grainy head

Transcriptional Regulation

The development of the posterior spiracles of Drosophila may serve as a model to link patterning genes and morphogenesis. A genetic cascade of transcription factors downstream of the Hox gene Abdominal-B subdivides the primordia of the posterior spiracles into two cell populations that develop using two different morphogenetic mechanisms. The inner cells that give rise to the spiracular chamber invaginate by elongating into 'bottle-shaped' cells. The surrounding cells give rise to a protruding stigmatophore by changing their relative positions in a process similar to convergent extension. In the larvae the spiracular chamber forms a very refractile filter, the filzkorper. The opening of the spiracular chamber, the stigma, is surrounded by four sensory organs; the spiracular hairs. Clones labeling the spiracular hairs show that each one is formed by four cells related by lineage, two neural and two support cells, the typical structure of a type I external sensory organ. When the larva is buried in the semi-liquid medium on which it feeds, the stigmatophore periscopes out of the medium allowing the larva to continue breathing. The genetic cascades regulating spiracular chamber, stigmatophore, and trachea morphogenesis are different but coordinated to form a functional tracheal system. In the posterior spiracle, this coordination involves the control of the initiation of cell invagination that starts in the cells closer to the trachea primordium and spreads posteriorly. As a result, the opening of the tracheal system shifts back from the spiracular branch of the trachea into the posterior spiracle cells (Hu, 1999).

Downstream of Abd-B the cascade can be subdivided into various levels. The activation of six genes -- cut, empty spiracles (ems), nubbin (nub), klumpfuss (klu), and spalt (sal) -- does not require expression any of the other genes studied, suggesting that these six genes are at the top of the cascade under Abd-B regulation. The cut, ems, nub, and klu genes are expressed in the spiracular chamber in overlapping patterns. The sal gene is not expressed in the spiracular chamber but in the cells that surround it and will form the stigmatophore. The exclusion of sal from the spiracular chamber is partly due to repression by cut, because in cut mutants sal is expressed at low levels in the internal part of the spiracle. Downstream of these putative Abd-B targets other genes are activated. These include the transcription factors grainyhead (grh), trachealess (trh) and engrailed (en) (Hu, 1999).

The spiracle phenotypes in mutants for the early Abd-B downstream genes have been analyzed. In sal mutants the stigmatophore does not form, resulting in embryos with a normal spiraclular chamber that does not protrude. Conversely, mutations in ems and cut affect the spiracular chamber but not the stigmatophore. Mutations for ems result in a spiracular chamber that lacks a filzkorper and is not connected to the trachea. In cut mutants the filzkorper is almost completely missing, but the trachea is still connected to the spiracular chamber and the spiracular hairs are also missing. In trh mutants, where the tracheal pits do not form and there is no tracheal network, the spiracular chamber cells still invaginate, forming a filzkorper. However, this filzkorper is shorter than that of the wild type probably due to a secondary requirement of trh, which is also expressed in the spiracular chamber cells. These results show that the spiracular chamber, the stigmatophore, and the trachea develop independently of one another. No phenotypes for either klu or nub could be detected, indicting that although these genes are expressed in the spiracle, they are either redundant or their function is not required for spiracle morphogenesis (Hu, 1999).

To follow the movements of the spiracular chamber cells as they invaginate, constructs were examined that were made with particular enhancers of the cut, ems, and grh genes, each of which drive expression of beta-gal in a subset of cells that express the cut gene at stage 11. These enhancers do not drive the whole spiracular expression of their genes, but are good tools for studying cell specification and the morphogenetic movements of the posterior spiracle cells. The expression of cut in the posterior spiracle is controlled by at least three different enhancers, two of which have been used in this study. From stage 13, the ct-A4.2 enhancer marks the precursors of the four spiracular hairs. The grh-D4 enhancer of the grh gene is expressed in a single group of cells in this area. The expression of ems in the spiracle is driven by at least by one enhancer: ems-1.2. From stage 11 this enhancer marks a group of cells abutting the tracheal pit. Double stainings of the cut-D2.3, ems-1.2, and grh-D4 lacZ constructs show that they are expressed in non-overlapping subsets of cells. The correlation of the expression of these three constructs allows the fate mapping of the spiracular chamber primordium when it is a two dimensional sheet of cells. The different spatial expression of these enhancers at stage 11 shows that the two-dimensional sheet of cells is already patterned and that the cells invaginate to precise positions during development (Hu, 1999 and references therein).

The connection of the posterior spiracle to the trachea is a regulated event. In mutants for the Drosophila FGF and FGF-receptor homologs branchless and breathless the tracheal pits do invaginate, but since they do not migrate toward one another, they do not form a continuous network. In contrast, in btl mutants, the posterior spiracle connects normally to the A8 spiracular branch of the trachea. In mutants for Abd-B the stigma of A8 does not slide posteriorly, but stays in the same position as in anterior abdominal segments, where the spiracular branch attaches to the outside epidermis. The contribution of the ems gene to coordination of morphogenetic movements has been examined. The spiracle-trachea connection occurs in cut and sal mutants but not in ems mutants. In ems mutants, invagination of the spiracle cells adjacent to the trachea does not occur, but more posterior cells of the spiracle invaginate normally. The elongation does not occur simultaneously in all cells, but starts in the more anterior ones and, in general, the invaginating cells keep contact with the external surface of the embryo. This results in the cells that have invaginated earlier being deeper in the spiracular chamber and more elongated. The defective invagination in ems mutants results in a spiracle without a lumen and with the tracheal opening located outside it. The results show that cell elongation and formation of a lumen are two independently controlled processes. The spiracles provide a good model for the study of cellular and molecular mechanisms controlling cell shape and cell rearrangements, two mechanisms which are used during the morphogenesis of a variety of organisms (Hu, 1999).

Targets of Activity

Activation of the Torso RTK (receptor tyrosine kinase) at the poles of the embryo activates a phosphorylation cascade that leads to the spatially specific transcription of the tailless gene. The Torso response element (TOR-RE) in the tll promoter indicates that the key activity modulated by the TOR RTK pathway is a repressor present throughout the embryo. The TOR-RE has been mapped to an 11-bp sequence. The proteins GAGA and NTF-1 (also known as Elf-1, product of the grainy head gene) bind to the TOR-RE. NTF-1 can be phosphorylated by Rolled, also known as MAPK (mitogen-activated protein kinase). tll expression is expanded in embryos lacking maternal NTF-1 activity. These results make NTF-1 a likely target for modulation by the TOR RTK pathway in vivo. Thus activation of TOR RTK at the poles of the embryo leads to inactivation of the repressor (GRH) and therefore, to transcriptional activation (by activators present throughout the embryo) of the tll gene at the poles of the embryo (Liaw, 1995).

