To investigate the role of NURF301 in chromatin remodeling, the entire NURF301 open reading frame was introduced into a baculovirus vector for protein expression. NURF301 and the other three NURF subunits were expressed individually and each of the epitope-tagged NURF components was immunopurified. ISWI, NURF55, and NURF38 were expressed highly in Sf9 cells. By contrast, recombinant NURF301 was poorly expressed and subject to proteolysis. Nevertheless, it was possible to reconstitute recombinant NURF complexes from individual subunits and purify the full complex from partial reconstitutes by glycerol gradient centrifugation. The reconstituted NURF complex clearly displays nucleosome-dependent ATPase activity. The reconstituted NURF complex also catalyzes nucleosome mobility. Mononucleosomes deposited by salt gradient dialysis on a 359 bp hsp70 promoter fragment adopt one of four major positions (N1-N4 nucleosomes). Native NURF catalyzes the movement of nucleosomes from the N1 and N2 positions to the preferred N3 position, and induces a slight positional change of N4 nucleosomes. NURF reconstituted from individual subunits has approximately the same activity as native NURF purified by conventional chromatography. Subcomplexes containing combinations of NURF subunits were generated and it was found that a complex reconstituted from individually purified ISWI and NURF301 could catalyze ATP-dependent nucleosome sliding. Neither NURF301 alone nor a mixture of ISWI, NURF55, and NURF38 changed nucleosome distributions when assayed at equivalent concentrations (Xiao, 2001).
To improve expression of NURF301, Sf9 cells were coinfected with recombinant baculovirus for all four NURF subunits. SDS-PAGE showed that coexpression greatly reduces proteolysis of NURF301 and increases the yield of the recombinant complex by 100-fold or greater. Recombinant NURF purified from coinfected cells showed nucleosome-stimulated ATPase activity; little or no stimulation was observed when nucleosomes were substituted by free DNA or core histones. Recombinant NURF purified from coinfected cells catalyzes nucleosome sliding in a similar manner to native and reconstituted recombinant NURF complexes (Xiao, 2001).
The ISWI ATPase by itself is capable of sliding nucleosomes, although sliding is less efficient and lacks positional specificity when compared to native ISWI complexes. Recombinant ISWI is less efficient than coexpressed, recombinant NURF in nucleosome-stimulated ATP hydrolysis. Moreover, sliding of nucleosomes to the preferred N3 position is not observed even when ISWI concentrations are increased by an order of magnitude over NURF. Taken together, these results indicate that efficient and accurate nucleosome sliding requires contributions from both ISWI and NURF301 (Xiao, 2001).
To investigate whether NURF301 provides a structural framework for assembly of the NURF complex, its interactions with the three smaller NURF subunits were analyzed, using S35-labeled NURF301 protein and ISWI, NURF55, and NURF38 proteins purified from baculovirus-infected cells. NURF301 can bind directly to each of the other NURF subunits, but no stable interactions were detected among ISWI, NURF55, and NURF38. The results indicate that NURF301 provides a scaffold to organize ISWI, NURF55, and NURF38 within the NURF complex (Xiao, 2001).
These interaction studies were extended by examining whether NURF can interact stably with its substrate -- nucleosomes. Recombinant NURF complex immobilized on beads binds to reconstituted nucleosomes (359 bp) and to nucleosome core particles (146 bp, lacking linker DNA). Nucleosome binding is ATP independent. To analyze the role of NURF301 in nucleosome binding, segments of the NURF301 coding region were fused to glutathione-S-transferase (GST). Three regions in NURF301 show prominent binding to nucleosomes or core particles, as revealed by GST-301 interactions. The results demonstrate that NURF301 is important for nucleosome binding, although a role for the other NURF subunits is by no means excluded (Xiao, 2001).
HMGA proteins can induce structural changes in nucleosomes and also alter DNA conformation to assemble stereospecific, multiprotein DNA complexes at enhancers. An N-terminal deletion of amino acids 1-121 in NURF301 was constructed that eliminates just the HMGA domain (DeltaN301). Cells were then co-infected with baculovirus expressing the four NURF subunits, NURFDeltaN301, wild-type ISWI, NURF55, and NURF38, and purified the protein complex was purified. Deletion of the N-terminal HMGA domain does not impair assembly of NURF. However, the NURFDeltaN301 complex shows reduced binding to nucleosomes as well as nucleosome core particles. Moreover, the reduced binding to nucleosomes is correlated with significant reduction in the ATPase activity. Strikingly, the NURFDeltaN301 complex also shows impaired activity in the nucleosome sliding assay. Nucleosome movements to the N3 position are significantly reduced for NURFDeltaN301 over a 10-fold concentration range; however, movement of the N4 'end' nucleosome is not significantly affected. It is concluded that the HMGA domain of NURF301 makes an important contribution to nucleosome sliding (for nucleosomes located away from DNA fragment ends) (Xiao, 2001).
To explore additional functions for NURF and NURF301, an analysis of proteins that interact with NURF was undertaken by identifying coimmunoprecipitating polypeptides in embryo nuclear extract fractions. Peptide sequencing of a 70 kDa species among numerous polypeptides that were substoichiometric with respect to NURF subunits reveal an unambiguous match with the embryonically expressed isoform of the GAGA transcription factor, GAGA519. A control experiment using beads coated with a nonspecific antibody showed no coimmunoprecipitation of GAGA factor. The association of GAGA factor and NURF was confirmed in binding assays. Binding to recombinant NURF complex was observed for S35-labeled GAGA519 and the alternatively spliced GAGA581 isoform, which is expressed at later stages in Drosophila development (Xiao, 2001).
