A dissection of the teashirt and tiptop genes reveals a novel mechanism for regulating transcription factor activity

Abstract

In the Drosophila eye the retinal determination (RD) network controls both tissue specification and cell proliferation. Mutations in network members result in severe reductions in the size of the eye primordium and the transformation of the eye field into head cuticle. The zinc-finger transcription factor Teashirt (Tsh) plays a role in promoting cell proliferation in the anterior most portions of the eye field as well as in inducing ectopic eye formation in forced expression assays. Tiptop (Tio) is a recently discovered paralog of Tsh. It is distributed in an identical pattern to Tsh within the retina and can also promote ectopic eye development. In a previous study we demonstrated that Tio can induce ectopic eye formation in a broader range of cell populations than Tsh and is also a more potent inducer of cell proliferation. Here we have focused on understanding the molecular and biochemical basis that underlies these differences. The two paralogs are structurally similar but differ in one significant aspect: Tsh contains three zinc finger motifs while Tio has four such domains. We used a series of deletion and chimeric proteins to identify the zinc finger domains that are selectively used for either promoting cell proliferation or inducing eye formation. Our results indicate that for both proteins the second zinc finger is essential to the proper functioning of the protein while the remaining zinc finger domains appear to contribute but are not absolutely required. Interestingly, these domains antagonize each other to balance the overall activity of the protein. This appears to be a novel internal mechanism for regulating the activity of a transcription factor. We also demonstrate that both Tsh and Tio bind to C-terminal Binding Protein (CtBP) and that this interaction is important for promoting both cell proliferation and eye development. And finally we report that the physical interaction that has been described for Tsh and Homothorax (Hth) do not occur through the zinc finger domains.

Figure 1. Schematic of Tsh and Tio Constructs, Genomic Integration and Protein Confirmation

(A) A schematic diagram of all Tsh and Tio constructs that were generated and used in this study. The location of the PLDLS and Zn domains are found at the top of the panel. (B) PCR analysis to confirm the proper location of each construct after phiC31 integration. Note that for each construct the expected bands of 227 and 454bp are obtained. (C) A western blot showing expression of each construct in S2 cells. Note that we were able to detect stable proteins for all constructs except for Tribolium Tsh/Tio.

Figure 2. Frequencies of Ectopic Eyes Induced by Tsh and Tio Variants

(A) A confocal image of a third instar disc containing an ectopic eye within the antenna. Anterior is to the right. (B,C) Light microscope images of adult flies containing ectopic eyes (arrows). (D,E) Graphs documenting the frequencies of ectopic eye formation induced by each Tsh and Tio variant.

Figure 3. Contribution of Each Zn Finger to Ectopic Eye Size

(A–D) Confocal images of third instar eye discs. Genotypes of each disc are at the bottom right of each panel. Visualized molecules are listed at the top right of each panel. Anterior is to the right. (E) Graph documenting the size ectopic eyes that are induced by each Tsh and Tio variant. Measurements were taken with ImageJ software.

Figure 4. Location of Tissue Outgrowths Induced by Tsh and Tio Variants

(A–K) Confocal images of third instar eye discs. Genotypes of each disc are at the bottom right of each panel. Visualized molecules are listed at the bottom left of panel A. Anterior is to the right. Dotted lines indicate examples of tissue outgrowths. (L) A summary diagram of the location of ectopic tissue growths. Blue indicates location of ectopic tissue growth that is induced by both Tsh and Tio full-length molecules. Yellow indicates location of ectopic tissue growth that is induced specifically by the Tio full-length molecule. Green indicates location that both Tsh and Tio induce cell proliferation and ectopic eye formation.

Figure 5. Interactions Between Tsh, Tio and Hth are not Mediated by the Zinc Finger Domains

Western blots demonstrating that interactions between Hth and Tsh and Tio occur through the non-zinc finger domains. The nuclear lysate rows demonstrate that each protein is made and stable in Kc167 cells. The negative control lanes demonstrate that the proteins are not non-specifically immunoprecipitated. The last row indicates that Hth was immunoprecipitated in each experiment. The second to last row (immunoblot) indicates that both full-length and zinc finger deletion Tsh and Tio proteins interact specifically with Hth.

Figure 6. CtBP Interacts with Tsh and Tio and is Expressed in the Developing Eye

(A) N-terminal sequences of Tsh and Tio. The PLDLS domain is highlighted in red. (B,C) Western blot and co-IP from S2 cells demonstrating that Tsh binds to CtBP through the PLDLS domain. (D,E) Western blot and co-EP from S2 cells demonstrating that Tio binds to CtBP through the PLDLS domain. (F–H) Confocal images of wild type third instar eye-antennal disc stained with antibodies directed against Elav (green) and CtBP (purple). The arrow indicates area of CtBP expression that overlaps with that of Tsh and Tio. Anterior is to the right.

Figure 7. Loss of CtBP Leads to De-Repression of dac and an Acceleration of the Furrow

(A–H) Confocal images of third instar larval eye discs. Visualized molecules are listed at the bottom right of each panel. (A,D) The arrows indicate areas of dac de-repression within CtBP clones. The arrowheads indicate area of normal dac expression in wild type tissue. (G,H) The arrows denote the acceleration of the furrow within CtBP clones. The arrowheads denote the normal furrow in wild type tissue. Anterior is to the right.

Figure 8. A Novel Internal Mechanisms for Regulating Transcription Factor function

(A) A schematic that summarizes the relative contributions that each Zn makes towards the processes of retinal development. Note that for Tsh Zn3 counteracts the effects of the other zinc fingers.Also note that such an internal balancing act does not exist for Tio. These differences may explain why Tio is a more potent inducer of ectopic eyes than Tsh. (B) A schematic that summarizes the relative contributions that each Zn makes towards the processes of cell proliferation. Note that while Zn1 and Zn3 counterbalance the activity of Zn2 within Tsh no such competing mechanism exists for Tio.