GAGA Factor Expedites Development in Drosophila

The GAGA factor (GAF), produced by Trithorax-like gene, Trl, in Drosophila [1] operates by binding to its DNA recognition sequence, having a consensus sequence, GAGAG [2]. As a transcription factor, it has been found in the promoters and enhancers for the modulation of expression of various genes like the homeotic genes and developmental genes, like the Ultrabithorax (Ubx), engrailed (en), fushi-tarazu (ftz) even-skipped (eve), hsp70, hsp 26, H3/H4, Adh, E74, actin-5C, and 1-tubulin [1,37]. Besides these roles, the factor is also involved in remodelling of the chromatin, in functions pertaining to the Polycomb responsive element (PRE), and insulator or boundary elements [5,9–14]. This factor shows a high proportion of pleiotropism owing to its localization on the promoter regions of a multitude of genes [8,9]. The gene, Trl is presumptively produced maternally [10] hence, a better understanding of its target genes is made by studying the phenotypes using combination of hypomorphic and hypomorphic or null Trl alleles. The expression of Trl is fundamental to all the stages and all tissues of the fl y, even if the levels of mRNA change signifi cantly in these stages [11]. Till date, the studies elucidating the roles of GAGA factor have been made in the embryonic stages. Studies on larval and imago stages have been very scantily made by knocking down or over-expressing different genes. Such experiments have clearly pointed that GAGA factor is an active player in the wing disc and salivary gland development. Immuno-fl uorescence studies of the salivary glands polytene chromosomes in Drosophila showed that this factor binds to a large number of euchromatic genes; hence it points out a role of GAGA factor in the maintenance of open, transcriptionally active chromatin regions.


Introduction
The GAGA factor (GAF), produced by Trithorax-like gene, Trl, in Drosophila [1] operates by binding to its DNA recognition sequence, having a consensus sequence, GAGAG [2]. As a transcription factor, it has been found in the promoters and enhancers for the modulation of expression of various genes like the homeotic genes and developmental genes, like the Ultrabithorax (Ubx), engrailed (en), fushi-tarazu (ftz) even-skipped (eve), hsp70, hsp 26, H3/H4, Adh, E74, actin-5C, and 1-tubulin [1,[3][4][5][6][7]. Besides these roles, the factor is also involved in remodelling of the chromatin, in functions pertaining to the Polycomb responsive element (PRE), and insulator or boundary elements [5,[9][10][11][12][13][14]. This factor shows a high proportion of pleiotropism owing to its localization on the promoter regions of a multitude of genes [8,9]. The gene, Trl is presumptively produced maternally [10] hence, a better understanding of its target genes is made by studying the phenotypes using combination of hypomorphic and hypomorphic or null Trl alleles. The expression of Trl is fundamental to all the stages and all tissues of the fl y, even if the levels of mRNA change signifi cantly in these stages [11]. Till date, the studies elucidating the roles of GAGA factor have been made in the embryonic stages.
Studies on larval and imago stages have been very scantily made by knocking down or over-expressing different genes.
Such experiments have clearly pointed that GAGA factor is an active player in the wing disc and salivary gland development.
Immuno-fl uorescence studies of the salivary glands polytene chromosomes in Drosophila showed that this factor binds to a large number of euchromatic genes; hence it points out a role of GAGA factor in the maintenance of open, transcriptionally active chromatin regions.
The GAGA factor was for the fi rst time identifi ed [3], as an in vitro activator of the promoter of the gene, Ultrabithorax in Drosophila. Later on, it found to bind to GAGA elements (having stretches of GAGA or CTCT) over the hsp70 heat shock promoter and H3/H4 histone gene promoter regions [3,[12][13][14]. Still further reports showed it to have crucial roles in the activation of genes and in regulating the chromatin structure [3,10,[14][15][16].
The GAGA factor also show an interaction with the promoter sequence upstream of genes, E74 [17], his3, hid,hsp26, and hsp70 [12] in Drosophila. However, the activation of transcription is found only for the genes, Kr and Ubx. Exceptionally, Kr gene is actively transcribed when the GAGA factor binds to the anchoring site located downstream to the target Kr gene.

Summary
The development in Drosophila is a concerted mechanism, occurring via the interplay of a constellation of genes and factors, operating in intricate synchrony. These factors, produced at precise points in their developmental cycle, operate via the activation, through binding to the various transcription factors. The GAGA factor (GAF) is such a product of the trithorax-like gene, Trl, which binds to a consensus DNA sequence for the modulation of the homeotic gene functions. Besides this, the factor has a role in chromatin remodelling; through binding with the Polycomb responsive element (PRE). The protein has a unique structural conformation with a zinc-fi nger DNA-binding, a BTB/POZ and a polyglutamine-rich Q domain. It has a unique role of acting as an anti-repressor of the gene, Kruppel, releasing the repression on it by the other DNA binding proteins. This report accomodates the interplay in which the GAGA factor is involved in the Drosophila embryonic development.

