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Nucleic Acids Research, 2003, Vol. 31, No. 9 2344-2352
© 2003 Oxford University Press

Multiple, dispersed human U6 small nuclear RNA genes with varied transcriptional efficiencies

Angela M. Domitrovich and Gary R. Kunkel

Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128, USA

*To whom correspondence should be addressed. Tel: +1 979 845 6257; Fax: +1 979 845 9274; Email: g-kunkel{at}tamu.edu

Received January 17, 2003; Revised and Accepted March 2, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Vertebrate U6 small nuclear RNA (snRNA) gene promoters are among the founding members of those recognized by RNA polymerase III in which all control elements for initiation are located in the 5'-flanking region. Previously, one human U6 gene (U6-1) has been studied extensively. We have identified a total of nine full-length U6 loci in the human genome. Unlike human U1 and U2 snRNA genes, most of the full-length U6 loci are dispersed throughout the genome. Of the nine full-length U6 loci, five are potentially active genes (U6-1, U6-2, U6-7, U6-8 and U6-9) since they are bound by TATA-binding protein and enriched in acetylated histone H4 in cultured human 293 cells. These five all contain OCT, SPH, PSE and TATA elements, although the sequences of these elements are variable. Furthermore, these five genes are transcribed to different extents in vitro or after transient transfection of human 293 cells. Of the nine full-length U6 loci, only U6-7 and U6-8 are closely linked and contain highly conserved 5'-flanking regions. However, due to a modest sequence difference in the proximal sequence elements for U6-7 and U6-8, these genes are transcribed at very different levels in transfected cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Previous investigation of vertebrate small nuclear RNA (snRNA) gene promoters has yielded important information regarding mechanisms of eukaryotic transcription (1). Whereas most of the U-type snRNA genes are transcribed by RNA polymerase II (pol II), the U6 gene is transcribed by RNA polymerase III (pol III). Most elements for both types of vertebrate promoters are shared, with the paradoxical exception that U6 genes contain a TATA-like element which directs pol III specificity (2,3). The shared elements include an snRNA promoter-specific proximal sequence element (PSE) at approximately –60, bound by the multisubunit SNAPc protein, and an octamer element (OCT) in the distal, or enhancer-like region located ~220 bp upstream of the start site. The distal region usually contains at least one other element adjacent to OCT, and in many cases this is an SPH element that is bound by the zinc finger transcription factor, SBF/Staf (4,5). The distal promoter region stimulates transcription up to 100-fold, and part of the mechanism for this activation is through a protein–protein interaction involving the POU domain of Oct-1 and the SNAP190 subunit of SNAPc (6–10). Furthermore, for the previously characterized human U6 gene (hereafter referred to as U6-1), this protein–protein contact is facilitated by a positioned nucleosome between the distal region and the PSE (11,12).

Vertebrate U6 and 7SK genes were the first examples of what is now known to be a distinct subclass of pol III promoters that do not contain intragenic control regions, but instead have control elements located in their 5'-flanking regions (type 3 genes) (13–17). All such type 3 promoters contain PSE and TATA-like elements at conserved locations upstream of the start site. Much current interest is focused on this promoter class because they can be employed to produce relatively high levels of synthetic RNAs in cultured cells, such as the small interfering RNAs used to induce RNAi-mediated knock-down of specific mRNA targets (18–22).

The genomic organization of human U1 and U2 loci is well known. Approximately 10–30 copies of true genes encoding U1 and U2 snRNAs are clustered on chromosomes 1 and 17, respectively, with widespread conservation of flanking sequences (23,24). However, whereas the promoter structure for the human U6-1 gene is well known and many details of the transcription initiation complex on this U6 promoter are emerging (17,25,26), the existence of other full-length human U6 genes has not been described. Early work estimated a total of approximately 200 human U6 genes plus pseudogenes (27), and it became apparent that the large majority of this number are U6 pseudogenes containing various nucleotide substitutions and truncations. In the present work, we have used the human genome database to identify several more active U6 genes and have compared promoter elements and relative transcriptional activities of these genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Identification of potential human U6 genes
A BLAT search [BLAST-like Alignment Tool; UC-Santa Cruz genome server (28)] from the UC-Santa Cruz genome bioinformatics site (http://genome.ucsc.edu; last search from the June 2002 freeze) was performed using the 107 nt that correspond to the full-length human U6 snRNA as the input sequence. The search resulted in seven candidates that scored 107 out of 107, and two matches of 106 out of 107. These nine genes were named randomly except that the originally isolated gene is referred to as U6-1. The two matches that scored 106 out of 107, U6-7 and U6-8, contain only four T residues at the 3' termination region instead of five that were included in the query sequence.

