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An atypical RNA pseudoknot stimulator and an upstream attenuation signal for −1 ribosomal frameshifting of SARS coronavirus

The −1 ribosomal frameshifting requires the existence of an in cis RNA slippery sequence and is promoted by a downstream stimulator RNA. An atypical RNA pseudoknot with an extra stem formed by complementary sequences within loop 2 of an H-type pseudoknot is characterized in the severe acute respiratory syndrome coronavirus (SARS CoV) genome. This pseudoknot can serve as an efficient stimulator for −1 frameshifting in vitro. Mutational analysis of the extra stem suggests frameshift efficiency can be modulated via manipulation of the secondary structure within the loop 2 of an infectious bronchitis virus-type pseudoknot. More importantly, an upstream RNA sequence separated by a linker 5′ to the slippery site is also identified to be capable of modulating the −1 frameshift efficiency. RNA sequence containing this attenuation element can downregulate −1 frameshifting promoted by an atypical pseudoknot of SARS CoV and two other pseudoknot stimulators. Furthermore, frameshift efficiency can be reduced to half in the presence of the attenuation signal in vivo. Therefore, this in cis RNA attenuator represents a novel negative determinant of general importance for the regulation of −1 frameshift efficiency, and is thus a potential antiviral target.

INTRODUCTION
The À1 ribosomal frameshifting is a translational regulation mechanism adopted by a variety of viruses to synthesize two or more proteins at a fixed ratio starting with a single translation initiation site from the same open reading frame (ORF) (1)(2)(3)(4). In response to the programmed frameshifting signals in messenger RNA (mRNA), the ribosome is induced to move one base backward in the 5 0 direction, and then continues translation in the new À1 reading frame. The ratio between the two protein products from frameshifting can be determined by the frameshift efficiency of the stimulation signals and can be a key factor for the propagation of virus within the host. It has been suggested to be a potential antiviral target for the interference of viral propagation (5).
Efficient induction of eukaryotic À1 ribosomal frameshifting requires two in cis RNA elements (1,6). The first one is a hepta-nucleotides' slippery site sequence of X XXY YYZ, where the recoding occurs. Analysis indicated X can be any three identical nucleotides whereas Y represents three A or U, and Z is A, U or C. In addition, a downstream stimulator RNA structure located 6-7 nt away from the slippery site is also needed for efficient frameshifting. This downstream RNA stimulator is usually an H-type RNA pseudoknot in which nucleotides from a hairpin loop form base pairs with singlestranded region outside the hairpin. This leads to a topology featuring two helical stems of base pairing region (stems 1 and 2) connected by two single-stranded loops (loops 1 and 2) with a quasi-continuous RNA double-helical structure. However, not all RNA pseudoknots can stimulate À1 frameshifting and non-pseudoknot RNA element has been characterized to be responsible for inducing À1 frameshifting in HIV-1 (7,8).
The reported À1 frameshift efficiency induced by different RNA pseudoknots range from 1 to 4% for Beet western yellows virus (BWYV) in plant cells to 25 and 50% for mouse mammary tumor virus (MMTV) and avian infectious bronchitis virus (IBV), respectively, in animal cells (6,9,10). There is no clear picture to correlate frameshift efficiency with a specific RNA pseudoknot stimulator. However, it is thought that resistance of a pseudoknot against deformation by a marching ribosome can cause the elongating ribosome to pause (3,(11)(12)(13). This will position the A-and P-site transfer RNAs (tRNAs) over the slippery site and, thus, increases the probability for ribosome to slip its A-and P-site tRNAs in the 5 0 direction by one base and resume the translation in the new reading frame. Parameters known to affect frameshift *To whom correspondence should be addressed. Tel: +886 4 22840468, ext 218; Fax: +886 4 22853487; Email: kychang@dragon.nchu.edu.tw The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org efficiency include sequence identity of the slippery site and its distance to the stimulator RNA, stability of the stimulator and interactions among the ribosome, the mRNA template and the associated tRNAs (1). Recently, the sequence identity of the spacer and the E-site tRNA have both been shown to modulate frameshift efficiency (14,15) as well. In addition, host factors have also been implicated to involve in efficiency modulation (16,17).
The severe acute respiratory syndrome (SARS) is an acute respiratory illness caused by a human coronavirus (HCV). Both SARS CoV and IBV belong to Coronaviridae (18)(19)(20). The IBV is known to use a À1 frameshifting mechanism to generate RNA-dependent RNA polymerase (RdRp) crucial for viral RNA replication. The 5 0 portion of the IBV genomic RNA that encodes the RdRp contains a slippery site of the sequences UUUAAAC, and is followed by an H-type RNA pseudoknot with an in-frame stop codon embedded within stem 1 of the folded pseudoknot. An elongating ribosome will fall from the viral RNA template without synthesizing RdRp if À1 frameshifting did not occur in front of this in-frame stop codon. Therefore, defining the biologically relevant mRNA secondary structure, which can regulate the À1 frameshift efficiency of the SARS CoV, may provide useful information for anti-SARS strategy. In this work, we report the identification of an atypical RNA pseudoknot of SARS CoV as an efficient RNA stimulator in promoting À1 frameshifting in vitro, and explore the role of different stem regions in-frameshift efficiency determination. In addition, we will also demonstrate that particular viral RNA sequences upstream of the slippery site possess an attenuation effect on the overall À1 frameshift efficiency. It, thus, suggests that À1 ribosomal frameshifting can be attenuated by an RNA element upstream of the slippery site in addition to being promoted by a downstream RNA stimulator.

Construction of reporter genes and mutagenesis
Plasmid encoding the gene for ORF 1ab junction region of SARS CoV, pCRII-SARS 12 265-13 653 was a gift from Professor Pei-Jer Chen and was used as the template for PCR cloning of the cDNA of viral RNA fragments. The p2luc reporter was a gift from Professor John Atkins at the University of Utah (21) and the pRL-SV40 vector was purchased from Promega. Forward and reverse DNA primers, respectively, carrying the SalI and BamHI restriction sites and appropriately designed annealing sequences were used for PCR amplification of the desired cDNA encoding SARS CoV viral RNAs from pCRII-SARS 12 265-13 653 . The amplified inserts were then sub-cloned into the SalI/BamHI sites of p2luc using standard procedures and the resulting recombinant vectors were transformed into DH5a cell for the maintenance and selection by ampicillin. Frameshifting stimulator sequences of the minimal IBV and MMTV pseudoknot were chemically synthesized. They were amplified by forward and reverse primers, respectively, containing BsrGI and BsaAI site, and ligated into the BsrGI/BsaAI site (1392/1426) of restriction enzymes treated pRL-SV40 vectors.
All of the base pairing disruption and restoration mutants were constructed by using the quick-change mutagenesis kit from Stratagene according to the manufacturer's instructions. In contrast, PCR-based ligation approach with appropriate primers was used to assemble different chimera, which carried the attenuation signal (the sequences are available upon request), and will be described briefly. Initially, two complementary oligonucleotides containing the sense and the antisense sequences corresponding to the 3 0 end and 5 0 star region of the two fragments planned to be jointed were synthesized chemically. They were then used as forward (sense) or reverse (antisense) primers in combination with the corresponding reverse or forward primer carrying appropriate restriction sites to amplify the desired pre-jointed fragments from pCRII-SARS 12 265-13 653 . The two individual PCR products with partially overlapping sequences were then assembled by the PCR-based ligation procedure (22), and then sub-cloned into the BamHI/EcoRI sites of p2luc vectors. The identities of all cloned and mutated genes were confirmed by DNA sequencing analysis.

RNA structure probing
RNA transcripts spanning the SARS 13 377-13 475 region of SARS CoV were generated by in vitro transcription using T7 RNA polymerase. The purified RNA of desired length was then dephosphorylated by shrimp alkaline phosphatase and 5 0 end labeled with [g-32 P]ATP using T4 polynucleotide kinase, and separated by a 12% sequencing gel. All the RNase protection experiments were performed in 50 ml reaction volumes containing 50 000-70 000 c.p.m. of 5 0 end labeled RNA in the presence of RNase cleavage buffer (30 mM Tris-HCl, pH 7.5, 3 mM EDTA and 200 mM NaCl), and 10 mM MgCl 2 was included in the same buffer for RNase V1 experiments. Before the addition of probing enzymes, the RNAs were denatured by heating at 70 C for 5 min followed by instant cooling on ice and brought back to 20 C for structural mapping. The following amounts of RNases were added for each reaction: 0.0088 (1000·)-0.044 (200·) mg RNase A (USB), 0.05-10 U RNase T1 (USB), 0.5-15 U RNase T2 (USB) and 0.01-01 U RNase V1 (Amersham Pharmacia). The hydrolysis RNA ladders were obtained by the incubation of RNA in the hydrolysis buffer at 85 C for 10 min and parallel RNA sequencing products were obtained by the treatment of unfolded RNA with RNases T1 and A. They were used as markers for the assignment of guanines and pyrimidines, respectively. All the reactions were incubated at 20 C for 10 min with the exception that RNase V1 was incubated for 15 min. The reactions were terminated by phenol-chloroform extraction, and precipitated, washed with 70% ethanol, and dried by vacuum. Finally, the cleavage products were loaded into a 10% denaturing gel with different running time to resolve different parts of RNA, and visualized by using phosphorimagery.

