of transcriptional network machinery in rod photoreceptor development and homeostasis.
Vertebrate rhodopsin proximal promoter consensus sequence
Approximately 200 bp around the NRE were aligned using CLUSTALW and then manually adjusted to optimize Pax-like cis-elements. Logos were created using WEBLogo3.0. The accession numbers for the sequences used in the alignment are described in Additional file 7: Table S1 and the multiple sequence alignment is in Additional file 8.
DNA expression constructs
pCS2-Otx5 was obtained from A. Viczian. pCS2-LNrl was subcloned from pMT-LNrl . The pCS2-Rax2b expression construct was generated by releasing the full length cDNA from the yeast one hybrid vector pGADT7-Rec2 with EcoRI and XhoI and ligating into the same sites of pCS2. pCS2-Rax2a (Rx-L; NM_001095716) was generated by PCR from a cDNA clone (XL073a16) obtained from NIBB and subcloned into pCS2. pCS2-Rax1a (Rx1; NM_001088218) and pCS2-Rax1b (Rx2a; AF001049) were generated by RT-PCR from Xenopus laevis adult retina total RNA and subcloned into pCS2. pCS2-hNrl and pCS2-hCrx were generated by PCR from cDNA clones obtained from A. Swaroop and S. Chen, respectively. hNrl was subcloned into the BamHI and Xba I sites, and hCrx into the BamHI and Not I sites in the pCS2 vector. All constructs were verified by sequencing. See Additional file 9: Table S2 for a list of primers.
Promoter reporter constructs
Four WT Xenopus rhodopsin promoter luciferase reporter constructs, pGL2-XOP (−5361/+41, -503/+41, -145/+41, -128/+41), were described previously . The deletion constructs containing disrupted regions within the XOP(−503/+41) proximal promoter (Ret1Δ, BAT1Δ) were described previously . The Ret1/BAT1Δ (−136/-91) deletion mutant was generated as described previously . The pGL2 -503DWN plasmid was described previously [pXOP(−503/+41)luc(5800)] . The pGL2 -503UP construct was generated as follows. A 5.8 kb PCR product of the Xenopus rhodopsin exons and 3′ region was amplified using the -503DWN plasmid as a template and cloned into the Xma I and Mlu I sites of the pGL2 basic plasmid. The XOP(−503/+41) sequence was amplified by PCR and inserted into the Nhe I and Bgl II sites just downstream of the 5.8 kb rhodopsin sequence. Mutations in the cis-elements (ATTA to AGGA) were introduced in both XOP(−5361/+41) and XOP(−503/+41) following the QuikChange XL Site Directed Mutagenesis protocol (Stratagene). The promoter constructs were verified by sequencing. The primers used in cloning are found in Additional file 9: Table S2.
Flp-In stable cell lines
A DNA fragment that included the FRT sites and hygromycin gene was PCR amplified using the pcDNA5 FRT TOPO vector as template (Invitrogen). The purified PCR fragment was cloned into the Sal I and Bam HI sites of the pGL2-basic vector (Promega), which we named pGL2-FRT. The XOP(−503/+41) and XOP(−5361/+41) promoter fragments were then excised from their pGL2 reporter constructs with Xma I and Bam HI and cloned into the same restriction sites in the modified pGL2/FRT vector. Flp-In 293 T-REx cells (Invitrogen), containing the stably integrated FRT target sites upstream of the Zeocin gene, were co-transfected with the pOG44 plasmid, containing the Flp recombinase gene (Invitrogen), and the pGL2-FRT-XOP(−503/+41) or pGL2-FRT-XOP(−5361/+41) plasmids using Fugene 6 following the manufacturer’s protocol (Roche Diagnostics). Cells were passaged 48 h after transfection. Hygromycin B (150 μg/ml) was added to the culture medium the next day. Twelve individual stable clones expressing XOP(−503/+41)-Luciferase or XOP(−5361/+41)-Luciferase were picked 14 days after selection and expanded. Cell lines were also tested for loss of Zeocin (100 μg/ml) resistence. Only the dually selected clones were used for reporter assays. The promoter constructs were verified by sequencing. The primers used in cloning are found in Additional file 9: Table S2.
