BIOLOGICAL OVERVIEW

Signals from the BMP family member Decapentaplegic (Dpp) play a role in establishing a variety of positional cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although a reasonably clear picture is available of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. Three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, are described that are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. These genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, it has been demonstrated that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals (Reim, 2003).

Cell identities that are determined by Dpp include the dorsal epidermis and peripheral nervous system (PNS) in dorsolateral regions of the ectoderm, the dorsal vessel, dorsal somatic and visceral muscles in the dorsal mesoderm as well as those of the extra-embryonic amnioserosa in the dorsalmost region of the embryo. In addition to promoting dorsal epidermal and PNS fates in the dorsolateral ectoderm, Dpp acts to suppress the formation of neurons of the central nervous system in the same area (Reim, 2003).

For a better understanding of these activities, it is important to consider that Dpp exercises some of its functions sequentially at different stages of development, during which dpp changes its own pattern of expression. In particular, during blastoderm and gastrulation stages, Dpp acts in a dose-dependent fashion to establish positional information in dorsal and lateral areas of the embryo and to specify amnioserosa tissue. Although dpp is expressed uniformly around ~40% of the dorsal circumference of the embryos during this stage, the activity of Dpp is modulated along the dorsoventral axis by diffusion of secreted gene products as well as by positive and negative regulators of the signaling pathway. Negative regulators include Short gastrulation (Sog) and Brinker (Brk), both of which are expressed ventrolaterally. Whereas Sog and its vertebrate homolog Chordin are secreted molecules that inhibit BMP signaling via binding to the ligand, Brk appears to be a nuclear factor that interferes with the signaling output via binding to regulatory sequences of Dpp target genes. By contrast, specification of amnioserosa fates in the dorsal 10% of embryonic cells requires maximal signaling activities that involve Sog as a positive regulator of Dpp in conjunction with Twisted gastrulation (Tsg) as well as a second, uniformly-distributed BMP ligand, Screw (Scw). Dpp, Sog and Tsg are thought to be present in a diffusible trimolecular complex that serves to carry and release active Dpp prefentially into dorsalmost areas where tsg is expressed (Reim, 2003).

After gastrulation, dpp expression ceases in the developing amnioserosa and becomes restricted to a broad stripe of cells in the dorsolateral ectoderm along the elongated germ band. During this period, the dorsally migrating cells of the mesoderm reach the dpp-expressing area of the ectoderm, thus allowing Dpp to induce dorsal mesodermal cell fates across germ layers. In addition, Dpp is thought to act in the continuing patterning processes within the dorsolateral ectoderm during this stage that lead to the specification of tracheal as well as particular epidermal and sensory organ progenitors. Both in the dorsal mesoderm and dorsolateral ectoderm, Dpp must act in combination with additional patterning molecules that provoke differential responses of cells to the Dpp signal. For example, in the dorsal mesoderm, the presence or absence of Wingless (Wg) activity determines whether cells will respond to Dpp by forming heart and dorsal somatic muscle progenitors versus visceral muscle progenitors (Reim, 2003).

In order to obtain more insight into the mechanisms of how Dpp signals pattern the embryo and how they are integrated with other patterning processes, it is crucial to study the regulation of Dpp target genes. To date, detailed molecular studies have been described for three targets that are induced during early embryogenesis, namely the homeobox genes zerknüllt (zen), tinman (tin) and even-skipped (eve). zen is required for the specification of the amnioserosa downstream of Dpp. Accordingly, the expression of zen in a dorsal on/ventral off pattern, although initially Dpp-independent, requires low levels of Dpp activity for its maintenance and high Dpp activities for its subsequent refinement to areas of the prospective amnioserosa. Likewise, tin is required for the specification of all dorsal mesodermal tissues and eve for the normal differentiation of specific pericardial cells and dorsal somatic muscles in a Dpp-dependent manner. All three genes have in common the presence of multiple binding sites for intracellular Dpp effectors, the Smad proteins Mad and Medea, in their regulatory regions: the Smad proteins are essential for mediating the inductive activity of Dpp. However, in addition to these Smad-binding sites, each of these genes has a characteristic set of additional regulatory sequences that, at least in part, explains its particular spatial and tissue-specific response to Dpp signals. For example, zen contains binding sites for Brk in addition to the Smad sites. It appears that the antagonistic activities of the Brk and Smad sites and the differential ratios of Brk versus active Smad proteins along the dorsoventral embryo axis determine the ventral border of Dpp-dependent zen domain during cellularization stages. The Smad sites but not the Brk sites are also required for zen induction in the prospective amnioserosa during the cellularized blastoderm stage. The mesodermal Dpp targets tin and eve require Smad-binding sites and, in addition, binding sites for Tin, which serve to target the Dpp response to the mesoderm. Further, the Dpp-responsive enhancer of eve contains functionally important binding sites for regulators that restrict its activity to segmental subsets of dorsal mesodermal cells, including the Wg effector Pangolin (Reim, 2003).