In vitro transcription/translation and frameshift assay
The T7-coupled transcription/translation (TNT) system (Progema) was used for the generation of the shifted and the non-shifted protein products according to the manufacturer's instructions. In each assay, a 25 ml reaction containing 500 ng of DNA template, 12.5 ml of reticulocyte lysate and 0.8 ml of 10 mCi 35 S-labeled methionine (NEN) was incubated at 30 C for 1.5 h. The samples were then resolved by 12% SDS-PAGE and exposed to PhosphorImager screen for quantification after drying. The frameshift efficiency was calculated by dividing the counts of the shifted product by sum of the counts for both shifted and non-shifted products, with correction of the methionine number in each product. Control experiments with the plasmids of 100-1000 ng produce similar results for frameshift efficiency (data not shown). For the generation of capped mRNA templates, the mMESSAGE mMACHINE kit (Ambion) was used according to the manufacturer's instructions.

Mammalian cell culture and luciferase assay
Human embryonic kidney HEK-293T cells were cultured in the DMEM supplemented with 10% fetal bovine serum. The transient expression was performed by LIPOFECTIN (Gibco BRL) transfection of 500 ng of reporter plasmids into 12-wells cultured cells. The cells were then assayed for the transient expression of the reporter gene 24 h after transfection using dual-luciferase assay of cell lysates, and normalized by b-galactosidase activity as an internal control. All the in vivo experiments were repeated three times with four to six assays for each reaction. Luciferase activity measurements for both in vitro reticulocyte lysate and in vivo transfected 293T cell lysates were performed by using the dual-luciferaseÔ reporter assay (Promega) according to the manufacturer's instructions on an ABI TR 717 luminometer. Calculation of luciferase-based frameshift efficiency was performed with control plasmids to calibrate the ribosome drop-off effect as described previously (21).

RESULTS
The À1 frameshifting stimulator of SARS CoV contains an atypical H-type pseudoknot with an extra stem-loop in loop 2 Sequence alignment of the junction regions of ORF 1ab of SARS CoV and other coronaviruses indicated the existence of conserved UUUAAAC slippery site followed by a stretch of viral RNA sequences with the potential to form an H-type RNA pseudoknot ( Figure 1). This region of the SARS CoV RNA genome, SARS 13 369-13 520 (for residues 13 369-13 520) was thus cloned into p2luc vector to examine its À1 frameshifting activity (21). To faithfully monitor frameshift efficiency, we also arranged the terminus of the viral sequences under investigation to keep the TAA sequences in the N-terminal part of firefly luciferase ORF in frame to act as a stop codon in the presence of À1 frameshifting. This leads to premature termination of the frameshifted product and, thus, prevents the underestimation of frameshift efficiency because this manipulation can minimize the ribosome drop-off effect while translating the full-length firefly ORF (21). A different vector, pRL-SV40 was also used as the second reporter. Frameshift assay using either of the reporters indicated that the selected viral RNA sequences possess a frameshift efficiency of 60% (data not shown), which suggests that the SARS 13 369-13 520 RNA contains an efficient À1 frameshifting stimulator in vitro.
Further examination of sequence contents of the SARS 13 369-13 520 RNA revealed a potential stem-loop structure within the loop 2 region of this IBV-type pseudoknot. Furthermore, their primary sequences were found to be conserved among isolated SARS CoV sequences, and similar stem-loop structures were also identified in the loop 2 region of bovine coronavirus and mouse hepatitis virus pseudoknot ( Figure 1). To confirm the existence of the predicted atypical pseudoknot, enzymatic structure probing experiments were performed on SARS 13 377-13 475 RNA and some of the results are shown in Figure 2A. As can be seen, the distribution of cleavage patterns for ribonuclease probes sensitive to singlestrand region (such as RNase T2, T1 and A in lanes 7-12 of Figure 2A) is in agreement with the existence of loop regions, whereas the cleavage pattern by RNase V1 (lanes 5 and 6) that prefers cutting of duplex and stacked conformations can be localized to the three predicted stem regions (S1, S2 and S3). However, co-existence of cleavage by both RNase V1 and single-strand sensitive probes can be found for nucleotides localized to the junction region between S2 and S3. This is probably caused by a dynamic conformational equilibrium in this region as non-denaturing gel analysis suggests that the A B SARS 13 377-13 475 RNA adopts a single conformation (data not shown). Nevertheless, these probing data are consistent with the existence of a stem conformation within the loop 2 in this atypical IBV-type pseudoknot as summarized in Figure 2B. Recently, the same S3 conformation has also been proposed and verified independently (23,24).
Disruption of the base pairing region in S3 of an atypical pseudoknot impairs À1 frameshifting stimulation activity of the SARS 13 369-13 520 RNA. To define the contribution of different stem regions of this atypical IBV-type pseudoknot in the stimulation of À1 frameshifting activity, we mutated those nucleotides mapped onto form the duplex conformations. Mutants expected to disrupt a stem and those expect to reform the stem by compensatory base changes were then measured for their efficiency in promoting À1 frameshifting in vitro ( Figure 3A). As shown in lanes 1-3 of Figure 3B, frameshift efficiency drops from 60% for wild-type construct to <1% for both 5 0 and 3 0 mutants that prevent the formation of base pairing in S1. In contrast, frameshift efficiency for the mutant with compensatory base changes to restore the base pairing scheme in S1 (lane 2 of Figure 3C) rises back to the wild-type level (55%). Similar results are observed for S2 with mutants destroying or reforming base pairs in the mapped duplex region (lanes 4 and 5 of Figure 3B and Lane 3 of Figure 3C). These experiments complement structural mapping data and strongly support the idea that it is the formation of the two typical H-type pseudoknot stems, S1 and S2, and not their sequence identities are required for efficient À1 frameshifting stimulation.
Interestingly, lower sensitivity to base pairing disruption is observed for mutants located in the mapped S3 region. Mutants with disruption of base pairing in the upper or and in the lower stem region of S3 both led to a substantial decrease in frameshift efficiency when compared with the wild-type construct (from 60 to 36 or 37%, respectively, in lanes 6 and 7 of Figure 3B). However, restoring the base pairing scheme in either portion by compensatory base changes restored frameshift efficiency to the wild-type level including the up-restoration mutant that even displays a completely different sequence composition (lanes 4 and 5 in Figure 3C), suggesting that the formation of base pairing and not the sequence contents in S3 is involved in the efficient frameshifting activity of this atypical IBV-type pseudoknot. Intriguingly, frameshift efficiency does not reduce further when more bases were mutated to disrupt all the potential base pair in S3 (lanes 8 and 9 of Figure 3B), implicating the existence of a residual frameshift activity for the remaining pseudoknot scaffold. Furthermore, a pseudoknot mutant lacking the S3 but harboring an 8 nt loop 2 possesses a frameshift efficiency of 53% (lane 10 in Figure 3B). As the topology and activity of this mutant are similar to those of the minimal IBV pseudoknot that lacks 75% of its wild-type loop 2 sequences (25), which indicates that S3 is not absolutely required for high frameshift efficiency. To rule out the possibility that the observed frameshift efficiency variation in S3 mutants is the outcome of their differences in ribosome loading efficiency of uncapped mRNA templates, separately transcribed capped mRNAs were also used to examine their in vitro frameshift efficiency as shown in Figure 3D. The data confirm the result from transcription-coupled translation approach used above, although lower frameshift efficiency for the wild-type construct is observed. Together, these data suggest that the roles of S3 in À1 frameshifting stimulation may be different from those of S1 to S2. Viral RNA sequences upstream of the slippery site can downregulate À1 frameshift efficiency in vitro Recently, analysis of a SARS CoV viral RNA genome containing a longer viral construct has revealed a very different frameshift efficiency from those observed in this work (26). To resolve this issue, we constructed a series of longer viral RNAcontaining reporters with stepwise extension of viral insert in the 5 0 direction upstream of the slippery site and measured their frameshift efficiencies. As shown in Figure 4A, frameshift efficiency of these viral RNA constructs are all lower than that of the shorter SARS 13 369-13 520 RNA construct. Analysis of these data suggested that the proximal 150 nt region upstream of the slippery site, that is, the sequences 13 222-13 368 possess most of the observed downregulation effect because addition of the other 475 nt further upstream of it (the sequences 13 222-12 748) only lead to a 2% decrease in frameshift efficiency (compare lane 7 with lane 3 of Figure 4A). Together, it argues that the SARS 13 222-13 368 viral RNA may possess a signal for the attenuation of À1 frameshift efficiency.
To confirm that the decrease in frameshift efficiency is contributed by the SARS 13 222-13 368 viral RNA sequence, a reporter containing a large viral insert but lacking the potential attenuation signal (sequences 13 222-13 368), SARS 12 748-13 368 D att was constructed. This construct possesses a frameshift efficiency of 55% although much longer protein is translated upstream of the slippery site and, thus, confirming the attenuation activity of the removed domain (compare lanes 2-4 of Figure 4B). Furthermore, the downregulation effect almost disappears for a chimera construct that has the potential attenuation signal fused downstream to the pseudoknot stimulator, arguing that this attenuation signal need to be positioned upstream of the slippery site to execute its function in vitro (lane 5 of Figure 4B). To examine whether the observed downregulation effect is mediated by polypeptide encoded by the viral RNA or by the RNA itself, an ORF-shifted mutant was generated and tested for its attenuation activity. In this construct, an A residue and two U residues are separately inserted into three different positions of the SARS 13 222-13 368 viral RNA to shift the reading frame, and consequently change the identity for 43 of the 50 amino acids encoded by the wild-type viral RNA sequences ( Figure 5A). The result in Figure 5A indicates similar downregulation effect for this construct when compared with the wild-type construct. Taken together, these data provide strong evidences that the upstream RNA itself as well as its orientation to the slippery site are crucial for the cis downregulation effect of À1 frameshift efficiency in vitro, and the most stable structure of SARS 13 222-13 368 RNA predicted by RNA structure version 4.11 (27) is presented in Figure 5B with a calculated free energy of À37.7 kcal/mol.