The Dorsal morphogen is a transcription factor that activates some genes and represses others to establish multiple domains of gene expression along the dorsal/ventral axis of the early Drosophila embryo. Repression by Dorsal appears to require accessory proteins that bind to corepression elements in Dorsal-dependent regulatory modules called ventral repression regions (VRRs). A corepression element has been identified in decapentaplegic (dpp), a zygotically active gene that is repressed by the Dorsal morphogen. This dpp repression element (DRE) is located within a previously identified VRR and close to essential Dorsal-binding sites. A factor from Drosophila embryo extracts has been identified that binds to the DRE but not to mutant forms of the DRE that fail to support efficient repression. This protein also binds to an apparently essential region in a VRR associated with the zerknullt (zen) gene. One of the DREs in the dpp VRR overlaps the binding site for a potential activator protein suggesting that one mechanism of ventral repression may be the mutually exclusive binding of repressor and activator proteins. The DRE-binding protein is identical to NTF-1 (equivalent to Elf-1, the product of the grainyhead gene), a factor originally identified as an activator of the Ultrabithorax and Dopa decarboxylase promoters. NTF-1 mRNA is synthesized during oogenesis and deposited in the developing oocyte where it is available to contribute to ventral repression during early embryogenesis. Previous studies have shown that overexpression of NTF-1 in the postblastoderm embryo results in a phenotype that is consistent with a role for this factor in the repression of dpp later in embryogenesis (Huang, 1995).

GRH was isolated on the basis of its binding to element I, a proximal promoter site that has a major role in nervous system expression of Dopa decarboxylase (Bray, 1989). Nevertheless, GRH does not mediate the nervous system expression of Dopa decarboxylase, even though GRH has been shown to bind to the promoter site (Bray, 1991). GRH binds to and regulates the proximal promoter of Ubx (Dynlacht, 1989), and a neurogenic enhancer region of ftz, 1.7 kb upstream of the structural gene (Dynlacht, 1989). GRH also regulates engrailed (Soeller, 1988).

GRH is a cofactor in the repression of decapentaplegic and zerknüllt. Repression by Dorsal appears to require accessory proteins that bind to corepression elements in Dorsal-dependent regulatory modules called ventral repression regions (VRRs). A corepression element in dpp is located within a previously identified VRR and close to essential Dorsal-binding sites. One of the Dorsal response elements in the dpp VRR overlaps the binding site for a potential activator, suggesting that one mechanism of ventral repression may be the mutually exclusive binding of repressor and activator proteins. The Dorsal response element binding protein is identical to GRH. (Huang, 1995). GRH protein binds to and regulates an essential ventral repression region associated with the zerknüllt gene (Huang, 1995).

The Drosophila Proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including the upstream regulatory element (URE), a DNA replication-related element, and E2F recognition sites. In addition to DRE and E2F sites, the PCNA promoter contains three CFDD (common regulatory factor for DNA replication and DREF genes recognition) sites. Among these three, at least site 1 could be demonstrated to play an important role in promoter activity in both cultured cells and living flies. In addition to the PCNA gene, multiple CFDD sites have been found in promoters of the DNA polymerase and DREF genes. In nuclear extracts of Drosophila embryos, a protein factor, the URE-binding factor (UREF), has been detected that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies reveal that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allows isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (Grh/NTF-1). The nucleotide sequence required for binding to Grh is indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to Grh reacts with UREF in embryo nuclear extracts. From these results it is concluded that GRH is identical to UREF. Although Grh has been thought to be involved in regulation of differentiation-related genes, this study demonstrates for the first time the involvement of a Grh-binding site in the regulation of the DNA replication-related Proliferating cell nuclear antigen gene (Hayashi, 1999).

The genetic programs that control patterning along the gut dorsoventral (DV) axis have remained largely elusive. The activation of the Notch receptor occurs in a single row of boundary cells that separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor. These results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer, one which, in turn, may be required for apical Notch receptor localization, suggests a simple mechanism by which the specification of a single cell row might be controlled (Fusse, 2002).

In order to investigate whether Notch signaling in the large intestine of wild-type embryos is activated beyond the boundary cells but actively repressed dorsally and ventrally, flies that carry the chimeric Notch receptor/transcription factor fusion construct N-Gal4/VP16 were used and the range of Notch signaling was determined. Upon heat shock, this fusion protein, which is membrane bound, becomes ubiquitously expressed in the embryo. In cells in which the Notch receptor is activated by ligand binding, the intracellular Gal4-VP16 transcription factor moiety is cleaved off and is able to subsequently activate reporter gene expression in cells that carry a UAS-lacZ construct. The ß-Gal expression pattern of such embryos reflects the range of Notch signaling. When anti-ß-Gal stainings of embryos that were heat shocked and carried the N-Gal4/VP16 and UAS-lacZ constructs was performed, ß-Gal expression was observed exclusively in the lateral boundary cells of the large intestine, demonstrating that Notch signaling is restricted to the boundary cells only. To further test this, flies were used carrying a lacZ-reporter construct in which multiple Su(H) binding sites from the Enhancer of Split m8 gene were combined with binding sites for the transcription factor Grainyhead (Grh). In cells, in which Notch signaling is active and Grh is expressed, Su(H) cooperates with Grh to yield high levels of reporter gene expression, whereas reporter gene expression is repressed in cells in which Notch is inactive. Determining the activity pattern of this construct in the hindgut using anti-ß-Gal antibody stainings demonstrates that activation of the reporter gene occurs exclusively in the boundary cells of the large intestine, consistent with the N-Gal4/VP16 data (Fusse, 2002).

These results demonstrate that Notch signaling induces the expression of the rho and kni/knrl genes and that both components are required, in turn, for the expression of Crb. It has been suggested recently that Su(H) functions as a core of a molecular switch by which the transcription of Notch target genes is regulated. In the absence of Notch signaling, Su(H) functions as a repressor, and, in the presence of Notch signaling, Su(H) can cooperate synergistically with other transcriptional activators to induce transcription of target genes. The finding that boundary cell-specific reporter gene expression can be induced in the hindgut by using a model Notch response element [composed of binding sites for Su(H) and the widely expressed activator Grainyhead] suggests the possibility that the localized activation of the rho and kni/knrl genes could rely on the same factors and the same molecular switch mechanism that has recently been proposed for this element and for Notch-dependent atonal and single minded expression. In evolutionary terms, the gut is most likely one of the most ancient organs that evolved in multicellular organisms. Consistently, the morphological processes involved in the development of the gastrointestinal tract of animals are highly similar. It remains to be shown whether or not the evolutionarily conserved regulators of the Notch signaling cascade also determine dorsoventral aspects of gut development in other animals, including vertebrates (Fusse, 2002).

An epidermal barrier wound repair pathway in Drosophila is mediated by grainy head

Wounded Drosophila embryos were used to define an evolutionarily conserved pathway for repairing the epidermal surface barrier. This pathway includes a wound response enhancer from the Ddc gene that requires grainy head (grh) function and binding sites for the Grh transcription factor. At the signaling level, tyrosine kinase and extracellular signal-regulated kinase (ERK) activities are induced in epidermal cells near wounds, and activated ERK is required for a robust wound response. The conservation of this Grh-dependent pathway suggests that the repair of insect cuticle and mammal skin is controlled by an ancient, shared control system for constructing and healing the animal body surface barrier (Mace, 2005).