Three novel genes have been discovered that respond to Dpp signals in the prospective amnioserosa, dorsal ectoderm and dorsal mesoderm, and are good candidates for being direct targets of the Dpp signaling cascade. The three genes, Dorsocross1 (Doc1), Dorsocross2 (Doc2) and Dorsocross3 (Doc3), which are present in a gene cluster, are closely related members of the T-box family of genes and presumably arose by relatively recent duplications from a common ancestor. The Dorsocross (Doc) genes are expressed in essentially identical patterns within several areas that receive high levels of Dpp signals, including the prospective amnioserosa during the cellularized blastoderm stage, the dorsolateral ectoderm and dorsal mesoderm during germ band elongated stages and areas that span the compartment border in wing discs. Doc expression in the prospective amnioserosa depends on dpp and zen, whereas the metameric expression in the dorsolateral ectoderm and dorsal mesoderm depends on a combination of dpp and wg. Genetic analysis demonstrates that the three Doc genes have largely redundant functions during amnioserosa development, as well as during dorsolateral ectoderm and dorsal mesoderm patterning. A focus was placed on the role of the Doc genes in the amnioserosa and dorsolateral ectoderm. They are essential for full differentiation and maintenance of the amnioserosa, including the arrest of cell proliferation in this tissue. Owing to the requirement of a functional amnioserosa for normal germ band retraction, loss of Doc activity produces embryos with a permanently extended germ band. Hence, Doc genes are functionally related to U-shaped and similarly expressed genes. These genes of the 'u-shaped group' include hindsight (hnt/pebbled), serpent (srp), tail-up (tup), u-shaped (ush), epidermal growth factor receptor (Egfr) and insulin-like receptor (InR); all are components of a regulatory network that controls normal development and functioning of the amnioserosa. In addition to the amnioserosa, Doc genes are required for the normal patterning of the dorsolateral ectoderm, which includes the repression of wg and ladybird (lb) expression within this area. These findings provide valuable insight into the mechanisms of how Dpp signals are executed during the development of the amnioserosa and the patterning of dorsolateral areas of the embryonic germ band (Reim, 2003).

The closely related T-box sequences, genomic clustering and virtually identical expression patterns of the three Dorsocross genes suggest that they are derived from relatively recent duplications of a common progenitor gene. Accordingly, the observation that loss of Doc1 or Doc3 does not cause any of the embryonic phenotypes seen upon loss of all three genes indicates that there is a large degree of functional redundancy among these three genes. Phylogenetic analysis with the extended T-box domain sequences shows that the Doc genes are most closely related to the vertebrate Tbx6 genes, whose expression in the paraxial mesoderm is reminiscent of the expression of the Doc genes in the dorsal somatic mesoderm. However, the limited reliability of the branches separating the Tbx6, VegT and Tbx2 subfamilies in the phylogenetic tree analysis, the absence of Drosophila orthologs of VegT and Tbx4/5 genes, as well as shared features of expression in the somatic and/or precardiac and cardiac mesoderm seem to support the alternative possibility that the Doc, Tbx6, VegT and Tbx4/5 genes arose from a common ancestral gene by gene amplifactions after the divergence of the insect and vertebrate lineages (Reim, 2003).

