Human Molecular Genetics, 2001, Vol. 10, No. 4 371-382
© 2001 Oxford University Press
Comparative genome analysis delimits a chromosomal domain and identifies key regulatory elements in the 
globin cluster
1MRC Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK, 2Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA, 3Faculteit der Geneeskunde en Gezondheids-Wetenschappen, Erasmus Universiteit, dr. Molewaterplein 50, Rotterdam, The Netherlands, 4School of Professional and Continuing Education, University of Hong Kong, Pokfulam Road, Hong Kong, 5Division of Pediatric Hematology/Oncology, Children’s Hospital, University of Texas Medical Branch, Galveston, TX, USA and 6Institut fuer Genetik und Allgemeine Biologie, Universitaet Salzburg, Austria
Received 4 October 2000; Revised and Accepted 21 December 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have cloned, sequenced and annotated segments of DNA spanning the mouse, chicken and pufferfish
globin gene clusters and compared them with the corresponding region in man. This has defined a small segment (
135–155 kb) of synteny and conserved gene order, which may contain all of the elements required to fully regulate globin gene expression from its natural chromosomal environment. Comparing human and mouse sequences using previously described methods failed to identify the known regulatory elements. However, refining these methods by ranking identity scores of non-coding sequences, we found conserved sequences including the previously characterized globin major regulatory element. In chicken and pufferfish, regions that may correspond to this element were found by analysing the distribution of transcription factor binding sites. Regions identified in this way act as strong enhancer elements in expression assays. In addition to delimiting the globin chromosomal domain, this study has enabled us to develop a more sensitive and accurate routine for identifying regulatory elements in the human genome. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Understanding the function of large stretches of genomic sequences is a major challenge of the post-genomic era: 4 years after its completion, two-thirds of the yeast genome has still to be assigned a function (1). The knowledge gap is even greater for mammalian genomes. A critical resource will be the availability of genomic regions that have been extensively characterized at a number of levels, allowing genomic sequence to be related to function. We have previously characterized the structure (2), epigenetic modifications (3–6) and function (summarized in refs 2 and 7) of a contiguous (376 kb) segment of DNA extending from the telomeric repeats of human chromosome 16p. This gene-rich region contains the
-like globin genes (tel-
-2-1-cen), which are expressed in a tissue- and developmental stage-specific manner, embedded within a variety of widely expressed genes (Fig. 1).
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Observations of the normal human
globin cluster and its mutants in vivo, together with experimental analysis of constructs containing various segments (1.5–120 kb) of the cluster in cell lines and transgenic mice, have identified some key cis-acting regulatory elements but, to date, it has not been possible to achieve fully regulated expression of the human globin genes in transgenic mice (summarized in ref. 7). Nevertheless, the globin genes can be expressed at high levels from a single copy of human chromosome 16 in an interspecific hybrid with a mouse erythroid cell background (7–9). Therefore, it appears that the transgenic constructs analysed to date may not span the entire chromosomal ‘domain’, defined here as a region containing all of the cis-acting elements required for fully regulated globin gene expression. Characterization of such domains will be of importance in understanding globin gene regulation and for understanding the general relationship between chromosome structure and function.
A comparison of the sequences spanning the human cluster with the syntenic regions of other organisms might delimit such a chromosomal domain. Presumably critical cis-acting regulatory elements will have remained together throughout evolution or, alternatively, equivalent elements would have been recruited. Therefore, cross-species comparisons may also highlight the critical regulatory elements within such a region.