Pipsqueak (Psq)
Psq, essentially regulates development via recognition of the GAGA sequences. However, it needs the GA stretch to be longer than the GAF [2]. Psq has been found to co-localize with GAF for numerous loci in the polytene chromosomes

Batman or ban
The batman or ban co-localizes with GAF and causes the activation and repression of homeotic genes [41]. Besides ban, the proteins, Corto and the sin3-associated polypeptide SAP18, form complexes with GAF's BTB/POZ domain [47,48] and causes histone deacetylation. GAF interacts with the large subunit, NURF 301 of the NURF [49,50], dSSRP1 subunit of FACT [51,52]. Heterochromatin formation is marked by K9-methylated histone H3 and its binding protein HP1, and has a tendency to spread into neighbouring regions [57,58]. The process of reassembling of the nucleosome, following the replacement of histone, keep on removing K9-methylated histone H3 at d1 and prevent the spreading of the heterochromatin ( Figure 2).  [69][70][71], whose product is normally limited to the germ cells. In some cases, the somatic environment can modulate spatiotemporal regulation of germ cell migration as in mutants of the hopscotch gene, encoding a Janus kinase, causing a premature migration of primordial cells [72]. This suggests that the GAGA factor does infl uence the migration of primordial cells via their contact with the somatic cells.

GAGA in wing development
The over-expression of GAF has been found to alter the gene expression of many genes in the wing disc. Depletion in GAGA, caused by the deletion at 69B was found to consistently reduce the size of the wing by about 10 %. Using NubbGAL4

GAGA factor relieves the repression of tailless
The development of both the anterior and posterior poles, which are the terminal domains of Drosophila embryos, is specifi ed by the maternal terminal system [78]. One of such gene is tailless, which is crucial for the development of terminal structure like telson and the posterior gut as well as head portions as head structures and the brain development [79][80][81][82]. The syncitial blastoderm stage of the embryo expression this transcript at the poles occur after the indirect activation by the maternally produced Torso receptor tyrosine kinase pathway at the embryonic termini. It partly relieves the repression caused by the HMG transcription repressor, Capicua and the co-repressor, Groucho [83,84]. The other repressor that it negates is the BTB domain zinc fi nger protein, Tramtrack69 [85]. A successful functional tailless ensures the normal expression of of other gap genes such as Kruppel and Knirps, and later on genes like hunchback , brachyenteron , and forkhead [79][80][81][82]. The reduction of concentration of the repressor causes a loss in a well-defi ned edge of expression domains [86,87]. It is seen that if the binding affi nity of GAGA factor to the tor-RE is low, and multiple tor-REs are present in the tll cis-regulatory region [85,88] the boundary of tll expression gets poorlydefi ned.  [94,95]. The density of histone H3 increases when the Zelda decreases in wild-type embryos [96], the Zelda thus, dictate the expression of the initial set of zygotic genes, transcribed post fertilization and also binds to the locus for genes that need to be activated later such that a precise sequence of gene activation ensues during gastrulation.

The Vertebrate homologue of GAGA factor
Although the essential and conserved role of PcG/trxG homolog was clearly proven in the Drosophila melanogaster, the vertebrate homologue for the Drosophila GAGA factor was unknown until the recent studies [55]. The recognition sites for the GAGA factor called the GAGA boxes were found in many genes in the vertebrates including the hox complexes but a putative GAGA factor was yet to be discovered The Evx2 and Hoxd13 genes have tracts rich in GA in mouse, human, and zebrafi sh and functions in blocking the enhancer in both transgenic fl ies and cultured human cells [106]. Mutating the GAGA binding sequence prevents it from functioning as an insulator [106]. The murine Hox clusters with Histone free regions associate with the GAF recognition sites and regulate the binding of Th-POK. Thus, the mammalian GAGA factor act in nucleosome reorganization at the Hox clusters providing a platform for binding regulatory proteins, organizing chromatin regulatory activities of the chromatin, including the formation of boundaries.

Conclusion
The evidence cited in this report, clearly indicates that the GAGA factor is involved at several levels in gene expression regulation. Hence, it would be improper to consider it as a simple transcription factor or anti-repressor. Its role as a structural protein on the chromatin conformation, from its primary to its tertiary structure, depicts a potential role as a transcriptional activator and repressor. The actual role of GAF in maintaining the secondary and tertiary structure still remains more speculative than quantitative; even the functional signifi cance of GAF multimers still remains cryptic. The multimers may affect the topology of the regulatory region where they bind, changing the rotational phase of the nucleosomes to enable the proteins to co-interact. The purifi cation of GAF and the fully