Subcloning of variant U6 genes
High Fidelity thermostable DNA polymerase (Clontech) was used to synthesize PCR products from variant U6 genes using human placental DNA and the following primers that typically spanned an ~500 bp region of genomic sequence for each variant: CHU62+208, 5'-GGAGATGGACACGGGAAGACTATGACG-3'; HU62-300, 5'-GGAGGCCTCACCCCCTAC CTCGGCCGC-3'; CHU64+198, 5'-GAGAATCATGTAGACCAAGTTTTAGG-5'; HU64-300, 5'-AAATTGAGTCA TCTGACAGAAATTATC-3'; CHU67+200, 5'-AGCTCGC GAGCAGGAAAATGCAGGCCC-3'; HU67-300, 5'-AAG TCCGCGGCACGAGAAATCAAAGCC-3'; CHU68+200, 5'-GTTTCGTCATTGGGTGCCACACGTTTT-3'; HU68-300, 5'-AGCTCCGCGGCACGAGAACTCAAAGCC-3'; CU69+434, 5'-CTTGGACATCAGAAAATGGAAGAA-3'; U69-320, 5'-TTCCTCGGTAACAACCAGTCGCCT-3'. The CU6-9+434 primer was situated further downstream than the other candidates in order to avoid a region of predicted repetitive DNA. PCR products were ligated into the pGEM-T vector (Promega) and carried in Escherichia coli XL1-Blue cells (Stratagene). The sequences of all variant U6 promoter templates were verified by dideoxy sequencing. A few nucleotides differed from the sequence reported in the human genome database, but none of these changes occurred in potential promoter elements or the U6 ‘coding’ sequence.

Construction of variant maxigenes
Using plasmid DNA from the above variants, PCR mutagenesis was performed using PfuTurbo polymerase (Stratagene) and the QuickChange protocol as suggested by the manufacturer. Primers that spanned the +72 to +101 region of the U6 transcribed sequence were used to insert 9 bp containing an XhoI site in this region (U6MAXITOP, 5'-ATGACACG CAAATTCCCTCGAGGCGTGAAGCGTTCCATA-3'; and U6MAXIBOT, 5'-TATGGAACGCTTCACGCCTCGAGGG AATTTGCGTGTCAT-3'). This is the same insertion introduced into the U6-1 maxigene in earlier studies (29,30).

Construction of plasmids containing the U6-7 and U6-8 proximal region switch
Using U6-7 and U6-8 maxigene plasmid DNAs and the QuickChange protocol, a HindIII site was inserted at position –70 immediately upstream of the PSE in order to facilitate swapping of proximal and distal promoter regions. Following XhoI–HindIII digestion, DNA fragments were purified from agarose gels, ligated, and the resulting plasmids used to transform E.coli XL1-Blue cells. The resultant plasmids are referred to as 7/8/7 and 8/7/8. This nomenclature means that the plasmid 7/8/7 contains the distal sequence of U6-7, the proximal sequence of U6-8 and the downstream sequence of U6-7. PCR mutagenesis using PfuTurbo polymerase was used to convert the promoter of the U6-7 maxigene to one containing the U6-8 PSE. Mutagenic primers spanned the promoter from –79 to –32 and contained the following sequences: U68PSETOP, 5'-AGCGCTCGATTCGGTCAC CGTAAGTAGAATAGGTGAGAAACTCCCGTG-3'; and U68PSEBOT, 5'-CACGGGAGTTTCTCACCTATTCTACT TACGGTGACCGAATCGAGCGCT-3'. This resulting plasmid is referred to as U6-7-8PSE. Another plasmid in which two nucleotides were added to the U6-7 maxigene promoter was synthesized using PCR mutagenesis with primers from –30 to +2 (U67PLUS2TOP, 5'-TATAAGACCTGGGGA TCCGGACTTATTTGCGT-3'; and U67PLUS2BOT, 5'-AC GCAAATAAGTCCGGATCCCCAGGTCTTATA-3'). The additional ‘CT’ nucleotides were inserted at position –13. This plasmid DNA is called U6-7plus2.

Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) using cultured human 293 cells was carried out as described previously (31). Polyclonal anti-TATA-binding protein (TBP) antibodies were prepared in rabbits using His6-tagged human TBP expressed in E.coli. IgG was purified from the serum using UltraLink protein A resin (Pierce). Polyclonal IgG against acetylated histone H4 (cat. no. 06-598) and normal rabbit IgG were purchased from Upstate Biotechnology. Antiserum against human SNAP43 was provided by N. Hernandez [Cold Spring Harbor Laboratories; CS49, anti-CSH375 peptide (32)]. After immunoprecipitation, cross-link reversal and DNA purification, gene-specific DNA sequences were detected using PCR and electrophoresis on 15% polyacrylamide gels. The following primers were used: U61-117, 5'-CTTGGGTAGTTTGCAGTTTTAAAA-3'; CU61-1, 5'-GGT GTTTCGTCCTTTCCACAAGAT-3'; U62-120, 5'-TGTT CTGCAACATACCACTGTAGG-3'; CU62-1, 5'-GACTCA AGGGTTACTGTCACACCT-3'; U63-121, 5'-CAACCT TTTATCATTAAGTATGCTG-3'; CU63-1, 5'-CCAACAT CCATTCTTGATAAAACC-3'; U64-120, 5'-TTCCTGC GATTCAAACTTAACTGG-3'; CU64-1, 5'-AAATGTAA CCTATTTTTAAATATTAGCACA-3'; U65-120, 5'-ATA CCTGGCATATGTTAGGCACTC-3'; CU65-1, 5'-TTGTCA AGACTTCTTTGCGTTTGAG-3'; U66-120, 5'-GTATACA GATCACTCAAAGGAAAC-3'; CU66-1, 5'-CCCATGGTT TAACATTTTTAAAGAAC-3'; U67-120, 5'-GTTACATGA AATTCTCCTAAAGGC-3'; CU67-1, 5'-GCAAATAAGTC CGTCCCCAGGTCT-3'; U68-120, 5'-TGCATGAAATTC TCCCAAAGGCTC-3'; CU68-1, 5'-GTTGAGAAGAAGTC ACCCACAGGC-3'; U69-131, 5'-CGTTAGTTGCATTACA CATTGGGC-3'; and CU69-1, 5'-GAATATTGAGTTCCAC CACCAGCT-3'.

Transfections and primer extensions
Human 293 cells were transfected with the appropriate U6 maxigene reporter plasmid using the calcium phosphate procedure, and total RNA was isolated and analyzed by primer extension as described previously (30). Products were separated by electrophoresis on 10% polyacrylamide gels containing 8.3 M urea. Relative band intensities were quantitated using a STORM PhosphorImager (Molecular Dynamics).