Animals have evolved biological armor, an epidermally derived integument, to protect their bodies from physical injury and dehydration and have evolved control pathways to regenerate this barrier after wounding. A key component of this barrier in mammals is the stratum corneum of the skin, and a key component of the barrier in insects is the cuticle. In invertebrates, the immediate barrier response to wounding involves the formation of a temporary plug at wound sites, along with the activation of melanization and cross-linking enzymes that encapsulate invading microbes and help seal wound openings. In vertebrates, the immediate humoral response to vascular wounding results in the activation of proteases leading to the formation of a fibrin clot to help seal wound openings (Mace, 2005).

In both invertebrates and vertebrates, introducing infectious microbes through wounds results in the induction of the innate immune pathways. In two branches of these pathways, Toll-family transmembrane receptors or Imd-dependent signals trigger a signaling cascade that allows transcription factors from the NF-kappaB family to enter the nucleus, where they directly activate the transcription of genes that provide a first line of defense against pathogens (Mace, 2005).

Another response to epithelial wounds is mediated by wound healing pathways that re-epithelialize the breach. Genes that are required for regenerating the epithelial sheet after laser-induced or mechanically induced wounds in the Drosophila epidermis include those encoding Rho, Cdc42, and Jun N-terminal kinase (JNK). Additional Drosophila genes have been implicated in the process of epithelial repair by means of their requirement for epidermal dorsal closure during late embryogenesis. These include genes encoding the Drosophila Jun and Fos transcription factors as well as Dpp, Ras, and Puckered. Homologs of most of these proteins, in addition to many others, are associated with the process of epithelial regeneration in vertebrates (Mace, 2005).

By comparison, the genetic pathways that respond to aseptic breaks in the barrier integument and provide for its regeneration are poorly understood. Although the integuments of both mammals and insects depend on a dense, highly cross-linked matrix of proteins and other macromolecules, heretofore there has been no reason to suspect common genetic control pathways in the repair of mammal skin and insect cuticle (Mace, 2005).

Drosophila embryos that lack all Hox gene function in a body region, or that are mutant for the Hox-interacting gene spen, develop ectopic sclerites (hard, melanized cuticular structures) in the trunk of first instar larvae. These sclerites were proposed to be ectopic head skeleton, but the sclerites in these mutant larvae often look like the cuticular scar tissue that often surrounds the healed hole generated by a sterile needle in late-stage embryos (Mace, 2005).

Therefore, whether the sclerites observed in spen or Scr Antp double-mutant larvae were associated with breaks in the epidermal integument was tested. To assay this, rhodamine-labeled dextran was injected into the perivitelline space of stage 17 wild-type, spen mutant, and Scr Antp mutant embryos. As a positive control, rhodamine-dextran directly was injected into the body cavity of a wild-type embryo. It was observed that the fluorescent dye penetrated the body cavity of spen mutant and Scr Antp double-mutant embryos but not control wild-type embryos. It is concluded that there are localized failures of epidermal integrity in late-stage embryos that lack the function of spen or both Scr and Antp (Mace, 2005).

Next, whether two genes required for normal cuticular sclerotization were activated in the wound regions that developed scars was tested. The genes were Ddc, which encodes dopa decarboxylase, and pale (ple), which encodes tyrosine hydroxylase. These proteins contribute to the formation of the larval and adult cuticular skeleton in epithelial cells through the production of catecholamines that are converted to quinones by phenol oxidases. The reactive quinones then cross-link protein polymers and chitin polymers to generate the largely impermeable integument of insects. First instar larvae that are doubly mutant for Ddc and ple have almost no melanization and sclerotization of the head skeleton. A key regulatory step in the localized deposition of hard, dark cuticle is exerted at the transcriptional level of these genes, given that the hard skeleton-producing cells of the late embryo and early larva accumulate abundant Ddc and ple transcripts (Mace, 2005).

In aseptically wounded late embryos, transcripts from both Ddc and ple accumulate to high levels in the epidermal cells near the wound site. Transcripts from these genes can be detected within 30 min after injury, suggesting that these genes are direct targets of a wound-induced signal transduction pathway. Transcription of Ddc and ple is also abundant in the defective epidermal regions that develop sclerotic scar tissue in spen mutants and Scr Antp double mutants. Control in situ hybridizations were done to eliminate the possibility that the increased Ddc and ple signals at wound sites were an accessibility artifact in late embryos (Mace, 2005).

To dissect transcriptional regulatory inputs involved in the activation of epidermal wound response genes, the regulatory regions of Ddc were analyzed. The expression pattern provided by a 7.5-kb segment of DNA that included a hemagglutinin-tagged Ddc protein coding sequence was examined. This 7.5-kb region provides the normal Ddc expression pattern during embryogenesis and is also activated near wound sites in late-embryonic epidermal cells (Mace, 2005).

Deletion analyses of lacZ and/or green fluorescent protein (GFP) reporter constructs fused to the hsp70 basal promoter show that sequences between -1.4 kb to the Ddc transcription start are sufficient for a wound response, but sequences from -381 base pairs (bp) to the transcription start are not. The -1.4-kb Ddc-GFP reporter is activated over many cell diameters near wound sites, and the extent of activation increases with larger wounds and longer incubations after injury. The graded nature of the response suggests that a signal is produced at the injury boundaries that activates the wound response enhancer in a dose-dependent fashion in nearby epidermal cells. The -1.4-kb Ddc-GFP reporter was also tested in Scr Antp double-mutant embryos and it was found found that the wound response reporter is activated in regions where cuticular scars develop. Similar results were obtained in spen mutants (Mace, 2005).

To determine whether the aseptic wound response pathway (as defined by the Ddc wound response enhancer) and the infectious wound response pathways overlap, tests were performed for activation of the -1.4-kb Ddc-GFP wound response reporter after aseptic wounding of zygotic mutants of the innate immunity signaling pathway genes Toll, tube, imd, and 18-wheeler as well as in zygotic mutants of the innate immunity transcription factor genes rel, Dif, and Dl (Dl and Dif were tested as a double-mutant combination). In all of these mutant backgrounds, the -1.4-kb Ddc-GFP reporter was activated near wounds, as it was in wild-type embryos. Toll maternal/zygotic mutants and tube maternal/zygotic mutants were also tested, and activation of the wound response reporter was observed at the breaks in the epidermal integument that occur in these mutants. (Mace, 2005).

Previous studies have indicated that the JNK pathway is required for the process of wound healing in embryos and adults. It was found that the -1.4-kb Ddc-GFP wound response reporter is still activated at aseptic wound sites of zygotic mutants in either Drosophila JNK, Jun, or Fos and is also activated in cells at and near the dorsal epidermal leading edge 'wound boundaries' that form when dorsal closure fails in these mutant backgrounds. The wound response reporter is not activated in dorsal epidermal leading edge cells in wild-type embryos. It is concluded that the zygotic functions of JNK, Jun, and Fos are dispensable for the activation of the -1.4-kb Ddc wound response reporter, even in the cells where the zygotic functions of these genes are required for the migration and sealing of epithelial cell sheets during dorsal closure (Mace, 2005).