Downregulation effect of the attenuation signal is general in vitro and also active in vivo
We also evaluated the generality of downregulation effect of this attenuation signal towards other À1 frameshifting stimulators by placing it upstream of the minimal RNA pseudoknot from IBV and MMTV, respectively (25,28) ( Figure 6A). Results show this upstream attenuation sequence also downregulates À1 frameshifting promoted by RNA stimulator derived from IBV and MMTV (compare A B Figure 4. An RNA attenuation signal is characterized upstream of the slippery site. (A) Results of 12% SDS-PAGE analysis of À1 frameshifting of different upstream extension constructs with the calculated efficiency shown in the bottom of the gel. The lower and higher bands within each well belong to the shifted and the non-shifted products, respectively. The schematic diagram on top of the gel shows the relative position of the stimulator pseudoknot, the slippery site (filled box) and the extended upstream and downstream viral sequences for the constructs under analysis. (B) The attenuation signal need to work in cis and its orientation to the slippery site is crucial for its downregulation activity. Cartoons are used to illustrate the relative orientation of the attenuation signal, the slippery site and the stimulator within the inserts under analysis. The identity of insert within each reporter construct and the calculated frameshift efficiency are shown in top and bottom of the gel, respectively. lanes 2-5 of Figure 6A). However, a weaker downregulation effect is observed when the attenuation signal is coupled to the minimal MMTV pseudoknot comparing with the minimal IBV pseudoknot. Therefore, the attenuation effect of this upstream signal can vary with the identity of the downstream stimulator. Finally, capped mRNA templates with or without the attenuation signal for the minimal IBV construct used above as well as a SARS viral construct carrying residues 12 265-13 652 and lacking any luciferase-related segment were also created to analyze their in vitro frameshift efficiencies. The results shown in Figure 6B reveal parallel results with the previous study in Figures 4A and 6A, and further rule out the involvement of ribosome loading difference in the observed attenuation effect. Furthermore, it demonstrates that such attenuation activity is not an artifact caused by the reporter system used because the inhibitory effect also functions within an all-viral context (compare lanes 2 with 3 of Figure 6B). To evaluate the biological significance of this attenuation signal in the regulation of À1 frameshifting, we constructed several p2luc-based reporters for inserts possessing or lacking SARS 13 222-13 368 viral RNA without shifting the stop codon located before firefly luciferase ORF to utilize the dualluciferase activity to monitor frameshift efficiency in vivo (21). The in vitro frameshift efficiency of these constructs were calculated from dual-luciferase assay result of the TNT products of both plasmids (lane 1 in Figure 6C), and reveals parallel results with the frameshift efficiency reported above (lanes 2 and 3 of Figure 4A). Similarly, calculation from the in vivo dual-luciferase assay result for cells transfected with the reporter constructs indicates that the frameshift efficiency reduces almost in half in the presence of attenuation signal comparing with the one lacking the attenuation signal (lane 2 in Figure 6C). Thus, this upstream attenuation signal also functions in vivo.

Manipulation of loop 2 conformation can modulate the À1 frameshift efficiency of IBV-type pseudoknot
The pseudoknot of short S1 with a bent helical junction such as the one in MMTV and the IBV-type pseudoknot of long S1 with a stringent length-requirement of 11 bp represent two distinct classes of frameshift-stimulating pseudoknot (25,(28)(29)(30). In either case, the ability to resist deformation by the translocating ribosome is thought to determine the frameshift efficiency stimulated by a specific pseudoknot (8,11,13,31). Alternatively, optimal contacts between the activity in ribosome and the stimulator for its efficient unwinding may also play roles in pseudoknot-stimulated À1 frameshifting (32). The impairment of À1 frameshifting for mutants with the disruption of S1 or S2 in SARS CoV pseudoknot can be rationalized by the formation of two separated hairpins and the coupled destruction of pseudoknot configuration. In contrast, disruption of the base pair in S3 is expected to be less important as the long loop 2 of the IBV-type pseudoknot has been thought to play a connection role to link the two stems (25). This is consistent with the wild-type like frameshift efficiency for the mutant lacking S3 in this study. Interestingly, the minimal IBV pseudoknot lacking most of its wild-type loop 2 sequences promotes À1 frameshifting better than its wild-type counterpart (10,25), whereas reverse trend is observed here for similar study on the SARS CoV pseudoknot. However, the medium frameshift efficiency for the two IBV-type mutants generated by complete base pairing disruption of S3 in SARS CoV pseudoknot suggest that the integrity of these base pairs in S3 may contribute extra À1 frameshifting activity in a delicate manner. We also note that existences of two extra base pair in the up-restoration mutants does not further enhance frameshift efficiency, implicating only a threshold value of stability is required. Taken together, they indicate that different secondary structure compositions within the loop 2 of IBV-type pseudoknot may modulate the frameshift efficiency of this type of pseudoknot, and implicate that the frameshift efficiency might be tunable by modulation of the S3 formation in vivo. Alternatively, the formation of S3 may also serve to prevent formation of unfavorable conformations that may interfere with frameshifting process as the long loop 2 could be merely constraint imposed by the encoded amino acids required for the RdRp activity. The atypical pseudoknot found in this work is different from those in transmissible gastroenteritis virus (TGEV) and HCV 229E, which also contain an additional stem essential for À1 frameshifting (33,34). The additional stem of 229E pseudoknot is formed by complementary sequences between the very long loop 2 and the 3 0 end sequences following stem 2, and is probably stacked onto stem 2 to form a quasi-continuous helix of 24-25 bp. In contrast, the S3 of SARS CoV pseudoknot could stack into the bottom of S1 to form a quasi-continuous helix of 25-26 bp interrupted by two separated A-bulges. Although our mapping data cannot provide information about stacking configuration among these stems in the SARS CoV pseudoknot, helical junction geometry between S1 and S2 obviously will be coupled to the positioning of S3. As modulation of frameshift efficiency by the modification of junction geometry via tertiary interactions has been observed in helical junction of BWYV pseudoknot (35,36), the modulation of helical junction configuration by the additional S3 in SARS CoV pseudoknot could also play an important role here. Furthermore, the existence of single-strand cutter cleavage for residues connecting S2 and S3 in probing data also suggests a dynamic nature of the junction region. Therefore, reduced frameshift efficiency in mutants potentially disrupting partial or all of the base pair in S3 can thus be caused by changes in junction geometry with the weakening or diminishing of S3 in these mutants.
Upstream attenuation sequence is a novel determinant for À1 frameshifting The significant decrease in À1 frameshift efficiency by upstream attenuation signal appears unexpected as the major viral factors characterized to determine frameshift efficiency all involve sequences between the slippery site and the downstream stimulator. Interestingly, recent works on HIV-1 and barley yellow dwarf virus (BYDV) both reported the involvement of sequences upstream of the slippery site in modulation of overall À1 frameshift efficiency (37,38). However, only short sequences immediately upstream of the slippery site are involved in the case of HIV-1, and the modulation can go either way for enhancement or attenuation without a defined activity (37). In contrast, a conserved hairpin upstream of the slippery site was proposed to slow down the marching ribosome and thus enhance À1 frameshift efficiency in the case of BYDV (38). Recently, mutational study on the nucleotide adjacent to the slippery site of the SARS CoV also reported an increment of À1 frameshift efficiency in vivo (24). Our finding is thus unique in which, it specifies an RNA element of negatively regulatory activity with general application, and suggest that it is an integral part for the À1 frameshift efficiency determinant of SARS CoV although it is separated from the slippery site with an intervenient spacer. Although sequence alignment and secondary structure prediction for both SARS 13 222-13 368 and SARS 13 318-13 368 RNA with the corresponding regions of other coronavirues reveal no strong consensus, the sequences of SARS 13 318-13 368 RNA do conserve among different strains of SARS CoV (data not shown). The existence of such an attenuation signal may provide the virus a regulation point for the tuning of RdRp synthesis in response to environmental changes. In contrast, a change in spacer sequence identity will lead to a fixed frameshift efficiency although it also reduced the efficiency. Furthermore, it may also change the property of the encoded amino acid composition in the N-terminal region of RdRp with dramatic impacts on the activity of RdRp.
With respect to the mechanisms responsible for the function of this negative determinant of À1 frameshifting, the factors known to affect frameshift efficiency should be examined in the future. However, it is less possible that it is mediated by the encoded polypeptide because 14 out of 18 amino acids encoded within SARS 13 318-13 368 RNA were changed in the out of frame mutant and no dramatic change in attenuation activity has been observed. In contrast, it is more likely that the attenuation signal can contact with and affect the factors capable of modulating the frameshift efficiency. Indeed, interferences of dynamic interactions/contacts between pseudoknot and the ribosome or auxiliary factors in mutants of BWYV pseudoknot have been proposed to be responsible for observed frameshift efficiency variation comparing with that of its wild-type counterpart (36). However, it is tempting to speculate that the function of this attenuation element may be related to the extra S3 of downstream atypical pseudoknot. However, the fact that the attenuation signal also downregulates À1 frameshifting promoted by other stimulators suggests its function is general. Furthermore, the requirement for an orientation upstream of the slippery site and the stimulator in vitro indicates that function of the attenuation signal is probably not mediated via interactions with the downstream determinants of À1 frameshifting. Further biochemical analysis of the attenuation element will be informative for revealing how it works and providing insight of the mechanism of À1 ribosomal frameshifting.