In vitro transcription in HeLa S100 extracts
S100 extracts were prepared from HeLa S3 cell pellets (purchased from National Cell Culture Center) as described previously (33). Transcription reactions were carried out using 500 ng of each plasmid DNA as described previously (34). RNAs were separated by electrophoresis on 10% polyacrylamide–8.3 M urea gels, visualized by autoradiography or quantitated using a STORM PhosphorImager (Molecular Dynamics).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Identification of multiple full-length human U6 snRNA genes
By using a BLAT search, with the 107 nt human U6 snRNA sequence as the input, we identified nine loci containing the same ‘coding’ region as the known human U6 snRNA gene (U6-1) (Table 1). Since some of the human genome sequence is still only of ‘draft’ quality, it is possible that this list is not yet complete. Except for U6-7 and U6-8, these loci are dispersed in the genome. U6-7 and U6-8 are separated by only 1014 bp on chromosome 14, and are encoded on opposite strands in convergent orientation. U6-2 and U6-9 are the only other loci located on the same chromosome, but are separated by 128 kb. Five of the nine (U6-1, U6-2, U6-7, U6-8 and U6-9) contain the upstream promoter elements, SPH, OCT, PSE and TATA, which were identified previously in the U6-1 promoter (Fig. 1; flanking sequences for all nine U6 loci in the Supplementary Material). However, we note that the upstream region of U6-9 contains a poor match to the consensus PSE, especially within the highly conserved ACCGT sequence near the 5' end of this element (Fig. 1). The locations and spacing between these promoter elements are very similar. However, the sequences between the promoter elements do not contain any similarity to the U6-1 flanking sequences. In fact, among all of these loci, only the U6-7 and U6-8 5'-flanking regions contain identifiable sequence similarity outside of the promoter elements (see Supplementary Material). The OCT and SPH elements are switched in the U6-2 and U6-9 gene promoters compared with U6-1 (Fig. 1). The other variants, U6-3, U6-4, U6-5 and U6-6, did not appear to contain any upstream promoter elements, but did contain the same full-length U6-1 ‘coding’ region.


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  		Table 1. Chromosomal locations of full-length human U6 gene loci
 


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  		Figure 1. Sequences of the promoter elements for the variant U6 promoters. (A) Distal regions containing the SPH and OCT sequences for the U6-1, U6-2, U6-7, U6-8 and U6-9 promoters. The letters in upper case indicate matches to the consensus sequence delineated at the top. The SPH consensus is from Schaub et al. (51). Each dash mark between the SPH and OCT elements represents one nucleotide. Note that the relative positions for the SPH and OCT elements are switched in the U6-2 and U6-9 promoters. (B) Proximal promoter regions containing the PSE and TATA sequences for the U6-1, U6-2, U6-7, U6-8 and U6-9 promoters. The letters in upper case indicate matches to the vertebrate PSE consensus sequence (52). Each dash mark between the PSE and TATA elements represents one nucleotide.

 
TBP and acetylated histone H4 bind to a subset of the variant U6 genes
To investigate the transcriptional potential of the variant U6 promoters, the presence of TBP or acetylated histone H4 (AcH4) was determined using ChIP. A wealth of data has demonstrated that TBP is essential for transcription initiation, including TATA-less and TATA-containing promoters recognized by pol III (35). Furthermore, the presence of hyperacetylated histones is a strong indicator of an actively transcribed gene (36,37). Sheared chromatin from formaldehyde-treated human 293 cells was immunoselected with anti-TBP, anti-AcH4 or normal IgG antibodies. Only a subset of the nine full-length human U6 gene loci (U6-1, U6-2, U6-7, U6-8 and U6-9) was detected at significantly higher levels by PCR in the DNA from anti-TBP and anti-AcH4 immunoselected chromatin compared with that selected by the normal IgG antibodies using primers spanning the region from –120 to –1 of the promoters (Fig. 2). In contrast, the PCR signals from anti-TBP, anti-AcH4 and normal IgG antibodies were the same background level for the U6-3, U6-4, U6-5 and U6-6 loci (Fig. 2). These last four variants are the ones lacking known external promoter elements. It is possible that the absence of a PCR product could reflect especially poor cross-linking efficiency at that promoter. However, the exact correlation of TBP and AcH4 signals at the various U6 loci is striking.



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  		Figure 2. TATA-binding protein and acetylated histone H4 interact with U6-1, U6-2, U6-7, U6-8 and U6-9 promoters but not with the other potential full-length U6 genes. (A) Sheared chromatin from human 293 cells that had been cross-linked with formaldehyde was immunoselected with anti-TBP antibodies or normal IgG antibodies, and DNA was purified following reversal of cross-links. The relative amounts of the U6 promoter sequences in the antibody-selected DNA samples were determined by electrophoresis of radiolabeled PCR products on a 15% gel. As an input control, ‘5% total’ represents 5% of the unselected chromatin DNA compared with the amount of the antibody-selected chromatin DNA assayed by PCR. (B) Sheared chromatin from human 293 cells that had been cross-linked with formaldehyde was immunoselected with anti-AcH4 antibodies or normal IgG antibodies, and DNA was purified following reversal of cross-links. The relative amounts of the U6 promoter sequences in the antibody-selected DNA samples were determined by electrophoresis of radiolabeled PCR products on a 15% gel. As an input control, ‘2.5% total’ represents 2.5% of the unselected chromatin DNA compared with the amount of the antibody-selected chromatin DNA assayed by PCR.

