BIOLOGICAL OVERVIEW

brahma, a member of the trithorax group (TRX-G) was initially isolated as a suppressor of Polycomb (Kennison, 1988). While Polycomb mutants fail to silence Antennapedia complex (ANTP-C) genes, trithorax mutants reverse this phenotype by failing to activate the same genes in a genetic game of "spy versus spy." brahma mutants cause developmental defects similar to other mutants which fail to express homeotic genes after embryogenesis, such as held out wings and loss of humeral bristles (Brizuela, 1994).

What is brahma's role in gene activation? To answer this question we first digress into the arcane world of the gene activation assay. There are two kinds. One uses naked DNA, not enshrouded with proteins, and the other uses chromatin, a complex of DNA and proteins in which proteins serves to restrict the access of transcription factors to their targets, the promoters of genes. Research carried out in yeast is enlightening since many of the 12 proteins found in the SWI/SNF complex of yeast have been purified and characterized. When SWI/SNF (pronounced switch-sniff) complex proteins are tested on naked DNA, the proteins act as a DNA dependent ATPase, that is they require naked DNA to harness the energy of ATP, which is required for some aspect of their function. The involvement of yeast SWI/SNF in overcoming the repressive affects of chromatin has recently been documented (Kruger, 1993). This study shows that mutating histones H3 and H4 (See Drosophila Histone H4 and Histone H3) partly relieve the requirement of the yeast SWI/SNF complex for transcription.

Brahma is homologous to yeast SWI2, a DNA-stimulated ATPase. Although both Brahma and yeast proteins have helicase domains, in no case has it been found that the helicase is functional. When they do work, helicases use the energy of ATP to unwind DNA. Despite the lack of a functional helicase domain the SWI/SNF yeast complex is still able to activate gene transcription. Although the ATPase activity is activated by naked DNA, SWI/SNF can activate gene transcription from DNA enshrouded with protein, that is SWI/SNF is able to overcome the repressive effect of chromatin to activate gene transcription (Tamkin 1995).

The second SWI2 homolog of Drosophila, ISWI, can be contrasted to Brahma, as it exists in a different gene activating complex. The ATPase activity of ISWI is activated by chromatin and not DNA; its concentration in the cell is 1000 times greater than that of Brahma, and ISWI's molecular weight is lower than that of Brahma because it lacks a C-terminal bromodomain, an important sequence domain that probably functions in protein-protein interaction. In addition, the protein complex that includes ISWI (NURF) is lower in molecular weight than the complex containing Brahma (Tsukiyama, 1995).

brahma, like yeast SWI/SNF, is part of a large protein complex. The newly identified SNR1 of Drosophila forms part of this complex. SNR1 is a homolog of yeast SNF5. Thus it is not an isolated protein that acts to activate gene transcription from chromatin but a large multi-protein complex. Both Brahma and SNR1 proteins are present in this complex as measured by their co-immunoprecipitation from Drosophila extracts (Dingwall, 1995).

It appears then, that there are two molecular machines for gene activation in Drosophila: 1) the SWI/SNF type complex, where ATPase activity depends on DNA and 2), the NURF complex, where ATPase activity relies on chromatin. Both of these protein complexes can activate transcription from chromatin, the protein enshrouded complex between DNA and protein. To date, these two complexes have yet to be fully explored. Very likely they each play a distinctive role in development, roles research efforts have yet to describe fully.

Loss of maternal brahma function blocks oogenesis; individuals homozygous for extreme brm alleles die as late embryos with no obvious pattern defects (Brizuella, 1994). Since it has not been possible to generate embryos lacking both maternal and zygotic brm function, the exact role of brm in embryonic development is not clear. Information concerning the role of brm after embryogenesis has been derived primarily from the analysis of hypomorphic brm alleles. Individuals trans-heterozygous for certain combinations of brm alleles survive to adulthood and exhibit developmental abnormalities similar to those arising from reduced expression of Antp-C and Bx-C genes, including the transformation of first legs to second legs and the fifth abdominal segment to a more anterior identity (Bruzuella, 1994). Because the effect of complete loss of brm function had not been examined, it was unclear whether brm is also involved in other processes. To clarify the role of brm in Drosophila development, mosaic analysis has been used to determine the null phenotype of brm mutations. As an alternative approach, site-directed mutagenesis was used to generate dominant-negative brm mutations and investigate the functions of evolutionarily conserved domains within the Brm protein (Elfring, 1998).

Using mosiac analysis, it has been found that the complete loss of brm function decreases cell viability and causes defects in the peripheral nervous system of the adult. The size and frequency of experimental clones in the head and thoracic segments are significantly reduced relative to the controls. In contrast, the frequency and size of control and experimental clones in the abdomen are similar. These data indicate that brm is essential for the development of imaginal tissues but not abdominal histoblasts. It is also possible that sufficient BRM RNA or protein persists after clone induction to allow the development of the abdominal segments. Examination of the phenotype of brm clones reveals unanticipated defects in the adult peripheral nervous system. The mechanosensory bristles of brm clones in the head, thoracic, and abdominal segments are either duplicated, stunted, or fused. In many cases, the sockets are also malformed, absent, or duplicated. No homeotic transformations have been observed in experimental clones in any of the abdominal segments. How might brm control the fate or proliferation of the cells that form mechanosensory organs? The simplest explanation is that brm regulates the activity of one or more neurogenic genes. Another possibility is that brm mutation disrupts regulation of the cell cycle. This is suggested by recent studies demonstrating that the human BRG1 and HBRM proteins cooperate with retinoblastoma tumor suppressor proteins to regulate cell cycle progression (see Retinoblastoma-family protein) (Elfring, 1998).