extfieldsofstudy: Biology Medicine

text:
A Novel Cysteine-rich Domain of Sep15 Mediates the Interaction with UDP-glucose:Glycoprotein Glucosyltransferase*

Selenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 co-purifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. In contrast, a Sep15 homologue, designated selenoprotein M (SelM), does not form a complex with GT. Sequence analysis of members of the Sep15 family identified a novel N-terminal cysteine-rich domain in Sep15 that is absent in SelM. This domain contains six conserved cysteine residues that form two CxxC motifs that do not coordinate metal ions. If this domain is deleted or the cysteines are mutated, Sep15 no longer forms a complex with GT. Conversely, if the cysteine-rich domain of Sep15 is fused to the N-terminus of SelM, the resulting chimera is capable of binding GT. These data indicate that the cysteine-rich domain of Sep15 exclusively mediates protein-protein interactions with GT.

Selenium is present in 25 human proteins in the form of selenocysteine, which is the 21st natural amino acid in the genetic code and is encoded by the UGA codon (1,2). Recent studies provide strong evidence that dietary selenium supplements reduce the incidence of cancer in animal models and human clinical trials (3)(4)(5). Low molecular weight selenium compounds and selenium-containing proteins have been implicated in this chemopreventive effect. However, the mechanism by which selenium suppresses tumor development remains to be established.
One candidate protein that may mediate the chemopreventive effects of selenium is the 15-kDa selenoprotein (Sep15) 3 (6,7). The Sep15 gene is located at position p31 on human chromosome 1. This region has been shown to undergo loss of heterozygosity, which is frequently associated with tumor progression, suggesting a potential tumor suppressor role for this selenoprotein (8,9). In addition, two polymorphisms found at positions 811 (C/T) and 1125 (G/A), the latter of which is located within the selenocysteine insertion sequence element, have been identified within the 3Ј-untranslated region of the human Sep15 gene. The nucleotide identity at these polymorphic sites has been shown to influence the efficiency of selenocysteine incorporation in a selenium-dependent manner (10).
The precise biological function of Sep15 has not been firmly established; however, this selenoprotein co-purifies with the essential regulator of the calnexin cycle designated UDP-glucose:glycoprotein glucosyltransferase (GT) (11). The calnexin cycle is a quality control pathway localized to the endoplasmic reticulum that specifically assists in the folding of N-linked glycoproteins (12). Once a newly synthesized protein enters the endoplasmic reticulum, a glycan core of 14-oligosaccharides (Glc 3 Man 9 GlcNAc 2 ) is covalently linked to appropriate asparagine residues. N-Linked glycoproteins are immediately recognized by glucosidase I and II, the first components of the calnexin cycle, which sequentially trim the two outermost glucose moieties from the glycan core (13). Once processed, membrane-bound calnexin and its lumenal homologue calreticulin bind to the glycan (14 -17). This triggers the binding of ERp57 (a lumenal protein disulfide isomerase) to calnexin and calreticulin and accelerates folding by catalyzing disulfide bond exchange (18 -22). Regardless of the folded status of the bound glycoprotein, glucosidase II cleaves the remaining glucose residue and the complex disassociates, releasing the glycoprotein. If the glycoprotein is properly folded, it is rapidly exported from the endoplasmic reticulum by vesicular transport to the Golgi complex. Misfolded proteins are re-glucosylated by GT, a 175-kDa lumenal enzyme that catalyzes the transfer of a glucose moiety from UDP-glucose to the distal mannose of the glycan core (23). This event creates a retention signal that re-initiates the calnexin cycle.
In this quality control pathway, GT functions as the folding sensor that assesses the folded status of glycoproteins. Although we do not understand how GT senses structural fidelity, several important clues have been revealed. (i) Sensing involves the innermost (and normally buried) N-GlcNAc glycosidic bond of the glycoprotein (24,25). (ii) GT preferentially monoglucosylates partially folded late folding intermediates as compared with their fully denatured counterparts (26 -28). (iii) GT is capable of distinguishing folded from misfolded domains within a single polypeptide (29,30). (iv) Localized folding defects in otherwise correctly folded glycoproteins or individual domains are recognized by GT (30,31). (v) GT monitors the tertiary and quaternary structures of glycoproteins (32).
Given the sequence diversity of glycoproteins, it is unlikely that the recognition mechanism used by GT involves a specific primary structure motif. A more robust mechanism, involving common biophysical properties of misfolded proteins, seems more plausible. Accordingly, the distribution of glycans and their spatial relationship to normally surface-inaccessible hydrophobic residues influence the order in which polypeptide segments achieve their native conformation (12,27,28). GT preferentially recognizes hydrophobic motifs proximal to glycosylation sites in glycopeptides (33). The efficiency of glucosylation can be positively correlated with the spatial proximity of site-directed mutations to each glycan, lending support to the idea that normally buried hydrophobic residues found adjacent to glycans mediate interactions with GT (31).
Our initial findings of the interaction between mammalian Sep15 and GT (11) raised the possibility that Sep15 might be a misfolded glycoprotein. Although Sep15 does not contain an endoplasmic reticulum retention sequence, it is neither secreted or glycosylated, suggesting that this selenoprotein might function as a co-factor that assists GT in assessing structural fidelity. Sep15 deficiencies observed in tumors may also impair GT-mediated regulation of the calnexin cycle.
In this report, we characterize interactions between members of Sep15 and GT protein families. Isothermal titration calorimetry was used to demonstrate that Sep15 forms a 1:1 complex with GT with an apparent K d value of 20 nM. Moreover, co-immunoprecipitation revealed that Sep15 binds to both human GT isozymes. Although selenoprotein M (SelM) shares significant sequence homology with Sep15 and has also been localized to the endoplasmic reticulum, it does not form a complex with GT. The interaction between Sep15 and GT is exclusively mediated by a novel cysteine-rich domain located at the N-terminus of Sep15. This conserved domain is present in other functional Sep15 homologues and contains six conserved cysteine residues that do not coordinate metal ions. When the cysteine-rich domain of Sep15 is removed, it no longer forms a complex with GT. Site-directed mutagenesis was used to demonstrate that the structural integrity of the cysteine-rich domain is an essential factor in the ability of Sep15 to bind GT.
A fusion protein of Sep15 and SelM from mouse (designated Sep15::SelM) was generated by fusing the cysteine-rich domain of Sep15 to the N-terminus of SelM. The cysteine-rich domain of Sep15, together with its native signal peptide, was PCR amplified using the following primers: 5Ј-TATAGACTAGACTCGAGGGCAGACCGCAG-3Ј and 5Ј-GCAGACTTCAAGGATGAATTCTGCATACAGCTTTTTGG-3Ј. This PCR product was digested with XhoI and EcoRI and ligated into the pCI-neo vector. SelM-U48C was PCR amplified without its signal peptide using the following primers: 5Ј-CTTGTGGCTCCAGC-CGAATTCACCACCAACTACCGACC-3Ј and 5Ј-ATGCGGCCGC-TACAGGTCGTCGTGTTCTGAAGCTTCCTCG-3Ј. The PCR product was digested with EcoRI and NotI and ligated into the pCI-neo vector containing the cysteine-rich domain of Sep15.
Mammalian expression vectors encoding protein C (PC)-tagged human GT1 and GT2 have been described previously (34). The gene encoding Sep15 from Drosophila melanogaster was PCR amplified from an expressed sequence tag clone using the following primers: 5Ј-TGC-TCGAGCAAATTGGCGCCGAATTGACGGCCGCCGACTG-3Ј and 5Ј-CAGTTTTGGGTATGGGTACCTTAGATCCTGTTGGTT-CGCAGG-3Ј. The PCR product was digested with EheI and KpnI and ligated into Baculovirus expression vector pFastBac-HTa (Invitrogen). A previously described cDNA clone (35) served as the template to introduce a C-terminal hexahistidine-tag into Drosophila GT. To improve secretion from insect cells the native signal sequence was replaced with the honeybee melittin signal peptide. Moreover, the C-terminal endoplasmic reticulum retention sequence was removed. Primer sequences are available upon request.
Native Gel Electrophoresis-Recombinant Drosophila Sep15 and GT proteins were expressed in Sf9 cells and purified by immobilized metalaffinity chromatography. Complexes of Sep15 and GT were prepared by incubating 5 g of GT dissolved in 10 mM Tris-HCl (pH 7.5) and 10 mM CaCl 2 with the indicated amounts of Sep15 for 10 min at 37°C. Samples were subjected to non-denaturing PAGE using Tris-glycine gels (Invitrogen) and transferred to polyvinylidene difluoride membranes. The Sep15-GT complex was visualized by Western blotting with Sep15specific antibodies.
Isothermal Titration Calorimetry-The apparent association constant for Sep15 binding to GT was determined using isothermal titration calorimetry. Protein solutions of Drosophila Sep15 and GT were dialyzed for 2 days against 25 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 1 mM CaCl 2 . The concentrations of both proteins were determined using Beer's law. This experiment consisted of a series of 8-l injections of Sep15 into a cell containing 1.4 ml of GT equilibrated at 20°C. Heats associated with Sep15 binding to GT were measured by an isothermal titration calorimeter (Microcal). The apparent association constant derived from these data were determined using software included with the calorimeter.
Metal Ion Analysis-The concentrations of twenty biologically relevant metal ions were determined by inductively coupled argon plasma emission spectrometry at the Chemical Analysis Laboratory, University of Georgia. Protein samples dissolved in 50 mM sodium phosphate (pH 7.0), 300 mM NaCl, and 250 mM imidazole were analyzed. Metal ion concentrations were calculated by subtracting the ion content of the buffer from that of the protein sample.