The Drosophila grainy head (grh) gene encodes two major transcription factor isoforms (Grh-N and Grh-O). Grh-N is expressed in regions of the central nervous system, whereas Grh-O is expressed in barrier epithelia such as the embryonic epidermis, the foregut, the hindgut, and the tracheal system. Zygotic mutants in grh die at the embryonic/larval transition with weak epidermal cuticle, malformed head skeletons that are composed of discontinuous grainy sclerites, and abnormal tracheal trunks. Clones of grh mutant cells in the adult epidermis have defects in pigmentation, cell polarity, and epidermal hair differentiation (Mace, 2005).

The similarity of the grh mutant phenotype to Ddc and ple mutants prompted a determination of whether grh function is required for activation of -1.4-kb Ddc wound response element, which has two evolutionarily conserved Grh binding sites. In aseptically wounded grh mutant embryos, the -1.4-kb Ddc wound response reporter is at most weakly activated in a few cells immediately adjacent to the wound border. This is consistent with the abnormal wound healing in grh mutant larval cuticle. When compared with wild-type wounds, the grh mutant wound sites are deficient in normal cuticle regeneration, as well as in displacement of the melanized plug that forms immediately after wounding. A similar phenotype is seen in wounded Ddc mutants, although the remaining plug is less melanized (Mace, 2005).

The -1.4-kb Ddc wound response enhancer has consensus transcription factor binding sites for Grh, NF-kappaB/Rel proteins, adenosine 3',5'-monophosphate response element-binding protein (CREB)-A proteins, and AP-1-like/basic-leucine zipper proteins, which are candidate sites required for the function of the wound response enhancer. Transgenic embryos for the -1.4-kb Ddc-GFP reporter with point mutations in the six consensus sites for NF-kappaB family proteins show normal wound-induced activation, whereas a similar reporter with point mutations in the single CREB-A and the three AP-1-like consensus sites show a marked reduction in wound-induced activation compared with wild-type reporter controls (Mace, 2005).

Further deletion analyses define a minimal Ddc epidermal wound response element from -472 bp to the start of transcription, in which sequences from -472 to -381 bp are required for wound response function. The -0.47-kb Ddc wound response element from D. melanogaster has only five blocks of marked sequence conservation (a perfect match of 6 bp or greater) with a D. virilis Ddc promoter proximal fragment from base pairs -392 to +13, which provides a wound response when attached to reporter genes in D. melanogaster embryos. The blocks of sequence conservation are each 12 to 13 bp and they include one Grh site, one AP-1 consensus site, one ETS consensus site, a GGGGGATT motif (which overlaps with one of the NF-kappaB consensus sites), and the TATA box region. In D. melanogaster, the conserved GGGGGATT motif, AP-1-like site, and ETS site are all within the interval of -472 to -381 bp that is required for wound response function. The conserved Grh site, which is closer to the promoter, is required for the -472-bp element function, given that its mutation abolished wound response reporter activation (Mace, 2005).

To identify potential wound response enhancers at the D. melanogaster ple gene, the sequences from 10 kb upstream to 10 kb downstream of the ple transcription start were scanned for clusters of evolutionarily conserved Grh, AP-1, ETS, and GGGGGATT sites, and two regions were selected. One is a 3.0-kb DNA fragment beginning 2.9 kb upstream of the ple transcription start, which contains two Grh sites, two AP-1 sites, four ETS sites, and three GGGGGATT sites. The second is a 995-bp fragment just upstream of the ple transcription start, which conserves one Grh, one AP-1, and three ETS sites but no GGGGGATT motif. Both fragments were tested in red fluorescent protein (DsRed) reporter construct and the 3.0-kb ple fragment was found to robustly activate reporter expression around aseptic wounds, whereas the 995-bp fragment shows a very weak and slow response (Mace, 2005).

The involvement of mitogen-activated protein (MAP) kinases in epithelial injury response prompted a test for receptor tyrosine kinase or MAP kinase activation in cells that induce wound response enhancers. Using antibodies directed against phosphotyrosine (p-Tyr), an increase was found in p-Tyr staining in the cells near aseptic wounds, as well as in the wounded thorax of spen mutants, when compared with controls. This increase in p-Tyr correlates well with cells that activate the -1.4-kb Ddc-GFP wound response construct, although at the times both could be tested (2 hours postwounding), some cells showed activation of the wound response enhancer without a detectable increase in p-Tyr (Mace, 2005).

Regulation of post-embryonic neuroblasts by Drosophila Grainyhead

The Drosophila post-embryonic neuroblasts (pNBs) are neural stem cells that persist in the larval nervous system where they proliferate to produce neurons for the adult CNS. These pNBs provide a good model to investigate mechanisms regulating the maintenance and proliferation of stem cells. The transcription factor Grainyhead (Grh), which is required for morphogenesis of epidermal and tracheal cells, is also expressed in all pNBs. This study shows that grh is essential for pNBs to adopt the stem cell program appropriate to their position within the CNS. In grh mutants the abdominal pNBs produced more progeny while the thoracic pNBs, in contrast, divided less and produced fewer progeny than wild type. Three candidates were investigated to determine whether they could mediate these effects; the neuroblast identify gene Castor, the signalling molecule Notch and the adhesion protein E-Cadherin. Neither Castor nor Notch fulfills the criteria for intermediaries, and in particular Notch activity is dispensable for the normal proliferation and survival of the pNBs. In contrast E-Cadherin, which has been shown to regulate pNB proliferation, is present at greatly reduced levels in the grh mutant pNBs. Furthermore, ectopic expression of Grh is sufficient to promote ectopic E-Cadherin and two conserved Grh-binding sites were identified in the E-Cadherin/shotgun flanking sequences, arguing that this gene is a downstream target. Thus one way Grh could regulate pNBs is through expression of E-cadherin, a protein that is thought to mediate interactions with the glial niche (Almeida, 2005).

The transcription factor Grh is present in all the postembryonic neuroblasts (pNBs). The characteristics of its expression pattern in the ventral ganglion, which reflect the segmental differences in the number of neuroblasts that persist in this part of the larval CNS, are summarized here. (1) Many more Grh-expressing cells can be detected in the thoracic region (these are reported to have 23 pNBs per hemisegment) than in the abdominal neuromeres (where only 3 pNBs, vm, vl and dl, persist). (2) Grh expression in the abdominal region disappears by late third instar (96 h after-hatching) corresponding to the time when the abdominal pNBs cease dividing and die. (3) Grh is also detected in smaller cells associated with the pNBs, which appeared to be the ganglion mother cells (GMCs) based on their position and co-labelling with a cell division marker phospho-Histone H3 (pH3). (4) Grh expression does not persist in the post-mitotic progeny of pNB lineages which can be labelled with Gooseberry-proximal (Gsb-p, transiently expressed in the progeny of some lineages and overlapped with Grh in the GMC but not in other cells). The more mature progeny also express high levels of Prospero, which is present at much lower levels in the Gsb-p expressing cells and the GMCs. The expression of Grh in the pNB and GMC suggests that it could have a role similar to that of neuroblast identity genes. Therefore, whether it confers specific properties on the thoracic and abdominal pNBs of the ventral ganglion was investigated (Almeida, 2005).

