Department of Pathology and Laboratory Medicine, Emory Vaccine Center, School of Medicine, Emory University, Atlanta, GA 30322, USADepartment of Biological Science, California State University Fullerton, Fullerton, CA 92834, USAInstitute of Molecular Evolutionary Genetics, Department of Biology, Pennsylvania State University, University Park, PA 16802, USA
r t i c l e i n f o
rticle history:eceived 9 April 2009ccepted 30 May 2009vailable online 26 June 2009
eywords:
a b s t r a c t
We have studied the genomic structure and evolutionary pattern of immunoglobulin kappa deleting ele-ment (KDE) and three kappa enhancers (KE5′, KE3′P, and KE3′D) in eleven mammalian genomic sequences.Our results show that the relative positions and the genomic organization of the KDE and the kappaenhancers are conserved in all mammals studied and have not been affected by the local rearrangementsin the immunoglobulin kappa (IGK) light chain locus over a long evolutionary time (∼120 million years of
mmunoglobulin kappa light chainappa deleting element (KDE)ecombination signal sequence (RSS)appa enhancerranscription factor
mammalian evolution). Our observations suggest that the sequence motifs in these regulatory elementshave been conserved by purifying selection to achieve proper regulation of the expression of the IGK lightchain genes. The conservation of the three enhancers in all mammals indicates that these species mayuse similar mechanisms to regulate IGK gene expression. However, some activities of the IGK enhancersmight have evolved in the eutherian lineage. The presence of the three IGK enhancers, KDE, and otherrecombining elements (REs) in all mammals (including platypus) suggest that these genomic elements
amm
were in place before the m
. Introduction
The expression of immunoglobulin kappa (IGK) light chain geness restricted in the B-cell lineage. This cell-type specific expressions regulated by the interaction of DNA-binding proteins with spe-ific promoter and enhancer sequences (Falkner and Zachau, 1984;enardo et al., 1987; Sen and Baltimore, 1986). Three enhancerlements have been described and functionally characterized inhe IGK-encoding locus of humans and mice, but whether theseequences are conserved in other mammalian species remainsnknown (Gimble and Max, 1987; Liu and Garrard, 2005; Meyer etl., 1990; Xiang and Garrard, 2008). The first enhancer, the KE5′, isocated in the intronic region between the immunoglobulin kappaoining (IGJK) and constant (IGCK) genes. The other two enhancersre located at the proximal (KE3′P) and distal regions (KE3′D) ofhe 3′ end of the IGCK gene (Inlay et al., 2002; Lenardo et al., 1987;
u et al., 1996). These enhancers contain specific nucleotide motifs
hat bind to specific transcription factors (Lenardo et al., 1987;ongubala et al., 1992; Schanke and Van Ness, 1994).
∗ Corresponding author at: Department of Pathology and Laboratory Medicine,mory Vaccine Center, School of Medicine, Emory University, Atlanta, GA 30322,SA. Tel.: +1 404 727 7259; fax: +1 404 727 8795.
During B-cell differentiation, the immunoglobulin light chaingene rearrangements occur in an orderly fashion starting withkappa chain gene rearrangements and proceeding to lambda chaingene rearrangement (Alt et al., 1980). Usually only one type ofimmunoglobulin light chain gene is expressed in a particular B-cell. In about 90% of cases the lambda-encoding genes undergorearrangements only when the recombination events of the kappa-encoding genes lead to non-functional products (Hieter et al., 1981;Korsmeyer et al., 1982; van der Burg et al., 2001). If the rearrange-ment between the immunoglobulin kappa variable region gene(IGVK) and IGJK produces a non-functional IGVK-IGJK product, thelocus undergoes segmental deletion through a rearrangement withthe kappa deleting element (KDE), which is located downstreamof the IGCK gene (Siminovitch et al., 1985). The KDE has beendescribed in humans and mice (Graninger et al., 1988; Langerak etal., 2004; Siminovitch et al., 1985), but whether these sequences arepresent in other mammals is currently unknown. Most of the KDE-mediated IGK gene rearrangements occur either via recombiningelement (RE) located in the IGJK-IGCK intron or via recombinationsignal sequence (RSS) located immediately 3′ end to the IGVK genes(Graninger et al., 1988; Langerak et al., 2004; Siminovitch et al.,
1985). Recent studies have demonstrated that KDE can recombineto the RSS flanking IGJK genes (Seriu et al., 2000). An alternativerecombination mechanism that can delete the entire IGJK clusterby means of a rearrangement between RSS of IGVK and intronic REhas also been reported (Feddersen et al., 1990).