Sep15, but Not SelM, Interacts with Human GT Homologues-Sep15
is a resident protein of the endoplasmic reticulum that has been previously shown to form a complex with GT when purified from rat and mouse liver (11). Two human GT homologues (GT1 and GT2) have been identified (36). These enzymes are highly homologous and display 83% sequence identity in their proposed C-terminal catalytic domains. However, their N-terminal non-catalytic domains are more diverse (49% sequence identity), and only GT1 displays measurable catalytic activity. To assess whether Sep15 functions as a protein co-factor that binds both GT isozymes, mouse Sep15 was co-expressed with PC-tagged GT1 or GT2 in NIH 3T3 cells. Sep15 co-immunoprecipitated with GT1 and GT2 (Fig. 1A), indicating that both human GT isozymes are capable of binding Sep15 in vivo. The reduced intensity of the Sep15 signal observed with GT2 is consistent with its lower level of expression as compared with that of GT1.
The recently described selenoprotein SelM (37) shares 31% sequence identity with Sep15 (Fig. 2, A and B). Both proteins encode a single selenocysteine residue. Primary sequence alignments illustrate that SelM has an elongated C terminus, whereas Sep15 possesses an extended cysteine-rich N terminus. The N-terminal sequence of Sep15 is conserved among functional Sep15 homologues and is conversely absent in sequence homologues of SelM (Fig. 2C). As observed with Sep15, selenoprotein SelM is also localized to the endoplasmic reticulum. Given the common intracellular location of Sep15 and SelM and their respective sequence homology, we speculated that SelM might also bind to GT. However, co-immunoprecipitation experiments indicate that mouse SelM does not form a complex with either human GT1 or GT2 (Fig. 1B).

Cysteine-rich Domain of Sep15 Mediates the Interaction with GT-
The absence of an N-terminal extension in SelM suggested that this region of Sep15 may potentially mediate protein-protein interactions with GT. To test this hypothesis, the cysteine-rich N-terminal extension of mouse Sep15 was fused to the N terminus of mouse SelM. When expressed in NIH 3T3 cells, this mouse protein chimera co-immunoprecipitates with human GT1 (Fig. 3). These data suggest a functional role for the N-terminal extension of Sep15 in the formation of a complex with GT.
Sequence analyses of the N-terminal domain of Sep15 proteins identified six conserved cysteine residues (Fig. 2C). Four of these six cysteine residues form a pair of CxxC motifs. To establish a structural and/or functional role for these cysteine residues and evaluate their potential involvement in constituting the GT-binding site, all cysteine residues were individually replaced with serine residues. Co-immunoprecipitation experiments demonstrate that formation of the Sep15-GT complex was abolished for each mutant (Fig. 4). These data indicate that the N-terminal extension of Sep15 forms a distinct cysteine-rich domain and that the structural integrity of this domain is required for Sep15 to form a complex with GT. Sep15 and GT Form a 1:1 Complex-One technical obstacle to investigating protein-protein interaction between Sep15 and GT is the naturally occurring selenocysteine residue encoded within the Sep15 gene. Selenocysteine-containing proteins are frequently difficult to express and purify. However, the Sep15 protein of Drosophila is not a selenoprotein and encodes two cysteine residues in place of the CxU motif (Fig. 2B). To examine the binding between Drosophila Sep15 and GT, both recombinant proteins were expressed and purified from Sf9 cells.
Formation of the complex between Sep15 and GT was analyzed by polyacrylamide gel electrophoresis under non-denaturing conditions (Fig. 5). Western blotting using antibodies specific for Sep15 detected a low molecular weight band corresponding to unbound Sep15 in addition to a higher molecular weight band that co-migrates with GT. Several minor low molecular weight bands were also detected, indicating that Sep15 may form homomultimers and/or is degraded under these experimental conditions. The band intensity of the Sep15-GT complex  continued to increase as the molar equivalents of Sep15 and GT were increased from 0.2 to 2.0, at which point the amount of unbound and GT-bound Sep15 became equivalent.
The stoichiometry of the Sep15-GT complex was precisely determined by isothermal titration calorimetry (Fig. 6). Sep15 forms a 1:1 complex with GT with an apparent K d value of 20 nM. The mixed species Sep15::SelM chimera also bound tightly to GT (supplemental Fig. S1). In contrast, no interactions were observed for SelM and Sep15 without its N-terminal cysteine-rich domain. These isothermal titration calorimetry results were confirmed by measuring the intensity of the strongest methyl resonance by one-dimensional 15 N-edited proton NMR spectroscopy (supplemental Fig. S2). A decrease in the strongest methyl resonance intensity in the proton NMR spectrum of the protein mixture in comparison to the added strongest methyl resonance intensities of the individual proteins indicates specific protein-protein interactions (38). Consistent with co-immunoprecipitation and isothermal titration calorimetry data, these NMR data indicate that both Sep15 and the Sep15::SelM chimera form complexes with GT. Conversely, SelM and Sep15 without its corresponding cysteine-rich domain do not bind to GT. Taken together these complementary techniques demonstrate that the cysteine-rich domain of Sep15 mediates that formation of a tight 1:1 complex with GT.
Formation of the Sep15-GT Complex Is Not Mediated by Coordinated Metal Ions-Four of the six conserved cysteine residues in the cysteinerich domain of Sep15 form two CxxC motifs, which are typically involved in metal ion coordination. To establish whether the cysteinerich domain of Sep15 does coordinate metal ions, we further analyzed Drosophila Sep15. When expressed in bacterial or insect cells, purified Sep15 protein was colorless. Moreover, UV-visible spectrophotometric analysis revealed only a single peak centered at 280 nm, indicating the absence of certain co-factors and iron-sulfur clusters. Sep15 protein samples were also analyzed for the presence of twenty biologically relevant metal ions by inductively coupled argon plasma emission spectrometry. Only Co 2ϩ was detected (0.24 molar equivalents), which is most likely non-specifically bound during protein purification by immobilized metal-affinity chromatography using cobalt-based resin. These data suggest that the cysteine-rich domain itself does not coordinate metal ions.
It has been previously reported that the catalytic activity of GT is dependent upon the presence of divalent cations (preferably Ca 2ϩ ) (39). Although metal ions were not detected in Sep15 protein preparations, it is possible that metal ions mediate the interaction between Sep15 and GT. To address this possibility, complex formation between Sep15 and GT was analyzed by polyacrylamide gel electrophoresis under non-denaturing conditions in the absence and presence of 1 mM Ca 2ϩ , Cu 2ϩ , Fe 2ϩ , and Zn 2ϩ . However, the Sep15-GT complex was observed independently of the presence of metal ions (data not shown). These results indicate that formation of the Sep15-GT complex is not mediated by coordinated metal ions.
Sep15 Does Not Affect Glucosylation of Thyroglobulin-The association of Sep15 with GT suggests that this selenoprotein may modulate the enzymatic activity of GT and/or be involved in assessing structural fidelity. To test this hypothesis, GT activity in the presence of Sep15 was analyzed by monitoring the amount of incorporated [ 14 C]glucose into denatured thyroglobulin. The presence of Sep15 did not affect the GT activity toward this protein (supplemental Fig. S3). These results indicate that, at least in the case of thyroglobulin, Sep15 does not modulate GT activity and is not required for glucosylation under these experimental conditions.