By systematic deletions, the NURF-interacting sequences common to both GAGA519 and GAGA581 were determined to be in a conserved region containing the DNA binding zinc finger and flanking sequences. This finding, which is consistent with a study of the GAGA factor regions necessary for chromatin remodeling, is clearly distinguished from known interactions involving the multimerization (POZ) and transactivating (glutamine-rich) domains of GAGA factor and is also reminiscent of interactions between the zinc finger of mammalian ELKF and the SWI/SNF complex. In addition to the interactions with the GAGA factor, the NURF complex binds to S35-labeled GAL4-HSF and GAL4-VP16 activators containing the GAL4 DNA binding domain fused to strong activation regions of HSF and VP16. Binding was specific for HSF and VP16, since little or no binding was detected for the GAL4(1-147) DNA binding domain (Xiao, 2001).
To define region(s) of NURF301 that interact with GAGA factor, HSF, and VP16, the binding of GST-NURF301 segments to S35-labeled activators was analyzed in GST-pull-down assays. Two regions on NURF301 responsible for GAGA factor binding are also nucleosome binding regions. Whether the interaction of NURF301 with GAGA factor or the nucleosome is mutually exclusive is presently unknown. A separate region of NURF301 is responsible for binding GAL4-HSF and GAL4-VP16. Consistent with these results, the DeltaN301NURF complex binds to GAGA factor less efficiently (~26% of wild-type) but still binds to GAL4-HSF and GAL4-VP16. The binding of other NURF subunits to activators was also examined. ISWI, NURF55, and NURF38 show little or no binding to S35-labeled HSF and VP16. Interestingly, ISWI, but not NURF55 and NURF38, shows binding to GAGA factor, albeit at a level lower than binding to NURF (Xiao, 2001).
The Drosophila nucleosome remodeling factor NURF utilizes the energy of ATP hydrolysis to perturb the structure of nucleosomes and facilitate binding of transcription factors. The ATPase activity of purified NURF is stimulated significantly more by nucleosomes than by naked DNA or histones alone, suggesting that NURF is able to recognize specific features of the nucleosome. The interaction between NURF and nucleosomes is impaired by proteolytic removal of the N-terminal histone tails and by chemical cross-linking of nucleosomal histones. The ATPase activity of NURF is also competitively inhibited by each of the four Drosophila histone tails expressed as GST fusion proteins. A similar inhibition is observed for a histone H4 tail substituted with glutamine at four conserved, acetylatable lysines. These findings indicate a novel role for the flexible histone tails in chromatin remodeling by NURF, and this role may, in part, be independent of histone acetylation (Georgel, 1997).
What are the structural determinants of nucleosomes that are important for the activity of NURF? Based on the loss of the nucleosome-stimulated ATPase activity of NURF and the diminution of the DNase I footprint when the histone tails are removed by limited proteolysis, it is suggested that the flexible tails of the Drosophila core histones are critical elements for interaction with NURF. This conclusion is strengthened by the inhibition of NURF ATPase activity by GST-histone fusions. The effects of cross-linking the core histones in nucleosomes are also consistent with a contribution from the histone tails, although contributions from the globular domains of the nucleosome core histones cannot be excluded by this technique. Finally, a minor role for nucleosomal DNA is indicated by the modest inhibitory effects of DNA on the nucleosome-stimulated ATPase activity of NURF. These several determinants, individually insufficient for stimulating the ATPase activity of NURF, may be required in a combinatorial manner for achieving ATP-dependent perturbation of nucleosome structure. It will be of interest to relate the recognition of these determinants to one or more subunits of the NURF complex, and to analyze how this recognition is transduced to nucleosomal reorganization coupled with the utilization of chemical energy. Although a discrete supercomplex of NURF and a nucleosome has not been detected by native gel electrophoresis, it will also be important, when sufficient amounts of NURF become available for systematic studies, to define the interactions between NURF and nucleosomes quantitatively by biophysical methods, and to determine the histone composition of the remodeled nucleosome (Georgel, 1997).
The requirement for the Drosophila histone tails in nucleosomal interactions with NURF and the lack of strong binding specificity for structured DNA, a property of the SWI/SNF complex, provides further evidence for separate modes of action for the NURF and SWI/SNF chromatin remodeling complexes, which share related ATPase subunits ISWI and SWI2/SNF2, and the ability to alter chromatin structure in vitro in an ATP-dependent manner. Genetic studies have shown that the histone H2A/H2B tails and the histone H3/H4 tails are essential for viability in yeast. For histones H3 and H4, the tails are also important for repression of basal transcription, for telomeric and silent mating locus repression and for activation and repression of some genes. The H3 and H4 tails have been shown to bind in vitro with the yeast silencing information regulators SIR3 and SIR4, providing direct evidence that these extended regions may form specific binding sites for protein regulators of nucleosome structure and function. Tup1, a repressor of transcription of yeast a-cell specific genes, has also been demonstrated to interact directly with the tails of histones H3 and H4. Together with the present findings, these results suggest that the flexible tails of the histone octamer serve as common sites of interaction with several distinct nuclear protein complexes that affect nucleosome stability in a positive or negative manner (Georgel, 1997).
Other biochemical studies have demonstrated that the basic histone tails partially restrict binding of transcription factors to nucleosomal DNA. This restricted accessibility of nucleosomal DNA imposed by the histone tails can be alleviated upon neutralization of charged lysines by acetylation. However, as indicated by the ability of the GST-yH4 (Q5,8,12,16) mutant protein to retain competitive inhibition of the nucleosome-stimulated ATPase activity of NURF, the four acetylatable lysines of histone H4 in yeast do not seem to be of crucial importance for interaction with NURF, as measured by the ATPase assay. These lysine positions are strictly conserved in the Drosophila histone H4 tail and also undergo acetylation. Hence, the remaining conserved amino acid residues of the Drosophila histone H4 tail are likely to be involved in the interaction with NURF, and this interaction, at least for histone H4, could be independent of the state of lysine acetylation. It should be noted that these results do not exclude an interaction between NURF and other lysine residues of the histone tails that are not subject to acetylation. Nonetheless, the observed ability of hyperacetylated nucleosomes to stimulate the ATPase activity of NURF as well as normal nucleosomes, in the case of both HeLa cell and Drosophila histones, is consistent with the possibility that NURF may act independently of the histone acetylation pathway of nucleosome destabilization. It will be of interest to elucidate, by site-directed mutagenesis, the precise nature of the interaction between NURF and the histone tails, to understand the mechanism by which this interaction leads to nucleosomal reorganization and to define the parallel or sequential nature of the pathways of nucleosome reorganization by chromatin remodeling and histone modifying activities (Georgel, 1997).
