High mobility group proteins HMGD and HMGZ interact genetically with the Brahma chromatin remodeling complex in Drosophila

Many pleiotropic roles have been ascribed to small abundant HMG-Box (HMGB) proteins in higher eukaryotes but their precise function has remained enigmatic. To investigate their function genetically we have generated a defined deficiency uncovering the functionally redundant genes encoding HMGD and HMGZ, the Drosophila counterparts of HMGB1-3 in mammals. The resulting mutant is a strong hypomorphic allele of HmgD/Z. Surprisingly this allele is viable and exhibits only minor morphological defects even when homozygous. However, this allele interacts strongly with mutants of the Brahma chromatin remodeling complex, while no interaction was observed with mutant alleles of other remodeling complexes. We also observe genetic interactions between the HmgD/Z deficiency and some, but not all, known Brahma targets. These include the homeotic genes Sex combs reduced and Antennapedia, as well as the gene encoding the cell-signaling protein Rhomboid. In contrast to more general structural roles previously suggested for these proteins, we infer that a major function of the abundant HMGB proteins in Drosophila is to participate in Brahma-dependent chromatin remodeling at a specific subset of Brahma-dependent promoters.     

Drosophila cyclin E interacts with components of the Brahma complex

Cyclin E-Cdk2 is essential for S phase entry. To identify genes interacting with cyclin E, we carried out a genetic screen using a hypomorphic mutation of Drosophila cyclin E (DmcycE(JP)), which gives rise to adults with a rough eye phenotype. Amongst the dominant suppressors of DmcycE(JP), we identified brahma (brm) and moira (mor), which encode conserved core components of the Drosophila Brm complex that is highly related to the SWI-SNF ATP-dependent chromatin remodeling complex. Mutations in genes encoding other Brm complex components, including snr1 (BAP45), osa and deficiencies that remove BAP60 and BAP111 can also suppress the DmcycE(JP) eye phenotype. We show that Brm complex mutants suppress the DmcycE(JP) phenotype by increasing S phases without affecting DmcycE protein levels and that DmcycE physically interacts with Brm and Snr1 in vivo. These data suggest that the Brm complex inhibits S phase entry by acting downstream of DmcycE protein accumulation. The Brm complex also physically interacts weakly with Drosophila retinoblastoma (Rbf1), but no genetic interactions were detected, suggesting that the Brm complex and Rbf1 act largely independently to mediate G(1) arrest.     

The Drosophila Brahma (SWI/SNF) chromatin remodeling complex exhibits cell-type specific activation and repression functions

The Brahma (Brm) complex of Drosophila melanogaster is a SWI/SNF-related chromatin remodeling complex required to correctly maintain proper states of gene expression through ATP-dependent effects on chromatin structure. The SWI/SNF complexes are comprised of 8-11 stable components, even though the SWI2/SNF2 (BRM, BRG1, hBRM) ATPase subunit alone is partially sufficient to carry out chromatin remodeling in vitro. The remaining subunits are required for stable complex assembly and/or proper promoter targeting in vivo. Our data reveals that SNR1 (SNF5-Related-1), a highly conserved subunit of the Brm complex, is required to restrict complex activity during the development of wing vein and intervein cells, illustrating a functional requirement for SNR1 in modifying whole complex activation functions. Specifically, we found that snr1 and brm exhibited opposite mutant phenotypes in the wing and differential misregulation of genes required for vein and intervein cell development, including rhomboid, decapentaplegic, thick veins, and blistered, suggesting possible regulatory targets for the Brm complex in vivo. Our genetic results suggest a novel mechanism for SWI/SNF-mediated gene repression that relies on the function of a 'core' subunit to block or shield BRM (SWI2/SNF2) activity in specific cells. The SNR1-mediated repression is dependent on cooperation with histone deacetylases (HDAC) and physical associations with NET, a localized vein repressor.     