Null alleles of grh are embryonic lethal. Therefore, to investigate the role of Grh in the pNBs advantage was taken of the grh³⁷⁰ allele, which has a frame-shift in the CNS specific transcript resulting in termination upstream of the DNA binding and dimerisation domain of Grh. The grh³⁷⁰ animals survive to post-embryonic stages because Grh is still present in other tissues, although it is absent from the pNBs. This allele was used in trans to a deletion that removes the grh gene [Df(2R)Pcl7B], which produces a slightly more severe phenotype than grh³⁷⁰ homozygotes, indicating that the grh³⁷⁰ allele is not completely null for CNS function (Almeida, 2005).

The proliferation of pNBs in wild-type and grh³⁷⁰ was examined by labelling cells in S-phase with bromodeoxuridine (BrdU). When wild-type and grh³⁷⁰ larvae were fed BrdU, several differences in the pNB lineages were detected: (1) there were 1-2 extra ventral clusters per hemisegment in the abdominal segments in grh³⁷⁰; (2) there were more progeny in the abdominal clusters; (3) there appeared to be fewer progeny in the thorax. Thus removal of grh appears to result in complex phenotypes in the pNB stem cells, with opposite effects on abdominal and thoracic pNBs (Almeida, 2005).

To confirm the defects observed with BrdU labelling, the expression of Gsb-p and Prospero was characterized in the CNS of wild-type and grh mutant larvae. In the embryo, the paired-homeodomain transcription factor Gsb-p is present in eight lineages per hemi-segment, where it confers positional identity. In the larval CNS, it is also present in eight lineages per thoracic hemisegment (6 ventral and 2 dorsal) and in 2 of the 3 abdominal lineages (vm and vl). Gsb-p expression was detected in a similar number of larval lineages in wild-type and mutant CNS, and where it was expressed in the subset of progeny located closest to the pNB (Almeida, 2005).

In wild-type CNS, Gsb-p expression disappeared from the abdominal regions at about the time when the abdominal pNBs normally die, suggesting Gsb-p marks a transient stage in the development of the pNB progeny. In grh³⁷⁰ expression continues until much later times (>96 h after hatching) in the abdominal segments and more labelled cells are detected in each abdominal cluster. At their peak (approximately 72 h after hatching) there are on average 7-9 Gsb-p cells per cluster in the mutant compared with 3-4 in wild-type. These data confirm the BrdU labelling and indicate that the abdominal pNBs proliferate more extensively and for longer. In contrast, the thoracic clusters of Gsb-p expressing-cells become progressively smaller over the course of larval development. By late third instar there are many fewer Gsb-p expressing cells in each thoracic cluster than in wild-type, suggesting that mutations in grh result in reduced proliferation or premature differentiation in these lineages (Almeida, 2005).

To further investigate the change in the pNBs behaviour in grh mutant, Prospero expression was monitored at late L3 CNS. Prospero protein is present in the progeny of both wild-type and grh³⁷⁰ pNBs. However, in grh³⁷⁰ the density of Prospero expressing cells in thoracic segments was clearly decreased, consistent with the decreased Gsb-p expression and BrdU incorporation in these lineages. Within the anterior of each segment a subset of pNB lineages showed a more marked reduction in the number of Prospero expressing cells. This was similar to the effect on Gsb-p, where two of the six clusters in each hemi-segment showed a more profound reduction in size and suggested that the precise effects of grh mutation differ according to the lineage (Almeida, 2005).

The change in proliferation patterns was born out when mitotic activity was analyzed using anti- phospho-histone H3 (pH3) antibody to give a snap-shot of the number of cells in mitosis. There was increased pH3 labelling in the abdominal region of grh mutant CNS with on average 13 mitotic cells in A2-A6 of grh³⁷⁰ CNS compared to <1 mitotic cell in wild-type. In contrast, there were fewer mitotic cells present in the thoracic region confirming that grh³⁷⁰ leads to reduced proliferation in thoracic pNB lineages, in contrast to the effects in the abdomen. The changes in proliferation in the thoracic lineages could reflect delays in the re-activation of the pNBs or an alteration in the subsequent maintenance/proliferation. To investigate this an examination was performed to see at what stage Gsb-p-expressing progeny first appear in wild-type and grh³⁷⁰ larvae. In both cases, Gsb-p expressing progeny were first detected in the thoracic neuromeres of late L2 CNS (30-45 h after hatching) and in the abdominal neuromeres of early L3 CNS (50-60 h after hatching) (Almeida, 2005).

In summary, therefore, reduced proliferation was observed of the thoracic pNBs in grh³⁷⁰ larvae. This contrasts with the effects in the abdominal segments, where the pNBs continue proliferating for longer. These complex defects suggest that grh is likely to regulate pNBs through a number of different mechanisms. Three possible candidates for downstream effectors Castor, Notch and Cadherins were examined. The former is a transcription factor expressed in the embryonic neuroblasts prior to Grainyhead. The latter are cell surface proteins implicated in stem cell regulation in several other systems (Almeida, 2005).

Previous studies have shown that E-Cadherin is necessary for normal pNB proliferation. These studies show that expression of a dominant negative E-Cadherin in the neural and glial cells reduces the number of progeny produced by pNBs to <25% of wild type. Expression in the ensheathing glia alone led to more minor reduction, arguing that the protein is needed in both glia and pNBs. As reported previously, strong expression of E-Cadherin was detected in the pNBs and their adjacent progeny. In grh³⁷⁰ however, the levels of E-Cadherin in the thoracic region of the CNS were dramatically reduced. Several pNBs lack significant E-Cadherin expression all together, others retained some expression but at much lower levels compared to wild type. Similar effects were seen in clones mutant for another loss of function grh allele, grh^B32. In lineages homozygous for grh^B32 there was a variable reduction in E-Cadherin compared to neighbouring wild-type lineages (Almeida, 2005).

The effects on Cadherin contrast with those on Notch, where expression in the thoracic pNBs remains robust in grh³⁷⁰, arguing against an indirect effect resulting from changes in size. To further test this, it was asked whether Grh is sufficient to promote E-Cadherin expression when expressed elsewhere in the CNS. A pros::Gal4 driver line was used that directs high levels of expression in neurons and lower expression in the pNB lineages. When this was used to drive expression of the CNS isoform of grh, high levels of ectopic E-Cadherin were detected, particularly in many of the embryo-derived neurons that are normally devoid of E-Cadherin expression at these stages. Neither Castor nor Notch expression was altered under these conditions. Therefore Grh appears to be an activator of E-Cadherin expression. However, ectopic Grh was not sufficient to direct additional proliferation under the conditions tested (Almeida, 2005).