Drosophila NURF is an ISWI-containing ATP-dependent chromatin remodeling complex that regulates transcription by catalyzing nucleosome sliding. To determine in vivo gene targets of NURF, whole genome expression analysis was performed on mutants lacking the NURF-specific subunit NURF301. Strikingly, a large set of ecdysone-responsive targets is included among several hundred NURF-regulated genes. Null Nurf301 mutants do not undergo larval to pupal metamorphosis, and also enhance dominant-negative mutations in ecdysone receptor. Moreover, purified NURF binds EcR in an ecdysone-dependent manner, suggesting it is a direct effector of nuclear receptor activity. The conservation of NURF in mammals has broad implications for steroid signaling (Badenhorst, 2005).
The ligand dependency of the NURF-EcR interaction implies that NURF functions as a coactivator for the ecdysone receptor. Like other NRs, EcR has two transcriptional activation function (AF) domains, the conserved AF2 located within the ligand-binding domain and isoform-specific AF1s at the N terminus. NURF is able to pull down, in a ligand-dependent manner, a minimal construct that contains the entire AF2 domain. However, inactivation of AF2 either by C-terminal truncation, or by mutation of F645 (a conserved residue critical for interaction of mammalian NRs with coactivators) blocks interaction with NURF. These results extend the repertoire of transcription factors shown to interact with NURF. These data indicate that purified NURF is a coactivator that binds to the AF2 region of EcR, in addition to previously demonstrated interactions with the GAGA factor, GAL4-VP16, and HSF (Badenhorst, 2005).
The nucleosome remodeling factor (NURF) is one of several ISWI-containing protein complexes that catalyze ATP-dependent nucleosome sliding and facilitate transcription of chromatin in vitro. To establish the physiological requirements of NURF, and to distinguish NURF genetically from other ISWI-containing complexes, mutations were isolated in the gene encoding the large NURF subunit, nurf301. NURF is shown to be required for transcription activation in vivo. In animals lacking NURF301, heat-shock transcription factor binding to and transcription of the hsp70 and hsp26 genes are impaired. Additionally, NURF is shown to be required for homeotic gene expression. Consistent with this, nurf301 mutants recapitulate the phenotypes of Enhancer of bithorax, a positive regulator of the Bithorax-Complex previously localized to the same genetic interval. Finally, mutants in NURF subunits exhibit neoplastic transformation of larval blood cells that causes melanotic tumors to form (Badenhorst, 2002).
The gene encoding the large NURF subunit, nurf301, was mapped by chromosomal in situ hybridization to the cytological interval 61A. One homozygous lethal P-element line. l(3)ry122 nurf3011, was identified in which a P-element had inserted in the untranslated leader sequence of nurf301. Subsequently, a series of EMS-induced mutations was recovered that failed to complement nurf3011. The three alleles presented (nurf3012, nurf3013, and nurf3014) encode truncated NURF301 proteins due to the introduction of stop codons at amino acids 546, 750, and 1536, respectively. Homozygous nurf301 mutants (and heteroallelic combinations) were lethal at late-third larval instar or early pupal stages. Three homozygous viable EP insertion lines were identified in which P-elements had inserted upstream of the nurf301 transcription start site. These were used as substrates for imprecise excision to recover lethal excision lines that failed to complement nurf3011. The deficiency Df(3L)3643 was characterized by PCR and shown to remove the ATG of nurf301 and at least two flanking genes downstream of nurf301 (Badenhorst, 2002).
The normal expression of nurf301 is reduced in the P-element insertion line nurf3011. In this mutant, transcription of iswi or a predicted gene upstream of nurf301 (CG7020) is unaffected. Lethality of this line is caused by the P-element insertion; precise excision of the P-element restores viability. In all the nurf301 mutants, the protein levels of the three other NURF subunits (ISWI, NURF55, and NURF38) and the ACF1 subunit of ACF and CHRAC are unchanged. This indicates that the nurf301 mutations specifically compromise NURF activity without significantly affecting the other ISWI-containing complexes ACF or CHRAC (Badenhorst, 2002).
NURF had originally been purified from nuclear extracts as a factor required to disrupt chromatin assembled in vitro on the promoters of the Drosophila heat-shock gene hsp70. Using in vitro assays it was shown that NURF cooperates with the GAGA factor to mobilize nucleosomes on the promoter of the heat-shock genes, establishing a nucleosome-free domain over the promoter, thus exposing sites for the heat-shock transcription factor (Badenhorst, 2002).
Expression of the heat shock genes is affected in nurf301 mutant adult. RNA dot-blot analysis shows that heat-shock-induced transcription of hsp26 and hsp70 is impaired in nurf3011 larvae. Impaired transcription leads to reduced heat-shock protein accumulation. Although HSP70 is detected in wild-type siblings after 10 min heat-shock, no protein is expressed in mutant animals. After 20 min of heat-shock, HSP70 is highly expressed in wild-type larvae. In contrast, nurf301 mutant animals only express low levels of HSP70 (Badenhorst, 2002).
Consistent with these findings, binding of the heat shock transcription factor Heat shock factor (HSF) is impaired in nurf301 mutants. In wild-type animals, immunofluorescence of salivary gland polytene chromosomes shows that, with the exception of the hsp83 locus at 63B, HSF does not generally bind to polytene chromosomes in the absence of heat stress. HSF-binding to hsp83 has been shown to be qualitatively different from the other heat-shock loci. The hsp83 locus does not appear to be GAGA factor-dependent; no (GA.CT)n elements are present in the promoter, nor does GAGA factor accumulate at this locus. Instead, HSF-binding is proposed to be facilitated by other transacting factors (Badenhorst, 2002).
