Transcriptional enhancers and the evolution of the IgH locus

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Transcriptional enhancers and the evolution of the IgH locus Bradley G. Magor, David A. Ross, Lars Pilström and Gregory W. Warr Based on comparisons of the structure and transcriptional

E

regulation of the IgH locus in

nhancers are essential to the proximal to the promoter to permit tranmammals and a bony fish, Brad expression of antibody genes: scriptional activation, they must also be Magor and colleagues present a they regulate not only the prosituated in the locus such that they are not duction of mRNA, but also all excised during the recombination events. hypothesis in which enhancer VDJ recombination and class switching, as In the higher vertebrates these conditions evolution was the driving force in well as hypermutation of productively reare met by the existence of multiple enarranged VDJ regions1Ð3. While VDJ joining hancers Ð at least six in the mouse IgH locus (and a prerequisite for) the and class switching are both chromosomal (Fig. 1). Whereas the DQ52 enhancer that is development of the complex and recombination events, they use quite sepapositioned among the germline D regions15 flexible IgH locus seen in mammals. is excised by VDJ recombination, none of rate mechanisms and most likely evolved the enhancers are excised by class-switch separately. Whereas the target sequences for recombination. VDJ joining consist of highly conserved Integral to the class-switch process are several enhancers that reheptameric and nonameric sequence motifs, separated by 12 bp or 23 bp spacers, those for class switching utilize large tracts (up to side 3′ of the locus in mammals. Some, if not all of these enhancers several kb) of repeated short sequences (such as TGGGG or GAGCT), (hs3a, hs1,2, hs3b and hs4)10,11,16Ð18 regulate sterile transcript prosome of which resemble x (chi) sequences and can serve as targets duction at the heavy chain constant region genes (Fig. 2) and might for prokaryotic recombinases4. Not surprisingly, different proteins play an important role in regulating IgH locus expression at later recognize and cleave the target sequences for VDJ joining and class stages of B-cell development11. Recent studies on the organization of switching. The products of the RAG genes are essential for VDJ join- the IgH locus in lower vertebrates suggest that the primordial IgH ing5, whereas the proteins that recognize and cleave switch se- locus lacked enhancers that were appropriately placed to accomquences are presently not known. However, it is clear that RAG modate the class-switch process. gene products are not necessary for class switching5. The only shared aspect of VDJ joining and class switch recombination identified to date is their reliance on the same (non-B-cell restricted) The primordial IgH locus mechanisms of DNA double-strand break repair (including the Ku Before attempting to draw conclusions concerning enhancer evoluproteins and DNA-dependent protein kinase) to complete the tion, it is informative to survey the types of organization that are recombination process6Ð8. Thus, it seems that the ability to carry out seen in the IgH loci of todayÕs vertebrates. There is no clear evidence VDJ joining in B cells arose early in vertebrate evolution9. that the agnathan fish (lamprey and hagfish) express typical antiVDJ recombination, and the RAG genes, have been found in all Ig- bodies or possess Ig genes (reviewed in Ref. 19). The most primitive expressing vertebrates examined to date9. In contrast, class switch- of the fishes to display typical antibodies are the sharks and rays ing must have arisen later in vertebrate evolution, being absent in (elasmobranchs), which possess IgH loci of a unique ÔmulticlusterÕ fishes and present in the tetrapod lineage (amphibia, reptiles, birds arrangement, in which units primarily of (VH-D-D-JH-CH) are reand mammals). We argue here that the position of the enhancer in peated many times (reviewed in Ref. 19). Modern bony (or teleost) fish (reviewed in Refs 19, 20), and all the primordial IgH locus, as represented at the phylogenetic level of the bony fish (Fig. 1), would have precluded the development of higher vertebrates possess limited variations upon the well known class switching, and that additional appropriately positioned en- ÔtransloconÕ arrangement of (VH)n-(D)n-(JH)n-(CH)n, where the conhancers were necessary for class switching to emerge, as illustrated stant region genes can encode from two classes in the teleost fish21 to as many as five classes (and many subclasses) in the mammals for the mammalian IgH locus in Figs 1 and 2. A common feature of VDJ recombination and class switching is (Fig. 1). Teleosts, as members of the earliest vertebrate lineage to that both processes are preceded by the production of sterile (non- have the translocon IgH configuration, should provide useful inproductive) transcripts12,13. These transcripts originate at promoters sights into the organization of the primordial translocon IgH locus. upstream of the sites to be recombined (Fig. 2), under the regulation The best understood teleost IgH locus belongs to the channel catof transcriptional enhancers. It is widely held that the process of fish. This species has multiple VH genes in several families (at least transcription opens the DNA, thereby giving access to the respec- seven), multiple D segments and nine JH segments (Fig. 1). Howtive recombination complexes (reviewed in Ref. 14). While the en- ever, the only CH genes that have been identified encode m and a hancer(s) driving sterile transcript production must be sufficiently homologue of d (Ref. 21). PII: S0167-5699(98)01380-2