The genomic sequence flanking the E-Cadherin gene (shotgun, shg) was examined for consensus Grh binding sites using two different strategies. Grh binds as a dimer. In recent studies of Grh family proteins a consensus target-site was derived (WCHGGTT). Eight matches to this consensus are present in the genomic region spanning from 1 kb upstream of the shg transcript (another gene, CG10540, starts 944 bp upstream of shg) to 5 kb downstream. A second search using a weighted matrix revealed 13 matches within 5 kb of shg. A comparison of the two sets of putative sites identified four common matches: AAACAGGTTA (−300); AAACAGGTAA (+275); ATACTGGTTT (2650 bp downstream, Shg2); CAACAGGTAG (3131 bp downstream, Shg1). The latter two are 100% conserved between D. melanogaster and the five other Drosophila species for which sequence is available. To confirm that these two sites are recognised by Grh, a stringent assay was used where their ability to compete with a well-characterised, high affinity site, Gbe2 from the Dopa decarboxylase gene, was tested. Both sites were able to compete, reducing the amount of probe bound by 51% (Shg1) and 75% (Shg2) when present at 40× molar excess. The presence of these conserved sites indicates therefore that shg/E-Cadherin is likely to be a direct target of Grh. However, E-Cadherin cannot be the only target, since it was not possible to rescue the grh³⁷⁰ mutant phenotype by supplying E-Cadherin via an exogenous driver (GrhNB::Gal4/UAS::E-Cadherin) (Almeida, 2005).

Although the data show that Cadherin is regulated by Grh, they do not resolve unequivocally whether it is a direct target. Examination of genomic sequence revealed two binding-sites close to the shotgun/E-Cadherin gene that are conserved in other Drosophila species and that are bound by Grh in vitro. Future studies will show whether these sites are essential for shg expression. However, these results are exciting because they provide a link between grh function in the pNBs and in other tissues. Changes were observed in E-Cadherin levels in response to grh in other parts of the animal. There is also an interaction between shg and several genes that act together with grh in epidermal morphogenesis (although no direct genetic interaction was seen between shg and grh itself). Given that the precise levels of E-Cadherin proteins can be critical in shaping the sorting and interactions between cells it will be important to determine whether Grh is required for this regulation. It will also be important to establish whether Cadherins are targets of Grh in other animals, for example in mice where mutations in Grhl3 result in defects in neural tube closure and epidermal integrity (Almeida, 2005).

Despite the fact that Castor appeared a likely target for Grh no evidence was found that it is deregulated in the larval CNS of grh mutants. There was no ectopic Castor in thoracic pNBs at the stage when they first reactivate, as might be predicted if Grh was essential for castor repression. No repression of Castor was found when Grh was ectopically expressed with pros::Gal4. Therefore, Castor does not appear to be a primary target for the effects of Grh in the pNBs. However, in wild type CNS Castor was present transiently in many if not all of the pNBs and it remains possible that in grh mutants Castor is activated prematurely or for more prolonged periods in some of the pNBs. It was not possible to dissect in sufficient detail the timing of expression in individual thoracic lineages to resolve this. Nevertheless, it is clear from these studies that Castor is re-activated in post-embryonic lineages. This indicates that the temporal cascade of transcription factor expression does not extend simply into the post-embryonic stages. It is possible that the quiescent period during early larval stages could reset the temporal clock so that embryonic factors can be reused. However, the presence of Castor in some pNBs at the time when they first reactivate would argue against the post-embryonic series recapitulating the embryonic one, since Castor is expressed at late stage in embryonic pNBs (Almeida, 2005).

Whether or not Notch is regulated by Grh, it is certainly a prime candidate to maintain the pNB stem-cells. Previous studies had shown that Notch is present on the pNBs and an E(spl)mγ-GFP reporter showed definitively that Notch is activated in these cells. However, surprisingly, mutations in Notch failed to perturb any of the aspects of pNB behaviour that could be easily assayed. For example, one simple model suggested that signals from the progeny to the pNB mediated by Notch would prevent the pNB from differentiating prematurely. However, in Notch mutants the pNBs were found to persist as normal throughout larval stages. The abdominal pNBs also disappeared at the normal stage, indicating that the timing of their apoptosis is independent of Notch, even though Notch does regulate cell death elsewhere. Likewise, no evidence was found that Notch controls proliferation of the pNBs because the number of progeny produced and their maturation was unaffected by Notch mutations. It has not been possible however to evaluate whether the ultimate fates of the progeny are altered in Notch mutants so it remains possible that it regulates the neuronal or glial cell types produced. Now that lineage maps are being generated for the pNBs it should be possible to start investigating this possibility. Nevertheless, it is clear that the pNBs retain their stem-cell characteristics in the absence of Notch activity (Almeida, 2005).

The defects in the pNB lineages of grh mutants are position dependant. Thus, the thoracic pNBs produce fewer progeny whereas abdominal pNBs proliferate for a more prolonged period. In general, such A/P position dependent patterning is co-ordinated by the homeotic genes and indeed abdA has been shown to regulate the timing of cell death and hence the period of proliferation in the abdominal pNBs, as well as regulating the number of pNBs that persist in abdominal segments. However the phenotype of abdA mutants is significantly different from that of grh; for example the abdominal clusters are much larger and there are no defects in thoracic clusters, so it is unlikely that grh is upstream of abdA. Furthermore, in parallel studies Cenci (2005) has shown that the initiation of AbdA expression still occurs in grh mutants. Therefore it is more likely that grh acts in parallel to the homeotic genes to co-ordinate the pNB program (Almeida, 2005).

Protein Interactions

GRH functions as a heterodimer. GRH mutant proteins lacking the novel activation domain act as trans-dominant inhibitors of GRH-directed gene activation in tissue culture cells (Attardi, 1993). GRH cannot activate Ubx from the upstream promoter by direct interaction with TATA binding protein, but requires six tightly bound TBP-associated factors (Dynlacht, 1991).

The Polycomb group (PcG) of proteins represses homeotic gene expression through the assembly of multiprotein complexes on key regulatory elements. The mechanisms mediating complex assembly have remained enigmatic since most PcG proteins fail to bind DNA. The human PcG protein dinG interacts with CP2, a mammalian member of the grainyhead-like family of transcription factors, in vitro and in vivo. The functional consequence of this interaction is repression of CP2-dependent transcription. The CP2-dinG interaction is conserved in evolution with the Drosophila factor Grainyhead binding to dring, the fly homolog of dinG. Electrophoretic mobility shift assays demonstrate that the Grh-dring complex forms on regulatory elements of genes whose expression is repressed by Grh but not on elements where Grh plays an activator role. These observations reveal a novel mechanism by which PcG proteins may be anchored to specific regulatory elements in developmental genes (Tuckfield, 2002).