Within several minutes of heat-shock, the bulk of HSF forms trimers competent for specific DNA-binding, and binds to the heat-shock loci and a number of other sites. Prominent are the 87A and 87C loci which carry two and three copies, respectively, of the hsp70 gene. In nurf301 mutant animals, hsp83 accumulates HSF as in nonshocked conditions. After 2 or 5 min heat-shock, no binding of HSF to hsp70 loci is detectable. HSF-binding to the hsp70 loci can be detected only after 10 min of heat-shock, and HSF staining is reduced compared to wild-type animals. Impaired HSF-binding is accompanied by reduced accumulation of RNA polymerase II at the hsp70 loci (Badenhorst, 2002).
ISWI, the catalytic subunit of NURF, is required for expression of the homeotic gene engrailed (en). However, ISWI is also a component of two other chromatin remodeling complexes, ACF and CHRAC. To resolve which ISWI-containing complex is required for homeotic gene expression, expression of Ultrabithorax (Ubx) and engrailed (en) were examined in nurf301 mutant animals. When both copies of nurf301 are mutated, in homozygous mutant nurf3011 larvae, expression of the Ubx protein becomes undetectable. The normal expression of Ubx in the haltere and third leg discs of wild-type third instar larvae is absent in nurf301 mutant animals. Expression of the homeotic gene en requires nurf301. The normal expression of En in the posterior compartment of imaginal discs is abolished in nurf3012 mutants. Semiquantitative RT-PCR analysis confirms that Ubx and en transcript levels are reduced in nurf301 mutant animals. These results confirm that the defects in homeotic transcription seen in iswi mutants are caused by abrogated NURF function (Badenhorst, 2002).
A positive regulator of the Bithorax-Complex, E(bx), has been localized genetically to 61A, the same cytological interval as nurf301. However, unlike numerous regulators of the BX-C, E(bx) had not been cloned. Since NURF is required for expression of Ubx, whether nurf301 corresponds to E(bx) was tested. Both alleles of E(bx) were no longer extant, so whether the mutations that were isolated in nurf301 recapitulated the published morphological properties of E(bx) mutants was tested (Badenhorst, 2002).
nurf301 mutants, like E(bx), increase the severity of bithorax (bx) mutant phenotypes. bx is a DNA regulatory element required for correct expression of Ubx in regions that give rise to the third (T3), but not second thoracic segment (T2) of the adult fly. This expression distinguishes T3 from T2 identity. Loss or reduction of Ubx levels in bx mutant animals (Ubx6.28/bx34e and Ubx6.28/bx8 mutant combinations causes a homeotic transformation of the third thoracic segment to the anterior second thoracic segment. Thus, the third thoracic segment, which is normally vestigial and naked, is transformed into the second thoracic segment, increasing its size and causing sensory bristles to develop. Moreover, the haltere (T3) is transformed toward wing fate (T2), manifested by increases in size and the development of bristles. The strength of these transformations is increased when one copy of E(bx) also is removed. Mutation of one copy of nurf301 similarly enhances bx phenotypes. With one copy of either the nurf3011, nurf3012 or a deficiency that removes nurf301 -- Df(3L)3643 -- the strength of the transformation is enhanced. nurf301 enhances both bx34e and bx8 mutations (Badenhorst, 2002).
Although NURF is required for expression of the homeotic genes in imaginal discs, neither E(bx) nor nurf301 homozygous mutant larvae display obvious homeotic transformations of the larval cuticle. The absence of mutant larval cuticle phenotypes is likely due to the large maternal dowry of nurf301 transcript contributed to embryos. Larval cuticular patterning is established before these transcripts have dissipated. Attempts were made to generate embryos lacking the maternal nurf301 contribution through use of the dominant female sterile technique. Although germ-line clones were produced using the parental chromosome, it was not possible to recover germ-line clones using nurf3011. Like ISWI, NURF301 is required for ovary development (Badenhorst, 2002).
The defects in homeotic transcription seen in nurf301 mutant animals effectively duplicate those reported for animals lacking the catalytic ISWI subunit. However, ISWI is also required to maintain higher order chromosome structure. In iswi mutants the male X chromosome is grossly disrupted relative to autosomes as revealed by polytene chromosome preparations. In nurf301 mutants the male X chromosome is similarly affected. The male X chromosome, identified by anti-MSL2 staining, is reduced in length and breadth, as seen in iswi mutant animals. The male X chromosome in homozygous mutant nurf3012 animals is highly aberrant. Disorganized male X chromosome morphology also was observed in nurf3012/nurf3013 and hemizygous nurf3011/Df(3L)3643 mutant animals. The results demonstrate that the chromosome condensation defect caused by perturbed ISWI function is mediated through the NURF complex. Although the effects on male X chromosome structure suggest that NURF can influence global chromosome structure, NURF function is not required for heterochromatic gene silencing. Reduction in NURF301 levels has no effect on position effect variegation. This is consistent with findings that showed that iswi is not a modifier of PEV (Badenhorst, 2002).
During the course of this analysis it was noticed that nurf301 mutant animals display a high incidence of melanotic tumors. Melanotic tumors have previously been reported in a number of mutant backgrounds and are generally caused by neoplastic transformation of the larval blood cells. The circulating cells (hemocytes) of the larval blood or hemolymph provide one tier of the innate immune system of insects by encapsulating or engulfing pathogens. A number of mutations have been shown to trigger the overproliferation and premature differentiation of hemocytes. Tumors form when these cells aggregate, or invade and encapsulate normal larval tissues Badenhorst, 2002).
