Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila.

Apoptosis has essential roles in a variety of cellular and developmental processes. Although the pathway is well studied, how the activities of individual components in the pathway are regulated is less understood. In Drosophila, a key component in apoptosis is Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is required to prevent caspase activation. Here, we demonstrate that Drosophila CG42593 (ubr3), encoding the homolog of mammalian UBR3, has an essential role in regulating the apoptosis pathway. We show that loss of ubr3 activity causes caspase-dependent apoptosis in Drosophila eye and wing discs. Our genetic epistasis analyses show that the apoptosis induced by loss of ubr3 can be suppressed by loss of initiator caspase Drosophila Nedd2-like caspase (Dronc), or by ectopic expression of the apoptosis inhibitor p35, but cannot be rescued by overexpression of DIAP1. Importantly, we show that the activity of Ubr3 in the apoptosis pathway is not dependent on its Ring-domain, which is required for its E3 ligase activity. Furthermore, we find that through the UBR-box domain, Ubr3 physically interacts with the neo-epitope of DIAP1 that is exposed after caspase-mediated cleavage. This interaction promotes the recruitment and ubiquitination of substrate caspases by DIAP1. Together, our data indicate that Ubr3 interacts with DIAP1 and positively regulates DIAP1 activity, possibly by maintaining its active conformation in the apoptosis pathway.

Loss of insulator activity by paired Su(Hw) chromatin insulators.

Chromatin insulators are regulatory elements that block the action of transcriptional enhancers when interposed between enhancer and promoter. The Drosophila Suppressor of Hairy wing [Su(Hw)] protein binds the Su(Hw) insulator and prevents enhancer-promoter interaction by a mechanism that is not understood. We show that when two copies of the Su(Hw) insulator element, instead of a single one, are inserted between enhancer and promoter, insulator activity is neutralized and the enhancer-promoter interaction may instead be facilitated. This paradoxical phenomenon could be explained by interactions between protein complexes bound at the insulators.

The P-Ph protein-mediated repression of yellow expression depends on different cis- and trans-factors in Drosophila melanogaster.

The ph(P1) allele of Drosophila melanogaster encodes a chimeric P-Ph protein that contains the DNA-binding domain of the P-element transposase and the Ph protein lacking 12 amino-terminal amino acids. It has been shown that the P-Ph protein is responsible for the formation of a repressive complex on P elements inserted at the yellow locus. Here we demonstrate that an enhancer element can suppress the P-Ph-mediated inhibition of yellow transcription. However, an increase of P-element copy number at the yellow locus overcomes the enhancer effect. The mobilization of P-element transposition induced the appearance with a high frequency of Su(y) mutations that partially or completely suppressed the inhibitory effect of ph(P1) on yellow expression. The Su(y) mutations were localized at different sites on chromosomes. One strong Su(y) mutation, sn(eP1), was found to be induced by a 1.2-kb P-element insertion into the transcribed noncoding region of the singed locus. The Su(y) mutations resulted in a high level of transcription of the 1.2-kb P element that contained the sequences encoding one DNA-binding and two protein-protein interaction domains of the transposase. The effect of Su(y) mutations can be explained by the competition between the truncated transposase encoded by a 1.2-kb P element and the P-Ph protein for binding sites on P-element insertions.

Broken chromosomal ends can be elongated by conversion in Drosophila melanogaster.

The fate of the termini of X chromosomes broken in the regulatory region of the yellow gene was followed in heterozygotes with X chromosomes carrying a point mutation inactivating the yellow gene. Each generation had a loss of about 70 terminal base pairs from the broken chromosome. However, gene conversion restoring the correct sequence at the chromosomal terminus took place with a frequency of about 1 x 10(-2) per generation. The average length of the conversion track was 2.7 kb. No recombination events occurred. In addition, we found that the normal functioning of the yellow body and wing enhancers located at the tip of the chromosome required about 4 kb of additional upstream sequence.

P-Element insertion at the polyhomeotic gene leads to formation of a novel chimeric protein that negatively regulates yellow gene expression in P-element-induced alleles of Drosophila melanogaster.

Polyhomeotic is a member of the Polycomb group (Pc-G) of homeotic repressors. The proteins encoded by the Pc-G genes form repressive complexes on the polycomb group response element sites. The phP1 mutation was induced by insertion of a 1.2-kb P element into the 5' transcribed nontranslated region of the proximal polyhomeotic gene. The phP1 allele confers no mutant phenotype, but represses transcription of P-element-induced alleles at the yellow locus. The phP1 allele encodes a chimeric P-PH protein, consisting of the DNA-binding domain of the P element and the PH protein lacking 12 amino-terminal amino acids. The P-PH, Polycomb (PC), and Posterior sex combs (PSC) proteins were immunohistochemically detected on polytene chromosomes in the regions of P-element insertions.

P element sequences can compensate for a deletion of the yellow regulatory region in Drosophila melanogaster.

The effects of interactions between P element and yellow regulatory sequences on the control of yellow expression were studied. The y mutations used in the analysis lack a segment of upstream sequence that extends from position -146 bp to -70 bp, relative to the transcription start site of the yellow gene. This sequence has been found to be necessary for the function of the yellow promoter. The insertion of one or two P element copies at position -69 bp compensates for the deletion in the regulatory region and restores yellow expression. After mobilization of the P element, new phenotypes were selected and molecularly characterized. Two regions in the 5' part of the P element, from 23 bp to 71 bp and from 82 bp to 108 bp, can each partially compensate for the yellow deletion. In addition, deletion derivatives of the P element were themselves able to activate yellow transcription. All such P elements retain at least 108 bp of sequence at the 5' end and 15-17 bp at the 3' end. Thus, the region of the P element from 23 bp to 108 bp contains cis-regulatory elements that can influence the transcription of neighboring genes.

Insertions of hybrid P elements in the yellow gene of Drosophila cause a large variety of mutant phenotypes.

A series of yellow mutations associated with a great variety of tissue-specific phenotypes were obtained from several highly unstable Drosophila melanogaster strains carrying the gypsy-induced y2 allele. These mutations are caused by insertion of additional DNA sequences of variable size 69 bp upstream of the yellow transcription start site. These sequences are flanked by identical copies of a deleted 1.2-kb P element arranged in the same or inverted orientation. The central part of the inserted element consists of genomic sequences originating from different regions of the X chromosome. The mutant phenotype caused by these chimeric elements depends on the nature of the sequences present either in the P element or in the central part of the insertion, suggesting that these sequences are able to affect expression of the yellow gene. In addition, sequences present in the central region of the insertions strongly modify the effects of the gypsy-bound suppressor of Hairy-wing [su(Hw)] and modifier of mdg4 [mod(mdg4)] proteins on yellow transcription. Analyses of these mutations give new insights into the mechanisms by which su (Hw) and mod(mdg4) affect enhancer function.