In this study, we have analysed contiguous segments of DNA spanning the globin clusters of mouse, chicken and two species of pufferfish, enabling us to define a small segment of synteny and conserved gene order which has been maintained throughout evolution. In the human cluster, this conserved segment spans 135–155 kb including the globin genes, their major regulatory element (MRE or HS-40) and several widely expressed genes. When comparing the human and mouse sequences using previously defined routines (10–13), we did not clearly identify regulatory elements. However, by ranking sequence identities, the most conserved non-coding matches included the MRE, the globin promoter and a previously characterized, erythroid-specific DNAse1-hypersensitive site (HS-33): none of these elements could be identified by aligning human with chicken or pufferfish sequences. However, using prior knowledge of known erythroid-restricted transcription factor (TF) binding sites (14–16) we have shown that in chicken and pufferfish an globin enhancer lies in a position corresponding to the MRE of human and mouse. In addition to delimiting the globin chromosomal domain, these findings highlight the potential value and limitations of comparative analysis for interpreting primary DNA sequence.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Overall comparison of the extended sequences
Clones containing orthologues of the human
-like globin genes were identified and contigs extending in either direction from these recombinants were assembled (Materials and Methods). For comparison we sequenced long segments of DNA spanning the mouse (Mus musculus; 183 kb), chicken (Gallus gallus; 103 kb) and pufferfish [Spheroides nephelus; 167 kb and Fugu rubripes; 36 kb (data not shown)] globin gene clusters and compared these with the previously analysed human [376 kb (2,17)] sequence (Fig. 1 and Table 1).
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It has been shown previously that the
globin clusters of several species (e.g. man, rabbit and horse), lie in a telomeric position (18–20). In man, the cluster commonly lies only 150 kb from the 16p telomere (allele A) (18). However, the mouse cluster clearly lies at an interstitial position on chromosome 11 (21–23). The precise chromosomal location of chicken (linkage group E35; http://www.ri.bbsrc.ac.uk) is currently unknown. We found no telomeric or subtelomeric DNA repeats in the mouse, chicken or pufferfish globin regions.
The GC content of the clusters and their surrounding DNA is greater than the average for each respective species (Table 1). The clusters lie in the most GC-rich isochores (24) of the human (H3, 54% GC) and mouse (H2, 50% GC) genomes. The chicken cluster also lies in a GC-rich isochore (H2, 50% GC) although isochores with higher GC content (H3 and H4) are found in this species (25). The pufferfish cluster has a similar GC content to those of the average segments of its genome. Comparisons of the proportions of each sequence representing exons, introns and repeat elements (Table 1 and Fig. 1) showed relative compaction of the clusters in pufferfish and chicken due to fewer repeats (chicken and pufferfish) and smaller introns (pufferfish). In the pufferfish S.nephelus, the region of conserved synteny is compacted 5-fold, commensurate with the 7.5-fold smaller genome of the closely related fish F.rubripes (26).
Defining the segments of conserved synteny
We have previously reported the fully annotated sequence of the human globin gene cluster (2,17) (summarized in Fig. 1 and Table 2). Using a similar approach (legend to Fig. 1) we initially analysed and annotated sequences from the other species. This showed that in addition to the -like genes, several previously identified genes surrounding the human cluster were also found flanking the clusters of mouse, chicken and the pufferfish S.nephelus (Fig. 1 and Table 2).
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Comparison of the human and mouse sequences using PipMaker (27) showed extensive matches (
155 kb in human and 112 kb in mouse) between the two clusters (Fig. 1A). Similarly, homology was seen comparing the chicken (Fig. 1B) and pufferfish (Fig. 1C) sequences with human, but at a lower percentage identity. More detailed comparisons of the sequences (e.g. Fig. 2) showed that the clearest matches occurred in the syntenic, orthologous genes for which the number of coding exons, their sizes and sequences are well conserved (Fig. 2), as are their predicted proteins (data not shown). We also noted strong identity between the human and chicken sequences between coordinates 350859 and 352206 (in the human cluster). It seems most likely that this represents a new gene, although this was not predicted by any of the current programs and there are no expressed sequence tag (EST) hits associated with this putative gene. In summary, these analyses clearly demonstrated that sequence comparison between any two of the species studied detected all of the genes including their alternatively spliced exons.
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Defining the boundaries of conserved synteny
Closer inspection defined the 5' breakpoints in homology between the
clusters of human and other species (Fig. 1). Comparing human and mouse, homology ends following the IL-9 receptor (human gene 3), which is a functional gene in mouse (28) but a pseudogene in man (29). In addition, there is no match to POLR3K (human gene 3.1) in the mouse, suggesting that a further rearrangement within the region of synteny occurred after divergence of these species. In pufferfish the last point of homology extends to the end of C16orf8 (human gene 5). Beyond this, orthologues for POLR3K and C16orf33 (genes 3.1 and 4) are found, but rearranged with respect to the human cluster. At present there are insufficient sequence data to define the extent of homology upstream of the chicken cluster.