Melanotic tumors are observed both in EMS-induced nurf301 mutants that truncate NURF301, the P-element induced mutation that reduces nurf301 transcript levels, and allelic combinations of these mutants. Tumor penetrance is extremely high (100% for nurf3012 at 25°C). Consistent with tumor development, circulating hemocyte cell number was increased dramatically in hemolymph isolated from nurf301 mutant animals. A large percentage of animals lacking ISWI, the catalytic subunit of NURF, also displayed melanotic tumors confirming that disrupted NURF function induces tumor formation. In iswi mutant animals the number of circulating hemocytes is also increased. In both nurf301 and iswi mutant hemolymph, small aggregates of hemocytes are often observed. All hemocyte cell types are present, from small round cells (prohemocytes) to crystal cells and lamellocytes (Badenhorst, 2002).
In Drosophila, larval blood cell transformation and melanotic tumor formation can be induced by inappropriate activation of either of two distinct signaling cascades: the Toll or the JAK/STAT pathway. Inappropriate activation and nuclear-localization of the Drosophila NF-kappaB homolog Dorsal, caused either by constitutive activation of the Toll receptor or removal of the inhibitor, the Drosophila IkappaB Cactus, leads to melanotic tumors in third instar larvae. In the second pathway, gain-of-function mutations in Hopscotch (Hop), the Drosophila Janus Kinase (JAK), induce melanotic tumors. Hop gain-of-function mutants cause tumor development by triggering constitutive activation and DNA-binding by the Drosophila STAT transcription factor, STAT92E (Badenhorst, 2002).
To resolve whether the melanotic tumors seen in the nurf301 mutants were caused by misregulation of either the TOLL or HOP/STAT92E pathways, whether nurf301 mutants enhance tumor phenotypes seen in constitutively active Toll or Hop mutant lines was tested. Tumor incidence in animals carrying one copy of a gain-of-function Hop mutation -- hopTum-1 -- is increased by simultaneous reduction in NURF301 levels. In contrast, removal of one copy of NURF301 fails to enhance the Toll gain-of-function allele Tl10b. The results suggest that NURF acts as a negative regulator within the Drosophila JAK/STAT signaling pathway (Badenhorst, 2002).
Molecular signatures of both JAK and Toll activation have been defined. It is known that Hop gain-of-function mutants induce expression of a complement-like protein TEP1. Overactivation of the Toll pathway also induces TEP1 synthesis but primarily induces expression of antimicrobial peptides, including Drosomycin (Drs) and Diptericin (Dpt). Loss of nurf301 induces tep1 but fails to induce drs or dpt, demonstrating that NURF301 principally affects the Hop/STAT92E pathway. Whether nurf301 interacts genetically with other known components of the Hop/STAT92E pathway was tested. Certain mutations in unpaired (upd, also known as outstretched), which encodes a ligand for the Hop receptor, display a characteristic wings-out phenotype, due to decreased activation of Hop and consequently decreased STAT92E function. When NURF301 levels are simultaneously decreased in these mutant backgrounds, animals are mostly restored to the wild-type. These genetic interactions confirm that NURF301 acts as a negative regulator of the Hop/STAT92E pathway, at a point downstream of Hop. Hence, disruption of NURF could affect either STAT92E or the targets of STAT92E. In nurf301 mutants, levels of the STAT92E transcription factor are not elevated, suggesting that NURF acts to repress the activity of STAT92E or the expression of some STAT92E target genes (Badenhorst, 2002).
An important question is how NURF is recruited to target sites in vivo. Four genes were shown in this study to be dependent on nurf301 for expression: Ubx, en, hsp26, and hsp70. All contain multiple binding sites for the GAGA factor, which is genetically required for their correct expression. On the Drosophila hsp70 and hsp26 promoters, (GA.CT)n cognate elements (to which the GAGA factor binds) are required for HSF-binding. When these sequences are deleted, HSF-binding to transgenes in polytene chromosomes is impaired, consistent with the defects seen in nurf301 mutant animals. It is therefore compelling that recent biochemical studies show that NURF and the GAGA factor bind to each other in crude extracts, and that purified NURF301 and GAGA factor interact directly in vitro. The principal interacting domains map to an N-terminal region of NURF301 and a stretch flanking the Zn finger DNA-binding motif of GAGA factor. These data suggest that NURF is recruited by the GAGA factor through specific, direct interactions with the NURF301 subunit, to catalyze local sliding of nucleosomes at bx, en, hsp26, and hsp70 promoters, increasing accessibility to sequence-specific transcription factors and RNA polymerase II. Curiously, though, reduction of nurf301 levels fails to enhance phenotypes of mutations in Trithorax-like, the gene that encodes the GAGA factor (Badenhorst, 2002).
Conceptual models of the function of chromatin remodeling machines reinforce the view that these complexes are required exclusively during gene activation to expose or 'open-up' chromatin. However, nucleosome sliding could equally be harnessed to repress genes. Phenotypic analysis of the orthologous yeast ISW2 complex suggests that ISWI-containing complexes may function both to activate and repress genes. The yeast ISW complexes appear to be recruited to sites within the genome through direct interaction with DNA-binding proteins to activate and repress genes by repositioning nucleosomes. Drosophila ISWI has been shown to be associated with transcriptionally silent regions of chromatin in salivary gland nuclei, suggesting that it may be involved in repression in this tissue. Analysis of NURF function during larval blood cell development suggests that NURF can repress targets of the JAK/STAT signaling pathway. In nurf301 mutants the expression of the complement-like protein TEP1 is induced. It remains to be established whether tep1 is a direct target of NURF (Badenhorst, 2002).