HEK293 or HEK293T cells were seeded into 24 wells at 2.4 × 105 and 1.6 × 105 per well, respectively. The stable Flp-In 293 T-REx cell lines were seeded at 2.4 × 105. Cells were co-transfected the next day with Fugene 6 and various combinations of reporter gene and/or expression constructs at a total DNA concentration of 1 μg/well following the manufacturer’s protocol (Roche Diagnostics). After 48 h cells were harvested and lysed with 100 μl/well of Passive Lysis Buffer following manufacturer’s instructions for the Luciferase Reporter Assay System (Promega). Luciferase activity was measured in 10 μl of cell lysate using a Biotek Synergy II plate reader (Biotek Instruments, Inc.). Luciferase activity from mock transfected.
Total RNA was isolated from Xenopus embryo heads (stages 35/36, 39/40) or adult retinas using an RNAeasy kit (Qiagen) and analyzed using a 2100 Bioanalyzer (Agilent Technologies). Complementary DNA was synthesized from 1 μg total RNA with Superscript II RT polymerase and random hexamers (Invitrogen). cDNA was amplified over 33 cycles using Advantage 2 DNA polymerase (Clontech) and the appropriate primers for histone H4 and Rax family members (Additional file 9: Table S2).
Real time RT-PCR
Total RNA was isolated from harvested adult Xenopus retinas using an RNAeasy kit (Qiagen) and analyzed using a 2100 Bioanalyzer (Agilent Technologies). Complementary DNA was synthesized from 1 μg total RNA using the Quantitect Reverse Transcription kit (Qiagen). Real time PCR reactions were comprised of 1 μl of cDNA (diluted 1:2), 5 μl of SYBR green mix (Lightcycler 480 SYBR Green I Master kit, Roche Diagnostics), 0.5 μl each of sense and antisense primers (10 pmol) to target (see Additional file 9: Table S2 for list of primers). Real time PCR was performed in a Lightcycler 480 instrument (Roche Diagnostics). The cycling parameters were 95ºC for 10 min followed by 46 cycles at 95ºC, 60ºC, 72ºC and 80ºC for 15 s, 15 s, 15 s and 5 s, respectively.
Yeast one-hybrid assay
The MATCHMAKER one-hybrid system (Clontech) was used to isolate Xenopus transcription factors which might bind to a highly conserved sequence found in tetrapod L-opsin promoters, the ROP2 cis-element . The bait vector was generated by ligation of an oligonucleotide containing four tandem repeats of ROP-2 (5′-GCCAATTAAGAGAT-3′) into the Xma I and Sac II sites of the pHIS2 vector. The construct was confirmed by sequencing. A cDNA library of the adult Xenopus retina was prepared using poly A RNA and oligo(dT) primers according to MATCHMAKER protocol except that the denaturation time during PCR was increased to 25 s. A fusion library in pGADT7-Rec(2) vector was constructed by homologous recombination in yeast. Yeast were sequentially transformed first with the bait plasmid and then the library. Transformants (6 × 106) were screened on SD/-His/-Ura/-Leu supplemented with 10 mM 3-amino-1,2,4-triazole (3-AT). The screen yielded 30 primary transformants. The plasmid DNA containing the activation domain and cDNA inserts was isolated using standard techniques. The Y190 strain was transformed with these rescued library plasmids and used for mating the secondary screen. Of the 30 transformants, seven survived after mating with the original ROP2-containing Y187 strain on selective plates containing 40 mM 3-AT, but not in plates containing Y187 strain containing scrambled ROP2 sequence bait. The library cDNA inserts were rescued following standard protocols and sequenced.
Rhodopsin proximal promoter
Xenopus opsin promoter
- 5′ US:
5′ upstream sequence
Xenopus laevis neural retinal leucine zipper
Cone rod homeobox
Human neural retinal leucine zipper
Human cone rod homeobox
Retina and anterior neural fold homeobox
Red cone opsin promoter
Relative light unit
Ret1, NRE, cis-elements in the XOP promoter.
This work was supported by the National Institutes of Health Grants R01-EY11256 and R01-EY12975 (BEK.), Research to Prevent Blindness (Unrestricted Grant to SUNY UMU Department of Ophthalmology), Fight For Sight (FFS), Lions of CNY, F32-EY016644 (VM). We thank Drs. D. Amberg and B. Haerer for valuable assistance in the design and performance of the yeast one-hybrid screen, Y. Milgrom for critical evaluation of data analysis and members our lab for helpful discussions.
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