At the downstream end of the cluster (right hand boundary in Figs 1 and 2), the sequences of mouse, chicken and pufferfish diverge from the human sequence within a very narrow region between co-ordinates 171000 and 171186 of the human cluster (Fig. 1). Beyond this region, the sequences of each species appear quite different. In the mouse cluster, the only identifiable sequences lying adjacent to the cluster consist of repetitive elements related to retroviral sequences. The orthologue of LUC7L (human gene 16), which lies immediately to the centromeric side of the human cluster, is located on mouse chromosome 17 linked to a pseudoalpha gene but quite separate from the functional mouse cluster on chromosome 11 (30). Downstream of the genes in the pufferfish we identified a group of genes (FLJ20004, KIAA1395, HUC and G19P1) with homologues on human chromosome 19. In the chicken there appears to have been an inversion, removing the orthologues of the human genes 16–19 and placing genes 23–20 in an inverted orientation next to the genes (Fig. 1).
Despite the long period of evolution (500 million years) throughout which these species have diverged from each other, the extant globin clusters are arranged in a remarkably similar manner. In addition to the genes themselves, several genes lying upstream of the clusters have been conserved. It seems likely that the boundaries of the cluster, created independently in all four species, define the maximum limits of the chromosomal segment containing all of the cis-acting sequences required for fully regulated gene expression.
Comparison of the four globin clusters
To further our understanding of how the globin gene cluster is normally regulated, we analysed the -like genes in detail in each species (Fig. 3). Where known, the genes are arranged along each chromosome in the order in which they are expressed in development. Genes predominantly expressed in the embryonic period are found in human (), mouse () and chicken (
). Similarly, genes expressed in the fetal/adult period are present in all species, interspersed with pseudogenes. As in all mammals studied to date (31–35 and R. Hardison, unpublished data), apparently functional 
genes are found in mouse and man. Although they could not carry oxygen (33) their role, if any, is currently unknown. At present, the pattern of expression of the pufferfish -like globins is unknown. However, transcripts corresponding to A1 are represented multiple times in a Fugu cDNA database containing 3213 genes (http://fugu.hgmp.mrc.ac.uk), but no ESTs corresponding to A2 were found even though this appears to be a structurally normal gene. A2 is possibly expressed at a very low level or may be an embryonic gene. Another functional -like gene (A3) is present in F.rubripes on another chromosome lying 2 kb from the Fugu ß globin gene (T. McMorrow and S. Philipsen, unpublished data). It is interesting that the pufferfish -like globin genes lie in the reverse orientation with respect to the linked non-globin genes compared with the human, mouse and chicken clusters (Fig. 3). It is also interesting that this is the first observation indicating a physical separation between the and ß clusters in fish or amphibians; in previous reports these genes are linked in these orders.
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Evolution of CpG islands
We analysed the association of CpG islands with the
-like globin genes and the widely expressed genes flanking the cluster. In the human cluster equally prominent CpG islands were associated with each functional -like gene and with each of the widely expressed genes flanking the cluster (2) (Fig. 3). In the mouse, prominent CpG islands were seen associated with the widely expressed genes (e.g. the C16orf35 gene in Figure 3 and data not shown) but only minor enrichment of CpG dinucleotides was found at the -like genes themselves (Fig. 3). A very similar pattern was observed in the chicken cluster. However, no distinct CpG islands could be detected at either the globin genes or the widely expressed genes in the syntenic region of the pufferfish (Fig. 3 and data not shown) due to the high background of CpG dinucleotides found in fish (36).