A prominent feature of the Doc genes is their expression in areas that receive inputs from Dpp, including the dorsalmost cells in blastoderm embryos, the dorsolateral ectoderm and mesoderm in the elongated germband, and distinct domains spanning the compartment border of the wing disc. Indeed, genetic data, together with the co-localization of Doc transcripts with active Mad in dorsal embryonic tissues, favor the possibility that the Doc genes are direct targets of the Dpp signaling cascade. However, the Dpp signals are required to act in combination with additional regulators during each of these inductive events (Reim, 2003).

Robust and stable induction of Doc expression in a dorsal stripe requires the activity of the homeodomain protein Zen as a co-activator of Dpp signals. The zen gene features an early, broad expression domain along the dorsal embryonic circumference, which is initially Dpp independent but subsequently requires Dpp for it to be maintained. Thereafter, its expression refines into a narrow dorsal domain in a process that requires peak levels of Dpp. The activation of Doc expression occurs at the same time as the refinement of zen expression and within the same narrow domain, which also coincides with high phospho-Mad levels. Although the maintenance and refinement of zen by Dpp is zen independent, it is proposed that Zen synergizes with peak signals of Dpp to trigger Doc gene expression in a dorsal stripe. The requirement for this proposed interaction between zen and dpp would explain the failure of zen to activate Doc genes in an early, broad domain as well as the observed low levels of residual Doc expression in zen mutant embryos, that may be due to inputs from Dpp alone. Formally, this proposed mechanism would be analogous to previously described inductive events in the early dorsal mesoderm, where the synergistic activities of the homeodomain protein Tinman and activated Smads induce the expression of downstream targets such as even-skipped. The identification of functional binding sites for Zen and Smads in Doc enhancer element(s) will be necessary for demonstrating that an analogous mechanism is active during induction of Doc gene expression in a dorsal stripe. In the absence of such data, it cannot be completely ruled out that dorsal Doc expression is controlled indirectly by Dpp, possibly via the combinatorial activities of zen and another high-level target gene of Dpp. Since mutations in several other genes that are expressed in the early amnioserosa, including pannier (pnr), hnt, srp, tup and ush, do not affect Doc expression until at least stage 12, these genes can be excluded as candidates for early upstream regulators of Doc (Reim, 2003).

Unlike zen, which is expressed only transiently, Doc expression is maintained throughout amnioserosa development. Hence, the Doc genes provide a functional link between the early patterning and specification events in dorsal areas of the blastoderm embryo and the subsequent events of amnioserosa differentiation. The activity of zen is required for all aspects of amnioserosa development that have been examined to date, including normal activation of C15. By contrast, the data demonstrate that the Doc genes execute only a subset of the functions of zen, which include the activation of Kr and hnt, but not that of C15 and early race, in amnioserosa cells. This interpretation is consistent with the failure to obtain a significant increase of amnioserosa cells upon ectopic expression of any of the Doc genes in the ectoderm or throughout the early embryo (using e22c and nanos-GAL4 drivers, respectively). The residual expression of hnt in some amnioserosa cells of Doc mutant embryos could be due to direct inputs from zen itself or from a yet undefined zen downstream gene acting in parallel with Doc. Nonetheless, the strong reduction of hnt expression in Doc mutant embryos could largely account for their amnioserosa-related phenotypes, including the absence of Kr expression, the decline of race expression, premature apoptosis and failure of germ band retraction. All of these phenotypes have also been observed in hnt mutant embryos. However, it is likely that Doc gene activity is required for the activation not only of hnt but also of additional genes of the u-shaped group and that Doc genes exert some of their functions in parallel with hnt. Some evidence for this notion is derived from the observation that loss of Doc activity has a stronger effect on Kr expression than loss of hnt activity (Reim, 2003).