 
Variable transcriptional activity of the U6 genes
Potentially active U6 genes (U6-2, U6-7, U6-8 and U6-9) as well as the U6-4 locus (as a negative control) were amplified by PCR from genomic DNA using High Fidelity thermostable DNA polymerase (Clontech) and inserted into plasmid vectors. In order to determine if these variants of the U6 snRNA gene are transcriptionally active, both transient transfection and in vitro transcription assays were performed. Since human cells have high levels of endogenous U6 snRNA, U6 maxigenes were constructed that contain a 9 bp insert near the 3' end of the transcribed region of the various U6 genes. The same maxigene sequence was used previously and resulted in a stable and readily detectable RNA product from the U6-1 gene (29,30). Human 293 cells were transfected with the various U6 maxigene plasmids along with a plasmid encoding the chicken ß-tubulin gene in order to normalize for variable transfection efficiency. Whereas the U6-2 gene was transcribed at approximately the same level as U6-1 (Fig. 3A, lanes 1 and 2), the levels of U6-8 and U6-9 were reduced somewhat (Fig, 3A, lanes 1, 4 and 5). Interestingly, U6-7, which has a very similar 5'-flanking region to that of U6-8, has a much lower transcriptional activity level compared with U6-8 and U6-1 (Fig. 3A, lanes 1, 3 and 4). U6-4 produced no detectable transcripts, which correlates with U6-4 containing no identifiable promoter elements (Fig. 3A, lane 7). We did not investigate transcription of the U6-3, U6-5 and U6-6 variant genes since they are similar to U6-4 in that they lack any identifiable promoter elements and they are not bound by TBP or AcH4 in cells (Fig. 2).




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  		Figure 3. U6 genes with recognized promoter elements have transcriptional activity in transfected cells and in vitro. (A) Human 293 cells were transfected by the calcium phosphate technique with plasmid DNAs containing maxigenes of U6-1 or variant U6 promoters. Expression was detected by primer extension with a primer spanning only the maxigene insertion. ‘U6’ represents transcription from the U6 promoter, and ‘cß3’ represents transcription from a co-transfected chicken ß-tubulin plasmid used as a control to normalize for variable transfection efficiency. (B) The relative band intensities for (A) were quantitated by phosphorimaging. After background subtraction, the U6/cß3 ratio was calculated and normalized to the value from the U6-1 promoter which was set to ‘1’. The height of each bar represents the mean of at least four different transfections, and the error bars show the standard deviation from the mean. (C) A 100 µg aliquot of HeLa S100 extract was incubated with 500 ng of the pGEM, U6-1 or the variant U6 promoter plasmids. ‘U6’ represents transcription from the U6 promoter. The ‘control’ is tRNAHis, which results from guanylyltransferase radiolabeling of endogenous RNA in the extract and serves as a recovery control for each sample. (D) The relative band intensities for (C) were quantitated by phosphorimaging. After background subtraction, the U6/control ratio was calculated and normalized to the value from the U6-1 promoter which was set to ‘1’. The height of the bar represents the mean of at least three different transcription assays, and the error bars show the standard deviation from the mean.

 
In vitro transcription of the various full-length U6 genes was performed as another comparison of relative promoter efficiencies. In previous work using chromatin-free templates, the distal region of the U6-1 promoter was silent in direct transcription assays when it is present in its normal location (29,33). Therefore, the proximal promoter (PSE + TATA) will have the greatest effect on transcription in vitro. HeLa S100 extracts along with plasmid DNAs that contain U6-1 or the variant U6 gene plasmids were used in transcription reactions. The transcriptional levels of U6-1, U6-7, U6-8 and U6-9 were similar (Fig. 3C, lanes 2, 5, 6 and 7, and D). In contrast, the level for U6-2 was significantly less than that of U6-1 (Fig. 3C, lanes 2 and 3, and D). Also, the level of transcriptional activity was negligible for U6-4 (Fig 3C, lane 4, and D).

