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Gene name - Trithorax-like Synonyms - GAGA Cytological map position - 70E--70F Function - transcription factor Keywords - trithorax group |
Symbol - Trl FlyBase ID:FBgn0013263 Genetic map position - 3-[41] Classification - zinc finger domain Cellular location - nuclear |
Recent results suggest that the Drosophila transcriptional activator known as GAGA factor, or Trithorax-like, functions by influencing chromatin structure (Granok, 1995). Chromatin is the complex of DNA and proteins that bind DNA into a highly ordered structure. Before further discussion of Trithorax-like, a word about chromatin is in order. Chromatin gets its name from the affinity that this DNA-protein complex has for dyes used to stain chromosomes and cell nuclei. At the earliest stage in Drosophila development, the chromatin is transcriptionally silent. All developmental decisions are based on maternal proteins that exist in the highly ordered oocyte prior to fertilization. The transition to zygotic transcription occurs at the stage of mid-blastula transition.
The chromosomal protein Histone H1 is considered a linker histone, since it is involved, by self association, in generating the superhelical 30 nm fiber of chromatin in chromosomes. Pre-blastoderm chromatin does not contain histone H1, but instead is saturated with HMG-D, the Drosophila homolog of HMG1. As maternal HMG-D is depleted, (mid-blastula transition, at approximately cell cycle 10), histone H1 accumulates, coincident with the start of zygotic transcription. At this time, the nuclei become more compact; this is paralleled by a reduction in size of mitotic chromatin (Ner, 1994).
GAGA transcription factor has been shown to counteract chromatin repression at all levels, and by so doing, trigger the active transcription of genes subject to repression. Drosophila gene hsp70 has proven a useful tool to build a better understanding of the process. HSP70 is a so-called heat shock protein. It functions on an as needed, emergency basis to repair or discard proteins denatured by high temperatures. The main transcription factor regulating hsp70 is HSF, the heat shock transcription factor. The promoter of hsp70 contains sites for HSF and Trithorax-like/GAGA, a constitutively expressed transcription factor that binds to poly GA rich sites present in the DNA that codes for many Drosophila genes.
The hsp70 promoter binds two other factors in addition to GAGA and HSF: TFIID which serves as the TATA-binding protein complex, and RNA polymerase II. In HSP70's inactive state, polymerase has paused after synthesizing a short transcript. Pulsing the temperature results in a relief of pausing, the release of the polymerase protein and the completion and continuation of gene transcription.
What is the role of GAGA in the activation of transcription of HSP70? An artificial system had to be constructed in order to investigate the question. A hsp70 plasmid DNA containing the hsp70 promoter was constructed and the chromatin, consisting of histones was constructed without GAGA. When GAGA and an additional protein complex are added to this mixture the disruption of the chromatin structure ensues in an energy dependent process. In other words disruption of the chromatin structures requires transfer of energy from the breakdown of ATP (Tsukiyama, 1994).
Subsequent biochemical work has resulted in the purification of a Nucleosome remodeling multiprotein complex (NURF) responsible for the energy dependent remodling of chromatin. One of the constitutes is ISWI, a homolog of the yeast chromatin remodeling factor SWI2/SWF2. Thus addition of GAGA along with NURF is sufficient to remodel the chromatin, relieving its repressive effects, allowing for access to the gene of other transcription factors and initiation of transcription. The various effects of GAGA could be explained by its ability to rearrange nucleosomal positions. Chromatin remodelling by GAGA and other factors in vitro require activities that maintain a highly dynamic state of chromatin (Becker, 1995).
The role of GAGA is a model of how trithorax group proteins activate silenced genes. GAGA may turn out to have no essentially different properties from other transcription factors. It has helped however in a conceptual switch concerning understanding of gene activation. No longer is it sufficient to know which factors interact for transcriptional interaction, but the question is taken one step forward; now one must know what roles these factors play in overcoming the repressive effects of chromatin, resulting in gene activation.
One additional property of GAGA warrents mention. Many transcription factors dissociate from DNA during mitosis: the chromosomes become protected by a class of proteins called polyamines, basic proteins that have a high affinity for nucleic acid. GAGA remains associated with DNA during mitosis (Raff, 1994 and O'Brien, 1995). What is the special role of GAGA in preserving the continuity of the state of gene activation during mitosis and how does the loss of affinity of other transcription factors relate to the preservation of the differentiated state? For reviews on the role of GAGA in transcription, see Granok, 1995 and Becker, 1995.