Strong evolutionary conservation of amino acid sequence exists between the mammalian and Drosophila members of the Grainyhead-like family. The likelihood of a similar conservation of function led the idea of the existence of a Drosophila homolog of dinG. Database searches identified a sequence that has been termed dring (FlyBase term: Sex combs extra), which has 44% identity and 61% similarity to the dinG amino acid sequence and 50% identity and 68% similarity in the domain of the dinG protein which interacts with the GRH-like family. To determine whether the Drosophila factor Dring could interact with Grh, radiolabeled in vitro-transcribed and translated Grh was generated for GST chromatography assays. Grh was shown to be specifically retained on a GST-Dring matrix but not on GST alone, confirming the evolutionary conservation of this interaction (Tuckfield, 2002).

DinG can interact with CP2 and repress transcription from a CP2-dependent promoter. These data were generated in the context of a concatemerized consensus CP2 binding site. No physiological target genes of CP2-mediated repression have been identified in mammalian systems. In contrast, the regulatory regions in the dpp and tll genes involved in Grh-mediated repression have been clearly defined in vivo. In view of the significant homology between Grh and CP2 in the DNA binding domain, whether the CP2-dinG complex could form on the Grh-responsive element in the dpp promoter was examined. A probe containing the DRE-B region of the dpp promoter was studied in an EMSA in the presence of nuclear extract from the mammalian cell line JEG-3. Addition of this extract to the DRE-B probe resulted in the formation of a DNA-protein complex. This complex was ablated by the addition of either anti-CP2 or anti-dinG antiserum. To extend this observation, whether the GRH-DRING complex could assemble on the regulatory regions in the dpp and tll genes that are critical for GRH-mediated repression was examined. Probes containing the DRE-B region of the dpp promoter and the tor-RE element in the tll promoter were studied in an EMSA with Drosophila embryo extract in the presence and absence of anti-Grh antiserum or anti-dinG antiserum (which cross-reacts with the Drosophila DRING protein). The be2 element of the Ddc promoter (where Grh functions as a transcriptional activator) was also studied. A complex consisting of at least Grh and Dring formed on both the dpp and tll elements. In both settings, the complex was ablated (or shifted out of the gel) by anti-Grh and anti-dinG antisera. In contrast, the complex formed on the Ddc promoter was ablated by the addition of anti-Grh antiserum but remained unchanged in the presence of anti-dinG antiserum (Tuckfield, 2002).

Efficient and specific targeting of Polycomb group proteins requires cooperative interaction between Grainyhead and Pleiohomeotic

Specific targeting of the protein complexes formed by the Polycomb group of proteins is critically required to maintain the inactive state of a group of developmentally regulated genes. Although the role of DNA binding proteins in this process has been well established, it is still not understood how these proteins target the Polycomb complexes specifically to their response elements. The grainyhead gene, which encodes a DNA binding protein, interacts with one such Polycomb response element of the bithorax complex. Grainyhead binds to this element in vitro. Moreover, grainyhead interacts genetically with pleiohomeotic in a transgene-based, pairing-dependent silencing assay. Grainyhead also interacts with Pleiohomeotic in vitro, which facilitates the binding of both proteins to their respective target DNAs. Such interactions between two DNA binding proteins could provide the basis for the cooperative assembly of a nucleoprotein complex formed in vitro. Based on these results and the available data, it is proposed that the role of DNA binding proteins in Polycomb group-dependent silencing could be described by a model very similar to that of an enhanceosome, wherein the unique arrangement of protein-protein interaction modules exposed by the cooperatively interacting DNA binding proteins provides targeting specificity (Blastyak, 2006).

The iab-7 PRE lies next to the Fab-7 boundary, a chromatin domain insulator element between the neighboring iab-6 and iab-7 cis-regulatory domains of BX-C. Fab-7 ensures the functional autonomy of these cis-regulatory domains; iab-7 is inactive in the sixth abdominal segment (A6), where iab-6 is active, while iab-7 is activated in segment A7. A large set of internal BX-C deficiencies is available, making this region ideal for genetic studies (Blastyak, 2006).

Class II deletions, which remove only the boundary region, fuse the otherwise intact cis-regulatory elements iab-6 and iab-7. The consequence of this fusion is that in some A6 cells iab-6 is inactivated by iab-7, while in some other A6 cells iab-6 ectopically activates iab-7. As a result, A6 will become a mixture of cell clones with either A5 or A7 identity. Due to the fact that the Abd-B gene, the expression of which is controlled by these cis regulators, is haploinsufficient, such transformations are evident even under heterozygous conditions. Class I deletions, which remove both the Fab-7 boundary and the adjacent iab-7 PRE, transform A6 into a perfect copy of A7, suggesting that in the case of class II deletions it is the iab-7 PRE that mediates the inactivation of iab-6 in A6; thus, the inactivation may depend on Pc-G-mediated silencing. Indeed, if a class II deletion is combined with some, but not all, Pc-G mutations, the resulting phenotype is indistinguishable from that of class I deletions. Based on this result, it should be possible to identify mutations in factors that specifically interact with the iab-7 PRE as enhancers of the phenotype of class II deletions (Blastyak, 2006).

Accordingly, several X-ray mutagenesis screenings were performed with the class II allele Fab-7². Among the enhancer mutants, one complementation group, represented by five alleles in the collection, is described here. Two alleles are associated with a cytologically visible breakpoint in 54F, and deficiency mapping placed the locus between the proximal breakpoints of the Pcl11b and Pcl7b deletions. Previously, four complementation groups were isolated within this interval. Noncomplementation with alleles of one of the four complementation groups showed that new mutant alleles were isolated of the previously described gene grainyhead (grh). The previously isolated grh alleles, including the molecularly characterized amorphic allele B37, are also strong Fab-7² enhancers, indicating that loss-of-function grh mutations affect the function of the iab-7 PRE (Blastyak, 2006).

Genome-wide prediction has indicated that the occurrence of the same limited set of consensus motifs can fairly accurately predict the PRE function of a DNA sequence (Ringrose, 2003). This observation suggests that many, if not all, PREs use the same set of DNA binding proteins. One of the frequently occurring consensus sequences within PREs is a poly-T motif. Many, although not all, GRH binding sites are T rich, and the current studies indicate that at least in some cases the poly-T consensus sequence may be a binding site for this protein. However, like other DNA binding proteins involved in PRE function, GRH alone cannot explain the specificity of targeting, since its function is not limited to PREs. In other contexts, GRH acts as a transcriptional activator. The fact that an array of distinct sequence motifs is required to accurately predict PREs probably means that there is no single major targeting activity. Indeed, in the case of the engrailed PRE it was demonstrated that all binding sites of DNA binding proteins are equally important for silencing activity. Identification of GRH as a PRE-related DNA binding protein and, in particular, its cooperative interaction with another member of this group both in vivo and in vitro may help in understanding the targeting of PC-G to PREs during development (Blastyak, 2006).