The regulation of IGK expression has been mainly studied inumans and mice, and only a few studies have identified some ofhe regulatory elements in the rabbit and the horse IGK regionsEmorine et al., 1983; Ford et al., 1994; Schanke and Van Ness, 1994;iminovitch et al., 1987; Xiang and Garrard, 2008). However, it isurrently unknown whether all mammals use similar or differentequences to regulate IGK expression. In addition, when these reg-latory elements evolved in mammalian IGK locus is not known.he multiple genomic sequences of different mammalian speciesurrently present in the public databases provide an excellentpportunity to study the level of conservation of these regulatoryegions. In the present study, we used comparative genomics andioinformatics approaches to gain insights into the genomic struc-ure and evolution of sequences that regulate the IGK expression.
. Materials and methods
.1. Genomic localization of the IGJK, IGCK and theibose-5-phosphate isomerase (RPIA) gene
The genomic location of the IGJK and IGCK genes and theanking non-IG gene RPIA (ribose-5-phosphate isomerase) wereetermined in a previous study (Das et al., 2008a) for eight mam-alian species (i.e. human, mouse, rat, dog, cow, horse, opossum,
nd platypus). To identify the IGCK and RPIA genes in the chim-anzee, orangutan, and macaque genome sequences we performedBLASTn search using the human IGCK (accession no. AAI10395) andhe RPIA (accession no. AAH15529) sequences as queries. To iden-ify the IGJK genes, which are very short and cannot be detected byLAST searches, we manually screened 7 kb upstream of the IGCKene, taking into account the location of the RSS sequence at the′ of the IGJK gene. We confirmed that the identified sequences are
GJK genes using the IGJK-specific molecular markers (Das et al.,008a).
.2. Identification of the kappa enhancers
We identified the kappa enhancer elements in 11 mammalianenomes (>5× genome coverage) using a four step approach. Thenformation of the genome assembly of the 11 mammalian speciess given in Supplementary Table 1. We first performed BLASTnearches using the previously reported three enhancer sequencesrom humans and mice: the 5′ enhancer (KE5′), located at the 5′
nd of the IGCK gene, the 3′ proximal enhancer (KE3′P), locatedt the proximal region of the 3′ end of the IGCK gene, and the 3′
istal enhancer (KE3′D), located at the distal region of the 3′ endf the IGCK gene (Judde and Max, 1992; Liu et al., 2002; Schankend Van Ness, 1994). This way we identified sequences homolo-ous to the KE5′ enhancer in chimpanzee, orangutan, macaque,at, and dog genomes and sequences homologous to the KE3′Pnd KE3′D enhancers in chimpanzee, orangutan, macaque, rat,og, and horse genomes. In the second step, we aligned the
dentified enhancer sequences from primates and rodents usinghe ClustalW (Thompson et al., 1994) and DiAlign (available atww.genomatix.de) programs and searched for conserved motifssing CoreSearch (available at www.genomatix.de) and MEMEVersion 3.5.7) (Bailey et al., 2006). We used the default param-ters of these programs and inspected the alignments manuallyo maximize similarity. In the third step, the motifs identified inhe previous step and the genomic organization of the IGJK, IGCK,
nd RPIA genes were used to scan the horse genomic sequence,o identify the KE5′ enhancer, and the cow, opossum, and platy-us genomic sequences, to identify of all three enhancer elements.
n the fourth step, we searched the retrieved sequences for com-on restriction sites and the motifs in which common transcription
logy 46 (2009) 3171–3177
factors (TFs) can putatively bind. This way we confirmed that theidentified sequences were homologous to the human and mouse� enhancer elements. To find the common restriction sites weused the restriction site detection program in GEMS Launcher(available at www.genomatix.de). The programs P-Match (avail-able at www.gene-regulation.com) and MatInspector (available atwww.genomatix.de) were used to locate the DNA motifs for puta-tive TF-binding sites. These two programs utilize the TRANSFACdatabase (Wingender et al., 1996) to identify match in the DNAsequences.