DISCUSSION
Selenoproteins have not been previously shown to participate in the quality control networks of the endoplasmic reticulum. The observation that Sep15 co-purifies with the essential regulator of the calnexin cycle FIGURE 4. Binding of Sep15 to GT is dependent upon the structural integrity of the cysteine-rich domain. NIH 3T3 cells were co-transfected with plasmid-encoded PCtagged human GT1 (GT1-PC) and the empty control vector pCI-neo or plasmid-encoded His-tagged mouse Sep15-U93C or Sep15-C38SU93C, Sep15-C49SU93C, Sep15-C52SU93C, Sep15-C67SU93C, Sep15-C70SU93C, and Sep15-C71SU93C mutants. Samples were immunoprecipitated (IP) using anti-PC antibodies, resolved by SDS-PAGE, and detected by Western blotting (WB) with anti-PC (first panel) and anti-His (second panel) antibodies. Cell lysates were also directly probed using anti-PC (third panel) and anti-His antibodies (fourth panel). suggests a potential mechanism for the chemopreventive effects of dietary selenium (11).
In this study, we characterized interactions between members of Sep15 and GT families using a series of complementary genetic and biophysical techniques and found that this selenoprotein interacts with both human GT isozymes. The 1:1 stoichiometry of this complex has an apparent K d value of 20 nM as determined by isothermal titration calorimetry using Sep15 and GT from Drosophila. In contrast, SelM (a sequence homologue of Sep15) does not interact with GT. Multiple sequence analyses identified a novel cysteine-rich domain that is conserved among the N termini of Sep15 proteins, which is not found in the sequences of SelM proteins. If the cysteine-rich domain of Sep15 is deleted, the truncated Sep15 protein no longer interacts with GT. However, when the cysteine-rich domain of Sep15 is fused to the N terminus of SelM, the resulting Sep15::SelM protein chimera forms a complex with GT.
The sequence of the cysteine-rich domain is highly conserved among Sep15 homologues from plants to humans (40) (Fig. 2C), suggesting that interactions between Sep15 and GT have been conserved throughout evolution. Mutating any one of these six conserved cysteine residues disrupts the interaction between Sep15 and GT. As four of these six cysteine residues form a pair of CxxC motifs, we speculated that these motifs might coordinate metal ions, such as iron or zinc. However, metal ion analysis of purified Sep15 protein samples did not identify specifically bound metal ions. It is more likely that these conserved cysteine residues form intramolecular disulfide bonds. Site-directed mutagenesis of cysteine residues may disrupt the proper arrangement of these disulfide bonds and compromise the structural integrity of the cysteine-rich domain, which in turn inhibits protein-protein interaction between Sep15 and GT. Taken together, these data unequivocally demonstrate that the cysteine-rich domain mediates the interaction between Sep15 and GT. However, the corresponding regions of GT that are involved in forming the binding surface for Sep15 remain to be established.
The fact that Sep15 does not possess the endoplasmic reticulum retention signal and its N-terminal part is responsible for maintaining Sep15 in the endoplasmic reticulum (41) suggests that its location in this cellular compartment is the result of the interaction with GT.
The biological significance of protein-protein interactions between Sep15 and GT within the endoplasmic reticulum is poorly understood. One possibility is that Sep15 modulates the enzymatic activity of GT by functioning as a protein co-factor that assists this folding sensor in assessing the structural fidelity of misfolded glycoproteins. However, our data show that GT-mediated labeling of thyroglobulin is not directly affected by Sep15. Sep15 appears to occur exclusively in the complex with GT, whereas GT is present in both selenoprotein-bound and selenoprotein-free form (11). Thus, it is possible that Sep15 is not required for the GT function per se but may be required for the recognition of glycoproteins with incorrectly formed disulfide bonds. It is an attractive possibility that the unusual CxU motif of Sep15 may participate in the reduction, isomerization, or oxidation of disulfide bonds of glycoprotein substrates of GT.


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An Sp1/KLF binding site is important for the activity of a Polycomb group response element from the Drosophila engrailed gene

Polycomb-group response elements (PREs) are DNA elements through which the Polycomb-group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated with two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181 bp PRE from the Drosophila engrailed gene. Here we show that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc finger proteins. There are 10 Sp1/KLF family members in Drosophila, and nine of them bind to Site 2. We derive a consensus binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila.

INTRODUCTION
Polycomb-group response elements (PREs) and Trithoraxgroup response elements (TREs) are DNA elements that are the targets of the Drosophila Polycomb-group (PcG) and Trithorax-group (TrxG) of genes, respectively. PcG genes encode proteins that act as transcriptional repressors and TrxG genes encode transcriptional activators. The best-studied targets of the PcG and TrxG genes are the homeotic genes in Drosophila where PcG and TrxG genes do not initiate the pattern of gene expression, but rather serve as the molecular memory to keep genes in either the off or on state. At the homeotic genes, PREs and TREs are often closely associated or interspersed [for reviews see (1,2,3)].
Many PcG and TrxG genes encode proteins that work in complexes to modify chromatin. For example, there are two well characterized PcG complexes, PRC2, or the E(z)/Esc complex (4-7) and PRC1, a complex that contains the PcG proteins Polycomb (Pc), Polyhomeotic (Ph), dRing/Sex combs extra (Sce), (8) and Posterior sex combs (Psc) (9). Chromatinimmunoprecipitation experiments have shown that PcG proteins are specifically bound to PREs in vivo [e.g. (10,11)], however, it is not known how they get recruited to the DNA, since these complexes do not specifically associate with PRE sequences in vitro.
Much effort has gone into trying to understand what constitutes a PRE in order to understand how PcG protein complexes are recruited to the DNA and to predict the occurrence of PREs by sequence analysis (12). PREs are complex elements-no single DNA binding site can act as a PRE. Instead, PREs are made up of binding sites for many different proteins. To date most PREs studied at the molecular level have been shown to contain DNA binding sites (usually multiple copies) for the proteins Pleiohomeotic (Pho) and its partially redundant homolog Pleiohomeotic-like (Phol), Zeste, GAGA Factor (GAF)/Pipsqueak (Psq) [reviewed in (1)] and Dorsal Switch Protein 1 (Dsp1) (13). Ringrose et al. (12) have reported that clustered pairs of GAF/Psq, Zeste and Pho/Phol sites can predict the location of many PREs. However it has recently been shown that a combination of Zeste, Dsp1, GAF/Psq and Pho/Phol sites in the same number, orientation and spacing as in the native PRE is insufficient to restore full PRE activity (13). This suggests that all of the DNA binding sites necessary for PRE activity are still not known.
With the aim of identifying the binding sites and factors necessary for PRE function, our lab has been trying to define the components that constitute a minimal 181 bp PRE at À576 to À395 upstream of the Drosophila engrailed gene. This element was originally identified in a pairing-sensitive silencing assay (14), an assay used to detect the function of many PREs. When PREs are included in the vector pCaSpeR, they have an unusual effect on the eye color marker mini-white. Normally, when transgenic flies are made with pCaSpeR, flies homozygous for the transgene have a darker eye color than flies heterozygous for the transgene. However, when a PRE is included in the pCaSpeR vector, the eye color of homozygous flies is often lighter than that of heterozygotes. This phenomenon is dependent on the chromosomes being able to pair. Fragments of DNA that mediate pairing-sensitive silencing are called pairing-sensitive elements (PSEs). Not all PSEs have been shown to act as PREs and vice versa [for review see (15)]. The engrailed 181 bp element behaves both as a PRE and a PSE (16).
The 181 bp engrailed PRE contains 3 GAF/Psq sites, 2 Pho/Phol sites, 2 potential Zeste sites and 1 Dsp1 site that almost entirely overlaps the Pho/Phol site that we have studied by mutational analysis (Figure 1). In addition to these known sites, we have identified a number of other protein-binding sites required for pairing-sensitive silencing [ Figure 1, ref (16)]. Here we investigate the role of one of these protein-binding sites, Site 2, in PRE function and show that the Sp1/KLF family of proteins bind to this site. We find that Sp1/KLF binding sites are present in most well characterized PREs.

Analysis of PRE activity
The construction of the 181-bxd-Ubx-LacZ construct was described in Americo et al. (16). The MutSite2 181 PRE was amplified from the pCasper/MutSite2 construct as described in Americo et al. (16) using 5 0 and 3 0 primers containing Xho1 sites. The amplified product was cloned as an Xho1 fragment into the Xho1 site of the bxd-Ubx-lacZ construct. The base changes in Mutsite2 are shown in Figure 3. The pCasper/ MutPho construct described in Americo et al. (16) was used as the starting point to introduce mutations in the second potential Pho/Phol binding site in the 181 PRE. A 5 0 primer carried an XhoI site and the relevant Pho/Phol site mutations from (GAGATGGC to GAGCGTGC, the match to the Pho/Phol consensus binding site is on the lower strand). The 3 0 primer carried an Xho1 site. The amplified product was cloned into the Xho1 site of the bxd-Ubx-lacZ construct. All plasmids were sequenced over the 181 bp PRE to confirm the presence of the described mutations. The bxd-Ubx-lacZ 181 wild-type and mutated derivatives were injected into a ry 506 strain by Genetic Services Inc.
Yeast one-hybrid screen A yeast one-hybrid screen was performed using the Clontech yeast one-hybrid kit. Standard yeast techniques were used as recommended by Clontech. A double-stranded oligonucleotide comprised of Site 2 (ACTCGGTAACGCCCCGTGA) repeated five times bounded by EcoR1 and Xho1 restriction sites was cloned into the EcoR1, Xho1 cleaved pHisi-1 vector (Clontech) to create pHisi-1:Site2. The pHisi-1:Site2 plasmid was linearized and integrated into the yeast genome. A Drosophila 0-18 h cDNA library (Clontech) was transformed into the yeast strain with the integrated pHisi-1:Site2 plasmid. Positive colonies were isolated after 4-6 days of incubation at 30 C on selective media. Plasmid DNA was isolated and sequenced. In addition to the clones reported here, we sequenced 31 other potential positives, only two others had potential DNA binding domains. These proteins did not bind to Site 2 after in vitro transcription/translation.

Cloning of the zinc finger domains into the pT7link expression vector
The zinc finger domains of the Sp1/KLF proteins were generated either by PCR directly from the cDNA or by RT-PCR from genomic DNA. Primers were designed that would PCR amplify the entire zinc finger region of each protein with an additional 8 N-terminal amino acids. The primers contained BamHI and XhoI ends for cloning the zinc finger regions downstream of the b-globin 5 0 -UTR and ATG in the pT7link expression vector (provided by R. Treisman). Each of the clones was confirmed by sequencing and used for in vitro transcription/translation.

