Brachyenteron/T-related gene

Transcriptional Regulation

Tailless activatesTrg expression. Huckebein represses Trg in the posterior tip of the embryo. Trg expression is independent of forkhead, suggesting fkh and Trg might act in parallel in the development of the hindgut. When the proctodeum has fully invaginated, Serpent takes over the role of hkb in repressing Trg in the primordium of the posterior midgut (Kispert, 1994 and Murakami, 1995). Trg is not expressed in embryos lacking maternal signals from Torso or Trunk, indicating that Trg is under the control of maternal terminal genes (Murakami, 1995).

Tailless protein activates the seventh stripe of expression of at least three pair-rule genes: even-skipped, hairy and fushi tarazu. tailless alleles can be placed in the same order of phenotypic strength on the basis of the deletion of either external structures (A8 and anal pads) or an internal structure (hindugt). Furthermore, different tll alleles result in a gradation in levels of brachyenteron expression and these levels of Bry expression are correlated with the size of the differentiated hindgut (Diaz, 1996).

The terminal portions of the Drosophila body pattern are specified by the localized activity of the receptor tyrosine kinase Torso (Tor) at each pole of the early embryo. Tor activity elicits the transcription of two 'gap' genes, tailless (tll) and huckebein (hkb), in overlapping but distinct domains by stimulating the Ras signal transduction pathway. Quantitative variations in the level of Ras activity can specify qualitatively distinct transcriptional and morphological responses. Low levels of Ras activity at the posterior pole direct tll but not hkb transcription; higher levels drive transcription of both genes. Correspondingly, low levels of Ras activity specify a limited subset of posterior terminal structures, whereas higher levels specify a larger subset. When a constitutively active 1X RasV12 gene is expressed in torso mutant embryos, brachyenteron (byn) is expressed in a small terminal cap, whereas the domain of expression in 2X RasV12 is much broader. Because both the activation and repression of terminal byn expression is known to depend, respectively, on tll and hkb, it is surmised that higher levels of Ras activity are required at the posterior of wild-type (ras+) embryos to drive sufficiently high levels of Hkb expression to repress byn expression (a phenomenon not observed with even 2X RasV12 ectopic expression). 1X RasV12 forms the least terminal of the posterior terminal structures: the eighth abdominal dentical band and the posterior spiracles. The extent of restoration is considerably greater in 2X RasV12 embryos: these form additional terminal structures such as the anal tuft and anal pads.The response to Ras activity is not uniform along the body. Instead, levels of Ras activity that suffice to drive tll and hkb transcription at the posterior pole fail to drive their expression in more central portions of the body, apparently due to repression by other gap gene products. The levels of Huckebein and/or Kruppel through the embryo might be responsible for a failure to express hkb in response to moderate RasV12 activity. It is concluded that tll and hkb transcription, as well as the terminal structures, are specified by two inputs: a gradient of Ras activity, which emanates from the pole, and the opposing influence of more centrally deployed gap genes, which repress the response to Ras (Greenwood, 1997).

During early embryogenesis in Drosophila, Caudal mRNA is distributed as a gradient with its highest level at the posterior of the embryo. This suggests that the Caudal homeodomain transcription factor might play a role in establishing the posterior domains of the embryo, which undergo gastrulation and give rise to the posterior gut. By generating embryos lacking both the maternal and zygotic mRNA contribution, caudal has been shown to be essential for invagination of the hindgut primordium and for further specification and development of the hindgut. Mature embryos lacking cad activity (maternal and/or zygotic contributions) were examined to assess the requirement for cad in establishing the structures that arise from the posterior ~15% of the blastoderm embryo, namely the posterior midgut, Malpighian tubules and hindgut.