Different PSE elements govern the differential expression of U6-7 and U6-8 genes in transient transfection experiments
Since there is such a dramatic difference in the transcriptional levels of U6-7 and U6-8 in transfection assays, further experiments were performed to explore the reason for this. The 5'-flanking sequences of U6-7 and U6-8 are the most similar among the variant U6 genes. Indeed, the distal regions and TATA elements are identical. To facilitate promoter swapping experiments, a HindIII site was inserted at position –70 just upstream of both PSE regions of the U6-7 and U6-8 maxigene promoters by site-directed mutagenesis. Using this restriction site and the XhoI site that was already present in the maxigenes, we replaced the U6-7 proximal region (from –70 to +87) with the U6-8 proximal region (7/8/7 in Fig. 4A) and vice versa (8/7/8 in Fig. 4A). After exchanging the proximal regions, transcription from the 7/8/7 promoter was increased to a level comparable with that from the normal U6-8 promoter (Fig. 4B, lanes 2 and 3). Transcription from the 8/7/8 promoter was dramatically reduced to a level corresponding to that of the normal U6-7 promoter (Fig. 4B, lanes 1 and 4). Therefore, the relative transcriptional efficiencies of U6-7 and U6-8 are controlled by differences in the proximal promoters (–70 to –1). Six nucleotides are different between the U6-7 and U6-8 PSEs (Fig. 1B). The TATA box sequence is exactly the same for both genes. However, two extra nucleotides are located between the TATA box and transcriptional start site of the more active U6-8 promoter (Fig. 1B). Therefore, we investigated whether either of these alterations was responsible for the different transcriptional efficiencies found in transfected cells. PCR site-directed mutagenesis was used to convert the U6-7 PSE into the U6-8 PSE (U6-7-8PSE in Fig. 4A). The level of transcription was increased to the same as that for the wild-type U6-8 promoter (Fig. 4C, lanes 2 and 3). On the contrary, when two additional nucleotides were inserted at position –13 of the U6-7 promoter (U6-7plus2 in Fig. 4A), the transcriptional level was not increased, but decreased to a barely detectable signal (Fig. 4C, lane 4). We conclude that the marked difference in transcription between the U6-7 and U6-8 promoters is a result of variation in the PSE.




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  		Figure 4. Different PSE elements are responsible for the differential expression of the U6-7 and U6-8 genes. (A) Proximal promoter structures of the various U6-7 and U6-8 plasmids used in these experiments. The PSE for the U6-7 promoter is a striped box, while the PSE for the U6-8 promoter is solid. The HindIII and XhoI arrows show where a HindIII site or XhoI site was inserted by site-directed mutagenesis. The ‘+1’ represents the transcriptional start site. A thin line represents the U6-7 gene, while a thick black line represents the U6-8 gene. The ‘XX’ in the U6-7plus2 promoter represents the addition of two nucleotides in that region. (B) Effect of swapping U6-7 and U6-8 proximal regions. Human 293 cells were transfected by the calcium phosphate method with plasmid DNAs containing the U6-7 hind (lane 1), U6-8 hind (lane 2), 7/8/7 (lane 3) and 8/7/8 (lane 4) promoters. Expression was detected by primer extension with a primer spanning only the maxigene insertion. ‘U6’ represents transcription from the variant U6 promoters, and ‘cß3’ represents transcription from a co-transfected chicken ß-tubulin plasmid used as a control to normalize for variable transfection efficiency. (C) Transfection experiments were carried out as described in (B), except that the plasmid DNAs contained either the U6-7 and U6-8 promoter without the HindIII site or the U6-7-8PSE or U6-7plus2 promoters.