The Drosophila gene taranis encodes a novel trithorax group member potentially linked to the cell cycle regulatory apparatus

Genes of the Drosophila Polycomb and trithorax groups (PcG and trxG, respectively) influence gene expression by modulating chromatin structure. Segmental expression of homeotic loci (HOM) initiated in early embryogenesis is maintained by a balance of antagonistic PcG (repressor) and trxG (activator) activities. Here we identify a novel trxG family member, taranis (tara), on the basis of the following criteria: (i) tara loss-of-function mutations act as genetic antagonists of the PcG genes Polycomb and polyhomeotic and (ii) they enhance the phenotypic effects of mutations in the trxG genes trithorax (trx), brahma (brm), and osa. In addition, reduced tara activity can mimic homeotic loss-of-function phenotypes, as is often the case for trxG genes. tara encodes two closely related 96-kD protein isoforms (TARA-alpha/-beta) derived from broadly expressed alternative promoters. Genetic and phenotypic rescue experiments indicate that the TARA-alpha/-beta proteins are functionally redundant. The TARA proteins share evolutionarily conserved motifs with several recently characterized mammalian nuclear proteins, including the cyclin-dependent kinase regulator TRIP-Br1/p34(SEI-1), the related protein TRIP-Br2/Y127, and RBT1, a partner of replication protein A. These data raise the possibility that TARA-alpha/-beta play a role in integrating chromatin structure with cell cycle regulation.     

The enhancer of trithorax and polycomb corto interacts with cyclin G in Drosophila

Background

Polycomb (PcG) and trithorax (trxG) genes encode proteins involved in the maintenance of gene expression patterns, notably Hox genes, throughout development. PcG proteins are required for long-term gene repression whereas TrxG proteins are positive regulators that counteract PcG action. PcG and TrxG proteins form large complexes that bind chromatin at overlapping sites called Polycomb and Trithorax Response Elements (PRE/TRE). A third class of proteins, so-called “Enhancers of Trithorax and Polycomb” (ETP), interacts with either complexes, behaving sometimes as repressors and sometimes as activators. The role of ETP proteins is largely unknown.

Methodology/Principal Findings

In a two-hybrid screen, we identified Cyclin G (CycG) as a partner of the Drosophila ETP Corto. Inactivation of CycG by RNA interference highlights its essential role during development. We show here that Corto and CycG directly interact and bind to each other in embryos and S2 cells. Moreover, CycG is targeted to polytene chromosomes where it co-localizes at multiple sites with Corto and with the PcG factor Polyhomeotic (PH). We observed that corto is involved in maintaining Abd-B repression outside its normal expression domain in embryos. This could be achieved by association between Corto and CycG since both proteins bind the regulatory element iab-7 PRE and the promoter of the Abd-B gene.

Conclusions/Significance

Our results suggest that CycG could regulate the activity of Corto at chromatin and thus be involved in changing Corto from an Enhancer of TrxG into an Enhancer of PcG.     

The Drosophila SNR1 (SNF5/INI1) subunit directs essential developmental functions of the Brahma chromatin remodeling complex

The Drosophila melanogaster Brahma (Brm) complex, a counterpart of the Saccharomyces cerevisiae SWI/SNF ATP-dependent chromatin remodeling complex, is important for proper development by maintaining specific gene expression patterns. The SNR1 subunit is strongly conserved with yeast SNF5 and mammalian INI1 and is required for full activity of the Brm complex. We identified a temperature-sensitive allele of snr1 caused by a single amino acid substitution in the conserved repeat 2 region, implicated in a variety of protein-protein interactions. Genetic analyses of snr1(E1) reveal that it functions as an antimorph and that snr1 has critical roles in tissue patterning and growth control. Temperature shifts show that snr1 is continuously required, with essential functions in embryogenesis, pupal stages, and adults. Allele-specific genetic interactions between snr1(E1) and mutations in genes encoding other members of the Brm complex suggest that snr1(E1) mutant phenotypes result from reduced Brm complex function. Consistent with this view, SNR1(E1) is stably associated with other components of the Brm complex at the restrictive temperature. SNR1 can establish direct contacts through the conserved repeat 2 region with the SET domain of the homeotic regulator Trithorax (TRX), and SNR1(E1) is partially defective for functional TRX association. As truncating mutations of INI1 are strongly correlated with aggressive cancers, our results support the view that SNR1, and specifically the repeat 2 region, has a critical role in mediating cell growth control functions of the metazoan SWI/SNF complexes.     