One of the hallmarks of amnioserosa development is that the cells of this tissue never resume mitotic divisions after the blastoderm divisions. To a large extent, this cell cycle arrest is due to the absence of expression of cdc25/string in the prospective amnioserosa: this absence prevents the cells from entering M-phase and leads to G2 arrest. In addition, the expression of the Cdk inhibitor p21/Dacapo in the early amnioserosa is thought to contribute to the cell cycle arrest. Although a detailed description of the regulation of string and dacapo expression in dorsal embryonic areas is lacking, it has been reported that zen is required for repressing dorsal string expression -- this repression is expected to prevent further cell divisions. Notably, the observation that amnioserosa cells re-enter the cell cycle in Doc mutant embryos demonstrates that Doc genes are required for the cell cycle block in addition to zen. Whereas zen mutant embryos already feature ectopic cell divisions in dorsal areas from stage 8 onwards, in Doc mutants the amnioserosa cells resume mitosis only during and after stage 10, which is shortly after Zen protein disappears. Thus, it is hypothesized that the Doc genes take over the function of zen in repressing string and prevent cell divisions at later stages of amnioserosa development when Zen is no longer present. Overall, the phenotype of Doc mutant embryos suggests that amnioserosa differentiation, including cell cycle arrest and the development of squamous epithelial features, initiates in the absence of Doc activity but is not maintained beyond stage 11. Thereafter, cell division resumes and there is a reversal of the partially differentiated state. Apoptotic events are not observed prior to stage 11 in Doc mutants. However at later stages, many amnioserosa cells die prematurely and the remaining cells are difficult to distinguish morphologically from dorsal ectodermal cells (Reim, 2003).

Altogether, these studies have identified the Doc genes as new members of the u-shaped group of genes, which control amnioserosa development, and provide new insights into the regulatory pathways in amnioserosa development downstream of Dpp. In future studies, it will be necessary to define in more detail the specific roles of the remaining genes of the u-shaped group, particularly ush, srp, tup and C15, in this regulatory framework (Reim, 2003).

Unlike in the presumptive amnioserosa, not all cells in the dorsolateral ectoderm and dorsal mesoderm that receive high levels of Dpp induce Doc expression. Rather, Wg signals are required in combination with Dpp in these tissues, such that the Doc genes are induced at the intersections of transverse Wg stripes and the dorsally restricted domain containing high phospho-Mad levels. The Doc stripes extend beyond the peak levels of Wg on both sides of the Wg stripes, which indicates that the Doc genes are able to respond to relatively low levels of diffusible Wg. In addition, the absence of Doc expression in the dorsalmost cells of the ectoderm that receive Wg and Dpp signals indicates the presence of a negative regulator that prevents Doc induction in the ectoderm adjacent to the amnioserosa until stage 12. Together, these inputs restrict Doc expression to metameric quadrants that encompass the areas of the dorsolateral ectoderm between the tracheal placodes as well as the underlying mesodermal cells (Reim, 2003).

Some of the effects of wg are known to be mediated by its target gene sloppy paired (slp), including the feedback activation of wg in the ectoderm and the repression of bagpipe (bap) in the mesoderm. However, the residual (although strongly reduced) expression of the Doc genes in the germ band of slp mutant embryos argues against a role of slp in mediating the function of wg to induce the Doc genes. Hence, the Doc genes may be direct targets of the Wg signaling cascade in the ectoderm and mesoderm (Reim, 2003).

Observations show that one of the important functions of the Doc genes in the dorsolateral ectoderm is the repression of wg expression. Although the expression of Doc initially depends on wg, the Doc genes subsequently exert a negative feedback on wg expression, which leads to the previously unexplained interruption of the wg stripes during stage 11. Because the ventral extent of the ectodermal Doc domains correlate with the ventral borders of high levels of P-Mad, it is concluded that the dorsal limit of the ventral wg stripes at stage 11 is determined indirectly by Dpp via Doc (Reim, 2003).

