BIOLOGICAL OVERVIEW

Retained, also known as Dead ringer (Dri), is a founding member of a recently defined ARID family of DNA binding proteins whose members share a conserved DNA binding domain termed the A/T-rich interaction domain. This family includes the B-cell-specific factor Bright and the Drosophila factor Eyelid (Osa). dri is developmentally regulated, being expressed in a restricted set of cells including some neural cells and differentiating cells of the gut and salivary gland ducts. It is unlikely that Dri is a general transcription co-factor or chromatin modifier, as is Eyelid, since transcription of only a small number of the genes are disrupted in dri mutant embryos (Valentine, 1998 and Shandala, 1999).

Dri has been shown to be a sequence-specific DNA binding protein. The in vitro sequence specificity of Dri is strikingly similar to that of many homeodomain proteins: Dri preferentially binds the PuATTAA sequence (Gregory, 1996). It is therefore likely that the phenotypes exhibited by dri mutant embryos result from disruption to the expression of regulatory genes. In terms of specific molecular function, Dri has been shown to act in conjunction with Dorsal to recruit Groucho and repress the zerknullt minimal ventral repression region (VRR) element (Valentine, 1998). Nevertheless there are no major dorsal-ventral patterning defects in dri mutant embryos, although variable gastrulation defects are observed, consistent with some level of disruption to dorsal-ventral patterning (Shandala, 1999).

Terminal development is disrupted in the dri maternal and zygotic mutant embryos. Both head and tail defects are invariably observed in dri maternal and zygotic mutant embryos. One of the most consistent and striking phenotypes is severe disruption of the cephalo-pharyngeal skeleton. Germline and zygotic dri mutant embryos still have a recognizable dorsal bridge, dorsal and ventral arms and mouth hooks, but the H-piece and lateralgraten are missing or severely malformed. In addition, the atypical anterior position of pharyngeal muscles, visualized using anti-muscle myosin immunostaining, indicates that head involution does not proceed properly (Shandala, 1999).

The appearance of these defects prompted an examination of genes that play a role in the formation of terminal structures. Expression of the terminal gene tailless and the genes buttonhead, empty spiracles, orthodenticle and argos was examined. Of these genes, disruption to only argos (aos) and buttonhead (btd) expression was observed. In wild-type embryos, aos is initially expressed at stage 5 in two terminal domains and a domain that flanks the position of the cephalic furrow. In embryos lacking dri maternal and zygotic product, expression of aos in the terminal domains is almost completely eliminated while expression in the region of the cephalic furrow is maintained, both before and after division into two stripes at the time of cephalic furrow formation. Zygotic aos mutant embryos exhibit head defects that are similar to those observed in maternal and zygotic dri mutant embryos, indicating that the dri mutant head defects are likely to be the result of loss of anterior aos expression in the dri mutant embryos. Analysis of btd expression reveals a regulatory relationship that accounts for another consistent dri mutant phenotype, the appearance of ectopic cephalic furrows. btd expression is found to be partially derepressed in the trunk of dri germline and zygotic mutant embryos. The cephalic furrow arises where expression of the head specific gap gene btd overlaps the first stripe of expression of the primary pair rule gene eve. The repetitive appearance of ectopic cephalic furrows is therefore likely to be the result of the coincident ectopic trunk expression of btd with the more posterior eve stripes. The ectopic furrows do not progress, most probably due to the incomplete derepression of btd in this region (Shandala, 1999).

dead ringer is required for proper patterning of the abdomen. To test the basis for defects in patterning, genes required for segment formation in the Drosophila embryo were examined. Expressions of the axis patterning gene, bicoid; the gap genes hunchback, Krüppel, knirps and giant; the primary pair-rule genes even-skipped, hairy and runt, and the segment polarity genes wingless and engrailed were examined in embryos lacking germline and zygotic dri function. Most of these genes are expressed normally with respect to their role in segment formation. The variable disruption to abdominal segment formation correlates with a variable reduction in expression of engrailed and wingless (wg) in stripes 9-14. The most consistent effect on expression of the segmentation genes in the dri maternal and zygotic mutant embryos is a disruption to the expression of even-skipped (eve) stripe 4, observed in nearly all embryos lacking both maternal and zygotic dri product. Specifically, the ventrolateral portion of eve stripe 4, although initiated appropriately is not maintained in dri mutant embryos, leading to the subsequent aberrant appearance of wg stripes 7 and 8 and disruption to the parasegment 4 ventrolateral setal belts (Shandala, 1999).