A search for regulatory elements
It has been suggested that, in mammals, regulatory elements can be found by searching for ungapped alignments outside known exons (11–13,37). Most recently it has been suggested that regulatory elements should be found in non-coding regions with >70% identity over at least 100 bp (10). Comparing human and mouse sequences, two matches fulfilled these criteria (Fig. 2, CS1 and CS2). CS1 (co-ordinates 54332–54440) probably corresponds to an alternatively spliced exon of C16orf8 (human gene 5) as we subsequently identified an EST sequence (HS804279) which matches CS1 and exons 1 and 2 of DIST1. The other match, CS2 (113455–113584), is a conserved sequence in intron 3 of C16orf35 (human gene 7). Although this element does not contain an open reading frame it may contain part of a non-coding exon since, in both mouse and human, a portion of CS2 has been shown to match the 3' end of exon 1 of C16orf35 (referred to as the PROX gene in ref. 38). It is also possible that this region plays a regulatory role, although it appears not to regulate expression of the globin genes (7), does not coincide with any currently known HS and has no enhancer activity in transient assays. Therefore, in our initial search for regulatory elements we found only two potentially interesting sequences that probably correspond to alternatively spliced exons.
The criteria of Loots et al. (10) did not identify any of the previously characterized regulatory elements in the cluster (e.g. the MRE or the globin gene promoters). However, when gapped alignments between human and mouse were simply ranked by alignment scores (based on length and identity) the highest matches included CS1, CS2, MRE, the promoters of the and 
genes, HS-33 (a previously mapped erythroid-specific HS), a constitutive HS and only one unknown sequence (Table 3). To test this analysis further we analysed the human and mouse ß globin clusters in the same way. Again, the highest matches identified most of the previously described cis-acting elements regulating the human ß globin cluster (Table 3), validating this analytical routine.
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At other loci, sequence comparisons of human with chicken (270 million years) and pufferfish (400 million years) DNA sequences have identified regulatory elements (39,40). For the
globin clusters, comparing chicken and pufferfish with human sequences showed homology in the exons of syntenic genes (data not shown). The only other match to the human sequence in either of these species coincided with CS1 (in pufferfish) and CS2 (chicken and pufferfish), which may represent exons of highly conserved genes around the cluster. Therefore, preliminary comparisons of these sequences did not reveal candidates for regulatory elements.
Since none of the sequences associated with previously characterized regulatory elements or HSs appeared to be strongly conserved in chicken or pufferfish, we examined the distribution of binding sites for TFs known to play a role in globin gene regulation (41). Many of these sites (e.g. GATA binding sites) are very frequent in all sequences analysed here and were not enriched in known erythroid-specific HSs. In contrast, Maf recognition elements (MAREs; YGCTGASTCAY) (42) are relatively infrequent (1 in 3000–8000 bp) and yet the HSs corresponding to the MREs of man (HS-40) and mouse (HS-26), both located in intron 5 of C16orf35, each contain two MAREs positioned exactly 21 bp apart (Fig. 4). No other ‘paired’ sites, within 60 bp of each other, were found in human and only one other was found in mouse (coordinates 74892–74904).
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We next searched the chicken sequence and the only paired MARE site was again found in intron 5 of the C16orf35 gene. In this case, the sites were also 21 bp apart (Fig. 4). To analyse this further, we examined the position of HSs throughout the chicken cluster and found that the paired MAREs are associated with an HS in HD3 erythroid cells (Fig. 5). In pufferfish, one paired MARE site was found upstream of the MPG gene (gene s6). A second paired site was again found in intron 5 of the C16orf35 gene (gene s7). In this paired site, the MARE motif (Fig. 5, right) lies in the opposite orientation to that observed in other species and is 43 rather than 21 bp distant from the second site. Closer inspection revealed a similar distribution of TF binding sites between all four species in this region (Fig. 4).
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These findings strongly suggested that the paired MARE sites in intron 5 of the C16orf35 gene in chicken and pufferfish (Fig. 3) may serve a similar role to that of the human and mouse
MREs. We therefore tested whether these regions, with minimal sequence identity but conserved TF binding sites, have maintained any functionally similar role.