E(var)3-9 of Drosophila melanogaster encodes a zinc finger protein

The importance of a gene's natural chromatin environment for its normal expression is poignantly illustrated when a change in chromosome position results in variable gene repression, such as is observed in position effect variegation (PEV) when the Drosophila melanogaster white (omega) gene is juxtaposed with heterochromatin. The Enhancer of variegation 3-9 [E(var)3-9] gene was one of over a hundred loci identified in screens for mutations that dominantly modify PEV. Haploinsufficiency for E(var)3-9 enhances omegam4 variegation, as would be expected from increased heterochromatin formation. To clarify the role of E(var)3-9 in chromosome structure, the gene has been cloned and its mutant alleles characterized. The involvement of E(var)3-9 in structure determination was supported by its reciprocal effects on euchromatic and heterochromatic PEV; E(var)3-9 mutations increased expression of a variegating heterochromatic gene in two tissue types. E(var)3-9 mutations also had a recessive phenotype, maternal effect lethality, which implicated E(var)3-9 function in an essential process during embryogenesis. Both phenotypes of E(var)3-9 mutations were consistent with its proposed function in promoting normal chromosome structure. The cloning of E(var)3-9 by classical genetic methods revealed that it encodes a protein with multiple zinc fingers, but otherwise novel sequence.     

batman Interacts with polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes.     

A gene located at 71F in Drosophila melanogaster encodes a novel zinc-finger protein that interacts with protein kinase CK2

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are two hormones produced and secreted by the heart to control blood pressure, body fluid homeostasis and electrolyte balance. Each peptide binds to a common family of 3 receptors (GC-A, GC-B and C-receptor) with varying degrees of affinity. The proANP gene disrupted mouse model provides an excellent opportunity to examine the regulation and expression of BNP in the absence of ANP. A new radioimmunoassay (RIA) was developed in order to measure mouse BNP peptide levels in the plasma, atrium and ventricle of the mouse. A detection limit of 3–6 pg/tube was achieved by this assay. Results show that plasma and ventricular level of BNP were unchanged among the three genotypes of mice. However, a significant decrease in the BNP level was noted in the atrium. The homozygous mutant (ANP–/–) had undetectable levels of BNP in the atrium, while the heterozygous (ANP+/–) and wild-type (ANP+/+) mice had 430 and 910 pg/mg in the atrium, respectively. Northern Blot analysis shows the ANP–/– mice has a 40% reduction of BNP mRNA level in the atrium and a 5-fold increase in the ventricle as compared with that of the ANP+/+ mouse. Our data suggest that there is a compensatory response of BNP expression to proANP gene disruption. Despite the changes in the atrial and ventricular tissue mRNA and peptide levels, the plasma BNP level remains unaltered in the ANP–/– mice. We conclude that the inability of BNP to completely compensate for the lack of ANP eventually leads to chronic hypertension in the proANP gene disrupted mice.     

The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product

Mutations in the single-minded (sim) gene of Drosophila result in the loss of the precursor cells giving rise to the midline cells of the embryonic central nervous system. We have examined the structure of the sim product by sequencing a sim cDNA clone, and have also determined the subcellular localization of the protein and its developmental expression by staining embryos with an antiserum against a sim fusion protein. The results indicate that sim is a nuclear protein specifically expressed along the midline of the neuroepithelium, the same subset of cells that are missing in the mutant. No similarity is observed between sim and any known nuclear protein, but, surprisingly, it is similar to the Drosophila period (per) locus gene product, which controls the periodicity of biological rhythms.