Gel mobility shift assay
Recombinant proteins were synthesized in vitro using the TNT-coupled transcription/translation system (Promega). Gel mobility shift assays were performed as described previously (16) using 3 ml of the in vitro translation reaction.

Searching known PRE and PSE sequences for Site 2 and other known protein-binding sites
The sequences of known PREs and PSEs were searched for the presence of consensus binding sites for Sp1/KLF family members, Pho/Phol, GAF/Psq, Zeste and DSP1 proteins. The exact parameters of the consensus sequences are given in the legend to Table 1. Consensus sequences were localized using a sequence visualization software program named Gene Palette (www.genepalette.org), (17). Pho/Phol, GAF/Psq and one of the Zeste consensus sites were chosen based on Ringrose et al. (12). The Sp1/KLF consensus site is as described in the text. We used an additional version of the Zeste consensus site (BGAGTGV) as described by Mohrmann et al. (18). The DSP1 consensus site came from Dejardin et al. (13).

Site 2 is required for PRE activity of the 181 bp engrailed fragment
We have previously shown that Site 2 is required for pairingsensitive silencing of the 181 bp engrailed PRE (16). Here we ask whether Site 2 is also important for the PRE activity of that fragment. We used the bxd-Ubx-lacZ reporter construct (19) to test the effect of mutations in Site 2 on PRE activity. In the absence of a PRE, the bxd-Ubx-lacZ reporter construct expresses lacZ throughout the embryo, in both the ectoderm and the nervous system, late in development ( Figure 2A). When the 181 bp PRE from the Drosophila engrailed gene is included in this vector, lacZ expression is restricted to parasegment six and posterior segments [ Figure 2B and ref (16)]. We saw restricted expression patterns in 75% of our lines, a number consistent with what has been seen by other investigators as PREs are not active in all chromosomal insertion sites.
We next introduced mutations into either Site 2 or the two Pho/Phol binding sites in the 181 bp fragment in the context of the bxd-Ubx-lacZ vector. The effect on PRE activity, as Engrailed PRE (16)  181  2  3  1  2  1  P,G assayed by embryonic b-galactosidase expression patterns, was analyzed. Mutation of either the Pho/Phol sites or Site 2 caused a reduction in the percentage of lines that had PRE activity. Both mutated constructs gave results that were significantly different from the expected frequency of 75% PRE activity for the unmutated construct (P < 0.001 by a chi-squared test). There were still a small percentage of lines with restricted expression in the Pho/Phol (20%), and the Site 2 mutant transgene (29%) as well as in the vector only control (8%). This probably reflects the fact that the bxd enhancer and the Ubx promoter are poised to work with flanking genomic PREs and may contain weak PRE activity on their own (19). When we scanned the first 350 bp of the bxd sequence that borders the 181 bp element in our constructs for the known PRE DNA binding sites, we found 3 Pho/Phol sites, 3 Dsp1 sites, 1 Zeste site and 2 potential Sp1 sites. This may explain why mutation of Pho/Phol or Site 2 did not completely eliminate the PRE activity of the 181 bp engrailed fragment in this vector. Nevertheless, our results suggest that both Pho/Phol and Site 2 binding sites contribute to the activity of the engrailed PRE, and that neither alone is sufficient for PRE activity.

Members of the Sp1/KLF family bind to Site 2
In order to identify the protein(s) that interact with Site 2 we carried out a yeast one-hybrid screen using multimerized Site 2 as bait. We isolated four cDNA clones that showed specific binding to Site 2 but not to a mutated Site 2 ( Figure 3). Each of these four clones encoded a member of the Sp1/KLF family of zinc finger proteins [CG5669, CG12029 (two different length clones) and luna]. The consensus binding sequence for the Sp1/KLF family based on mutational analysis of a KLF family member (KLF4) binding site is (G/A)(G/A)GG(C/T)G(C/T), (20). The Site 2 sequence contains a perfect match to this consensus sequence (Figure 3). The Sp1/KLF family is an important group of proteins that in mammals have been shown to be involved in cell morphogenesis, differentiation and cancer. These proteins share a high degree of homology over 3 Cys2/His2 zinc fingers (>65% sequence identity with each other), located at or close to the C-terminal end of the protein. The N-terminal regions are generally unique [for reviews see (21,22)].
We identified three members of the Sp1/KLF family in our yeast one-hybrid screen, however our screen was not saturating. When we searched the Drosophila genome sequence for homology to the zinc finger region of these proteins we identified a total of 10 members of this class in Drosophila. The zinc finger regions of these members are highly conserved as summarized in Figure 4. The mammalian factors can be subdivided into three classes (Sp1/class I family, class II family and class III family) based on closer homology between members of the subfamily relative to members outside the subfamily [reviewed in (21)]. This homology sometimes extends to functional protein domains that lie outside the DNA binding domain. The Drosophila proteins can also be placed into different groups based on homology of the zinc fingers with the zinc fingers of the human SP1/KLF proteins. CG5669, dSp1 and Btd are more closely related to the Sp1/Class I proteins than to the KLF proteins. In fact, dSp1 has 97% amino acid identity with hSp8 zinc fingers and also has homology to hSp8 in the region N-terminal to the zinc fingers. For CG5669 and Btd it is hard to assign a homolog since they have significant identity with a number of Class I proteins. Sequence identities of CG5669 with the zinc fingers of Class I range from 76 to 87%, for Btd the range is 65-74%. CG12029, Luna and CG9895 have closer amino acid identity to the Class II human proteins. Luna is most closely related to  KLF6 and KLF7 and shows conservation of a putative activation domain at the N-terminus (23). Cabot (also known as CG4427) and Bteb2 seem to belong to the class III subclass showing 72-85% identity with human members of this class. Hkb and CG3065 are harder to place. CG3065 has 62-65% identity to members of the Sp1 family and 62-64% identity with KLF9 and KLF14 members of class III. Hkb is the most diverged member of this class of proteins. The highest degree of homology for Hkb is 53% identity with hSp5 and it is difficult to say whether it should be placed in this family at all. We note that Hkb binds only weakly to sequences that match the Sp1/KLF consensus ( Figure 5).
Each of the zinc finger regions of these 10 proteins were cloned in frame into an in vitro transcription/translation vector and expressed in vitro. The products were tested for binding to Site 2. Nine of the 10 Drosophila Sp1/KLF family member zinc fingers show binding to Site 2 and only CG3065 shows no binding ( Figure 5B). Binding to each of these factors is specific based on competition experiments (Figure 3 and data not shown). No binding was seen with product from the vector alone (data not shown). Binding of Luna, Cabot and Hkb to Site 2 is very weak. This result is not due to the production of inactive protein since, with the exception of Hkb, we see strong binding of these factors to other binding site sequences (see below Figure 5).
In order to get a better idea of the binding specificity of the Drosophila Sp1/KLF family members, we tested their binding to six additional oligonucleotides ( Figure 5). BTE, Sp1 and AP2 are sequences that have previously been used to test binding specificity of human KLF4 (20). Note that the oligonucleotide named Sp1 contains two matches to the consensus sequence. PRE D 1 and PRE D 2 are sequences within an Ubx PRE, PRE D (24) that most closely matched the Sp1/KLF binding site. Oligo A (16) is a sequence from the engrailed 181 PRE with only one mismatch with the Sp1/Klf consensus. Not surprisingly, like the mammalian Sp1/KLF family members, our data indicate that the Drosophila Sp1/KLF proteins bind with different affinities to the different sites. Both sequences within the consensus and bases that flank the consensus were important for binding. For example although Site 2 and PRE D 1 perfectly match each other in the Sp1/KLF core consensus sequence, the relative binding of the Sp1/KLF proteins differ between the two sites. Bteb2 binds strongly to Site 2 but weakly to PRE D 1 and conversely, Cabot binds strongly to PRE D 1 but weakly to Site 2. This result shows the influence of flanking sequences on DNA binding affinity. The exact base composition of the core consensus sequence is also important. The BTE and PRE D 1 core differ in only one base, a G versus an A in the second position and sequences flanking the consensus are identical for four bases on each side. Despite this, CG3065 binds more strongly to the BTE sequence than to PRE D 1.
We next tested sequences with mismatches within the consensus to get a better idea of the binding specificity of the Drosophila family members. PRE D 2, has one mismatch to the consensus, it has a C rather than a G or A in position 2. PRE D 2 did not bind any of the Drosophila Sp1/KLF family members. An AP2 binding site, that contained a C instead of a G or A at the first position did not bind any of the Sp1/KLF zinc fingers with high affinity. The importance of the first base is also seen with the Mutsite2 oligonucleotide ( Figure 3). Mutsite2 contains the sequence CGGGCGT, a mismatch in the first position of the consensus, and does not compete for binding with the Site 2 oligonucleotide. Oligo A, which differs from the consensus sequence at position 5 having an A instead of a C or T, did not bind any of the 10 Sp1/KLF zinc fingers. This data set tells us that the Sp1/KLF consensus derived by Shields and Yang (20) is largely predictive of which sites will be bound by the Drosophila factors. Shields and Yang (20) report a C/T for the seventh base of the KLF consensus binding site. However, their experiments showed that an A in the seventh position was also able to compete for binding of the KLF4 factor although not as well as a C or T in this position. This suggests that A can also be added to the seventh position. In addition, binding sites for the mammalian Sp1 class of factors would predict that a G in the last position of the consensus sequence is also a valid binding site (25). Note that the Sp1 oligonucleotide we used contained both the sequences GGGGCGG and GGGGCGA. This oligonucleotide was bound quite well by several factors. Based on our analysis here, the data of Shields and Yang (20), and what is known of the Sp1 binding site we propose that any base may function in the seventh position of the consensus making it (G/A)(G/A) GG(T/C)G.