Misregulation of TEP1 is seen in mutants that overactivate the Drosophila JAK/STAT (Hop/STAT92E) signaling cascade and induce the formation of melanotic tumors. Increased signaling through the Hop/STAT92E pathway leads to the overproliferation and aberrant differentiation of larval blood cells that subsequently invade and encapsulate normal host tissue. Animals lacking NURF301, or the catalytic ISWI subunit, exhibit an identical neoplastic transformation of larval blood cells. In the absence of NURF, the proliferation and differentiation of hemocytes, and the accumulation of lamellocytes, is triggered (Badenhorst, 2002).
The data suggests that NURF normally represses targets of the Hop/STAT92E pathway. Genetic epistasis places nurf301 downstream of Hop. Loss of NURF resembles gain-of-function mutations in hop, and targets of the Hop/STAT92E cascade are up-regulated in nurf301 mutants. Normally, Hop activation leads to the expression and posttranslational modification of STAT92E. Although nurf301 mutants activate the Hop/STAT92E pathway, the levels of the STAT92E transcription factor are unchanged in NURF mutant animals. It is suggested that NURF acts downstream of STAT92E. In resting cells, in the absence of Hop/STAT92E signaling, NURF could normally repress STAT92E target genes. When NURF is removed, repression is no longer maintained, and targets are transcribed mimicking the effects of Hop activation. However, STAT activity is also influenced by a number of inhibitory, STAT-binding proteins. Among these are the suppressor of cytokine signaling (SOCS) and protein inhibitor of activated STAT (PIAS) family of inhibitors. It is possible that NURF is required for expression of one such inhibitor, and that loss of NURF activates the Hop/STAT92E cascade by removing a STAT inhibitor (Badenhorst, 2002).
The involvement of NURF in larval blood cell development agrees well with recent literature implicating a number of chromatin-modifying or chromatin-associated complexes in hemocyte development and melanotic tumor formation. Mutations in modulo, which encodes an interacting partner of the coactivator CBP, cause melanotic tumors. Of particular significance, screens for mutations that cause hematopoietic defects identified domino (dom), which encodes a member of the SWI2/SNF2 family of DNA-dependent ATPase that is distantly related to ISWI. Mutations in domino cause overproliferation of hemocytes, like NURF mutants. However, unlike NURF mutants, hemocytes fail to enter the hemolymph and remain trapped in enlarged lymph glands that become melanized. It will be interesting to assess the relative contributions of the ISWI and DOM complexes in the regulatory hierarchy of larval blood development (Badenhorst, 2002).
A striking feature of male animals that lack either NURF301 or the catalytic subunit ISWI is the distorted, bloated morphology of the male X chromosome. This implicates NURF in the maintenance of male X chromosome morphology. In flies, X chromosome dosage compensation is achieved by up-regulating transcription from the male X chromosome. One characteristic of the male X chromosome is the specific acetylation of histone H4 at Lys 16 (H4-K16), which is believed to favor a looser chromatin structure that allows increased transcription. These patterns of acetylation are established by the male-specific expression of components of the MSL complex that are tethered on the male X chromosome and subsequently recruit the histone acetyl transferase MOF (Badenhorst, 2002).
Genetic studies demonstrate that H4-K16 acetylation antagonizes ISWI function on the X chromosome. Biochemical characterization of the ISWI-containing ACF and CHRAC complexes has revealed that they can assemble and slide nucleosomes to establish regular ordered arrays. Regular nucleosome arrays are presumed to provide better substrates for chromatin compaction and, thus, it was speculated that ACF and CHRAC might be the complexes that help compact the male X chromosome. However, NURF is the ISWI complex required for normal male X chromosome morphology. Unlike ACF or CHRAC, NURF disrupts regular, ordered arrays of nucleosomes. While it is possible that NURF is required for global aspects of higher order chromosome morphology that are needed to maintain normal male X chromosome structure, other local or transcription-based mechanisms could also account for the nurf301 and iswi phenotypes. The dosage compensation machinery is recruited to the male X chromosome at specific, high affinity sites or entry points and subsequently spreads into flanking chromatin. NURF may regulate chromatin accessibility at one or a number of these initiation sites. In the absence of NURF, entry of the dosage compensation machinery at such sites may be changed. Alternatively, NURF may control transcription of components of the sex-determination and dosage compensation pathway. Irrespective, the observed antagonistic relationship between ISWI function and H4-K16 acetylation suggests that the action of NURF on the X chromosome is correspondingly influenced by H4 lysine acetylation. This influence on NURF could be direct, as suggested by effects of acetylated H4 tail peptide on ISWI ATPase activity in vitro (Badenhorst, 2002).
ISWI is the catalytic subunit of at least three protein complexes that have demonstrated in vitro chromatin-remodeling activity: NURF, ACF, and CHRAC. Here, mutation of a NURF-specific component reproduces the published properties of mutations in iswi. Both iswi and nurf301 are required for homeotic gene expression, proper larval blood cell development, and normal male X chromosome morphology. The specific in vivo functions of the ACF and CHRAC complexes remain to be established (Badenhorst, 2002).
Clues to the function of ACF and CHRAC may be derived from studies of the human Williams Syndrome Transcription Factor-ISWI complex (WSTF-ISWI). The in vitro activities of the WSTF-ISWI complex are essentially identical to ACF. WSTF-ISWI is targeted to pericentric heterochromatin during replication and is believed to allow heterochromatin reassembly in the wake of the replication fork. It is tempting to speculate that ACF and CHRAC may have similar functions in Drosophila and could be implicated in the establishment of repressive chromatin structures after replication. As mutations that selectively compromise individual remodeling complexes become available, whole genome expression analysis will allow the relative contributions of specific complexes to gene activation and silencing events in vivo to be dissected (Badenhorst, 2002).
Drosophila NURF is an ISWI-containing ATP-dependent chromatin remodeling complex that regulates transcription by catalyzing nucleosome sliding. To determine in vivo gene targets of NURF, whole genome expression analysis was performed on mutants lacking the NURF-specific subunit NURF301. Strikingly, a large set of ecdysone-responsive targets is included among several hundred NURF-regulated genes. Null Nurf301 mutants do not undergo larval to pupal metamorphosis, and also enhance dominant-negative mutations in ecdysone receptor. Moreover, purified NURF binds EcR in an ecdysone-dependent manner, suggesting it is a direct effector of nuclear receptor activity. The conservation of NURF in mammals has broad implications for steroid signaling (Badenhorst, 2005).