Functional characterization of putative regulatory elements
It has previously been shown that the human and mouse MREs behave as strong enhancers in erythroid cells (15,43–46). We used a transient transfection assay to investigate the effects of the putative regulatory elements from pufferfish and chicken on the chicken A and pufferfish A2 promoter, respectively. Each promoter was inserted next to the renilla luciferase gene in the vector pRLnull and activity was measured in HD3 chicken cells. Results from the promoter construct were compared with those obtained when the putative MREs from either the pufferfish or chicken sequences were inserted immediately 5' to their respective promoters (Fig. 6). We also investigated whether CS2 had any enhancer activity in these assays, again by inserting the chicken and Spheroides CS2 sequence next to their respective globin promoters. In each experiment, the putative regulatory elements were inserted in both orientations (Fig. 6).
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The chicken sequence from intron 5 of the chicken orthologue of the C16orf35 gene increased luciferase activity almost 20-fold over levels observed in the promoter alone. The equivalent pufferfish sequence also increased activity >20-fold. In both cases the magnitude of the effect was orientation dependent (Fig. 6). No effects were seen for CS2.
To determine whether the MAREs were critical for these enhancer effects we introduced mutations into each site (M1 and M2) and also constructed double mutants (M12). We found that, in the chicken, mutating the 5' site reduces activity by a third and, in the pufferfish, to a sixth of wild-type levels. Mutating the second site has a slightly greater effect and the double mutant reduces expression still further. Although the double mutant still shows increased levels over the promoter construct, the MARE sites account for almost 90% of the enhancer activity.
Therefore, it appears that these poorly conserved elements, not readily identified by conventional sequence comparisons, may in fact underwrite some, or maybe all, of the role of the MRE in these species.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Comparison of the
globin clusters of five distantly related species has shown that synteny and conserved gene order extend over a short distance (135–155 kb). Inspection of the boundaries of this conserved chromosomal unit suggests that although the breakpoints lie in very similar locations, they have been defined by quite independent evolutionary events in each species. Whatever the mechanism(s), this is of considerable practical interest, as these breakpoints may delimit a chromosomal segment containing all of the critical cis-acting elements required for fully regulated expression of the -like globin genes. It is interesting that even the 135 kb region representing the human/pufferfish synteny contains all the previously mapped erythroid-specific HSs around the human globin cluster and broadly corresponds to a segment of chromatin which becomes hyperacetylated when the genes are fully active in erythroid cells (E. Anguita et al., in preparation). At present, none of the fragments containing the cluster that have been tested in transgenic mice span this entire region. Since all of these constructs are expressed suboptimally (7), it remains to be seen whether a fragment containing this entire region would be fully regulated in transgenic mice. In general, these findings suggest that cross-species comparisons may address the important question of whether chromosomes are organized into discrete structural and functional domains.
Current software is capable of finding coding regions of the human genome. Programs for identifying regulatory elements adjacent to the genes have been less refined, nevertheless primary sequence analysis readily detects CpG-rich islands which associate with the promoters of virtually all housekeeping genes and approximately half of all tissue-specific genes (47,48). Since CpG islands co-localize with the promoters of genes, they may be involved in regulating their transcription (49) and specifically in influencing expression of the -like globin genes (3). Although prominent CpG islands are associated with each of the widely expressed genes flanking the clusters (human, mouse and chicken), the CpG islands associated with the -like genes themselves have been substantially eroded during the evolution of mouse and chicken, demonstrating that they are not absolutely required for globin gene regulation in these species. Similar erosion of the CpG islands associated with tissue-specific genes has been noted before (49,50) and, therefore, this cross-species comparison confirms that CpG islands may be less useful for identifying tissue-specific genes in these species and adds to the evidence that CpG islands are not fundamentally important in regulating gene expression but reflect some other aspect(s) of genome structure, function or evolution as discussed by Antequera and Bird (49).
