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Mechanism and functional role of the interaction between CP190 and the architectural protein Pita in Drosophila melanogaster

ABSTRACT: The architectural protein Pita is critical for Drosophila embryogenesis and predominantly binds to gene promoters and insulators. In particular, Pita is involved in the organization of boundaries between regulatory domains, controlled by the expression of three hox genes in the Bithorax complex (BX-C). The best-characterized partner for Pita is the BTB/POZ-domain containing protein CP190. Here, we precisely mapped two unstructured regions of Pita that interact with the BTB domain of CP190. The deletion of the CP190-interacting regions did not significantly affect the binding of the mutant Pita protein to most chromatin sites. The expression of the mutant protein completely complemented the null pita mutation. However, the mutant Pita protein does not support the ability of multimerized Pita sites to prevent cross-talk between the iab-6 and iab-7 regulatory domains that activate the expression of Abdominal-B (Abd-B), one of the genes in the BX-C. The recruitment of the Pita region and the interaction with CP190 and the polytene chromosomes of larvae induces the formation of a new interband, which is a consequence of the formation of open chromatin in this region. These results suggested that the interaction with CP190 is required for the primary Pita activities, but other architectural proteins may also recruit CP190 in flies expressing only the mutant Pita protein.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE160007 | GEO | 2020/11/30

REPOSITORIES: GEO

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Project description:CP190 is a Drosophila protein of unknown function that exhibits cell cycle-specific localisation to the centrosome during mitosis and the nucleus during the interphase. CP190 associates with multiple sites on polytene chromosomes and its predicted amino acid sequence identifies four C2h2 zinc finger motifs along with an N-terminal BTB/POZ domain (similar to GAGA factor). Experimental studies have identified a 128 amino acid domain that direct centrosome association and a bipartite NLS. Interest in the function of CP190 was initially driven by an interest in centrosome function. Is is also fascinating to know why a protein that forms a core component of the centrosome also shares the attributes and distribution expected of a regulator of gene expression. CP190 mutants (EMS generated) were recently isolated whose recessive lethal phenotype indicates that CP190 is essential. The two best-characterised isolates, CP190[1] and CP190[2], are either nulls or severe hypomorphs (CP190 protein is undetectable on Western blots of total protein from homozygous mutatant third instar larvae). Both mutants can be rescued as homozygotes by expression of wild type CP190 from a transgenic second chromosome carrying a CP190 cDNA under the control of the polyubiquitin promoter. Stocks homozygous or hemizygous for either of these mutant alleles die as pharate adults that show no obvious externally visible defects. Mutant larvae show a slight developmental delay compared to their heterozygous siblings, but their brains and discs are of normal size and brain squash preparation show no obvious mitotic phenotype. Since CP190 is a chromatin-associated protein and has motifs that are typical of proteins involved in regulating gene expression, it seems likely that the gene expression profile of CP190 mutants will be aberrant. This experiment hopes to identify genes that exhibit aberrant expression in the mutant compared to the wild type. Target RNA were isolated from: homozygous CP190[1] 0 hour white prepupae (non tubby, red malpighian tubules) homozygous CP190[2] 0 hour white prepupae (non tubby, red malpighian tubules) homozygous red[1] e[1] 0 h white prepupae (source from which the mutated chromosome was obtained) For each CP190 mutant, total RNA preparations were made from three separate groups of 100 prepupae selected from independent cultures, similarly, six independent RNA samples were prepared from red[1] e[1] stock as 'wild type' controls, giving a total of 12 target RNA samples. Prepupae were collected from bottles every 30 min and frozen directly in liquid N2. Frozen samples were pooled and ground to a powder in liquid N2 before resuspension in TRIZOL and shipment to Flychip facility at the University of Cambridge, UK.

Project description:Thermomacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, a M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supra-normal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to up-regulation of 55 genes. Genome re-sequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism or transport. These mutations included 7 non-synonymous substitutions, 4 insertions and 1 deletion. One of the insertion mutations mapped to pseudogene, Msed_1517, and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that include the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula was naturally lacking this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low affinity high velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated spontaneous arsenate resistant mutants derived from CuR1 all underwent mutation in pitA and non-selectively became copper resistant. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching. The study comprises 5 samples, described in detail below. WT_CuR1: Differential transcriptional response of Metallosphaera sedula DSM 5348, WT, to the supra-normal copper resistant spontaneous Metallosphaera sedula mutant, CuR1 under normal growth conditions. This experiment was done to analyze the differential transcription of WT cells compared with CuR1 cells at mid log phase. WT-15_CuR1-15: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT and CuR1 15 minutes post copper challenge. The copper cultures were harvested 15 minutes after the shock. WT-60_CuR1-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT and CuR1 60 minutes post copper challenge. The copper cultures were harvested 60 minutes after the shock. WT-15_WT-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT 15 and 60 minutes post copper challenge. The copper cultures were harvested 15 and 60 minutes after the shock, respectively. CuR1-15_CuR1-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula CuR1 15 and 60 minutes post copper challenge. The copper cultures were harvested 15 and 60 minutes after the shock, respectively.

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Mechanism and functional role of the interaction between CP190 and the architectural protein Pita in Drosophila melanogaster

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