To characterize the physiological function of Drosophila NURF, a series of EMS-induced lesions was generated in the gene encoding the largest NURF subunit, Nurf301. Focus was placed on NURF301, since the large subunit is the only NURF-specific subunit and is obligatory for the assembly of NURF. Twelve EMS-induced lesions were isolated in Nurf301, all of which encode truncated NURF301 products. In addition to the previously reported male X-chromosome and melanotic tumor phenotypes, null Nurf301 mutants that truncate before the putative WAKZ motif display a non-pupariating phenotype. Mutants exhibit a slight developmental delay relative to heterozygous siblings, but larvae do not form pupae and can continue to survive in culture for up to 2 wk. In the few cases where white prepupae form, these retain an elongated larval form and fail to evert the anterior spiracles completely. Null Nurf301 mutants appear to undergo the larval molts normally. However, embryos contain a large dowry of maternally loaded Nurf301 mRNA, suggesting that Nurf301 mutants have sufficient NURF301 protein to support initial larval development, including the molt from L2 to L3 (Badenhorst, 2005).
In contrast to null Nurf301 alleles, Nurf301 mutations that truncate NURF301 after the WAKZ motif (Nurf3014, Nurf30110, Nurf30111, and Nurf30112 do pupariate, indicating that N-terminal fragments of NURF301 that extend beyond the WAKZ domain support pupariation. This agrees with in vitro data showing that these fragments contain sites of interaction with the other three NURF subunits and may be able to coordinate the assembly of a NURF complex. Heteroallelic combinations of these alleles survive to adult stages. However, flies exhibit developmental abnormalities and are sterile, indicating that the C-terminal regions of NURF301, while dispensable for pupariation, have functions (Badenhorst, 2005).
To define gene targets of NURF that are required for pupariation, whole genome expression profiles of null Nurf301 mutant (Nurf3012/Nurf3018) third instar larvae were compared with those of wild-type larvae. A set of 477 genes were identified for which there was a statistically significant (P < 0.05) change in expression between mutant and wild-type samples. Of these, 274 genes were decreased at least threefold in Nurf301 mutants, while 203 exhibited at least threefold elevated levels of expression in the mutant samples, suggesting that NURF may function both as an activator and repressor of transcription (Badenhorst, 2005).
Classification according to gene ontology of the 274 genes that require NURF301 for expression revealed that a sizeable number correspond to ecdysone target genes. An additional 30 of the 274 genes have no gene ontology classifications, but are known to be highly expressed at the larval/pupal transition and may be additional ecdysone targets. Taken together, this indicates that NURF is required for expression of targets of the ecdysone receptor. Similar classifications of the genes that show increased expression in Nurf301 mutants indicated that many are immune-related genes (Badenhorst, 2005).
An involvement of NURF in EcR signaling agrees well with the pupariation defects observed in Nurf301 mutants, which resemble the phenotypes of mutants in key downstream regulatory targets of the ecdysone receptor. To further confirm that NURF is required for ecdysone signaling, the expression in Nurf301 mutants was examined for all known ecdysone targets that had been annotated and included in the Affymetrix microarrays. In Nurf301 mutants, expression of a significant majority of these genes was reduced by greater than fivefold. The few genes for which there were no relative differences in expression are genes that are predominantly expressed at the prepupal/pupal transition (after the point of sampling; for example, Edg84A and Eip63F-1) or whose induction would be difficult to detect in whole animals because of a high background of tissues in which expression is constitutive or even repressed by ecdysone -- Eip71CD (Eip28/29) and Eip55E (Eip40) (Badenhorst, 2005).
Remarkably, although approximately equal numbers of genes exhibit elevated or reduced expression in Nurf301 mutants, among known ecdysone-responsive genes, Nurf301 mutation produced only decreases in expression. This suggests that NURF functions specifically as a coactivator in the ecdysone response. Finally, microarray analysis shows that transcript levels of known ecdysone synthetic enzymes are unchanged in Nurf301 mutants, indicating that NURF does not indirectly influence expression of responsive genes by affecting ecdysone levels (Badenhorst, 2005).
The altered expression of selected ecdysone targets was validated by analyzing transcript levels using Northern analysis and semiquantitative RT-PCR. Northern blotting confirms that Sgs1, Sgs3, and Eig71Ee are not expressed in null Nurf301 mutants (Nurf3012/Nurf3013 allelic combination) or Iswi mutants that lack the catalytic subunit of NURF. Similarly, semiquantitative RT-PCR confirms that expression of Eig71Ea, ImpE2, and Fbp1 is reduced in null Nurf301 or Iswi mutants. In contrast, transcript levels of EcR and usp are unchanged in Nurf301 mutants, indicating that the failure to express ecdysone target genes is not an indirect effect of reduced levels of the ecdysone receptor. As expected, allelic combinations with Nurf301 mutants that truncate after the putative WAKZ motif, and that are able to pupariate, do express ecdysone target genes (Badenhorst, 2005).
Lastly, as an additional demonstration that NURF is required for Sgs3 transcription, expression of an Sgs3-GFP reporter transgene was examined in null Nurf301 mutant animals. Sgs3-GFP is expressed in the salivary glands of Nurf3012/+ heterozygous animals but is not expressed in homozygous mutant Nurf3012 animals (Badenhorst, 2005).
The failure of ecdysone-responsive genes to be expressed in NURF mutants indicates that NURF is a coactivator of the Drosophila ecdysone receptor (EcR). Thus, whether NURF could physically interact with EcR was tested. In a pull-down assay vitro-translated EcR isoforms, EcR-A and EcR-B2, interact with Flag-tagged recombinant NURF. These interactions are dependent on the presence of added ecdysone (Badenhorst, 2005).