2.3. Identification of the KDE and the recombining element (RE)in the IGJK-IGCK intron
The RE in the IGJK-IGCK intron of the human � encodinglocus contains a palindromic heptamer signal sequence (CACAGTG)(Klobeck and Zachau, 1986; Siminovitch et al., 1985). To identifysequences in other mammalian genomes, which are homologousto the RE sequence of human, we first scanned the IGJK-IGCKintron sequences for the conserved CACAGTG motif. We thenanalyzed the level of sequence similarity between the reportedhuman RE and all the other mammalian sequences using 50 bpupstream and 50 bp downstream sequences of the CACAGTG motifand finally we searched for common restriction sites. To iden-tify the KDE sequence we performed BLASTn searches using thehuman (Klobeck and Zachau, 1986) and mouse (Siminovitch etal., 1987) KDE sequence as queries. Both of these sequencescontain a heptamer signal sequence (CACTGTG) and a nonamersequence (AGTTTCTGC) separated by a 23 bp spacer (Siminovitchet al., 1987). These searches identified potential homologous KDEsequences in all eutherian mammals. To identify KDE homologoussequences in non-eutherian mammals we scanned the genomicsequences of opossum and platypus for the presence of the con-served CACT(A)GTG motif between the IGCK and RPIA genes andwe analyzed the sequence similarities between eutherian and non-eutherian mammals using 50 bp upstream and 50 bp downstreamsequences of the CACT(A)GTG motif. Additionally, we consideredthe relative positions of the heptamer and nonamer sequences aswell as the relative positions of the KDE homologous sequences tothe enhancer sequences and the RPIA genes. Finally, we searchedfor common restriction sites in the 106 bp long sequence includingthe heptamer motif.
3. Results
3.1. Evolutionary conservation of the 5′ enhancer (KE5′) element
The cross-species comparison of ∼400 bp sequences of the IGJK-IGCK introns revealed that a ∼150 bp region is fairly conservedamong all mammalian species under study (Fig. 1). In this region sixcommon restriction sites are found in eutherian mammals, three ofwhich are also conserved in non-eutherian mammals. To character-ize the potential gene regulatory regions, we searched for putativeTF-binding sites (see Section 2). One class of such sites is the E-box, which contains the consensus sequence CANNTG (Murre etal., 1991; Yutzey and Konieczny, 1992). We identified three E-boxes(Schanke and Van Ness, 1994) in the 150 bp region of the IGJK-IGCKintron. Two (E1 and E2) of these E-boxes are conserved in all mam-malian species examined and the third (E3) is conserved only ineutherian mammals (Fig. 1). The E-boxes are the potential binding
sites for the twist subfamily of basic helix-loop-helix (bHLH) tran-scription factors (Virolle et al., 2002). We also identified a potentialNF�B (nuclear factor �B)-binding site. This site is located immedi-ately upstream of the E-box (Fig. 1) proximal to the IGJK genes. ThisNF�B-binding site is conserved in all eutherian mammals studied
S. Das et al. / Molecular Immunology 46 (2009) 3171–3177 3173
Fig. 1. Alignments of the most conserved regions of the KE5′ . Three conserved motifs are highlighted in colors. Common restriction sites and common putative transcriptionfactor binding sites are shown by horizontal bars below the alignments. The common restriction sites for eutherian mammals are indicated by the letter E and the restrictions lettero hree
(f
3
3XmeItbirbfabm
aaatb
3
g
ites common for both eutherian and non-eutherian mammals are indicated by therientation. The spacing between the two conserved regions is given in base pairs. T
Schanke and Van Ness, 1994). However, no potential NF�B site wasound in the homologous region in non-eutherian mammals.
.2. Conserved motifs in 3′ enhancer elements (KE3′P and KE3′D)
Our analysis indicated that the two-enhancer elements at the′ end of the IGCK gene (Inlay et al., 2002; Judde and Max, 1992;iang and Garrard, 2008), KE3′P and KE3′D, are conserved in allammals studied (Figs. 2 and 3). In both regions we identified sev-
ral motifs that are conserved throughout mammalian evolution.n particular, we found that the KE3′P enhancer sequence con-ains two conserved E-box motifs. One E-box (CAACTG) is conservedetween eutherian mammals and the other (CAT(C)CTG) is present
n both eutherian and non-eutherian mammals (Fig. 2). The KE3′Pegion contains five putative TF-binding sites. Among them, theinding sites for the macrophage-specific factor (PU.1), the inter-eron regulatory factor (IRF), and the bHLH factor are conserved inll mammals studied whereas a potential binding site of the pairedox (PAX) transcription factor is conserved only between eutherianammals.In the distal 3′ enhancer sequence (KE3′D), two E-boxes (CACCTG
nd CAGA(C)TG) and one NF�B site have been described in humansnd mice (Liu et al., 2002). Our analysis suggests that all three sitesre present in all 11 mammalian species under study (Fig. 3). Addi-ionally, we identified another putative TF-binding site for a TF thatelongs to the ETS family, which is conserved in all mammals.