The simplest interpretation of these results is that Dri can act either as an activator or as a repressor, depending on the context within which it finds itself. These different actions must depend on the combination of regulators acting on the respective position-specific cis-regulatory sequences. The differential regulation of aos and btd domains by dri occurs at a time when Dri is found in all somatic nuclei of the embryo, so that Dri must be acting to permit the proper function of other developmental regulatory factors. However, following gastrulation, dri expression becomes exquisitely tissue and stage specific (Gregory, 1996), raising the possibility that it may specify spatial-specific expression at later stages of development. It is presumed that Dri, like Osa, which is a member of the Trithorax Group of genes implicated in the modification of chromatin structures required for epigenetic regulation (Vazquez, 1999), is acting to establish stable chromatin structures. These structures may favor, but not be essential for, the formation of complexes in which other transcriptional regulators act, so that the absence of factors such as Dri introduces an element of chance into the stable formation of such complexes; consequently, there is an introduction of variability in gene expression and mutant phenotype, a phenomenon repeated observed for Dri (Shandala, 1999).

GENE STRUCTURE

cDNA length - 3.7kb

Exons - 12

Bases in 3' UTR - 877

PROTEIN STRUCTURE

Amino Acids - 901

Structural Domains

dead ringer (Gregory, 1996 and Shandala, 1999) is a founding member of a new family of proteins whose members share a conserved DNA binding domain, termed the ARID (A/T Rich Interaction Domain, Herrscher, 1995, Gregory, 1996). Members of this gene family include Drosophila osa (also referred to as eyelid; Treisman, 1997; Vazquez, 1999); yeast SWI1 (OíHara, 1988); the mammalian jumonji (Motoyama, 1997); Smcx (Agulnik, 1994a); Smcy (Agulnik, 1994b), and the MRF1 and MRF2 (Huang, 1996) genes, as well as genes encoding Retinoblastoma binding proteins RBP1 and RBP2 (Fattaey, 1993). Sequence comparisons show that DRI belongs to a subgroup within this family that exhibits an extended region of similarity either side of the ARID. This motif is referred to as the extended ARID (eARID; Kortschak, 1998). The eARID group, which is poorly characterized, includes DRI, human DRIL1 (Kortschak, 1998), mouse Bright (Herrscher, 1995) and proteins encoded by the C. elegans T23D8.8 and D. rerio dri1 and dri2 genes (Kortschak, 1998). There is some evidence that members of this group are implicated in transcriptional regulatory processes. The mouse dri ortholog Bright (for B-cell regulator of IgH transcription; Herrscher, 1995) encodes a B cell-specific protein that appears to bind the minor groove of a consensus MAR sequence (AT/ATC). Bright acts to displace a conserved human homeoprotein CUX (ortholog of Drosophila Cut) to activate the immunoglobulin heavy chain intronic enhancer, Em, specifically in B cells (Wang, 1999 and Shandala, 1999).

The Dead ringer protein from Drosophila is a transcriptional regulatory protein required for early embryonic development. It is the founding member of a large family of DNA binding proteins that interact with DNA through a highly conserved domain called the AT-rich interaction domain (ARID). The solution structure of the Dead ringer ARID (residues Gly262-Gly398) was determined using NMR spectroscopy. The ARID forms a unique globular structure consisting of eight alpha-helices and a short two-stranded anti-parallel beta-sheet. Amino acid sequence homology indicates that ARID DNA binding proteins are partitioned into three structural classes: (1) minimal ARID proteins that consist of a core domain formed by six alpha-helices; (2) ARID proteins that supplement the core domain with an N-terminal alpha-helix, and (3) extended-ARID proteins, which contain the core domain and additional alpha-helices at their N- and C-termini. Studies of the Dead ringer-DNA complex suggest that the major groove of DNA is recognized by a helix-turn-helix (HTH) motif and the adjacent minor grooves are contacted by an alpha-hairpin and C-terminal alpha-helix. Primary homology suggests that all ARID-containing proteins contact DNA through the HTH and hairpin structures, but only extended-ARID proteins supplement this binding surface with a terminal helix (Iwahara, 1999).

date revised: 30 Sept 99

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