The stages of gastrulation can be observationally followed by using expression of brachyenteron byn as a marker for the hindgut primordium. In the wild-type embryo, byn is expressed in a ring at the circumference of the amnioproctodeal plate. The edges of this ring come together as the posterior midgut primordium invaginates during stages 6 and 7; the ring of the hindgut primordium then sinks inward during stage 8 and is completely internalized by the end of stage 9. The zygotically expressed cad stripe and the posterior wg stripe are also expressed in the bordering ring (i.e., the hindgut primordium) of the invaginating amnioproctodeal plate. Strikingly, in cad-deficient embryos, the byn-expressing ring of hindgut primordium draws together, but fails to invaginate, remaining on the outside of the embryo. Thus, although internalization of the Malpighian tubule and posterior midgut primordia is normal in cad-deficient embryos, the gastrulation movements necessary for internalization of the hindgut primordium do not occur in embryos lacking cad activity (Wu, 1998).

The absence of the hindgut primordium from cad-deficient embryos suggests that Caudal regulates genes required for establishing and/or maintaining the hindgut primordium. tailless, forkhed, byn, bowl and wingless are likely targets for cad regulation, since all are required for some aspect of hindgut development: the hindgut is missing from both tll and fkh embryos, and severely reduced in wg, byn and bowl embryos. bowl, also called bowel, codes for a zinc finger transcription factor related to odd-skipped. Since maternally provided Caudal, which persists only through the blastoderm stage, is sufficient for essentially normal hindgut formation, the fact that all of these genes are expressed at the posterior of the embryo during the blastoderm stage means that they are potential targets for regulation by Caudal. The effect of absence of maternal and/or zygotic cad activity on the expression of these genes was assessed by in situ hybridization with appropriate probes. For tll, byn and bowl, absence of cad activity does not result in a detectable effect on expression. As described below, however, cad activity is essential for expression of fkh and wg. Both maternal and zygotic cad contributions are necessary for posterior wg expression. During early stage 5, just prior to its expression in 14 stripes that are required to establish the segmental pattern, wg is expressed in two domains at the anterior, and in a broad posterior stripe. This terminal wg stripe is located at approximately 8-12% EL, overlapping with the posterior of the zygotic cad stripe and with the position of the hindgut and Malpighian tubule primordia in the blastoderm fate map. Expression of the wg terminal stripe has been shown to be independent of other segmentation genes, but has not been otherwise characterized. All embryos from mutant cad germline mothers (even those expressing zygotic cad) fail to express the terminal stripe of wg. These results demonstrate that maternal cad activity is essential for the transcription of wg in the terminal stripe. Among embryos from wg heterozygous parents, approximately one-quarter (presumably those lacking only the zygotic component of cad expression) lack the terminal wg stripe. Thus both maternal and zygotic cad activities are required for expression of the terminal wg stripe (Wu, 1998).

The expression of the early cap of fkh also requires cad activity; approximately half of the embryos from mutant cad germline females mated to cad heterozygous males (i. e., cad m-z - embryos) show a dramatic reduction in both the size and intensity of the posterior cap of fkh expression. However, if cad is supplied either maternally or zygotically, fkh expression is normal. Thus expression of the posterior cap of fkh requires cad activity, which can be provided either maternally or zygotically. Later, by stage 10, fkh expression is as strong in cad-deficient as in wild-type embryos, indicating that this later expression is independent of cad activity. Since tll and hkb are also required to activate early fkh expression but are not themselves regulated by cad, cad must act combinatorially with these two genes to promote early fkh expression (Wu, 1998).

cad also regulates wg in combination with other genes. In addition to the demonstrated requirement for cad, expression of the posterior wg stripe requires positive input from fkh and tll, since the stripe is absent from the respective mutant embryos. Since embryos lacking either maternal or zygotic cad fail to express the posterior wg stripe, but still express fkh and tll, cad must act combinatorially with fkh and tll to promote formation of the posterior wg stripe. Expression of the terminal stripe thus requires the combinatorial action of cad, tll and fkh; the posterior limit of the stripe is known to be defined by repression by hkb (Wu, 1998).