The maintenance of wg after stage 10 has been shown to depend on two different positive feedback loops, one being active in the dorsal and the other in the ventral ectoderm. The dorsal feedback loop is mediated by the ladybird homeobox genes (lb=lbe and lbl), whereas the ventral loop is mediated by the Pax gene gooseberry (gsb). The Doc genes must interrupt one or both of these feedback loops, although it is not clear whether the primary block is at the level of the wg gene or at the level of the transcription factor-encoding genes lb and/or gsb. Another target for repression by the Doc genes in this pathway could be slp, which is required both dorsally and ventrally in wg feedback regulation. It is thought that lb is unlikely to be the primary target of Doc repression since the failure of wg repression temporally precedes the expansion of the lb stripes in Doc mutant embryos. Furthermore, the observation that the Doc genes can also repress wg in other tissue contexts such as the imaginal discs, where gsb, lb and slp are not components of a wg feedback loop, seems to favor the mechanism of a direct repression of the wg gene by Doc (Reim, 2003).

Taken together, these observations show that dynamic interactions among positive and negative feedback loops, which share wg as a common component, are involved in the dorsoventral and anteroposterior patterning of the embryonic ectoderm. The activity of the Doc genes in negatively regulating wg and lb, as well as their potential positive effects on yet unknown targets in the dorsolateral ectoderm, are expected to be important for the proper dorsoventral organization of the cuticle and sensory organs. In the mesoderm, the metameric expression domains of the Doc genes during stages 9-11 include the dorsal somatic and cardiac mesoderm. Notably, preliminary analysis has revealed defects in dorsal somatic muscle and dorsal vessel development in Doc mutant embryos, that are currently being examined in more detail. Finally, it is noted that the expression pattern of the Doc genes in the embryonic epidermis is very reminiscent of the pattern of expression and activity of another T-box gene, optomotor-blind (omb), in the pupal epidermis. Doc and omb expression overlap in the wing discs although, unlike omb, Doc expression is interrupted near the Wg domains. Furthermore, it has been reported that dominant mutations in the gene Scruffy (Scf) and their revertants genetically interact with omb during abdominal cuticle and wing patterning (Kopp, 1997). Because the breakpoints of two Scf revertants, Df(3L)Scf-R6 and Scf-R11, have been mapped directly upstream and downstream, respectively, of the Doc3 gene, it is tempting to speculate that the Scf phenotype is caused by rearranged Doc3. Future studies will clarify the relationship between Scf and Doc genes and establish whether the T-box genes Doc and omb functionally interact during patterning of the adult cuticle and wings (Reim, 2003).

GENE STRUCTURE

Sequence information from the Berkeley Drosophila Genome Project reveals that three novel, closely related T-box encoding genes are clustered within ~40 kb of genomic sequences at 66F1 to 66F2 on chromosome arm 3L. In reference to their peculiar patterns of expression in blastoderm embryos, these genes have been named, from proximal to distal, Dorsocross1 (Doc1; previously Tb66F2 -- see Lo, 2001), Dorsocross2 (Doc2) and Dorsocross3 (Doc3). The gene cluster also includes an unrelated predicted gene, CG5194, which maps between Doc2 and Doc3 (Reim, 2003).

cDNAs for the three Doc genes were isolated from an early embryonic cDNA library. Comparisons between cDNA and genomic sequences indicate that Doc2 encodes at least three different mRNA products, which appear to be generated from alternative transcription start sites. Among these, Doc2 variants A and B encode identical polypeptides, whereas variant C does not encode any long open reading frame. The data from Northern analysis indicate that the longest cDNAs obtained for each gene are close to full length if the polyA tails are taken into account (1.7 kb transcripts versus 1500 bp cDNA for Doc1, 2.0 kb transcripts versus 1759 bp cDNA for Doc2A, and 1.8 kb transcripts versus 1681 bp cDNA for Doc3). For Doc2, these data indicate that variant A (1.75 kb, presumably corresponding to the 2.0 kb transcripts) is expressed much more strongly than the other two variants. In addition, it is noted that splicing occurs at identical positions within the open reading frames of Doc1, Doc2 and Doc3, although most introns in Doc3 are much smaller as compared with those in the other two genes (Reim, 2003).