In summary, Trithorax-like belongs to the trithorax group of genes required for normal expression of homeotic genes. Trl is involved in modifying accessability of promoters by altering the nucleosome structure, so that other transcription factors can bind (Farkas, 1994). TRL causes nucleosome disruption in an energy-dependent reaction that requires other proteins as well (Wall, 1995). Trithorax group genes oppose the action of Polycomb group genes. The latter function to silence active genes. TRL is associated with specific regions of heterochromatin during all stages of the cell cycle, including mitosis. The continual association of TRL with promoters, even during mitosis, may help explain the continuity of the differentiated state, since most transcription factors dissociate from DNA during mitosis (O'Brien, 1995).
The function of GAGA is not restricted to that of a gene-specific transcriptional activator. Trl mutations are dominant enhancers of position-effect variegation, indicating that GAGA counteracts heterochromatic silencing (Farkas, 1994). GAGA has also been implicated in the functioning of the polycomb response elements (Strutt, 1997). Immunolocalization studies revealed a strong association of GAGA with the GA-rich centric heterochromatin throughout the cell cycle in early embryos (Raff, 1994). More recent studies suggested a mitosis-specific association of GAGA with GA-rich satellite DNA (Platero, 1998). This observation might be related to a variety of nuclear cleavage cycle defects, displayed by Trl mutants, that include asynchrony and failure in chromosome condensation and segregation (Bhat, 1996). Thus, GAGA is a multipurpose protein that mediates gene-specific regulation but also plays a global role in chromosome function.
The Drosophila GAGA factor self-oligomerizes both in vivo and in vitro. GAGA oligomerization depends on the presence of the N-terminal POZ domain. The formation of dimers, tetramers, and oligomers of high stoichiometry is observed in vitro. GAGA oligomers bind DNA with high affinity and specificity. As a consequence of its multimeric character, the interaction of GAGA with DNA fragments carrying several GAGA binding sites is multivalent and of higher affinity than its interaction with fragments containing single short sites. A single GAGA oligomer is capable of binding adjacent GAGA binding sites spaced by as many as 20 base pairs. GAGA oligomers are functionally active, being transcriptionally competent in vitro. GAGA-dependent transcription activation depends strongly on the number of GAGA binding sites present in the promoter. The POZ domain is not necessary for in vitro transcription, but in its absence no synergism is observed upon an increase in the number of binding sites contained within the promoter (Espin·s, 1999).
GAGA is known to enhance transcription from promoters containing d(GA…TC)n sequences, both in vitro and in vivo. To analyze the contribution of the presence of multiple binding sites to the transcription activity of GAGA, the rate of GAGA-dependent transcription activation from promoters containing an increasing number of GAGA binding sites was determined. For these experiments, the GAGA binding site found at the C-region of the engrailed promoter was multimerized and fused to a minimal promoter, which efficiently drives transcription of a G-less cassette. The constructs used in these experiments contain from 1 to 6 copies of this engrailed site. The extent of maximal activation obtained in the presence of GAGA strongly depends on the number of binding sites present at the promoter. No significant activation is observed from constructs containing only one or two GAGA binding sites, and only a moderated 3-fold activation is observed in the presence of three binding sites. However, a strong increase in activation, to about 8-9-fold, is seen from constructs containing five or six binding sites. This behavior depends on the presence of the POZ domain. When the transcription activity of the DeltaPOZ245 peptide is analyzed, a significant activation is observed in the presence of two GAGA binding sites, which increases only slightly, as does the number of binding sites. In this case, a low though reproducible activation is detected even in the presence of a single site. The synergism in transcription activation detected upon increasing the number of binding sites is consistent with the higher affinity of GAGA oligomers for fragments carrying multiple GAGA sites. Consistent with this hypothesis, this synergism depends on the presence of the POZ domain (Espin·s, 1999).