.3. Analysis of the KDE and the RE of IGJK-IGCK intron
We screened the downstream and upstream sequences of IGCKenes (see Section 2) to identify the KDE and the intronic RE
M. For putative TF-binding sites, the bars above and below the lines indicate theirE-box sequences are marked as E1, E2 and E3.
sequences. Our results suggest that the KDE contains a con-served nonamer sequence located at the 5′ end of a conservedheptamer sequence (CACTGTG) in all mammals studied (Fig. 4a).This configuration resembles the one previously reported for thehuman and mouse KDE (Siminovitch et al., 1987). In all mam-mals studied, the length of the spacer between heptamer andnonamer sequences is 23 bp. The only exception is the platy-pus KDE, which contains a 24 bp spacer between the heptamerand nonamer sequences. Whether the 24 bp spacer is char-acteristic of non-eutherian mammals cannot be deduced withcertainty, because the opossum genomic sequence is incom-plete in this region. A comparison of the restriction sites onKDE sequences showed that only TspRI and Tsp4CI sites arecommon in all mammalian species studied, whereas BisI, CviJI,and MaeI restriction sites are common in eutherian mam-mals.
In order to determine the exact recombination site in IGJK-IGCKintron we used the 106 bp genomic region (i.e. 50 bp upstreamand 50 bp downstream sequences of CACAGTG heptamer) con-taining human IGK intronic recombining element as a referencesequence. In humans, the IGK intronic RE contains a canonicalheptamer sequence CACAGTG (Klobeck and Zachau, 1986). Simi-lar to the human sequence one CACAGTG motif can be identified inthe IGJK-IGCK intron in all mammals studied (Fig. 4b). Sequenceanalysis showed that with the exception of the conserved hep-tamer sequence, the remaining sequence of the intron is highly
diverged. Only two common restriction sites (TspRI and Tsp4CIsites) could be found in the heptamer signal sequence (Fig. 4b).No nonamer-like conserved sequence is found either 12 bp or23 bp upstream and downstream from the conserved heptamermotif.
3174 S. Das et al. / Molecular Immunology 46 (2009) 3171–3177
Fig. 2. Alignments of the most conserved regions of the KE3′P. Three conserved motifs are highlighted with different colors. Common restriction sites and common putativetranscription factor (TF)-binding sites are shown by horizontal bars below the alignments. The “M” in the parenthesis indicates that the restriction sites are common in botheutherian and non-eutherian mammals, whereas “E” indicates that the restriction sites are common in eutherian mammals only. For putative TF-binding sites, the bars aboveand below the lines indicate their orientation. The E-box sequences are shown with boxes. Due to the incompleteness of the opossum genome sequence project the KE3′Psite could not be detected.
Fig. 3. Alignments of three conserved motifs of the KE3′D. Common restriction sites and common putative TF-binding sites are shown by horizontal bars below the alignments.The “M” in the parenthesis indicates that the restriction sites are common in both eutherian and non-eutherian mammals, whereas “E” indicates that the restriction sites arecommon in eutherian mammals only. For putative TF-binding sites, the bars above and below the lines indicate their orientation. The spacing between the two conservedregions is given in base pares. The E-box sequences are shown in boxes.
S. Das et al. / Molecular Immunology 46 (2009) 3171–3177 3175
Fig. 4. Sequence comparisons of the KDE and the IGJK-IGCK intronic RE. Common restriction sites are shown by horizontal bars below the alignments (M: common restrictionsites for both eutherian and non-eutherian mammals; E: common restriction sites for eutherian mammals only). (a) Sequence alignments of the KDE region. The conservedh opossa es areo
3e
tK(tc
4
maloge
TPm
D
S
HCOMMRDCHOP
eptamer and nonamer sequences are highlighted. Due to the incompleteness of thelignments of RE located in the IGJK-IGCK intron. The conserved heptamer sequencr downstream of the conserved heptamer motif.
.4. Conservation in the relative positions of KDE, RE andnhancer elements
The comparative analysis of IGK-encoding locus revealed thathe physical distance between RE-KE5′, KE5′-IGCK, IGCK- KE3′P,E3′P-KE3′D, and KE3′D-KDE can little vary from species to species
Table 1). However, the relative positions and the orientation ofhe KDE, RE, three enhancer elements, IGCK, and IGJK genes areonserved in all mammalian species studied (Fig. 5).