To annotate the human genome in full, it will be important to develop routines for identifying the key regulatory elements (e.g. promoters, enhancers and silencers) from the DNA sequence alone. Ultimately one might hope to identify other chromosomal elements; e.g. origins of replication, boundary elements and signals for nuclear sub-compartmentalization. At present it is not clear to what extent cross-species sequence comparisons can achieve this since, to date, very few studies have identified such elements by sequence analysis alone. Because many of the bona fide elements regulating globin expression in vivo in man and mouse have been previously characterized (reviewed in refs 7,44 and 45), this study allowed us to test the ability of current routines to identify such elements. Using criteria set out by others (10) we were unable to distinguish even the most important regulatory elements (the globin promoters and MREs) of the human or mouse globin clusters from many other non-coding regions. However, simply ranking the identity scores of conserved non-coding sequences identified many of the known regulatory elements in the globin cluster. Similarly, using this approach to analyse the human and mouse ß globin gene clusters we identified most of the known regulatory elements (Table 3), whereas previously defined criteria (10) identified only HS2 (51). Therefore, rather than setting fixed, arbitrary criteria, which may differ from one comparison to another, identifying the most highly scoring non-coding matches between two species, as set out here, may provide the most efficient guide to regulatory elements. This simple approach may be made even more informative by parallel analysis of several closely related species.
Although this approach was not informative in more distantly related species, using prior knowledge of the TF binding sites in the human and mouse MREs it was possible to identify enhancer elements at a conserved position (intron 5 of C16orf35) in chicken and pufferfish. Although it is not yet clear whether these enhancers are functionally equivalent to the MREs, our data strongly indicate that this type of regulatory element was present in this position in the ancestral globin cluster. These findings suggest that future cross-species searches for regulatory elements might also include routines searching for significant conservation of motifs corresponding to chosen TF binding sites of interest separated by non-conserved gaps.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of contigs
The human contig has been published previously (2,17). In mouse we started with the contig containing 2CF2, P3 and P48 (52). Gaps in this contig were closed with F1 827 and the contig was extended with P2 and o05238 (P1 clones from the ICRF P703 mouse P1 library). In chicken, we started with
G2, G5 and G7 (53,54). This was extended with D18260 and D0242 (Library 125, Resourcenzentrum, Germany). A cosmid library derived from F.rubripes genomic DNA was constructed in the pTCF cosmid vector (55). Cosmids A and F representing the F.rubripes globin locus were identified by screening this library with an A1 globin cDNA isolated from a cDNA library from peripheral blood. Subsequently, using probe 129 (coordinates 34062–34626 of the Fugu sequence) we isolated the P1s PACFF-74p9, -56M21 and -51D12 from the S.nephelus library (Genome Systems).
Isolation of probes
Human (6,56) and mouse (45,57) probes were as previously described or were amplified from primers (available on request) designed from the sequence. Probes to the chicken (C22/20), Fugu (129) and nephelus clusters were amplified using primers (available on request) designed from the sequence. Probes corresponding to A1 and ß globin (Fugu) were isolated from cDNA made from peripheral blood RNA from a 3- to 4-month-old fish screened with salmon globin cDNA probes.
Sequence analysis
Recombinants were sequenced as previously described (2). Some data were previously available for mouse (21,22,38,44, 45,58,59) and chicken (53,54,60,61) but new sequence contigs were assembled without reference to these. Each newly assembled sequence was masked for repeats and initially annotated by sequence homology using the BLAST suite (62) to search nucleotide [dbEST (63) and EMBL (64)] and protein [SwissProt and Trembl (65)] sequence databases and our unpublished cDNA sequences. In addition, sequences were analysed with the gene prediction programs GRAIL and GENSCAN (66,67). All sequences and analyses were processed using an automated annotation system and stored in ACEDB (http://www.acedb.org/). Sequences were compared by sequence identity using PipMaker (http://bio.cse.psu.edu/pipmaker). G+C% and the frequency of CpG dinucleotides plots were calculated using a 200 bp stepping window. Putative regulatory elements were identified by using BLASTZ (an integral part of PipMaker) to align genomic sequences that had been masked for known exons and repeats. Localized regions of sequence conservation, i.e. ungapped high scoring pairs (HSPs), were returned by PipMaker using the ‘concise text’ output option. A score for each ungapped HSP was calculated (+5 for each identical base and –4 for each mismatch) and used to rank the HSPs. Specific TF binding sites were identified using MacVector software and the Transfac database (http://transfac.gbf-braunschweig.de/TRANSFAC/index.html).