Several observations suggest that, to some extent, GAGA functions at the chromatin level, participating in the formation of an open chromatin structure. GAGA is the product of the Trithorax-like(Trl) gene which, being a member of the Trithorax group, antagonizes the chromatin-mediated repression that Polycomb genes induce upon the expression of the homeotic genes. A more direct link to chromatin structure is indicated by the fact that Trl is an enhancer of position effect variegation. Moreover, in collaboration with nucleosome remodeling factor, GAGA was shown to help nucleosome disruption at specific regions of the hsp70 promoter, encompassing GAGA binding sites. At present, little is known about the specific contribution of GAGA to chromatin remodeling, but GAGA appears to be particularly efficient in this respect. Although a direct interaction with the chromatin remodeling machinery cannot be excluded, the simultaneous interaction of GAGA oligomers with multiple adjacent sites could significantly contribute to the higher efficiency of GAGA in disrupting nucleosomes. In this context, it would be interesting to know whether a functional POZ domain is required for efficient nucleosome disruption. GAGA can also activate transcription in vitro, suggesting a possible interaction with the basal transcription machinery. These results indicate that the presence of several independent GAGA sites is required for efficient transcription activation in vitro, indicating that the oligomeric character of GAGA might also be functionally relevant in this context. Interestingly, in the case of the DeltaPOZ245 peptide, significant transcription activation is detected in the presence of a single binding site, and no synergism is observed upon increasing the number of GAGA binding sites. These results suggest that the synergism observed with full GAGA arises from specific features of the GAGA-DNA complex rather than from the simple recruitment of multiple GAGA molecules to the promoter (Espin·s, 1999).
The GAGA transcription factor of Drosophila is ubiquitous and plays multiple roles. Characterization of cDNA clones and detection by domain-specific antibodies has revealed that the 70-90 kDa major GAGA species are encoded by two open reading frames producing GAGA factor proteins of 519 amino acids (GAGA-519) and 581 amino acids (GAGA-581), that share a common N-terminal region which is linked to two different glutamine-rich C-termini. Purified recombinant GAGA-519 and GAGA-581 proteins can form homomeric complexes that bind specifically to a single GAGA sequence in vitro. The two GAGA isoforms also function similarly in transient transactivation assays in tissue culture cells and in chromatin remodeling experiments in vitro. Only GAGA-519 protein accumulates during the first 6 h of embryogenesis. Thereafter, both GAGA proteins are present in nearly equal amounts throughout development; in larval salivary gland nuclei they colocalize completely to specific regions along the euchromatic arms of the polytene chromosomes. Coimmunoprecipitation of GAGA-519 and GAGA-581 from crude nuclear extracts and from mixtures of purified recombinant proteins, indicates direct interactions. It is suggested that homomeric complexes of GAGA-519 may function during early embryogenesis; both homomeric and heteromeric complexes of GAGA-519 and GAGA-581 may function later (Benyajati, 1997).
Bases in 5' UTR - 177
Bases in 3' UTR - 100
Trl has two major structural domains: a zinc finger domain and an N-terminal BTB domain, also known as a POZ domain, responsible for transcriptional activation. Trithorax (Trx) itself has no BTB domain (Farkas, 1994 and Soeller, 1993).
Two other Drosophila proteins with zinc finger domains, Tramtrack and Broad Complex, also contain N-terminal domains highly related to that of TRL (Soeller, 1993).
To better define the molecular basis of the pleiotropic effects of Trithorax-like mutations, cDNAs were cloned that encode the GAGA isoforms of D. melanogaster and a distantly related species, D. virilis. The genomic organizations of both the D. melanogaster and D. virilis genes were characterized, and the expression patterns of isoform-specific mRNAs were analysed. The D. virilis GAGA isoforms show high similarity to their D. melanogaster counterparts, particularly within the BTB/POZ protein-interaction and the zinc finger DNA-binding domains. Interestingly, conservation clearly extends beyond the previously defined limits of these domains. Moreover, the comparison reveals a completely conserved block of amino acid residues located between the BTB/POZ and DNA-binding domains, and a high conservation of the C-terminus specific for one of the GAGA isoforms. Thus, sequences of as yet unknown functions are defined as rewarding targets for further mutational analyses. The high conservation of the GAGA proteins of the two species is in accord with the nearly identical genomic organization and expression patterns of the corresponding genes (Lintermann, 1998).