The failure of the hindgut to become internalized in caudal-deficient embryos raises the question of whether cad might regulate a zygotically expressed gene required for the invagination of the amnioproctodeal plate. One gene known to be required for gastrulation is fog; fog mutant embryos lack not only the posterior midgut, but, as revealed by anti-Crb staining, the Malpighian tubules and hindgut as well. In the blastoderm stage embryo, fog expression is first activated in the region that will become the ventral furrow; shortly thereafter, expression is initiated in a posterior cap, in the region that will become the amnioproctodeal invagination. In cad-deficient embryos, fog expression in the prospective ventral furrow is normal, but is significantly reduced in the posterior cap. Thus, cad is required for the normal level of expression of fog in the prospective amnioproctodeal plate; decreased fog expression in cad-deficient embryos is likely responsible for the failure of the hindgut primordium to be internalized during gastrulation. Since fkh or wg mutant embryos do not display detectable defects in gastrulation, fog is the only gene presently known to mediate the effects of cad on gastrulation. In fog mutant embryos, none of the posterior gut primordia invaginate, while in cad-deficient embryos the posterior midgut and Malpighian tubule primordium do invaginate; thus, consistent with the in situ hybridization results, a low level of fog activity is present at the posterior of embryos lacking cad (Wu, 1998).

In addition to cad, three other genes (fkh, byn and wg,), which are required at the posterior of the Drosophila embryo for formation of the hindgut, are related to genes found throughout the metazoa, known as HNF-3 (alpha, beta, and gamma), Brachyury (also known as T) and Wnt, respectively. In many cases, these homologs are expressed in portions of the Îblastopore equivalent' at the posterior of the embryo, that overlap with domains of expression of cad (Cdx). In C. elegans, a Wnt homolog is expressed, and required for proper posterior development, in the same posterior blastomere where the cad homolog pal-1 functions. In sea urchin, HNF-3 and Brachyury homologs are expressed in the vegetal plate just prior to gastrulation. In fish and frog, Caudal, Brachyury and Wnt (Wnt8 and Wnt11) are initially expressed around most or all of the blastopore lip while HNF-3 expression is dorsally localized. As gastrulation proceeds, the expression of these genes becomes more restricted and non-overlapping, with HNF-3 and Brachyury expression becoming localized to the notochord and Wnt8 expression retreating from the dorsal position and becoming exclusively ventral. Patterns of expression of HNF-3 and Brachyury consistent with this general description have been found in ascidians (phylum Urochordata), amphioxus (cephalochordates of the phylum Chordata), chick and mouse. Required roles for some of these genes have been demonstrated by analysis of mutants: mouse HNF-3beta knockouts reveal requirements in the formation of the node, notochord and head process; fish no tail and mouse T mutants reveal a requirement for Brachyury in migration of mesoderm through the primitive streak and in formation of the notochord. There is thus a constellation of conserved genes -- cad (Cdx), fkh (HNF-3), wg (Wnt8 and Wnt11) and byn (Brachyury) -- whose overlapping expression patterns in the blastopore equivalent suggests function in a related process. The phenotypes of the available mutations in these genes suggest that the common function is to specify cell fate at the blastopore; in most cases, essential parts of this fate are internalization and forward migration, two of the cellular movements that occur during gastrulation (Wu, 1998 and references).

