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.     

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.     

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.     

Physical interactions of Dmnk with Orb: implications in the regulated localization of Orb by Dmnk during oogenesis and embryogenesis.

The Dmnk (Drosophila maternal nuclear kinase) gene, encoding a nuclear protein serine/threonine kinase, is expressed predominantly in the germline cells during embryogenesis, suggesting its possible role in the establishment of germ cells. We report here that Dmnk interacts physically with Drosophila RNA binding protein Orb, which plays crucial roles in the establishment of Drosophila oocyte by regulating the distribution and translation of several maternal mRNAs. Considering similar spatiotemporal expression pattern of Dmnk and orb during oogenesis and early embryogenesis, it is suggested that Dmnk plays a role in establishment of germ cells by interacting with Orb. Although there are two forms of Dmnk proteins, Dmnk-L (long) and Dmnk-S (short) via the developmentally regulated alternative splicing, Orb can associate with both forms of Dmnk proteins when expressed in culture cells. However, immunohistochemical analysis revealed that Dmnk-S, but not Dmnk-L, can affect the subcellular localization of Orb in a kinase activity-dependent manner, suggesting differential functions of Dmnk-S and Dmnk-L in the regulation of Orb.     

Cortactin modulates cell migration and ring canal morphogenesis during Drosophila oogenesis

Cortactin is a Src substrate that interacts with F-actin and can stimulate actin polymerization by direct interaction with the Arp2/3 complex. We have isolated complete loss-of-function mutants of the single Drosophila cortactin gene. Mutants are viable and fertile, showing that cortactin is not an essential gene. However, cortactin mutants show distinct defects during oogenesis. During oogenesis, Cortactin protein is enriched at the F-actin rich ring canals in the germ line, and in migrating border cells. In cortactin mutants, the ring canals are smaller than normal. A similar phenotype has been observed in Src64 mutants and in mutants for genes encoding Arp2/3 complex components, supporting that these protein products act together to control specific processes in vivo. Cortactin mutants also show impaired border cell migration. This invasive cell migration is guided by Drosophila EGFR and PDGF/VEGF receptor (PVR). We find that accumulation of Cortactin protein is positively regulated by PVR. Also, overexpression of Cortactin can by itself induce F-actin accumulation and ectopic filopodia formation in epithelial cells. We present evidence that Cortactin is one of the factors acting downstream of PVR and Src to stimulate F-actin accumulation. Cortactin is a minor contributor in this regulation, consistent with the cortactin gene not being essential for development.     

Function of the Evx-2 gene in the morphogenesis of vertebrate limbs.

Vertebrate gene members of the HoxD complex are essential for proper development of the appendicular skeletons. Inactivation of these genes induces severe alterations in the size and number of bony elements. Evx-2, a gene related to the Drosophila even-skipped (eve) gene, is located close to Hoxd-13 and is expressed in limbs like the neighbouring Hoxd genes. To investigate whether this tight linkage reflects a functional similarity, we produced a null allele of Evx-2. Furthermore, and because Hoxd-13 function is prevalent over that of nearby Hoxd genes, we generated two different double mutant loci wherein both Evx-2 and Hoxd-13 were inactivated in cis. The analysis of these various genetic configurations revealed the important function of Evx-2 during the development of the autopod as well as its genetic interaction with Hoxd-13. These results show that, in limbs, Evx-2 functions like a Hoxd gene. A potential evolutionary scenario is discussed, in which Evx-2 was recruited by the HoxD complex in conjunction with the emergence of digits in an ancestral tetrapod.     

Caenorhabditis elegans DAZ-1 is expressed in proliferating germ cells and directs proper nuclear organization and cytoplasmic core formation during oogenesis

The deleted in azoospermia (DAZ) family genes encode potential RNA-binding proteins that are expressed exclusively in germ cells in a wide range of metazoans. We have previously shown that mutations in daz-1, the only DAZ family gene in Caenorhabditis elegans, cause pachytene stage arrest of female germ cells but do not affect spermatogenesis. In this study, we report that DAZ-1 protein is most abundantly expressed in proliferating female germ cells, in a manner independent of the GLP-1 signaling pathway. DAZ-1 is dispensable in males but it is expressed also in male mitotic germ cells. Detailed phenotypic analyses with fluorescence microscopy and transmission electron microscopy have revealed that loss of daz-1 function causes multiple abnormalities as early as the onset of meiotic prophase, which include aberrant chromatin structure, small nucleoli, absence of the cytoplasmic core, and precocious cellularization. Although the reduced size of nucleoli is indicative of a low translational activity in these cells, artificial repression of general translation in the germline does not phenocopy the daz-1 mutant. Thus, we propose that DAZ-1 in C. elegans plays essential roles in female premeiotic and early meiotic germ cells, probably via regulating the translational activity of specific target genes required for the progression of oogenesis.     

