Regulatory Divide Between Agnathans And Gnathostomes

In the case of the adaptive immune system, several critical innovations appear to have occured only after the divergence of the ancestors of jawed and jawless vertebrates, as already noted. Among these changes are alterations and expansions of the repertoires of transcription factors available to direct lymphocyte differentiation programs. There are numerous examples of transcription factors encoded by a single gene in invertebrates up to cephalochordates, but by a family of three or four members in bony vertebrates (reviewed in ref. 124). The expansion of these gene families and the evolutionary specialization of their expression patterns and "downstream" functions greatly enhances the potential regulatory sophistication in vertebrates. PU.l, and possibly Ikaros as well, are members of families that underwent threefold expansion well after the origin of vertebrates, between the divergence of agnathan and gnathostome vertebrates and the divergence of bony and cartilaginous fish.

PU.1 is a valuable probe for evolutionary shifts in transcription factor function because of its relation to the rest of the Ets family and because of its well-mapped domain structure. The PU.l/Spi (spleen focus-forming virus preferential integration site) subfamily of Ets factors uses an easily recognizable Ets domain to bind to DNA, but this version of the Ets domain sequence is distinctive enough to define one of the five major subdivisions of Ets factors (125). In PU.1 itself there are additional discrete, well-mapped "activation" domains carrying out separate functions. The region N-ter-minal to the Ets domain contains acidic and glutamine-rich transactivation domains and a PEST domain, each of which carries out interactions with different transcrip-tional partners in a modular fashion (117,118). As a positive regulator, PU.1 uses these interaction domains to synergize powerfully with other developmentally regulated factors for activation of different batteries of cell type-specific target genes. PU.1 can also act as an important negative regulator, for example, by a mutually inhibitory protein/protein interaction with hematopoietic GATA factors (126-129). GATA/PU.1 competition is thought to help regulate erythroid vs. myeloid specification of pluripo-tent precursors and could conceivably play a role in T-cell development as well (123,130). Thus, the complex and central function of PU.1 in the hematopoietic system depends on more than its DNA-binding Ets domain. Conservation of the structure of each of its non-Ets domains may be an indicator of conservation of PU.1 activity in a different regulatory context.

The PU.1 Ets domain is conserved throughout the vertebrate radiation. There is an indisputable PU.1/Spi family member even in the agnathan lamprey (Petromyzon mari-nus) (131). Cartilaginous fish have a whole family of three PU.1/Spi members, like mammals (123). By contrast, the PU.1 Ets domain cannot be detected in any invertebrate to date. No member of this subfamily of Ets factors is encoded in the Drosophila or C. elegans genome sequences, and even deuterostome invertebrates seem to lack such genes. Multiple attempts to detect a family member in sea urchin genomic DNA or cDNA have been unseccessful (M.K. Anderson, X. Sun, R. Pant, and E.V.R., unpublished data). Thus, the divergence of this PU.1/Spi subfamily from other Ets factor genes is probably a vertebrate-specific innovation.

Phylogenetic comparison yields another interesting result, namely, evidence for a further discontinuity, occuring after the origin of the PU.1/Spi Ets domain. The sequence of the transcription factor gene itself provides structural evidence for an evolutionary shift in function. The protein-interaction domains of lamprey Spi do not maintain the organization of any other PU.1/Spi family member (123,131) (Fig. 6, middle and bottom). However, in the cartilaginous fish Raja eglanteria, there is a PU.1 ortholog with a sequence that meticulously corresponds to the mammalian, avian, and teleost fish versions in every domain (Fig. 6, top), and another Raja family member that resembles, throughout its length, both PU.1 and the closely related factor, SpiB (132). This implies that the transcription factor interaction circuits in which PU.1 (and SpiB) take part are older than the chondrichthyan divergence, about 450 Mya, but that for the most part they arose since the divergence between the agnathans and gnathostomes

Lamprey DiseaseSkate Fish Infection

Fig. 6. A discontinuity in conservation of PU.l/Spi structural domains between lampreys and cartilaginous fish. Sequences are from refs. 131 and 132. (Top) Alignment of skate PU.l (sequence 1, gi: 11245498) with human PU.l (sequence 2, gi:36561). The same results are obtained using murine instead of human PU.1. (Middle) Alignment of lamprey PU.1 (sequence 1, gi:8748404) with mouse PU.1 (sequence 2, gi: 111187). Only the Ets domain is detectably similar. (Bottom) Alignment of lamprey PU.1 (sequence 1) with human SpiB (sequence 2, gi:36563). Some similarity is detectable in the PEST domain as well as the Ets domain. Alignments were done by BLASTP 2.1.2 using the BLOSUM62 matrix with penalties of 11 for gap opening and 1 for gap extension, a wordsize of 3, x_dropoff of 50 and "expect" value of 300.