 
SNAPc binds to the U6-9 promoter
As mentioned earlier, the U6-9 promoter contains a poor match to the PSE consensus. Especially noticeable is the lack of the ACCGT sequence present in the PSE consensus and the U6-1, U6-7 and U6-8 genes, as well as the absence of a long polypurine stretch near the 3' end of the PSE. Nevertheless, U6-9 is transcribed at high levels in transfection and in vitro transcription assays (Fig. 3). Therefore, we wanted to investigate the interaction of the human PSE-binding complex (SNAPc) to the U6-9 promoter. ChIP experiments were performed where the chromatin was immunoselected with anti-SNAP43 antiserum (one of the SNAPc proteins). It was shown previously that antibodies against any of the SNAPc subunits could be used to detect binding to the U6-1 promoter by this assay, and use of the anti-SNAP43 antibody appeared optimal (12). The U6-9 promoter sequence was readily detected by PCR in the DNA from anti-SNAP43 immunoselected chromatin compared with the background level seen with DNA selected by the normal IgG antibody (Fig. 5). A similar level of binding was found at the U6-1 promoter. As a negative control, the U6-4 promoter was not immunoselected with the anti-SNAP43 antiserum since the PCR signals from this antibody were the same as for the normal IgG (Fig. 5). The U6-4 promoter was not expected to bind SNAP43 since it does not have a PSE sequence. These results do not exclude the possibility that an incomplete SNAPc complex binds at the U6-9 promoter. Furthermore, a quantitative comparison of SNAPc occupancies at the U6-1 and U6-9 promoters is not possible with these experiments, since formaldehyde cross-linking efficiencies may vary at the different promoters. However, these results indicate that SNAPc binds to the U6-9 promoter within cells, even in the presence of what looks to be a poor cognate PSE site.



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  		Figure 5. SNAPc binds to the U6-9 promoter. Sheared chromatin from human 293 cells that had been cross-linked with formaldehyde was immunoselected with anti-SNAP43 antibodies or normal IgG antibodies. The relative amounts of the U6-4, U6-9 and U6-1 promoter sequences in the antibody-selected DNA samples were determined by electrophoresis of radiolabeled PCR products on a 15% gel. As an input control, ‘5% total’ represents 5% of the unselected chromatin DNA compared with the amount of the antibody-selected chromatin DNA assayed by PCR.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY MATERIAL
 REFERENCES
 
We characterized the transcriptional properties of six out of a total of nine full-length U6 snRNA loci in the human genome. Five human U6 genes that contain identifiable promoter elements found in the previously characterized U6-1 gene are potentially active. We believe that the four other full-length U6 loci are inactive in human 293 cells, since they are not bound by TBP or enriched in AcH4 in ChIP assays. Indeed, we verified that one of the latter, U6-4, was not transcribed in transient transfection or in vitro transcription assays.

It is possible that the human U6 gene set that we describe is not yet complete since the deposited human genome sequence is not fully refined. For example, when we began this project, the U6-9 gene was not recovered in our first BLAT search. Furthermore, this work has focused on full-length human U6 genes that match the major U6 snRNA sequence perfectly. Multiple U6-related loci that contain nucleotide substitutions or truncations are dispersed throughout the genome. At least one such variant is transcribed from an internal promoter (38–40). In addition, the functional U6 homolog, U6ATAC RNA, operates as a cofactor in the splicing of AT–AC type introns and is expressed at a reduced level in human cells (41).

Most of the full-length human U6 genes are dispersed in the genome, unlike the human U1 and U2 snRNA genes that are organized in homogeneous repeats (23,24). The major exception is the U6-7/U6-8 pair, in which the separation is only 1014 bp in a convergent orientation. Although U6-2 and U6-9 are separated by only 128 kb on chromosome 19, no apparent sequence similarity exists in their flanking regions outside of the promoter elements. It is striking that four full-length, perfectly conserved U6 loci are lacking identifiable promoter elements and are apparently not expressed in 293 cells. Possibly, these represent relatively recent genomic additions. Alternatively, these loci could be transcribed in selected cell types driven by as yet uncharacterized promoter elements. Indeed, differential expression of pol III-transcribed 5S RNA (e.g. Xenopus oocyte versus somatic) or tRNA genes (e.g. Bombyx tRNAAla) is well known (42–44).