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representative of the earliest vertebrate lineage to have a translocon-type IgH locus, other observations support our contention that an Em39-like enhancer was the primordial enhancer. First, sequence analyses of the µÐδ intergenic regions in human and mouse indicate the presence of several enhancer motifs that might be remnants of the primordial enhancer23. Second, the catfish Em39 functions in a manner equivalent (in strength and B-cell specificity) to that of the murine Em enhancer22,23. Third, IgH enhancers that have been analysed (including Em39) share some common enhancer motifs including mEboxes, octamers, mA and mB, although not all of these have been shown to be functional22Ð24.

Origins of the JH–m enhancer If we accept that the primordial enhancer was Fig. 1. Schematic comparison of the locations of transcriptional enhancers within the IgH loci placed similarly to the Em39 of the catfish, then of the mouse and a teleost fish (catfish). Exon clusters encoding the constant regions of various how did enhancers arise in the JHÐm intron and isotypes are shown as single yellow boxes. The relative positions of switch regions are indicated the 39 region of the locus? Moreover, in what by green squares. Enhancers are shown as red ovals. The naming of the mammalian enhancers order did these events occur? Enhancers are is taken from Refs 10, 11. In the older nomenclature hs1,2 corresponds to 39a; hs3a to Ca39; known to arise by several processes including and hs3b to hs3. duplication, transposition or mutation of preexisting sequences25Ð27. We propose that the position of the transcriptional enhancer The most parsimonious model that can be envisioned would (Em39) in the catfish locus can explain why additional isotypes (be- have the development of the JHÐm intronic enhancer occurring first yond m and d), expressible by class switching, did not arise in the (Figs 3, 4). There are two possible means by which a part (or the teleost lineage. The Em39 enhancer straddles the 39 end of the m gene whole) of Em39 could have inserted into the JHе intron: one inand extends into the mÐd intergenic intron22,23 (Fig. 3). In consider- volves transposition, the other recombination. In terms of transpoing the location of Em39, we note that it is well placed to act as a fa- sition, Henikoff28 noted previously the presence of a Tc1/Marinercilitating element for VDJ recombination, as does the Eµ enhancer in like transposon remnant in the catfish IgH locus. This element, mammals (Fig. 1). Em39 is also well placed to drive the long primary which is bounded by 85 bp inverted repeats, encompasses the 59 transcripts from which the m and d mRNAs are generated by alter- end of the Em39 enhancer (Fig. 3) and includes enhancer motifs native RNA processing21. However, it has been shown that the found in the mammalian IgH enhancers. Could movement of this mammalian intronic enhancer, when placed in a position analogous transposable element into the JHÐm intron during early vertebrate to that of Em39, drives hypermutation less effectively3. In this regard evolution have provided the primordial Eµ enhancer? Evidence that it is interesting to note that bony fish show restricted diversity in such a transposition occurred was sought by comparing the setheir antibody responses, and while the definitive reason for this is quence of the remnant transposon in Em39 with that of the JHÐm unknown, a lack of somatic hypermutation has been discussed as intron of mammalian species. Sequences similar to those of the remnant transposon (up to 60% identity in stretches of ~100 nuone possible cause20. If a downstream CH cluster were to undergo a recombination to cleotides) were found to flank the intronic enhancers of both mouse the JHÐm intron, as occurs in class switching, then an enhancer in the and rabbit. In addition to its postulated role in generating the inposition occupied by Em39 would be excised along with the m and d tronic enhancer, transposition might have been the mechanism by regions (Fig. 2), leaving the IgH locus without a functional enhancer which other enhancers entered the IgH locus: as discussed later, the to drive transcription of the rearranged gene. Thus, to acquire class hs1,2 enhancer most likely arose by such an event10. Alternatively, the translocation of Em39 to the JHÐm intron could switching there must have been in place the following: (1) enhancers that are not excised during recombination, and (2) en- involve simple recombination. In some human leukemias, the 59 hancers that are proximal to the CH genes that undergo recombi- end of the mÐd intergenic region has undergone recombination to nation (that is, close enough to drive sterile transcription and the 59 end of the JHÐm intron29. This homologous recombination occurs between a 442 bp direct repeat found in both introns. However, somatic hypermutation). Was the enhancer of the primordial translocon IgH locus situated such a recombination would delete the intervening m exons. As similarly to that of the catfish? In addition to the fact that teleosts are such, a duplicated m region would have been required downstream