PROTEIN STRUCTURE

Amino Acids - 391 (Doc1); 469 (Doc2); 424 (Doc3)

Structural Domains

Sequence comparisons show that the three Doc proteins share high degrees of similarity within their T-box domain sequences (>95% amino acid identities) as well as within short sequence stretches extending N- and C-terminally from these domains. The N-terminal regions of the polypeptides up to the T-box domains are moderately conserved (>40% amino acid identities), whereas the C-terminal regions contain only few short stretches of additional sequence similarity. Additional sequence comparisons with T-box domains from vertebrates and phylogenetic analysis show that the Doc T-box domains are most closely related to those from members of the Tbx6 subfamily of T-box (Papaioannou, 2001) proteins (Reim, 2003).

The EH1 motif in metazoan transcription factors

The Engrailed Homology 1 (EH1) motif is a small region, believed to have evolved convergently in homeobox and forkhead containing proteins, that interacts with the Drosophila protein Groucho (C. elegans unc-37, Human Transducin-like Enhancers of Split). The small size of the motif makes its reliable identification by computational means difficult. The predicted proteomes of Drosophila, C. elegans and human have been systematically searched for further instances of the motif. Using motif identification methods and database searching techniques, which homeobox and forkhead domain containing proteins also have likely EH1 motifs was examined. Despite low database search scores, there is a significant association of the motif with transcription factor function. Likely EH1 motifs are found in combination with T-Box, Zinc Finger and Doublesex domains as well as discussing other plausible candidate associations. Strong candidate EH1 motifs have been identified in basal metazoan phyla. Candidate EH1 motifs exist in combination with a variety of transcription factor domains, suggesting that these proteins have repressor functions. The distribution of the EH1 motif is suggestive of convergent evolution, although in many cases, the motif has been conserved throughout bilaterian orthologs. Groucho mediated repression was established prior to the evolution of bilateria (Copley, 2005).

Sequence motifs were sought in homeobox containing transcription factors taken from the proteins of human, Drosophila and C. elegans, by first masking known Pfam domains, and then using the expectation maximization algorithm implemented in the meme program. The first non-subfamily specific motif identified corresponded to previously known examples and new instances of, the EH1 motif, in 100 sites, with an E-value of < 10^-126. The same approach was applied to Forkhead containing transcription factors, identifying 25 sites with a combined E-value of < 10^-31. These motifs also appeared to conform to the consensus of the EH1 motif (Copley, 2005).

To further investigate the significance of this similarity, hidden Markov models (HMM) were constructed of the motif (EH1^hox & EH1^fh) which were then searched against the complete set of predicted proteins from human, D. melanogaster and C. elegans. The highest scoring non homeobox containing domain match of EH1^hox was a Forkhead protein (human FOXL1), and the second highest scoring non-Forkhead containing match of EH1^fh was to a homeobox containing protein (Drosophila Invected). In both cases, nearly all the high scoring hits were to proteins containing domains with transcription factor function. Among the best scoring matches of the EH1^hox searches were several T-box (TBOX), Doublesex Motif (DM), Zinc finger (ZnF_C2H2) and ETS containing proteins (Copley, 2005).

The presence of EH1 motifs within various homeobox, and to a lesser extent, forkhead-containing proteins has been widely reported, although not systematically studied. EH1-like motifs co-occurring with 3 major groupings of homeobox sub-types were found: the extended-hox class, typified by Drosophila Engrailed; the paired class, including Drosophila Goosecoid, and the NK class, including Drosophila Tinman. Related to the paired class homeobox domains, a number of genes containing PAIRED domains only were also found to contain EH1-like motifs. With only a few exceptions, the EH1-like motif occurs N-terminal to the homeobox domain and C-terminal to the PAIRED domain when present. A number of these proteins have been shown to interact with Groucho or its orthologs, e. g., C. elegans cog-1, Drosophila Engrailed and Goosecoid, and in high throughput assays Drosophila Invected and Ladybird late (Copley, 2005).