Sp1/KLF binding sites are present in many PREs and PSEs
We have found sequences that are bound by the Sp1/KLF family of proteins in both the engrailed 181 bp PRE and in PRE D of Ubx. Are Sp1/KLF binding sites a general feature of PREs as has been found for Pho/Phol, Dsp1, GAF/Psq and Zeste binding sites? To address this question we have searched the molecularly well-defined PRE (fragments under 1.5 kb) and PSEs sequences for the presence of the Sp1/KLF consensus sequence using the Gene Palatte sequence analysis program (see Materials and Methods). We searched using the consensus sequence described above [(G/A) (G/A) GG (T/C) G]. We note that this consensus is based on a small data sample and may not detect all potential Sp1/KLF binding sites. For example, some of the mammalian family members bind to a GT box [GGTGTGGGG, (26)] a sequence that is not contained in the Sp1/KLF consensus we used. We also included in our search the distribution of potential Pho/Phol, GAF/Psq, Zeste and Dsp1 sites in these PREs/PSEs. Of the fragments we examined, the 181 engrailed PRE, iab-7 PRE and the iab-8 PRE have all been shown to act as both PREs and to mediate pairing-sensitive silencing (PSS). PRE D has not been tested for PSS, but multimerized copies of subfragments of this PRE have been shown to mediate PSS (27). A 450 bp engrailed fragment located from À1944 to À1503 upstream of the engrailed transcription start site and fragments of DNA from escargot, even-skipped and proboscipedia mediate PSS. The minimal MCP element, a 142 bp fragment from the iab-5 regulatory region of the Abdominal-B gene, acts as a PRE in vectors designed to test for PRE activity but does not mediate PSS (28,29).
The distributions of the various binding sites in the PRE/PSEs are summarized in Table 1. Of the PREs and PSEs we examined, all except the minimal MCP element have potential Sp1/KLF binding sites and about one-half of them contain more than one potential Sp1/KLF consensus binding site. In addition all but the minimal MCP element contain potential Pho/Phol, GAF/Psq, Zeste and Dsp1 sites, often in multiple copies (see Table 1). The minimal MCP1 element seems to be atypical by not having Zeste or Sp1/KLF consensus binding sites. This MCP element was tested for PRE activity in a vector that, like the vector we used to test the PRE activity of the engrailed 181 bp PRE, contains Ubx sequences containing many potential Sp1/KLF binding sites as well as binding sites for other PRE-binding factors. When we searched the larger 823 bp MCP sequence that has been shown to have insulator and pairing activity, we detect a potential Sp1/KLF binding site and a match to the Zeste consensus binding site. It is interesting to note that the minimal MCP element lacks Zeste sites and does not mediate PSS. Zeste has long been known to be involved in transvection, mediating trans-interactions between chromosomes and it has been speculated that it may play a role in mediating interactions between the PRE and the promoter in PcG silencing (30).
The presence of potential Sp1/KLF sites in so many different PREs and PSEs suggests that Sp1/KLF binding sites may play a general role in PRE function. We note that the Sp1/KLF consensus sequence was not one of the sequence motifs found to be statistically enriched in PREs over random sequence by Ringrose et al. (12). However, this may be because it is so short and degenerate (RRGGYG). The Zeste binding motif is also very short and degenerate (YGAGYG) and was also not statistically enriched in PREs over random sequence (Ringrose et al. (12). Clearly the function of Sp1/KLF binding sites in other PREs will need to be tested, preferably in vectors that do not contain nearby Sp1/KLF consensus sequences.

Function of the Drosophila Sp1/KLF family members
The Sp1/KLF family of proteins in mammals is very complex. These proteins can be activators or repressors; some can do both depending on cellular and binding site context. Some proteins function ubiquitously; whilst others function only in specific cells, and some do both at different stages of development. For example, Sp1 and Sp3 proteins function redundantly early in embryogenesis although they have different functions later in development. There is also competition for binding sites. Post transcriptional modifications can also affect the function of these proteins [for reviews see (21,22)].
Of the 10 Drosophila Sp1/KLF members, four are well characterized genetically (btd, dSp1, hkb and cabot). Since these four genes are involved in the development of particular structures in the fly and are not ubiquitously expressed, we believe it unlikely that they play general roles in PcG repression. buttonhead (btd) is important for the development of head structures and mechano-sensory organs (31). dSp1 is located 50 kb from btd and may act with btd in mechanosensory organ development (32). hkb mutations have been studied extensively and are found to affect the specification of endoderm (33), salivary gland development (34), germ cell migration (35), as well as many other developmental processes. Overexpression of cabot affects sensory organ development (36). The function of the luna gene during embryogenesis has been studied using RNAi (23), and the phenotypes obtained do not mimic PcG phenotypes. However, analysis of luna mutants will be necessary to assess its role in PcG function.
We obtained a mutant allele of the Bteb2 gene from the Drosophila genome project. These flies contain an insertion of a piggyback transposon into the coding region of the Bteb2 gene and should produce a null mutation in this gene. These flies are homozygous viable and fertile and have no phenotypic defects. Thus, if the Bteb2 gene plays a role in PcG repression, its role is likely redundant with that of another member of the family. Mutations in the other Sp1/KLF family members have not yet been isolated.

DISCUSSION
Here we describe another binding site important for function of the engrailed 181 bp PRE in Drosophila and present evidence that Sp1/KLF family members can bind to that site. Consensus binding sites for this family of proteins have been found in most of the well characterized PREs implying that this binding site may play a general role in PRE activity.
Identifying which Sp1/KLF factor acts through Site 2 is not a easy task. Not only are a number of members of this class genetically uncharacterized there is also the possibility that there may be functional redundancy as is seen with Pho and Phol. The existence of a viable and fertile Bteb2 mutant suggests that functional redundancy will be observed with the Sp1/KLF family in Drosophila. Experiments using family member-specific antibodies in chromatinimmunoprecipitation experiments on PREs will help elucidate which Sp1/KLF family members play a role in PRE function in Drosophila.
What role the Sp1/KLF family of proteins play in recruiting the PcG complexes to the PRE remains to be elucidated. In fact, for most of the other proteins required for PRE function, their roles are not yet clear. Both GAF and Psq bind the sequence GAGAG (37,38), a sequence shown to be important for PRE function (28,30). Psq has been shown to be in a complex with PcG proteins isolated from the Drosophila cell line SL2 (39,40) and psq mutations enhance the mutant phenotypes of the PcG genes polyhomeotic (Ph) and Polycomb (Pc) in larval and adult tissues (39,41). This suggests that Psq may be important for PRE function. GAF [for review see ref (42)] has also been reported to co-purify with some PcG proteins (27) and has been shown by chromatin-immunoprecipitation experiments to be present at PREs (10). GAF is a member of the TrxG of genes but may also play a role in PcG repression (27,30,43). The DNA binding protein Pho has been shown to bind in vitro to a chromatinized PRE template only if GAF is present (44). GAF and Psq can interact through their BTB proteinprotein interaction domains and it has been proposed that they may function together in vivo (45).
Zeste has been shown to be important for both PRE and TRE activity. Zeste is a stoichiometric component of the biochemically purified PcG complex, PRC1 (9) suggesting a role in PcG repression and Hur et al. (30) report that Zeste is required for the PcG-mediated repression of an Ubx transgene. In contrast, experiments with the iab-7 PRE have shown that Zeste binding sites are important for the ability of this DNA to act as a TRE, not as a PRE (46).
Pho and Phol (47,48) have recently been shown to be required to recruit an Esc-E(z) complex to a PRE (49). In vitro, Pho interacts directly with E(z) and Esc whereas Phol interacts with Esc. Recruitment of the Esc-E(z) complex leads to methylation of lysine 27 of histone H3 by the SET domain of E(z). The methylated K27 recruits a Pc-containing complex through interaction of the chromo-domain of Pc with the methylated histone tails. Pho has also been shown to interact with Pc in vitro (50). Pho/Phol double mutants have a very strong PcG phenotype, much stronger than mutations in the genes encoding the other PRE-binding factors suggesting that Pho/Phol play a central role in PRE function. It has been proposed that Dsp1 facilitates the binding of Pho/Phol to the PRE (13).
The role that the Sp1/KLF family may play remains to be elucidated but it is intriguing to note that mammalian Sp1 has been reported to interact directly with YY1 (the mammalian homolog of Pho). This interaction requires the first one and a half zinc fingers of YY1, a region that is 96% identical between the Drosophila and mammalian proteins. The 158 amino acid C-terminal region of Sp1 (includes the three zinc fingers and one of the activation domains, domain D), can mediate the interaction leading to an increase in the level of correctly initiated transcripts (51,52). These data raise the possibility that Pho or Phol may interact with Sp1/KLF proteins at PREs.