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(a) Remnant transposon in the 5' region of catfish Eµ3' TM1

TM2

(b) A possible origin of Eµ by transposition

JH8 JH9

Cµ1 Cµ2 Cµ3 Cµ4 TM1

C δ 1 Cδ 2

TM2 Eµ3'

Transposition

Fig. 2. Schematic of the class-switch process showing the sites for initiation of sterile transcription. (a) Class switching is preceded by production of sterile transcripts (green arrows) that originate at promoters (green circles) upstream of the sites that participate in the recombination event. Sterile transcript production is regulated in part by enhancers (red ovals). Each constant region gene, except d, also has at its 59 margin a switch (S) region sequence (green square) and the small I exon (not shown). (b) Class-switch recombination produces a circular excision product and, in this example, a message encoding e-chain. Productive pre-mRNA transcripts (purple arrows) originate 59 of the recombined VDJ.

of the enhancer, before the translocation event, if a functional IgH locus were to remain. In mammals the m gene and all of its derivative CH clusters except d have class-switch region sequences (S), the class-switch-related I exon and a promoter in their 59 flank (reviewed in Ref. 14). Thus, it seems likely that these elements were assembled upstream of m before it was duplicated to generate the 39 CH gene clusters (Fig. 4). We cannot predict whether the transposition of an enhancer to the JHÐm intron preceded the development of the S region and its associated elements, although one attractive feature of this model is that transcriptionally active sites, transposons and S regions can all be targets for insertion of, or recombination with, exogenous DNA (Refs 30Ð33). Therefore, the integration of one of these elements into the JHÐm intron would be conducive to the acquisition of additional elements.

Origins of the 39 enhancers The acquisition of an enhancer in the JHÐm intron would eliminate the problem of enhancer deletion following class switching. However, the 39 enhancers in the mammalian locus are still essential for class switching to particular isotypes. In targeted deletion experiments, CognŽ et al.2 observed that elimination of the murine hs1,2 (39a) enhancer greatly depressed production of certain CH sterile

Fig. 3. The catfish IgH enhancer includes a remnant transposon. The enhancer motifs (red) that comprise the catfish Em39 enhancer extend from the area between the two m transmembrane exons (TM1 and TM2) into the mÐd intergenic region. (a) Location of the remnant Tc1/Mariner-like transposon sequence (blue) and its flanking 85 bp inverted repeats (green). This remnant transposon (shaded oval) encompasses several enhancer motifs that are integral to IgH enhancer function (octamers, mE5 and mB). Elements of the locus are not shown to scale. Organization of the transposon is based on Henikoff 28. (b) In the ancestral IgH locus, a functional transposon (shaded oval, containing three enhancer motifs in red) might have translocated to the JHÐm intron, carrying with it enhancer function.

transcripts, which resulted in a large reduction in switch rearrangements. Thus, development of the class switch process probably required the prior acquisition of 39 enhancers. We suggest that the multiple 39 enhancers seen in the mammalian locus arose by two pathways: one was the duplication process from which novel C region genes arose, and the other was transposition. In the first case, we would argue that the original duplication of the m gene also involved its 39 flanking region. Thus, the duplicated m gene that developed ultimately into a C gene of novel isotype, carried with it either part or all of the original Em39 enhancer, which in this manner would give rise to 39 enhancer function (Fig. 4). An evolutionarily more recent example of an enhancer undergoing duplication along with a C-region gene comes from the human a genes. The duplication that produced the human a1 and a2 genes also included the hs1,2 enhancer34. In terms of a transpositional origin of 39 enhancers, there seems little doubt that the hs1,2 enhancer entered the IgH locus by this mechanism. The mouse hs1,2 enhancer is flanked by inverted repeat sequences35, consistent with it having inserted into this region, perhaps as a transposon10. Furthermore, the homologous enhancer downstream of the rat IgH locus resembles hs1,2 in nearly every respect, except for the fact that its orientation has been inverted17. How then did the other 39 enhancers arise? One informative observation is that the mouse hs3a (Ca39) and hs3b enhancers, which flank hs1,2, are virtual mirror images of one another, and are themselves flanked by inverted repeats10. The insertion of the hs1,2 enhancer might have been associated with the duplication and inversion of this

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region10. The hs4 enhancer, by virtue of its shared transcription factor-binding motifs24, could share a common origin with the other enhancers, although there is insufficient information available to deduce its likely history within the IgH locus.