EH1-like motifs also occur N- and C-terminal to Forkhead domains. The N-terminal class consists of the Sloppy-paired genes of Drosophila and orthologous or closely related sequences: human FOXG1, and Drosophila CG9571; the C. elegans ortholog fkh-2 contains an EH1-like motif although a cysteine residue causes a low score. The C-terminal class consists of an apparent clade including the human FOXA, FOXB, FOXC and FOXD genes, although if the EH1 motif was present in the common ancestor of this clade, multiple losses must have later occurred. The situation is complicated somewhat by an EH1-like motif at the N-terminus of C. elegans unc-130, i. e., in the FOXD like family. The EH1 motif in slp1 has been shown to interact with groucho, and FOXA type genes have been shown to interact with human groucho orthologs (Copley, 2005).

Likely EH1 motifs co-occurring with T-Box domains in two distinct contexts. The motif occurs C-terminal to the T-box in the Drosophila Dorsocross proteins Doc1, Doc2 and Doc3. It is found N-terminal to the T-box in 11 proteins including mls-1 and mab-9 from C. elegans; H15, Mid/Nmr2 and Bi/Omd from Drosophila; in humans there are strong matches to TBX18, TBX20 and TBX22 and more marginal matches to TBX3 and TBX2. As far as is known, none of these proteins has been shown to interact with groucho or its orthologs, although several are known to act as transcriptional repressors: for instance, in murine heart development, Tbx20 represses Tbx2 which in turn represses Nmyc; the Dorsocross genes from Drosophila repress wingless and ladybird, and Doc itself is repressed by mid/nmr2. The human proteins TBX1 and TBX10, and Drosophila Org-1 (which are all closely related to those above) do not appear to contain EH1 motifs. The human T (brachyury) protein contains a motif broadly similar to the EH1 consensus: LQYRVDHLLSA in a comparable N-terminal location to those found in other T-box containing proteins. Although this motif scores poorly against EH1^hox, the homologous regions from other T orthologs provide a more persuasive case for the presence of a functioning EH1 motif in these proteins (Copley, 2005).

Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart

Identification of genomic regions that control tissue-specific gene expression is currently problematic. ChIP and high-throughput sequencing (ChIP-seq) of enhancer-associated proteins such as p300 identifies some but not all enhancers active in a tissue. This study shows that co-occupancy of a chromatin region by multiple transcription factors (TFs) identifies a distinct set of enhancers. GATA-binding protein 4 (GATA4), NK2 transcription factor-related, locus 5 (NKX2-5), T-box 5 (TBX5), serum response factor (SRF), and myocyte-enhancer factor 2A (MEF2A), referred to as 'cardiac TFs,' have been hypothesized to collaborate to direct cardiac gene expression. Using a modified ChIP-seq procedure, chromatin occupancy by these TFs and p300 were defined genome wide and unbiased support for this hypothesis is provided. This principle was used to show that co-occupancy of a chromatin region by multiple TFs can be used to identify cardiac enhancers. Of 13 such regions tested in transient transgenic embryos, seven (54%) drove cardiac gene expression. Among these regions were three cardiac-specific enhancers of Gata4, Srf, and swItch/sucrose nonfermentable-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3 (Smarcd3), an epigenetic regulator of cardiac gene expression. Multiple cardiac TFs and p300-bound regions were associated with cardiac-enriched genes and with functional annotations related to heart development. Importantly, the large majority (1,375/1,715) of loci bound by multiple cardiac TFs did not overlap loci bound by p300. These data identify thousands of prospective cardiac regulatory sequences and indicate that multiple TF co-occupancy of a genomic region identifies developmentally relevant enhancers that are largely distinct from p300-associated enhancers (He, 2011).

EVOLUTIONARY HOMOLOGS

Related mammalian genes include human Tbx6 and murine Tbx2. For information about Doc homologs see Optomotor blind: Evolutionary homologs

date revised: 7 July 2003 Dorsocross1, Dorsocross2 and Dorsocross3: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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