The striking conservation in expression (and likely in function) of cad suggests that the regulation of posterior terminal development in Drosophila by Caudal may represent a more ancient regulatory mechanism than the tor receptor and the two genes that it activates: tll and hkb. Of these three genes, a vertebrate homolog is known only for tll; the function of this vertebrate gene, Tlx, is related to that of Drosophila tll not in the posterior, but rather in the anterior, in the establishment of the brain. Thus the Torso receptor pathway and its activation of tll and hkb has probably been superimposed relatively recently (in evolutionary terms) upon a more ancient, Caudal-regulated network of gene activity controlling gastrulation and gut formation. The fact that the same four genes are expressed at both the blastopore equivalent of chordates and at the amnioproctodeal invagination of Drosophila suggests that these two highly dynamic domains are homologous. Given the regulatory hierarchy that is present in Drosophila, it is proposed that in embryos of the proximate ancestor to arthropods and chordates, the posterior was defined by a posterior-to-anterior gradient of Cad activity. Cad is thought to have then activated expression of downstream network of genes in control of invagination (gastrulation) and gut specification. Cad expression in the archenteron probably continued during evolution and played an essential developmental role, since this structure differentiated into the gut. Going beyond the bilaterian ancestor to chordates and arthropods, it is worth considering that this nexus of gene expression may have evolved even more basally in the metazoa. The foregoing, by homologizing the insect amnioproctodeal invagination with the echinoderm and vertebrate blastopore, does not fit with the classical definition of protostomes and deuterostomes. This view categorizes arthropods as protostomes, in which the mouth is derived from the primary invagination of gastrulation; chordates are categorized as deuterostomes, where the mouth arises from a secondary invagination. More recently, comparisons of gastrulation patterns in many different species, as well as construction of molecularly based cladograms, have called into question the utility of these classically defined groups. While there continues to be uncertainty in understanding of 'protostome' and 'deuterostome' phyla, the significant conclusion of the information presented here is that there may be a homology between the blastopore of vertebrates and the amnioproctodeal (posterior) invagination of insects (Wu, 1998 and references).

The Groucho corepressor mediates negative transcriptional regulation in association with various DNA-binding proteins in diverse developmental contexts. Groucho has been implicated in Drosophila embryonic terminal patterning: it is required to confine tailless and huckebein terminal gap gene expression to the pole regions of the embryo. An additional requirement for Groucho in this developmental process has been revealed by establishing that Groucho mediates repressor activity of the Huckebein protein. Putative Huckebein target genes are derepressed in embryos lacking maternal groucho activity and biochemical experiments demonstrate that Huckebein physically interacts with Groucho. Using an in vivo repression assay, a functional repressor domain in Huckebein that has been identified contains an FRPW tetrapeptide, similar to the WRPW Groucho-recruitment domain found in Hairy-related repressor proteins. Mutations in Huckebein’s FRPW motif abolish Groucho binding and in vivo repression activity, indicating that binding of Groucho through the FRPW motif is required for the repressor function of Huckebein. Thus Groucho-repression regulates sequential aspects of terminal patterning in Drosophila (Goldstein, 1999).

One proposed Hkb target is the snail (sna) gene, which is transcribed in the ventral-most portion of the embryo. sna expression is thought to be excluded from the posterior pole by hkb activity. Accordingly, sna and hkb expression domains abut in cellularizing wild-type embryos, whereas sna expression extends to, and includes, the posterior pole of hkb mutant embryos. In tor^D embryos, hkb expression expands towards the center of the embryo and the sna domain correspondingly retracts. By contrast, in gro^mat- embryos, the expression of sna does not respect the sna posterior border and spreads to the pole, overlapping extensively with the hkb expression domain. The expression of the T-related gene brachyenteron (byn; also called Trg) also seems to be repressed by Hkb. byn is not expressed at the most posterior region of wild-type (or tor^D) embryos, whereas it extends throughout the posterior cap of hkb mutant embryos, consistent with hkb setting the posterior limit of byn expression. However, it is found that byn is ectopically expressed at the posterior tip of gro^mat- embryos. Together, these results suggest that gro is, directly or indirectly, necessary for hkb repressor functions (Goldstein, 1999).