Fig. 6. A discontinuity in conservation of PU.l/Spi structural domains between lampreys and cartilaginous fish. Sequences are from refs. 131 and 132. (Top) Alignment of skate PU.l (sequence 1, gi: 11245498) with human PU.l (sequence 2, gi:36561). The same results are obtained using murine instead of human PU.1. (Middle) Alignment of lamprey PU.1 (sequence 1, gi:8748404) with mouse PU.1 (sequence 2, gi: 111187). Only the Ets domain is detectably similar. (Bottom) Alignment of lamprey PU.1 (sequence 1) with human SpiB (sequence 2, gi:36563). Some similarity is detectable in the PEST domain as well as the Ets domain. Alignments were done by BLASTP 2.1.2 using the BLOSUM62 matrix with penalties of 11 for gap opening and 1 for gap extension, a wordsize of 3, x_dropoff of 50 and "expect" value of 300.

approx. 50-100 My earlier. As already seen, this is the same phylogenetic interval in which the components of the adaptive immune system became established in vertebrates.

5.2. PU.1 and the Agnathan/Gnathostome Divide in Immune System Function

Cartilaginous fish have all the components of an adaptive immune system that have been examined so far. Not only do they have RAG genes, immunoglobulins, T-cell receptors, and MHC molecules, but they also have well-organized lymphoid organs, including a thymus that is homologous to that of bony vertebrates (133). Consistent with a conserved developmental role for PU.1, they have a well-differentiated PU.1/Spi transcription factor family, and this is expressed, as in mammals, in the tissues that harbor B-cell and macrophage cell types.

In contrast, agnathans still have not revealed any molecular traces of an adaptive immune system, although cell morphologies and tissues suggest some links to gnathos-tome counterparts. Searches for genes encoding adaptive immune system receptors, histocompatibility antigens, and recombinases in the lamprey have remained uniformly unsuccessful even with recent technologies (57). The lamprey does have round hematopoietic cells that have been referred to as possible lymphocytes, but there is no molecular evidence to distinguish them from any other kind of small, nongranular circulating cell (134).

The single, highly divergent PU.1/Spi family member in the lamprey could be associated with the lymphocyte-like cells (131), but the identities of any target genes are a complete mystery. A critical question is whether these cells share anything other than the expression of a PU.1/Spi family factor with mammalian lymphocytes; it seems unlikely from its structure that lamprey Spi can participate in most of the transcription factor interaction circuits that give mammalian hematopoietic cells their identities. There is some weak similarity between the lamprey Spi and mammalian SpiB within the PEST domain of the latter (Fig. 6, bottom). This is the domain in which interferon regulatory factors (IRF4 or interferon consensus sequence binding protein [ICSBP]) interact with PU.1 (or SpiB) to turn on common target genes. Initially, this kind of interaction was defined as important for immunoglobulin light chain gene expression in later stages of B-cell development (135), but more recently it has been shown to play an important role in macrophage gene expression as well (136,137). If this PEST domain interaction is significant, it could indicate that IRFs are the most ancient interaction partners of PU.1/Spi family transcription factors.

5.3. Use and Modification of Other Transcription Factor Innovations

Ikaros is another gene that is needed for early T-cell development in the mammalian fetus, and for all B-cell (and NK-cell) development. Although it is dispensible for myeloid and erythroid cell development, and thus is distinct from PU.1, its role in establishing a normal stem cell pool (113) implies that lymphocytes require some overlapping or common function(s) shared with stem cells. Ikaros, like PU.1, is a member of a small family of related genes, all members of which are found in vertebrates from mammals to cartilaginous fish (138,139) In all these animals, Ikaros family members appear to have homologous sites of expression, suggesting a linkage to hematopoietic cell development throughout jawed vertebrates. Only a single member of the family has been found to date in lamprey (139). Here the lamprey "Ikaros" sequence is much more similar to those of members of the family seen in jawed vertebrates than is the case for the lamprey Spi, suggesting that the function of Ikaros should be conserved in the lamprey even if the lack of related genes with overlapping functions limits the complexity of the regulatory patterns. As in the case of PU.1, however, no Ikaros family member has been found yet in sea urchins (J.P. Rast, personal communication), and only the sequences encoding individual hunchback-type zinc finger domains can be recognized in the genome sequences of flies or nema-todes. Thus it may be that the functions mediated by this transcription factor family are also vertebrate innovations.

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