The protein coding sequences of Trl class A transcripts are split between four exons, which are separated by three introns of 2.2 kb, 118 bp and 160 bp. Class B transcripts are derived by the use of an alternative splice site within exon IV. Transcripts of both classes thus share exons I to III and the 5' portion of exon IV. The BTB/POZ domain is encoded, in about equal shares, by exons I and II. In addition to the C-terminal half of the BTB/POZ domain, exon II also encodes a putative nuclear localization signal. The minimal DNA-binding domain of the GAGA factor consists of a single C2H2 zinc finger and two regions of basic amino acids located immediately N-terminal to the zinc finger, and is encoded by exons III and IV. The 3' end of exon III contains basic region I and the rest of the binding domain is located in that part of exon IV that is common to both transcript classes. This part also contains a third region of basic amino acid residues located C-terminal to the zinc finger, which seems to be dispensable for DNA bining. The polypeptide encoded by the 3' part of exon IV, which is specific for class A transcripts, is characterized by stretches of polyglutamine. Simlar regions of high glutamine content are also found in the polypeptide encoded by the class B-specific exon V. The C-terminal sequences specific for the D. melanogaster GAGA-581 (class B) and the D. virilis class B isoform show a significantly higher conservation than the C-terminal sequences specific for the D. melanogaster class A and D. virilis class A isoform. Since functional differences between the different isoforms must be based on these C-terminal sequences, the class B isoforms may have adopted specialized functions that are more sensitive to changes in the amino acid sequence (Lintermann, 1998).
A class A-specific probe detects a small transcript (2.5-kb) restricted to adult females and early embryos, suggesting that it represents a maternal mRNA. The pattern of class A and B transcript expression strikingly changes during development. While the class A transcripts dominate in early embryos, transcripts of the two classes are present in similar amounts at later stages of embryogenesis. In first and second instar larvae, the 3.4-kb class B mRNA is predominant. In third instar larvae the 2.5-kb class A transcript increases to a level comparable to that of the 3.4-kb class B transcript, but the 3.9-kb class B transcript is underrepresented. This situation changes in the pupa, where the ratios between the three transcripts are comparable to the ratios seen in late embryos. Class A transcripts are not detected in males; the 3.4-kb class B transcript is clearly the dominating species in males. The 3.9-kb class B transcript seems to be strongly underrepresented in both males and females. A similar sex-specific expression of class A and class B transcripts is observed in D. melanogaster and D. virilis (Lintermann, 1998).
The effect of GAGA protein on chromatin structure and promoter function has been the subject of much attention, yet little is known of the actual mechanism and the specific contributions of individual GAGA domains to its function. The DNA-binding activity of GAGA, as specified by the single zinc finger binding domain (Zn), has been examined in some detail; however, the functions of the POZ/BTB and glutamine domain (Q) remain poorly understood. Three separate activities of the Q domain of GAGA are reported: promoter distortion, single-strand binding, and multimerization. In vitro, GAGA binding to the hsp70 promoter produces extended DNase I protection and KMnO4 hypersensitivity. These activities require both the Zn domain and Q domain of GAGA, and appear independent of the POZ/BTB domain. GAGA also has a single-stranded DNA binding affinity, as does the Q-rich region alone. GAGA forms multimers both in vitro and in vivo, and the Q domain itself forms multimers. Protein-protein interactions mediated by the Q domain may, therefore, be at least partially responsible for the multimerization capabilities of GAGA (Wilkins, 1999).
The BTB/POZ domain defines a conserved region of about 120 residues; it has been found in over 40 proteins to date. It is located predominantly at the N terminus of Zn-finger DNA-binding proteins, where it may function as a repression domain, and less frequently in actin-binding and poxvirus-encoded proteins, where it may function as a protein-protein interaction interface. A prototypic human BTB/POZ protein, PLZF (promyelocytic leukemia zinc finger) is fused to RARalpha (retinoic acid receptor alpha) in a subset of acute promyelocytic leukemias (APLs), where it acts as a potent oncogene. The exact role of the BTB/POZ domain in protein-protein interactions and/or transcriptional regulation is unknown. The BTB/POZ domain from PLZF (PLZF-BTB/POZ) has been overexpressed, purified, characterized, and crystallized. Gel filtration, dynamic light scattering, and equilibrium sedimentation experiments show that PLZF-BTB/POZ forms a homodimer with a Kd below 200 nM. Differential scanning calorimetry and equilibrium denaturation experiments are consistent with the PLZF-BTB/POZ dimer undergoing a two-state unfolding transition. Circular dichroism shows that the PLZF-BTB/POZ dimer has significant secondary structure including about 45% helix and 20% beta-sheet. Crystals of the PLZF-BTB/POZ have been prepared that are suitable for a high resolution structure determination using x-ray crystallography. The data support the hypothesis that the BTB/POZ domain mediates a functionally relevant dimerization function in vivo. The crystal structure of the PLZF-BTB/POZ domain will provide a paradigm for understanding the structural basis underlying BTB/POZ domain function (Li, 1997).