GATA factors play an essential role in endodermal specification in both protostomes and deuterostomes. In Drosophila, the GATA factor gene serpent (srp) is critical for differentiation of the endoderm. However, the expression of srp disappears around stage 11, which is much earlier than overt differentiation occurs in the midgut, an entirely endodermal organ. Another endoderm-specific Drosophila GATA factor gene, GATAe, has been identified. Expression of GATAe is first detected at stage 8 in the endoderm , and its expression continues in the endodermal midgut throughout the life cycle. srp is required for expression of GATAe, and misexpression of srp resulted in ectopic GATAe expression. Embryos that either lack GATAe or have been injected with double-stranded RNA (dsRNA) corresponding to GATAe fail to express marker genes that are characteristic of differentiated midgut. Conversely, overexpression of GATAe induces ectopic expression of endodermal markers even in the absence of srp activity. Transfection of the GATAe cDNA also induces endodermal markers in Drosophila S2 cells. These studies provide an outline of the genetic pathway that establishes the endoderm in Drosophila. This pathway is triggered by sequential signaling through the maternal torso gene, a terminal gap gene, huckebein (hkb), and finally, two GATA factor genes, srp and GATAe (Okumura, 2005).

In the posterior terminal region of the blastoderm, prospective regions of the posterior endoderm and hindgut abut each other. byn is responsible for determination of the prospective hindgut. byn is expressed throughout the prospective posterior endoderm during early stage 5, but soon disappears in this region. It is the srp gene that represses byn in the prospective endoderm. Thus, the boundary between the endoderm and hindgut is established by the repressive activity of srp on byn. Whether GATAe also represses byn was examined, since GATAe continues to be expressed after endodermal srp expression ceases. Ectopic expression of byn in the prospective endodermal domain is observed in embryos that lack GATAe, although the area of ectopic expression is much smaller than that observed in the srp mutant, in which ectopic expression of byn is observed throughout the entire prospective endoderm. Moreover, when misexpressed in the prospective hindgut domain, both GATAe and srp strongly repress byn expression. Thus, GATAe is required to maintain the endodermal identity that is initially established by srp (Okumura, 2005).

Targets of Activity

brachyenteron expression is activated by tailless, but Trg does not regulate itself. Trg expression in the hindgut and anal pad primordia is required for the regulation of genes encoding transcription factors (even-skipped, engrailed, caudal, AbdominalB and orthopedia) and cell signaling molecules (wingless and decapentaplegic). In Trg mutant embryos, the defective program of gene activity in these primordia is followed by apoptosis (initiated by reaper expression and completed by macrophage engulfment), resulting in severely reduced hindgut and anal pads. In Trg mutants, orthopedia is not expressed in the proctodeum, while neural expression of otp is unaffected. Early eve expression is unaffected in Trg mutants. By stage 8, however, eve expression in the anal pad primordium is almost entirely absent from Trg mutants. Only anal pad expression of cad requires Trg; expression in Malpighian tubules and hindgut is not affected. dpp expression is absent in the hindgut. Although Trg is not expressed in the midgut or the Malpighian tubules, it is required for the formation of midgut constrictions and for the elongation of the Malpighian tubules. Females mutant for Trg are sterile, and no egg chambers in their ovaries progress beyond stage 7 of ovarian development (Singer, 1996).

By examining expression of arc in different mutant embryos, it was determined that transcription factors known to be required for patterning and maintenance of various developing epithelia control arc expression in those domains. tll and hkb, which are required to pattern the posterior 15% of the embryo, control arc expression in the posterior midgut primordium. fkh, which appears to act as a maintenance, or permissive, transcription factor, is required for expression of arc throughout the gut. byn, which is required for hindgut development and specifies its central domain (the large intestine), controls expression of arc in the elongating hindgut. Kr and cut, required for evagination and extension of the Malpighian tubule buds control expression of arc in the tubule primordia (Liu, 2000).