The ligand dependency of the NURF-EcR interaction implies that NURF functions as a coactivator for the ecdysone receptor. Like other NRs, EcR has two transcriptional activation function (AF) domains, the conserved AF2 located within the ligand-binding domain and isoform-specific AF1s at the N terminus. NURF is able to pull down, in a ligand-dependent manner, a minimal construct that contains the entire AF2 domain. However, inactivation of AF2 either by C-terminal truncation, or by mutation of F645 (a conserved residue critical for interaction of mammalian NRs with coactivators) blocks interaction with NURF. These results extend the repertoire of transcription factors shown to interact with NURF. These data indicate that purified NURF is a coactivator that binds to the AF2 region of EcR, in addition to previously demonstrated interactions with the GAGA factor, GAL4-VP16, and HSF (Badenhorst, 2005).
To confirm further that NURF functions in ecdysone signaling in vivo, genetic interactions between EcR and components of NURF were assayed. A dominant-negative EcR mutant (EcR-F645A) was expressed in follicle cells in the developing egg chamber of female flies. EcR-F645A is defective in transcriptional activation and interferes with ecdysone signaling, leading to a number of embryo defects, including malformed dorsal appendages. Decreasing the titer of a coactivator has been shown to enhance this phenotype. Mutation of a single copy of any of three NURF subunits increases the frequency and severity of these aberrations, consistent with NURF functioning as a coactivator for the ecdysone receptor (Badenhorst, 2005).
These results provide one of the first demonstrations of a biological requirement for an ISWI-containing chromatin remodeling enzyme (NURF) in steroid hormone-dependent transcriptional activation. To date, most studies of ATP-dependent chromatin remodeling during NR transactivation have focused on the SWI/SNF family of chromatin remodeling complexes. It has been shown that SWI/SNF enzymes are required for activation by the retinoid receptor heterodimer (RAR/RXR), glucocorticoid receptor, and estrogen receptor. Moreover, interaction studies reveal that SWI/SNF remodeling complexes can be targeted to NRs through interactions with the noncore subunits BAF250, BAF57, and BAF60a (Badenhorst, 2005).
However, there are many families of ATP-dependent chromatin remodeling complexes, including those based on the SWI2/SNF2, ISWI, INO80, and CHD1 catalytic subunits. Each group of remodeling enzymes has distinct mechanisms of operation. For example, SWI/SNF enzymes increase chromatin accessibility by DNA looping, whereas ISWI enzymes induce nucleosome sliding and the SWR1 category catalyzes histone exchange. It is important to determine whether NRs exclusively employ the SWI2/SNF2 branch or use a much wider repertoire of remodeling enzymes to exert their functions (Badenhorst, 2005).
This study provides evidence that the ISWI-containing chromatin remodeling enzyme NURF is a coactivator of a Drosophila NR, the ecdysone receptor. In addition to previous demonstrations of direct interactions between NURF and the GAGA factor and HSF, this study shows that purified NURF binds to EcR in an ecdysone-dependent manner, suggesting it is a direct effector of NR activity. These conclusions are broadly consistent with studies that indicated that ISWI complexes are required for NR-dependent transactivation in vitro. Addition of recombinant ISWI has been shown to stimulate activation of a chromatinized MMTV promoter by progesterone receptor. It has been suggested that ATP-dependent chromatin remodeling by an ISWI-containing remodeling complex (present in chromatin assembly extracts) is required to stabilize the binding of RAR/RXR to targets in chromatin (Badenhorst, 2005).
Inspection of the NURF301 coding sequence, and of the corresponding rat and human homologs, reveals that NURF301 contains two conserved NR (LxxLL) boxes. These motifs can mediate interaction between NRs and transcriptional coactivators and suggest a mechanism by which NURF can interact with, and be recruited by, EcR. To date no suitable antibody has been developed that allows direct visualizaion of recruitment of NURF to ecdysone-responsive promoters. However, chromatin immunoprecipitation (ChIP) using anti-ISWI antibodies shows that an ISWI-containing complex binds to the ecdysone response element of the hsp27 promoter. This ISWI ChIP signal is lost in Nurf301 mutants, suggesting that it is due to NURF recruitment (Badenhorst, 2005).
Analyses of Drosophila Iswi mutants have provided critical insights into the functions of ISWI-containing chromatin remodeling enzymes. These studies have provided the first demonstration that ISWI chromatin remodeling complexes are required to maintain male X-chromosome morphology for homeotic gene expression and metamorphosis. However, Drosophila ISWI is the catalytic ATPase subunit of at least three complexes: ACF, CHRAC, and NURF. As such, analysis of Iswi mutants alone does not allow the relative functions of these complexes to be discriminated. By focusing investigations on the NURF-specific subunit NURF301, this study has been able to define specific functions of NURF. The pupariation defects seen in Nurf301 mutants, and the reduced ecdysone target gene expression noted in Nurf301 mutants, highlights the critical function of NURF in EcR-dependent activation. As expected, defects seen in Nurf301 mutants are also observed in Iswi mutants. However, mutations in subunits specific to ISWI complexes other than NURF, for example, the ACF1 subunit of ACF and CHRAC, do not affect EcR function. Acf1 null mutants are semi-lethal but are able to produce viable adults (Badenhorst, 2005).
These studies on Drosophila NURF have important implications for NR function in mammals. Homologs of ISWI and the large subunit NURF301 exist in mouse and humans. Moreover, human NURF has been purified and exhibits identical biochemical properties as Drosophila NURF. The human homolog of a Drosophila NURF target, engrailed, is also a target of human NURF. Given this conservation of function, it will be of interest to determine if gene targets of mammalian NRs related to Drosophila EcR also require NURF for expression (Badenhorst, 2005).
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date revised: 20 January 2007
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