SWI/SNF complexes containing Brahma or Brahma-related gene 1 play distinct roles in smooth muscle development

SWI/SNF ATP-dependent chromatin-remodeling complexes containing either Brahma-related gene 1 (Brg1) or Brahma (Brm) play important roles in mammalian development. In this study we examined the roles of Brg1 and Brm in smooth muscle development, in vivo, through generation and analysis of mice harboring a smooth muscle-specific knockout of Brg1 on wild-type and Brm null backgrounds. Knockout of Brg1 from smooth muscle in Brg1(flox/flox) mice expressing Cre recombinase under the control of the smooth muscle myosin heavy-chain promoter resulted in cardiopulmonary defects, including patent ductus arteriosus, in 30 to 40% of the mice. Surviving knockout mice exhibited decreased expression of smooth muscle-specific contractile proteins in the gastrointestinal tract, impaired contractility, shortened intestines, disorganized smooth muscle cells, and an increase in apoptosis of intestinal smooth muscle cells. Although Brm knockout mice had normal intestinal structure and function, knockout of Brg1 on a Brm null background exacerbated the effects of knockout of Brg1 alone, resulting in an increase in neonatal lethality. These data show that Brg1 and Brm play critical roles in regulating development of smooth muscle and that Brg1 has specific functions within vascular and gastrointestinal smooth muscle that cannot be performed by Brm.     

Female Sterile Mutations on the Second Chromosome of Drosophila Melanogaster. II. Mutations Blocking Oogenesis or Altering Egg Morphology

In mutagenesis screens for recessive female sterile mutations on the second chromosome of Drosophila melanogaster 528 lines were isolated which allow the homozygous females to survive but cause sterility. In 62 of these lines early stages of oogenesis are affected, and these females usually do not lay any eggs. In 333 lines oogenesis proceeds apparently normally to stage 8 of oogenesis, but morphological defects become often apparent during later stages of oogenesis, and are visible in the defective eggs produced by these females whereas 133 lay eggs that appear morphologically normal, but do not support normal embryonic development. Of the lines 341 have been genetically characterized and define a total of 140 loci on the second chromosome. Not all the loci are specific for oogenesis. From the numbers obtained we estimate that the second chromosome of Drosophila contains about 13 loci that are relatively specific for early oogenesis, 70 loci that are specifically required in mid to late oogenesis, and around 30 maternal-effect lethals.     

SCAR is a primary regulator of Arp2/3-dependent morphological events in Drosophila

The Arp2/3 complex and its activators, Scar/WAVE and Wiskott-Aldrich Syndrome protein (WASp), promote actin polymerization in vitro and have been proposed to influence cell shape and motility in vivo. We demonstrate that the Drosophila Scar homologue, SCAR, localizes to actin-rich structures and is required for normal cell morphology in multiple cell types throughout development. In particular, SCAR function is essential for cytoplasmic organization in the blastoderm, axon development in the central nervous system, egg chamber structure during oogenesis, and adult eye morphology. Highly similar developmental requirements are found for subunits of the Arp2/3 complex. In the blastoderm, SCAR and Arp2/3 mutations result in a reduction in the amount of cortical filamentous actin and the disruption of dynamically regulated actin structures. Remarkably, the single Drosophila WASp homologue, Wasp, is largely dispensable for these numerous Arp2/3-dependent functions, whereas SCAR does not contribute to cell fate decisions in which Wasp and Arp2/3 play an essential role. These results identify SCAR as a major component of Arp2/3-dependent cell morphology during Drosophila development and demonstrate that the Arp2/3 complex can govern distinct cell biological events in response to SCAR and Wasp regulation.