Brachyury proteins regulate target genes through modular binding sites in a cooperative fashion

Brachyury proteins, a conserved subgroup of the T domain transcription factors, specify gut and posterior mesoderm derivatives throughout the animal kingdom. The T domain confers DNA-binding properties to Brachyury proteins, but little is known how these proteins regulate their target genes. A direct target gene of the Drosophila Brachyury-homolog Brachyenteron has been characterized. Brachyenteron activates the homeobox gene orthopedia (otp; Simeone, 1994) in a dose-dependent manner via multiple binding sites with the consensus (A/G)(A/T)(A/T)NTN(A/G)CAC(C/T)T. The sites and their A/T-rich flanking regions are conserved between D. melanogaster and Drosophila virilis. Reporter assays and site-directed mutagenesis demonstrate that Brachyenteron binding sites confer in part additive, in part synergistic effects on otp transcription levels. This suggests an interaction of Brachyenteron proteins on the DNA, that maps to a conserved motif within the T domain. Mouse Brachyury also interacts with Brachyenteron through this motif. The Xenopus and mouse Brachyury homologs activate orthopedia expression when expressed in Drosophila embryonic cells. It is proposed that the mechanisms to achieve target gene expression through variable binding sites and through defined protein-protein interactions might be conserved for Brachyury relatives (Kusch, 2002).

Based on its expression pattern, the homeobox gene otp is a likely candidate for a direct target of Byn. otp mRNA expression becomes detectable shortly after the onset of byn expression in the common primordium of hindgut and anal pads (in the following only referred to as the hindgut primordium). byn is necessary for the gut-specific otp expression and can activate ectopic otp expression when overexpressed by means of the GAL4/UAS system. The otp locus has been characterized and two transcription start sites were found, one for hindgut-specific transcripts, another ~5.8 kb further upstream for CNS-specific transcripts. During later stages of embryogenesis otp becomes expressed in the CNS, an aspect independent of byn (Kusch, 2002 and references therein).

To identify the hindgut-specific regulatory elements within the otp locus, various genomic fragments of the locus were fused to lacZ reporters and the transgenic embryos were monitored for ß-galactosidase expression in the hindgut. A 1.8-kb XhoI/EcoRI fragment ~950 bp upstream of the putative hindgut-specific transcription start site confers lacZ expression in the hindgut primordium in a pattern identical to endogenous otp. lacZ is strongly expressed throughout the hindgut including anal pads, at lower levels at the distal part of the hindgut tube close to the pads, and is not found in the small intestine of the hindgut. Thus, the element contains all the information for proper regulation of otp. A further subdivision of the 1.8-kb element yielded three regulatory fragments: a proximal, a central, and a distal. The proximal (P_1310-1817) and the central (C_872-1309 ) fragment were sufficient to direct hindgut-specific lacZ expression in an otp-specific pattern, indicating that the gut expression of otp is regulated by at least two independent Byn-responsive elements. The third, distal fragment (D_1-871) did not give detectable lacZ expression in the embryo (Kusch, 2002).

Does Byn directly regulate otp expression via the C_872-1309 and the P_1310-1817 fragments and what do possible Byn binding sites look like? To address these questions, DNase I protection experiments were performed and three protected regions were found in the C_872-1309 and two in the P_1310-1817 fragment. Three of these regions consist of two binding sites, which are arranged in tandem repeats (viii/ix, x/xi) or as nested inverted repeats (xiva and xivb). DNA around the repeats (e.g., the entire region between the binding sites xi and xii) became more sensitive to DNase I in the presence of Byn protein. Such hypersensitive sites indicate a compression of the major groove due to DNA bending and suggest a loop formation between the sites (Kusch, 2002) .

The comparison of the protected regions in the fragments P_1310-1817 and C_872-1309 identifies a consensus sequence for Byn binding. This consensus of (A/G)(A/T)(A/T)NTN(A/G)CAC(C/T)T is similar to the consensus for Brachyury binding. Astonishingly, seven regions were also found within the D_1-871 fragment that were protected by Byn from DNase I. Because D_1-871 does not confer lacZ expression in embryos and each of these sites deviates from the consensus sequence of the sites in the P and C fragments in at least one position, it is concluded that they must be significantly less efficient for transcriptional activation by Byn. These sites probably have low affinity to Byn as suggested by their deviations from the consensus (Kusch, 2002).