A dominant-negative brm mutation (DNbrm) was generated by replacing a conserved lysine in the ATP-binding site of the Brm protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the high molecular weight (2 million Daltons) Brm complex. Expression of the dominant-negative Brm protein causes peripheral nervous system defects, homeotic transformations, and decreased viability. Individuals bearing one or two copies of the dominant-negative Brm are viable, but frequently exhibit partial transformations of haltere to wing, as evidenced by an increase in haltere size and the appearance of ectopic bristles on the capitellum. Approximately one third of dominant-negative Brm adults exhibit this transformation, which is presumably caused by the decreased expression of the Ultrabithorax gene. Increasing the ratio of dominant-negative Brm to wild-type Brm to 2:1 is lethal. Thus, the dominant-negative brm mutation behaves as an antimorphic allele of brm. Expression of the dominant-negative Brm protein in patterns identical to the segmentation genes hairy or engrailed has no effect on embryonic viability or segmentation. The lack of an embryonic phenotype resulting from embryonic expression of the dominant-negative Brm protein may be caused by the high maternal expression of wild-type Brm protein, which is sufficient to allow embryogenesis to proceed to near completion in the absence of zygotic brm function. Expression of the dominant-negative protein in imaginal tissues after embryogenesis leads to greatly reduced viability. Individuals reared at 20ƒ display partial transformation of first leg to second leg, as evidenced by a reduction in the number of sex comb teeth on the first leg. This phenotype is also seen in adults trans-heterozygous for hypomorphic brm alleles and is presumably caused by decreased expression of the Sex combs reduced (Scr) gene. Adults reared at 20ƒ also display twinning of mechanosensory bristles, a phenotype similar to that observed in clones of brm2 tissue. Expression of the dominant negative protein also has dramatic effects on the size and morphology of the wing; mutant wings are reduced in size, and the L5 and the posterior cross-vein (PCV) are usually absent. Defects in the campaniform sensilla, a class of sensory organs important for flight, are also observed with high frequency. These defects fall into four classes: missing sensilla, duplication or triplication of sensilla, transformation of sensilla into bristles, and the appearance of ectopic sensilla. Ectopic sensilla and bristles are observed most frequently on the L3 vein. Three sensilla (L3-1, L3-2, and L3-3) and no bristles are normally found on this vein. By contrast, approximately one-half of mutant wings display one or two additional sensilla on L3. Ectopic bristles are observed on this vein in approximately one-fifth of mutant wings (Elfring, 1998).

GENE STRUCTURE

Bases in 5' UTR - 55

Exons - five

Bases in 3' UTR - 297

PROTEIN STRUCTURE

Amino Acids - 1638

Structural Domains

Brahma has both a helicase domain that functions as a DNA-dependent ATPase and a bromodomain (Tamkun, 1995). The homology to Drosophila ISWI is high in the helicase domain, but ISWI, with a lower molecular weight than Brahma lacks the C-terminal bromodomain (Elfring, 1994).

The brm gene encodes a 1638 residue protein that is similar to SNF2/SWI2, a protein involved in transcriptional activation in yeast, suggesting possible models for the role of BRM in the transcriptional activation of homeotic genes. In addition, both BRM and SNF2 contain a 77 amino acid motif (the bromodomain) that is found in other Drosophila, yeast, and human regulatory proteins and may be characteristic of a new family of regulatory proteins (Tamkun, 1992).

In humans there are two SWI2 homologs, BRG1 and hbrm. BRG1 is present in a high-molecular-mass conplex of 2MD, similar in size to the yeast SWI/SNF complex. Two separate complexes contain either BRG1 or hbrm. The purified BRG1 of hbrm complexes contain 9-12 polypeptides. Like the yeast SWI/SNF complex, the complexes purified possess an ATP-dependent nucleosome disruption activity and are able to failitate transcription factor binding to mononucleosomes. Three proteins of the complex containing BRG1 have been purified, and termed BAFs (BRG1 associated factors). Two of the three, BAF170 and BAF155 are SWI3 homologs. They contain a tryptophan-repeat domain present in two or three tandem repeats in the myb family of proteins, but do not demonstrate DNA binding activity. A second conserved region is a predicted leucine-zipper region, possibly serving as a dimerization domain. BAF60 is a homolog of yeast SWP73, a novel SWI/SNF protein, and is homologous to a C.elegans protein. The subunit composition of BRG1 complex is different when purified from several different cell types (Wang, 1996a).

date revised: 22 October 98 

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