The trithorax group (trxG) consists of a genetically defined class of genes responsible for maintaining the active expression state of homeotic genes. Through the differential expression pattern of homeotic genes, cells become programmed to support specific structures and functions. Mutations in any member of the trxG result in reducted homeotic gene expression and in homeotic body pattern transformations. For the reciprocal process, the genes of the Polycomb group (PcG) are necessary for maintaining the repressed state of homeotic gene expression. In Drosophila, both groups are part of what is called a "cellular memory" mechanism. Their task is not to initiate the differential expression pattern of homeotic genes, which in Drosophila is performed by the early-acting patterning factors encoded by maternal and segmentation genes, but to maintain the genetic expression status stably and heritably over developmental time (for reviews, see Refs. 1, 2). Subsequently, it was found that many other developmentally important genes, whose expression patterns needs to be tightly and faithfully maintained, are also targets of this type of regulation in addition to the homeotic genes.
Molecular analysis of several members of the two groups indicate involvement of their protein products at the level of chromatin structure. Each group acts in large multiprotein complexes to fulfill its functions. Although PcG proteins cooperate to generate silenced chromatin structures, to keep genes inactive, the proteins of the trxG cooperate to counteract the PcG-repressed chromatin by modifying chromatin structures for transcriptional activation.
1. The Trithorax protein and its targets
The gene trithorax (trx), the name-giving member of the group, was identified by its requirement for normal expression of multiple homeotic genes of the bithorax and Antennapedia complex. Different homeotic genes and their promoters are affected differently in trx mutants, suggesting a coordinated spatial and temporal requirement for trx to achieve normal expression patterns (3, 4). Several parts of homeotic gene expression are dispensable of trx function and probably require other factors of the trxG, like ashl, for maintenance (5, 6).
The trx gene expresses a complex pattern of messenger RNA transcripts which encode two large protein isoforms (4, 7, 8). The TRX proteins are characterized by containing several evolutionarily conserved protein motifs, a novel variant of the nuclear receptor-type DNA-binding domain, a 130-to 140-residue motif known as the SET domain, and a C4HC3 zinc-finger motif, known as the PHD finger. The SET domain is also found in ASH1 and in other chromatin-associated proteins, including Enhancer of Zeste, a member of the PcG (6, 9). The SET domain was functionally analyzed in yeast on a protein having this conserved part. It was demonstrated that the domain is necessary for transcriptional silencing and for other cellular processes dependent on defined chromatin structures (10). The PHD finger is found in a large class of nuclear proteins, most of which function as adapters between specific activator proteins and other components of the transcription machinery. A potential role in mediating protein-protein interactions has been proposed, but the precise molecular function of the domain remains unknown. Although no direct sequence-specific DNA-binding activity has been demonstrated for TRX in vitro, it was found by immunostaining that the protein binds to approximately 75 chromosomal sites on polytene chromosomes (7, 11). Interestingly, many TRX binding sites overlap with binding sites of PcG proteins at chromosomal elements known as PcG response elements (PRE), suggesting a functional cross talk between these counteracting factors (11, 12).
2. Brahma and the chromatin-remodeling machines
The trxG member brahma (brm) was isolated in a genetic screen searching for dominant suppressors of homeotic transformations produced in Polycomb mutants (13). The idea behind this screen was to identify additional genes involved in inactivating the expression of homeotic genes. Indeed, it was found that brahma mutants cause developmental defects similar to other mutants that fail to express homeotic genes adequately (14). Molecular analysis showed that the Brahma protein is homologous to the yeast SWI2 protein, a DNA-dependent ATPase (15) and also part of a multiprotein complex with similarities to the well-known SWI/SWF complex from yeast.
Identification of the function of Brahma and its relationship to yeast components involved in transcriptional activation was the key to isolating other components associated in what are now called chromatin-remodeling machines (16). ISWI is another SWI2 homologue of Drosophila and is found in several multiprotein complexes involved in opening chromatin structures in an energy-dependent fashion (17, 18). Thus far, four different machines involved in chromatin-remodeling processes have been isolated in Drosophila: (1) the BRM-SWI/SNF complex, whose ATPase activity is DNA-dependent; (2) the NURF complex, whose ATPase activity relies on chromatin; (3) CHRAC, where energy is used to increase DNA accessibility in chromatin; and (4) ACF which, like the previous two, is also an ISWI-containing and ATP-utilizing chromatin-assembly and remodeling factor (for a review, see Ref. 19). The GAGA factor was originally identified biochemically as a DNA-binding transcriptional activator (20). GAGA factor cooperates with complexes like NURF to remodel chromatin. The recent isolation of mutations in the GAGA factor-encoding gene revealed a phenotype similar to trx. Hence, the gene was called Trithorax-like (TRL) and was classified as a trxG member (21).
3. Vertebrate homologues
Several members of the Drosophila trxG have counterparts in humans and mice. The mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, Htrx), a TRX homologue, was found through its involvement in the majority of infantile acute lymphocytic and mixed lineage leukemias (for a review, see Ref. 22). Disrupting the murine Mll gene by gene targeting causes homeotic transformations of the vertebrae in heterozygotes and loss of homeotic gene expression, supporting the notion that Mll is a functional equivalent of trx (23). Moreover, conserved components of chromatin-remodeling complexes were found in mammalian systems. Two genes, mbrm and brg1, are homologues of brm and also part of a SWI/SNF-like complex (24-26). Another SWI/SNF component is encoded by the human HSNF5 (Ini1) gene, and the putative Drosophila homologue snr1 interacts genetically with trx and brm (16, 27).
Initially, the genetic definition of the trxG been an entry point to identify genes involved in maintaining homeotic gene expression. It is becoming increasingly clear, however, that several members of the group fulfill much broader tasks and are parts of the general transcriptional activation complexes, thus allowing an assessment of the molecular interactions of these important regulatory factors that control transcriptional and eventually, other chromosomal processes at the level of chromatin structure.