A cooperative interaction between GAF (Trithorax-like) and PHO has been demonstrated (Mahmoudi, 2003). In contrast to the case of GRH and PHO, cooperation between GAF and PHO is independent of the physical interaction between the two proteins and requires a nucleosomal context. Although the physical basis of this cooperative interaction is not understood, it also suggests that cooperativity may be an important principle in the organization of nucleoprotein assembly at PREs (Blastyak, 2006).

What could be the impact of cooperativity on PC-G targeting? Theoretically, one of the most significant problems encountered by a DNA binding protein is the huge excess of potential binding sites in the genome, including both functional sites and pseudosites. It can be assumed that if any of the DNA binding proteins involved in targeting are present in limited amounts in the nucleus, then their binding occurs only at the highest-affinity sites, where a combination of certain binding sites facilitates their cooperative binding. Several observations contradict this simple model. First, if the amount of these DNA binding proteins were limited, their mutations would be expected to result in strong haploinsufficient phenotypes, which is not the case. Second, studies on the DNA binding proteins EVE, FTZ, and GAF demonstrated that in vivo they also bind to genes that are not controlled by them. These functionally irrelevant sequences may represent pseudosites, and the relatively low level of binding at these sites may indicate a low binding affinity. Thus, it appears that restricted binding site occupancy of DNA binding proteins is not necessary for specificity in gene regulation. Likewise, even though the DNA binding proteins present on PREs may bind to nonfunctional sites, it is likely that the functionally relevant high-affinity sites are distinguished from pseudosites in vivo by the unique arrangement of distinct, stably bound cooperative partners. However, although in this model of targeting of PRC1 to the iab-7 PRE, cooperativity at the level of the DNA binding proteins is critically required for binding stability, by itself it is insufficient to provide the required specificity of the targeting process (Blastyak, 2006).

In contrast to the DNA binding components, other constituents of the silencing complex appear to be limiting factors. This is suggested by the fact that most Pc-G genes were identified either on the basis of their characteristic haploinsufficient phenotypes or on the basis of their dominant genetic interaction with other known Pc-G members. The number of potential PRE sequences is also relatively small, as a genome-wide survey estimated it to be not more than a few hundred in Drosophila. This brings us to the question of how the abundant DNA binding proteins link the limited amount of PC-G complexes to the low-frequency target sites with high specificity (Blastyak, 2006).

The first clue comes from studies showing that all of the PRE DNA binding proteins have the ability to interact with various PC-G proteins that are all subunits of the same preformed protein complex, PRC1. These interactions appear to be weak by themselves, as illustrated by the fact that although the occurrence of these interactions can be demonstrated by using short protocols like immunoprecipitation, the resulting complexes do not survive nonequilibrium methods used for traditional biochemical purification of protein complexes. The consequence of the cooperativity at the level of DNA binding proteins is that the otherwise weak interaction surfaces are integrated into a stable composite surface that can serve as a high-affinity docking site for the limited amount of PRC1 complex. In the model, this second level of cooperativity would provide targeting specificity (Blastyak, 2006).

Notably, the same DNA binding proteins involved in PC-G targeting can separately participate in weak interactions with various other protein complexes involved in processes unrelated to, or the opposite of, Pc-G-dependent silencing, such as TFIID-dependent transcription or chromatin remodeling by SWI/SNF. Based on the available data, interaction surfaces of any such complex are not shared by these DNA binding proteins, and according to this model, their concerted recruitment to PREs is unlikely. Also, in agreement with the experimental data, this model predicts that in the absence of DNA none of the DNA binding proteins will be able to interact stably with the complex to be recruited. The integration of several weak protein-protein interaction modules into a single entity is a prerequisite for the complex to dock on chromatin (Blastyak, 2006).

It has been shown that transcription through the iab-7 PRE displaces PC-G proteins and results in concomitant recruitment of the TRX and BRM proteins. Thus, iab-7 PRE appears to be a switchable element and the potential, for example, of PHO to interact with protein partners having a function that is the opposite of PC-G silencing might be realized under certain circumstances. There is insufficient data to explain the mechanism underlying this switch. One possibility is that binding of some DNA binding proteins to DNA or to their interacting partners is modified by posttranslational modifications, as it was shown in the case of the human homologue of Grh. According to the model, even the modification of a single actor (e.g., GRH) can radically influence the overall assembly configuration of the targeting complex and might be responsible for the dynamic nature of the iab-7 PRE (Blastyak, 2006).

This model shows remarkable similarity to the functional and structural organization of enhanceosomes. For example, multimerization of the binding sites of any of the DNA binding proteins involved in beta interferon (IFN-ß) enhanceosome formation does not reproduce faithfully the virus inducibility of the intact enhancer. Instead, these synthetic enhancers respond promiscuously to inducers that are normally not involved in regulation of the IFN-ß gene. The molecular basis of the selective inducer response of the enhanceosome is established by the following cooperative interactions. First, in their original context, the mutually cooperative interactions at the level of DNA binding proteins promote binding stability. Second, on the resulting spatially arranged protein surface, each DNA binding protein contributes to the recruitment of a protein complex through interactions with one of its subunits. It is concluded that the integration of different, hierarchical levels of cooperativity could be a general principle in the targeting of protein complexes to chromatin (Blastyak, 2006).

The validity of the enhanceosome model has already been demonstrated by in vitro reassembly of the IFN-ß enhanceosome with well-defined recombinant components. In vitro studies with a nucleosomal template have provided valuable insights into the role of PRC1 in regulation of the chromatin structure. However, in this experimental system the excess of PRC1 and nonspecific DNA binding of PRC1 complex members overcomes the problem of targeting. An initial attempt to reconstitute cooperativity at the level of DNA binding proteins failed, possibly because the simultaneous presence of several other DNA binding proteins is required for cooperative assembly. Until these components of PREs are identified, it is likely that PC-G targeting cannot be faithfully reconstituted in vitro. Hopefully, the identification of as-yet-unknown DNA binding protein components of PREs, together with the conceptual framework presented here, will facilitate these studies (Blastyak, 2006).

Recent results showed that in vivo stable recruitment of PC to the Ubx PRE critically depends on the presence of the E(Z) protein. E(Z) is a member of a PC-G complex, which is distinct from PRC1, and possesses histone methyltransferase activity. These findings led to a model wherein, upon binding of the EZ complex, its enzymatic activity could provide the mark for the specific targeting of PRC1. Hence, recruiting of PRC1 would only indirectly depend on sequence-specific DNA binding proteins, as they primarily act as recruiters of the E(Z) complex, but not PRC1. Contrary to the predictions of this model, it was found that although mutations in PRC1 complex members are similarly strong dominant enhancers of the Fab-7² phenotype as grh and pho, amorphic E(z) alleles in heterozygous condition are not. Thus, the current results indicate a rather intimate link between these DNA binding proteins and PRC1 complex members. However, it is still possible that in a nucleosomal context the histone mark could provide an additional constituent for binding whose presence can be critical in vivo in certain tissues. Certain PC-G group members have a tissue-specific phenotype, and GRH is also not ubiquitously expressed, which supports this notion (Blastyak, 2006).

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