Whether the Byn sites and their arrangement or spacing are conserved was investigated. For this purpose, the sequences of the otp loci of Drosophila melanogaster and Drosophila virilis were compared. Seven of the Byn binding sites and their flanking sequences are highly conserved between the two species; only their spacing is somewhat different. Instead of two Byn tandem sites (viii/ix, x/xi), in D. virilis only two individual sites with spacing similar to the tandems in D. melanogaster are found. Also most of the sites in fragment D are not strictly conserved, although the total number of Byn sites in D. virilis (14) is about the same as in D. Melanogaster (15). The large conserved regions flanking the Byn sites are A/T rich and presumably facilitate DNA-bending, but do not appear to contain obvious binding sites for other factors. Thus, the high number of binding sites and their embedding in flexible DNA stretches are a conserved feature of the otp regulatory region (Kusch, 2002).

All three high affinity (type A) Byn sites show a high homology to a half of the palindromic Brachyury binding site. Thus, it is very likely that vertebrate Brachyury proteins can recognize Byn sites and are able to activate otp transcription. To test this hypothesis, mouse Brachyury and Xenopus Xbra were tested in Drosophila embryos by means of the GAL4/UAS system. Of the two proteins, Brachyury is able to ectopically activate otp expression in the developing salivary glands of the late embryo. Both Brachyury homologs can activate transcription, although to a lesser extent than Byn, and therefore are apparently able to recognize the Byn sites in the otp gene (Kusch, 2002).

Byn binding sites of putatively low affinity (type B, on fragment D) exert synergistic effects on target gene expression when combined with putatively high affinity sites. Likewise, the synthetic combination of type A and type B sites reveals a strong synergism in transcriptional activation. Furthermore, extended regions between Byn sites become hypersensitive to DNase I suggesting an interaction between different Byn sites during transcriptional activation, possibly by the physical interaction of Byn itself. X-ray analysis of Xbra T domains shows that two Xbra molecules are close enough for a dimerization on the synthetic 24-bp palindrome of the Brachyury consensus. However, this contact in the crystal could be attributable to the two inverted binding half sites in the palindrome rather than being a biologically relevant dimerization. Therefore, a possible Byn-Byn interaction was tested in a cellular context, and the Byn open reading frame (ORF) was fused to the activation (GAD-Byn) and to the DNA binding domain (GBD-Byn) of yeast GAL4, respectively. The constructs were introduced into an appropriate yeast strain and, in case of a Byn-Byn interaction, should allow growth on a selective medium due to the formation of a functional GAL4 activator. In fact, combinations of constructs containing the Byn T domain (amino acids 84-276) were able to confer growth when combined in the cells, supporting the idea that Byn proteins interact via their DNA-binding domain. To narrow down the interaction region, N- and C-terminal deletions of the Byn T domain were generated. These truncations revealed that the central region of the T domain (amino acids 151-205) is sufficient for the interaction. This region contains a stretch of amino acids that forms most of the contact surface of the two Xbra T domains in the crystal, and therefore, most likely, is also being used for dimerization in vivo. The conservation of this region of the Brachyury-type T domain suggests that all members of the Brachyury subgroup can dimerize. Indeed, a murine Brachyury-GAL4 fusion construct can interact with the Drosophila Byn interaction domain, indicating that the ability to dimerize is a general feature of Brachyury proteins (Kusch, 2002).

Surprisingly, in the regulatory region of otp, no single Byn site was found that would be sufficient to activate transcription on its own. Also in other cases in which natural target sites for T domain transcription factors have been described, only single half-sites have been identified by virtue of their high similarity to the original Brachyury consensus. For Xbra, recognition motifs matching the Byn consensus have been found in the efgf locus. Also the T domain proteins Tbx-2, Mga, and VegT/Tpit recognize binding motifs similar to high affinity Brachyury half-sites. Thus, rather bind than to a palindrome Brachyury site, proteins seem to bind a 12-bp site in vivo. Possibly, the palindrome is even disadvantageous for the animal and might cause an exceedingly stable occupancy of the site by the protein (Kusch, 2002).