A novel zinc finger protein, ZID (standing for zinc finger protein with interaction domain) was isolated from humans. ZID has four zinc finger domains and a BTB domain, also know ans a POZ (standing for poxvirus and zinc finger) domain. At its amino terminus, ZID contains the conserved POZ or BTB motif present in a large family of proteins that include otherwise unrelated zinc fingers, such as Drosophila Abrupt, Bric-a-brac, Broad complex, Fruitless, Longitudinals lacking, Pipsqueak, Tramtrack, and Trithorax-like. The POZ domains of ZID, TTK and TRL act to inhibit the interaction of their associated finger regions with DNA. This inhibitory effect is not dependent on interactions with other proteins and does not appear dependent on specific interactions between the POZ domain and the zinc finger region. The POZ domain acts as a specific protein-protein interaction domain: The POZ domains of ZID and TTK can interact with themselves but not with each other, or POZ domains from ZF5, or the viral protein SalF17R. However, the POZ domain of TRL can interact efficiently with the POZ domain of TTK. In transfection experiments, the ZID POZ domain inhibits DNA binding in NIH-3T3 cells and appears to localize the protein to discrete regions of the nucleus (Bardwell, 1994).
Specific DNA binding to the core consensus site GAGAGAG has been shown with an 82-residue peptide (residues 310-391) taken from the Drosophila transcription factor GAGA. Using a series of deletion mutants, it was demonstrated that the minimal domain required for specific binding (residues 310-372) includes a single zinc finger of the Cys2-His2 family and a stretch of basic amino acids located on the N-terminal end of the zinc finger. In gel retardation assays, the specific binding seen with either the peptide or the whole protein is zinc dependent and corresponds to a dissociation constant of approximately 5 x 10(-9) M for the purified peptide. It has previously been thought that a single zinc finger of the Cys2-His2 family is incapable of specific, high-affinity binding to DNA. The combination of an N-terminal basic region with a single Cys2-His2 zinc finger in the GAGA protein can thus be viewed as a novel DNA binding domain. This raises the possibility that other proteins carrying only one Cys2-His2 finger are also capable of high-affinity specific binding to DNA (Pedone, 1996).
GAGA is a nuclear protein encoded by the Trithorax-like gene in Drosophila that is expressed in at least two isoforms generated by alternative splicing. By means of its specific interaction with DNA, GAGA has been involved in several nuclear transactions including regulation of gene expression. The GAGA519 isoform has been studied as a transcription factor. In vitro, the transactivation domain has been assigned to the 93 C-terminal residues that correspond to a glutamine-rich domain (Q-domain). It presents an internal modular structure and acts independently of the rest of the protein. In vivo, in Drosophila SL2 cells, Q-domain can transactivate reporter genes either in the form of GAGA or Gal4BD-Q fusions, whereas a GAGA mutant cannot (where the Q-domain has been deleted). These results give support to the notion that GAGA can function as a transcription activating factor (Vaquero, 2000).
Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases. Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members. S. pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome. The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction. Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner. Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1. It is proposed that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes (Geyer, 2003).
These results identified BTB/POZ proteins as components of Pcu3p/Pip1p ubiquitin ligase complexes. Four pieces of evidence suggest that BTB/POZ domain proteins are functionally equivalent to the SKP1/F box adaptor dimers determining the substrate specificity of CUL1-associate SCF complexes: (1) all three BTB/POZ proteins present in the fission yeast genome interact with Pcu3p/Pip1p complexes; (2) BTB/POZ domains are structurally related to SKP1; (3) N-terminal residues invariably conserved in all CUL3 homologs, including Pcu3p, cluster in the same region of CUL1 that mediates its interaction with SKP1/F box adaptor dimers. Both the Btb3p/Pcu3p interaction and Pcu3p-dependent Btb3p degradation depend on the integrity of this conserved N-terminal region. (4) Btb3p is ubiquitylated in vitro in a Pcu3p-dependent manner, a finding reminiscent of CUL1-dependent ubiquitylation and degradation of F box proteins. Taken together, these findings strongly suggest that the BTB/POZ domain proteins ubiquitously present in eukaryotes define a family of substrate-specific adaptors for CUL3. Since fission yeast encodes three different BTB/POZ domain proteins, all of which interact with Pcu3p and Pip1p, it may form a minimum of three distinct RING/cullin 3/BTB complexes (Geyer, 2003).
date revised: 30 August 2000
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