. Discussion
The immunoglobulin-encoding locus has been the subject ofultiple gene rearrangements during the evolution of mammals
nd these rearrangements have resulted in variation in the total
ength of the locus, the number of component genes, and theirrientation (Das, 2009; Das et al., 2008a,b). Our analysis also sug-ests that most of the non-coding sequences are highly divergedven in closely related species. In contrast, the relative positions
able 1hysical location of RE, KE5′ , IGCK, KE3′P, KE3′D, and KDE in the genome of differentammalian species.
um genome sequence project the potential KDE could not be detected. (b) Sequencehighlighted. No nonamer-like conserved sequence is found either in the upstream
and the genomic organization of the regulatory elements of theIGK-encoding locus (KDE, RE, and the three enhancers) are con-served in all mammals studied (Fig. 5). These observations suggestthat although the non-coding sequences of the locus evolve moreor less neutral and mutations accumulate in random (Nei, 2007),the motifs in these regulatory elements evolve under purifyingselection due to functional constraints. These constraints are mostprobably related to the proper regulation of expression and rear-rangements of the immunoglobulin genes.
The presence of all three enhancers and the conservation of spe-cific TF-biding sites in all mammals suggest that these species mayuse similar mechanisms to regulate the expression of the IGK genes.However, some TF-binding sites can be detected only in eutherianmammals [e.g., the NF�B binding site and the distal E-box in the 5′
enhancer (KE 5′) (Fig. 1), and one E-box sequence and the PAX tran-scription factor binding site in the KE3′P enhancer (Fig. 2)]. Thesefindings suggest that some activities of the � enhancers might haveevolved in the eutherian lineage.
The recombination signal sequences in immunoglobulin genescontain conserved heptamer and less conserved nonamer motifs,separated by either 12 ± 1 or 23 ± 1 bp spacer (Akira et al., 1987).The recombination generally takes place between one RSS with a12 bp spacer and one with a 23 bp spacer (12/23 joining rule). Thecomparative analysis of KDE shows that like IGJK genes it is com-posed of conserved heptamer and nonamer sequences, separatedby 23 bp spacer sequence. However, in platypus the length of thespacer is 24 bp. In contrast to canonical RSS, the RE in IGJK-IGCKintron is composed of an isolated conserved heptamer (CACAGTG)motif without an obvious nonamer sequence. The � gene deletion isgenerally mediated by the site-specific recombination event eitherbetween KDE and RE of IGJK-IGCK intron or between KDE and RSS
of IGVK gene (Beishuizen et al., 1997; Moore et al., 1985), whileonly few � gene deletions are mediated via an alternative dele-tion mechanism (Beishuizen et al., 1994; Feddersen et al., 1990).There is evidence that in some cases KDE can also recombine to theRSS of IGJK gene (Seriu et al., 2000). The possible recombination
3176 S. Das et al. / Molecular Immuno
Fg(t
ebtslttml
igmtt
ig. 5. Relative positions of the KDE, RE, three enhancer elements, IGCK, and IGJK,enes. The 3′ end of the IGK-encoding locus is flanked by the non-IG gene RPIAribose-5-phosphate isomerase). Arrows indicate the transcription orientation ofhe genes.
vents in IGK locus are summarized in Fig. 6. Some of these recom-inations are atypical (cannot be formed directly either because ofhe inverted positions of their respective RSS or because their RSSpacer lengths do not obey the 12/23 joining rule) and occur in veryow frequency (Langerak et al., 2004). However, the conservation ofhe KDE element and the RSSs with which it recombines suggesthat all mammals may use all the different pathways of rearrange-
ent to achieve proper selection of functional immunoglobulinight chain proteins.
The presence of the KDE, RE, and three enhancer elements (Fig. 5)n all mammalian IGK locus (including platypus) suggest that these
enomic regions must have been shaped before the radiation of theammalian lineages from their common ancestor. The conserva-
ion in the genomic organization of the regulatory sequences andhe conservation in the location of the regulatory elements relative
Fig. 6. Schematic diagram of possible recombination events in IGK locus.
logy 46 (2009) 3171–3177
to transcription unit indicate that all mammalian species possiblyuse the similar molecular apparatus for the regulation of IGK lightchain expression.
Acknowledgements
We thank Max Cooper, Jan Klein, Parimal Majumder, MasayukiHirano, Masafumi Nozawa, and Sayaka Miura for their valuablecomments and suggestions. This work was supported by theNational Institutes of Health [grant GM020293-35 to M. N.] andby the California State University Fullerton [start-up money to N.N.].
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.molimm.2009.05.180.
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