Concluding remarks We have proposed that the failure to evolve the class-switch process in the IgH locus of vertebrates below the phylogenetic level of amphibia was linked to the absence of appropriately placed enhancers capable of facilitating this process. Determination of the genomic structure and enhancer function of the IgH locus of amphibians (the earliest diverged and still surviving vertebrate lineage to possess class switching) should test our models of enhancer translocation during IgH evolution.

Original work of the authors cited here was supported by awards from the National Science Foundation MCB 9406249 and MCB 9722996.

Brad Magor ([email protected]) is at the Hopkins Marine Station, Stanford University, Pacific Grove, CA 93950, USA; Lars Pilstršm is at the Dept of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Uppsala S-751 23, Sweden; David Ross and Gregory Warr ([email protected]) are at the Dept of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29424-2211, USA. References 01 Serwe, M. and Sablitsky, F. (1993) EMBO J. 12, 2321Ð2327

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Fig. 4. A model for the evolutionary expansion of the IgH locus, showing the acquisition of the elements necessary for the emergence of class switching. (a) Em39 drives VDJ recombination and expression of the m and d genes. (b) A duplication of the m gene gives rise to d. (c) Em39 drives VDJ recombination and the expression of m and d. Acquisition of the enhancer in the JHÐm intron, and of the switch region (S) precede the expansion to a class switch competent locus. (d) Duplication and transposition of the S/m/ Em39 unit gives rise to a C-region gene, in the 39 region of the locus, which can participate in class switching.

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Does properdin crosslink the cellular and the humoral immune response? Wilhelm J. Schwaeble and Kenneth B.M. Reid Properdin, the only known positive regulator of complement activation, is produced by cells of the roperdin was first described by C5 convertase complexes [C3bBb and Louis Pillemer and co-workers (C3b)nBb]2,3. Its essential role is demonstrated monocyteÐmacrophage, T-cell and by the inability of properdin-depleted sera to in 1954 as a novel plasma comgranulocyte lineages. Complement activate complement via the alternative pathponent that activates compleactivation products have an way. Properdin is present in plasma at quite ment in the absence of immune complexes1. This discovery, in particular the physiologilow concentrations (5Ð15 mg ml21). The effiestablished role in the cellular ciency by which this component promotes cal potential that was ascribed to properdin, immune response. However, here, complement activation is illustrated by the generated considerable interest. However, observation that the addition of purified PillemerÕs views were not generally acWilhelm Schwaeble and Kenneth properdin can restore alternative pathway cepted by immunologists at the time as the Reid discuss current data activation in properdin-deficient sera in a interpretation of his experimental data condemonstrating that leukocytes form dose-dependent fashion (with complete troversially contradicted the existing dogma, restoration requiring approximately 50% of established by Paul Ehrlich, according to a positive feedback loop of activation the normal serum concentration). Thus, local which complement activation was strictly an by properdin release. changes of properdin levels could signifiimmune-complex mediated process. Howcantly modulate the steady state of alternaever, more than a decade later, PillemerÕs tive pathway activation. hypothesis was verified by the subsequent Properdin is a polydisperse mixture of cyclic polymers formed characterization of no less than six plasma components that form an antibody-independent route of complement activation, known as the by head to tail associations of a single chain glycoprotein (approxialternative pathway2. Properdin has been identified as the essential mately 53 kDa) as dimers, trimers and tetramers, in a constant ratio positive regulatory component of this activation route. of 20 : 54 : 26 (Ref. 4). The functional activity of the polymers increases with their size, such that the tetramer is estimated to be ten times as active as the dimer5. Properdin monomers are absent from Function and structure of properdin plasma and cell culture supernatants, whereas polymers are present Properdin ensures optimal rates of alternative pathway activation by at constant ratios6. This implies that the formation of polymers is virtue of its ability to bind and stabilize the inherently labile C3 and a carefully controlled intracellular process that maintains a well

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