Identification of DNAse1-hypersensitive sites
Nuclei were prepared from 1 x 108 chicken HD3 (erythroid) cells and DNAse1-hypersensitive sites were identified as previously described by Higgs et al. (4).
Plasmid constructs
A 500 bp fragment containing the chicken A-globin promoter (coordinates 52393–52862) was amplified using the primers 5'-CCTTCCCGGGGGTTGCACCTCTGTGTT-3' and 5'-GTGCTCCTGAACCTACAG-3'. The PCR product was kinased and ligated into the SmaI site of the pRL-NULL plasmid (Promega); the orientation and sequence were confirmed. Similarly, a 500 bp fragment immediately 5' of the pufferfish A2 globin gene was amplified and cloned (primers 5'-CCTTCCCGGGGCCAAACTGGTGCTCAATTT-3' and 5'-TTGTTGTTGGGGGTGTTTTT-3'; coordinates 988906–99337). Primers at the 5' end of the insert (with respect to the luciferase gene) contain an SmaI site allowing the subsequent insertion of the following PCR products: the putative chicken RE (primers 5'-TTGTTGTTGGGGGTGTTTTT-3' and 5'-TTGTTGTTGGGGGTGTTTTT-3'), chicken CS2 (primers 5'-GAACTGAAATGCCACCAACC-3' and 5'-CCACACTCATTCTGGTTACCC-3'), putative pufferfish RE (primers 5'-CTGTAGAAAGTGCTTAGAAGTGAA-3' and 5'-GCAAAGTAGTCTTCTTTACATTTTT-3') and pufferfish CS2 (primers 5'-TTTGCAGCACGTTTATCCAA-3' and 5'-GATTCACAGAATCCGCTGGT-3'). Mutations were introduced into the putative MRE by PCR-based, site-directed mutagenesis (QuikChange; Stratagene). All modifications were confirmed by sequence analysis. All PCRs were performed using Pfu DNA polymerase (Stratagene).
Transient transfection and functional assays
Chicken HD3 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum transfected by electroporation using a 950 µF capacitor array charged at 300 V. For each transfection, 1 x 106 cells were resuspended in 0.25 ml of RPMI 1640 with 10 µg of a test construct in pRL-NULL and 5 µg of a transfection control plasmid pCMVßGal. After discharge, cells were left on ice for 10 min before resuspending in growth medium and incubating at 37°C for 2 days. Subsequently, luciferase activities were determined according to the manufacturer’s instructions (Promega). Relative ß galactosidase activity in lysates was measured using O-nitrophenyl-D-galactopyranoside (0.67 mg/ml) as substrate in a 0.1 M phosphate buffer pH 7.0 containing 10 mM KCl and 1 mM MgSO4 incubated at 37°C for 15–45 min. The A420 was determined after stopping the reaction by the addition of 0.3 M sodium carbonate. All constructs were tested in triplicate in at least three independent transfection experiments.
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ACKNOWLEDGEMENTS |
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We are grateful to Professor Sir D.J. Weatherall for continued support and encouragement. We are grateful to Dr G. Elgar for helpful advice. We thank Drs W.G. Wood, R.J. Gibbons and V. Buckle for their comments on the manuscript. We are grateful to Julianne Ellu for providing HD3 cells. We also thank H. Ayyub for excellent technical assistance. Mouse cosmids were provided by Dr Q. Zhao and chicken recombinants were provided by Dr J. Dodgson. J. Flint and J. Frampton are Wellcome Trust Senior Fellows. B.A. was supported in part by the National Institutes of Health.
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FOOTNOTES |
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+ Present address: Clinical Genetics, NCI, Rockville, MD 20852, USA
§ To whom correspondence should be addressed. Tel: +44 1865 222393; Fax: +44 1865 222500; Email: drhiggs@molbiol.ox.ac.uk
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REFERENCES |
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