Within the regulatory region of otp an unusually high degree of tolerated variability for Byn binding sites is found. Within the 15 characterized sites two types were distinguished, A and B. Type A sites (to which the sites x, xii, and xiii belong) have a consensus, which differs only by two positions from the Brachyury consensus. This consensus has been selected from DNA molecules that bind optimally to Brachyury by binding site selection, a method that enriches for high-affinity binding sites. It is therefore assumed that the type A sites constitute high-affinity sites for Byn. More importantly, type A sites are essential for strong transcriptional up-regulation. Site x is required for 80% of the transcriptional activation by the C fragment, site xii is absolutely essential for C, and variants of the P fragment without site xiii have lost the ability to activate transcription. Finally, two type A sites, in tandem or in inverted orientation, are sufficient to activate Byn-dependent transcription (Kusch, 2002).

The Byn binding sites of type B yield a more relaxed consensus that encompasses the high-affinity consensus. They are significantly less efficient to activate Byn-dependent transcription than type A sites. For instance, the set of seven type B sites in the D fragment (i to vii) is not sufficient for transcription on its own, and the sites ix and xi contribute little to the activity of fragment C. Based on these observations it is proposed that the type B sites constitute low-affinity sites for Byn. In contrast to type A sites, a type B doublet is not able to activate transcription in the synthetic Byn response elements, neither in tandem nor in inverted orientation. Transcriptional activation is observed only in combination with a type A site, which is inverted to the type B site (Kusch, 2002).

What could be the basis for the ability of the Byn T domain to bind to such variable target sequences? Due to the high homology between Xbra and Byn and between their target sites, it is reasonable to assume that the crystal structure analysis of the Xbra T domain could serve as a model for Byn. The basic residues of the highly conserved motif CAC(C/T)T are contacted by amino acids of the T domain and constitute a sequence-specific binding interaction. Nucleotide exchanges within this stretch would disrupt the hydrogen bonds to the protein and lead to significantly lower binding efficiency. This is the case for the variant site Deltaxii-4, which is only marginally protected against DNase by Byn. A number of type A sites do not have the 3'-T of CAC(C/T)T, a feature which presumably contributes to their low binding efficiency. At the 5' end of the Byn consensus the RWW-motif defines a particular conformation of the double helix rather than a specific base sequence. At these positions the T domain contacts the phosphoribosyl backbone of the nucleotides, stabilizing the protein-DNA interaction, but allowing a higher variability of the bases. Thus, the structural data for the T domain give a good explanation how Brachyury proteins can accommodate the variability of their target sequences. It is reasonable to assume that T domain transcription factors, not belonging to the Brachyury-type subgroup, also recognize a wide range of target sites with variable affinities (Kusch, 2002).

This study shows that Byn binds to different types of target sites cooperatively and thereby activates a certain level of otp expression. Particular arrangements of such high- and low-affinity sites could also serve another purpose: the dose-dependent activation of target genes. There are several indications in Drosophila that the dose of Byn is relevant and informative. Byn is expressed in a dorso-ventral gradient in the blastoderm and allelic series of byn mutants exhibit graded defects in the embryos. This is fully consistent with the finding that above a certain threshold of Byn effector, the cis-regulatory region of otp strongly responds transcriptionally. In other species, Brachury-type transcription factors also give very clear dose-dependent responses. In vertebrates, Brachyury is distributed in an antero-posterior gradient in the embryo, and the formation of distinct embryonic structures depends on certain thresholds of Brachyury. It is conceivable that target gene responses depend on the levels of Brachyury expression as well as the amount and combination of high- and low-affinity sites within their regulatory regions (Kusch, 2002).

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