The NuA4/TIP60 histone-modifying complex and Hr78 modulate the Lobe 2 mutant eye phenotype

Abstract

Gene regulation is important during tissue formation, but redundant systems make it difficult to study in vivo. The protein Jazf-1 is a member of the NuA4/TIP60 histone-modifying complex, and a transcriptional repressor has been suggested to be important for Drosophila melanogaster eye development. We used the GAL4-UAS system to determine the impact of altering gene expression. GAL4-UAS manipulations of Jazf-1 in the eye caused variable and not fully penetrant phenotypes. Increased expression of Jazf-1 has been shown to suppress a Lobe ² small eye phenotype. We found that Lobe ² produces a sensitive background for an in vivo assay to monitor gene regulatory complexes. Depleting Jazf-1 and other NuA4/TIP60 complex members significantly enhanced the eye phenotype. We also tested Hr78, which directly interacts with Jazf-1, and found it inversely modifies the Lobe ² phenotype. An Hr78 mutation predicted to uncouple the Jazf-1 interaction but still capable of interactions with transcriptional activators further enhanced the Lobe ² mutant phenotype, suggesting the loss of a repressing complex. We believe that Hr78 is acting as an anchor for repressing and activating complexes and the NuA4/TIP60 complex helps repress genes that can negatively impact eye formation in the context of Lobe ² .

Keywords: GAL4/UAS; Hr78; Jazf-1; enhancer; suppressor.

Figure 1

Jazf-1 locus with GFP expression. (a) Schematic representation of GFP inserted into the Jazf-1 locus. RA, RE, and RF coding sequences will splice in the GFP cassette. Jazf-1 RB is incapable of splicing in the GFP cassette. (b–g) Jazf-1 expression in Drosophila melanogaster eyes and ocelli photoreceptors. (b–d) UAS:GFP responder and a Jazf-1:GAL4 driver were inserted in the Jazf-1 locus. (e–g) UAS:GFP responder inserted in a different location with not responder. Brightfield (b and e), GFP (c and f), and merge (d and g) images demonstrate that expression from the Jazf-1 locus but not responder controls in the eye and ocelli (white arrows).

Figure 2

Schematic representation of overexpression and knockdown strategy in the eye. (a) Overexpression of a gene of interest in the eye. 1. (a) Gal4 encoding gene with an eyeless-promoter will express the Gal4 transcription factor in the eye (The Driver). 2. The UAS is integrated into the endogenous gene promotor via a transposable element that is stably integrated into the DNA. The Gal4 transcription factor increases the expression of the endogenous gene of interest (The Responder). 3. The overexpression of the endogenous gene of interest increases the gene product. (b) Knockdown of a gene of interest in the eye. 1. (a) Gal4 encoding gene with an eyeless promoter will express the Gal4 transcription factor in the eye (The Driver). 2. The UAS is linked to a shRNA with a complementary sequence to the gene of interest that is to be silenced. 3. The shRNA targets mRNA for degradation based on complementary sequence recognition. 4. Degradation of mRNA leads to decreased protein expression.

Figure 3

Jazf-1 overexpression and Jazf-1-RNAi driven in the eye. (a) UAS-Jazf-1 responder and eyeless-GAL4 driver. (b) Jazf-1-RNAi responder and eyeless-Gal4 driver as heterozygotes do not display any obvious phenotype. (c–f) Examples of Jazf-1-RNAi; eyGAL4 homozygotes display variable phenotypes. (c) Extra structures to the posterior of the eye (white arrows). (d and e) Broken sections and a smaller eye field. (f) Arrows point to two different eyes, one fully formed (#), and the other absent (*) in the same organism.

Figure 4

Lobe² and Jazf-1 overexpression in the eye. (a–c) Representative images of D. melanogaster eyes. (a) Example of a line containing an eyGAL4 driver with no responder. (b) Lobe² allele. (c) Lobe² allele with an eyGal4 driver and UAS:Jazf-1 responder. Jazf-1 overexpression suppresses the eye phenotype of the Lobe² allele. (d) Graphical representation of the eye sizes measuring the dorsal-ventral axis. All measurements are the average of 10 females; error bars are the standard deviation. A Student’s T-test was conducted between the Lobe² and the Lobe² allele rescued with a Jazf-1 overexpression in the eye (p = 5 × 10⁻⁷).

Figure 5

Lobe² is enhanced by NuA4/TIP60 complex member knockdowns. (a–c) Representative images of Drosophila eyes. (a) Lobe² with a knockdown of Jazf-1. (b) Lobe² with Xbp-1 knockdown. (c) Lobe² with Nipped-A knockdown. (d) Graphical representation of the eye sizes measuring the dorsal-ventral axis with members of the NuA4/TIP60 complexed knocked down. No appreciable difference occurred with a single driver and responder present. On a Lobe² background, knockdown of NuA4/TIP60 complex members enhanced the small eye phenotype. All measurements are the average of 10 females; error bars are the standard deviation. A Student’s T-test was conducted between the Lobe² and those with Lobe² and a knocked down NuA4/TIP60 complex member showed a significant enhancement in the eye phenotype with all the members (*p < 0.0005).

Figure 6

Lobe² and Hr78 expression level changes influence the overall size of the eye. (a and b) Images of phenotypes resulting from Hr78 gene expression level changes with the Lobe ² allele. (a) Hr78-RNAi in the eye does not significantly change the size of the eye. (b) Hr78-RNAi on a Lobe² partially restores the eye. (c) Graphical representation of the eye sizes measuring the dorsal-ventral axis with variations in Hr78 expression with Lobe ² alleles. All measurements are the average of 10 females, error bars are the standard deviation. A Student’s T-test was conducted between the Lobe² and those with Lobe² and variations of Hr78 expression levels.

Figure 7

(a) Hr78² heterozygous eyes. (b) Lobe ² and Hr78 ² heterozygotes. Eyes show a decrease in the size of the eye compared with Lobe ² by itself. (c) Graphical representation of the eye sizes measuring the dorsal-ventral axis. Hr78 ² heterozygotes display a typical eye size. When placed on an Lobe ² mutant background, the size decreases, including some with no eye formed. All measurements are the average of 10 females; error bars are the standard deviation. A Student’s T-test was conducted between the Lobe ² and the Lobe ² ; Hr78 ² mutants displayed a significant enhancement in the eye phenotype (p = 0.0033).

Figure 8

Proposed model of regulation. Hr78 acts as an anchor for chromatin modifiers. The NuA4/TIP60 complex can bind through Jazf-1 to Hr78 via the AF-2 domain. Activators can bind to Hr78, tentatively including nej/CBP through the AF-1 domain. Contact with the NuA4/TIP60 complex allows for histone modifications that typically repress expression. While the activators will enhance gene expression. The Lobe² allele allows Lobe protein to be expressed in the eye and usually shrinks the eye. Changes in the chromatin modification will enhance or suppress this eye phenotype because they modify the expression changes associated with the Lobe² allele.