Among the expressed U6 genes, the PSE/TATA spacing is highly conserved, consistent with previous experiments that demonstrated drastically reduced transcription from the U6-1 promoter when >2–3 bp were either deleted or inserted between these elements (30,45). Furthermore, whereas the relative orientation and spacing between OCT and SPH elements in the distal region are flexible in the various human U6 genes, the spacing between the distal region and upstream edge of the PSE is quite conserved. The latter observation is consistent with the precise positioning of a nucleosome in this region for the U6-1 gene (11,12). Only the U6-9 gene promoter deviates from this model, with an SPH element located ~30 bp in a more proximal position. Nevertheless, the OCT element in the U6-9 promoter is still located comparably with that in the U6-1 promoter. It will be interesting to compare the chromatin structures of these two genes.

Except for the distal regions of U6-7 and U6-8, the sequences of the PSE, TATA and SPH elements vary among the full-length active U6 genes. Previous work has demonstrated the central role of the PSE in governing transcriptional efficiency of mammalian U6 genes. SNAPc binds with higher affinity to the mouse U6 PSE than the human U6-1 PSE in an electrophoretic mobility shift assay, and, correspondingly, the mouse U6 promoter is stronger in vitro (46,47). Reduced transcription from the U6-2 promoter in vitro probably reflects a relatively poor proximal promoter, within either the PSE, the TATA box or both (Fig. 3C and D). Our in vitro transcription system responds poorly to activation by the distal region when it is positioned normally (29,33). Comparable expression levels of U6-2 and U6-1 maxigenes in transfected cells may indicate a greater fold stimulation by the U6-2 distal region in the context of its own proximal promoter (Fig. 3A and B). However, results with the U6-9 gene are puzzling. The U6-9 PSE is an especially poor match to the consensus (Fig. 1), but in vitro transcription of U6-9 is similar to that of U6-1, U6-7 and U6-8 (Fig. 3C and D). SNAPc may bind the non-consensus U6-9 PSE with sufficient affinity, or the TATA box is especially efficient, and compensates for a poor PSE. Another possibility is that the U6-9 promoter may contain a strong BURE [BRFU (Brf2) recognition element] located downstream of the TATA box, a novel control element recently recognized in pol III type 3 promoters (48).

We were intrigued by the large difference in expression of the U6-7 and U6-8 genes when introduced separately into cells since these closely linked genes contain very similar 5'-flanking sequences with identical distal elements and TATA boxes. Only six nucleotides differ between the U6-7 and U6-8 PSEs, five of which are transitions, and no changes occur in the ACCGT core sequence (Fig. 1). We show that the difference in transcriptional efficiency results from the modest variation in the PSE (Fig. 4). It is surprising that these two promoters have comparable efficiencies in vitro because we would have expected transcription in the extract to manifest differences at the proximal promoter. Possibly the difference in transcription is only noticeable with a chromatin-assembled template or when some regulatory factor is missing in the S100 extract. A particularly interesting model is that SNAPc bound to the U6-8 PSE is better poised for activation compared with that bound at the U6-7 PSE. The latter model is reminiscent of the situation proposed for the Drosophila PSE-binding protein. In that case, a difference in the PSE between the U1 and U6 promoters results in selection of either the pol II or pol III transcriptional apparatus, possibly through alternative conformations of the PSE-binding protein (49,50). Further experimentation with these human U6 promoters could yield additional insight into the mechanisms of basal and activated transcription of snRNA gene promoters.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at NAR Online.


   ACKNOWLEDGEMENTS
 
We thank two undergraduate students, Heather Hooten and Derek Kelly, for assistance with PCR cloning of some genomic DNA fragments and plasmid DNA isolation. Nouria Hernandez (Cold Spring Harbor Laboratories) kindly supplied the anti-SNAP43 antiserum used in some ChIP assays. HeLa S3 cell pellets used for S100 extract preparations were supplied by the National Cell Culture Center (Minneapolis, MN). DNA sequencing was performed at the Gene Technologies Laboratory, Institute of Developmental and Molecular Biology, Texas A&M University. This work was supported by grants from the National Science Foundation (MCB-9808214) and the Robert A. Welch Foundation (A-1469).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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