spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 19 April 2006
doi: 10.1242/dev.02372

Development 133, 1979-1989 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Acar, M.
Right arrow Articles by Bellen, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Acar, M.
Right arrow Articles by Bellen, H. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Senseless physically interacts with proneural proteins and functions as a transcriptional co-activator

Melih Acar1,*, Hamed Jafar-Nejad2,3,*, Nikolaos Giagtzoglou3, Sasidhar Yallampalli2, Gabriela David2, Yuchun He3, Christos Delidakis4 and Hugo J. Bellen1,2,3,{dagger}

1 Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA.
2 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
3 Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA.
4 Institute of Molecular Biology and Biotechnology, FORTH and Department of Biology, University of Crete, Heraklion, GR-71110, Greece.


Figure 1
View larger version (40K):

[in a new window]
  		Fig. 1. Zn-finger domains of Sens mediate DNA binding, repression and activation. (A) Schematic of the ac-luc reporter used in the S2 cell transcription assay. E1, E2 and E3 represent the E-boxes, and S represents the S-box in the 470 bp ac proximal enhancer. (B) Alignment of the fly (D. m) Sens Zn-finger (ZF) domains with the corresponding Zn-finger domains of Homo sapiens (H.s) Gfi1 and Caenorhabditis elegans (C.e) PAG-3 shows that Zn-finger domains of GPS proteins and the linker regions that connect them are highly conserved. Stars represent the cysteines in the C2H2 structure. Squares represent the amino acids that are predicted to contact DNA in C2H2-type Zn-finger domains. Circles represent the amino acids in linkers that have the potential to be phosphorylated. Blue boxes denote divergent amino acids. (C) EMSA assay using a previously characterized probe (R21, see Materials and methods) and Sens with different types of Zn-finger mutations. Sens loses its ability to bind to DNA if the cysteines in Zn finger 1, 2 or 3 are mutated. The amino acids that were predicted to contact DNA in Zn fingers 2 and 3 but not in Zn finger 1 seem to be crucial for DNA binding. Zn finger 4 seems to be dispensable for DNA binding. (D) Activation assays in S2 cells show that all Zn fingers are involved in the synergism with proneural proteins to upregulate the expression of the ac-luc reporter. Sens fails to synergize with proneural proteins when either Zn finger 2 or 3 is mutated, but exhibits some synergism upon mutating Zn finger 1 or 4. (E,F) Repression assays on the ac-luc reporter using wild-type and Zn-finger mutant Sens expression constructs. Sens loses its ability to repress the ac-luc reporter at low levels if Zn finger 1, 2 or 3, but not 4, is mutated.

 

Figure 2
View larger version (116K):

[in a new window]
  		Fig. 2. Zn fingers 2 and 3 are indispensable for the bristle-inducing ability of Sens. (A) Thorax of a wild-type (Canton S) fly. (B) Ectopic expression of wild-type sens with Eq-Gal4 driver induces the formation of many extra bristles. Ectopic expression of Sens with a mutant Zn finger 1 or 4 with Eq-Gal4 driver can also induce extra bristle formation (C,F). Sens loses its ability to induce ectopic bristle formation if either Zn finger 2 or 3 is mutated (D,E). Midline bristle loss is observed in flies expressing Sens, Sens-1CC and Sens-4CC, which is due to a closure defect during thorax development when Sens retains activity. Sens-2CC and Sens-3CC do not display the dorsal closure phenotype.

 

Figure 3
View larger version (50K):

[in a new window]
  		Fig. 3. A sens genomic transgene can partially rescue the adult PNS phenotypes of sens mutant flies when Zn finger 1 is mutated. (A) Bristles fail to develop in sensE2 mitotic clones generated by the C684-Gal4 driver (see Materials and methods). (B) sens-g is able to rescue all bristles in sensE2 mitotic clones (bristles in clones are marked with yellow). (C) sens-1CCg is able to rescue some of the bristles in sensE2 mitotic clones. (D) sens-3CCg fails to rescue the bristle loss in sens mutant clones. Arrows in C,D indicate lost macrochaetae; the arrowhead in C indicates a rescued macrochaetae. Mutant areas in A-D are marked with white lines. (E) Quantification of number of post-orbital bristles formed in flies that have large sens mutant clones generated by the eyeless-FLP system. Error bars represent the standard error of the mean. y w was used as the wild-type (WT) strain. For each genotype, post-orbital bristles of 7-24 flies were counted. The differences among `no transgene', sens-2CCg and sens-3CCg are not statistically significant, neither are the differences among wild type, sens-g and sens-4CCg. However, the bristle number for sens-1CCg is significantly different from all other genotypes and again shows partial rescue (P<0.0001).

 

Figure 4
View larger version (70K):

[in a new window]
  		Fig. 4. Sens with a phosphomimetic mutation in linker 2 is able to induce bristle formation but does not bind the R21 probe. (A) EMSA assay using wild-type Sens and mutant Sens versions with phosphomimetic mutations in the linker regions that connect the Zn fingers. R21 oligonucleotide, to which Sens binds strongly, was used as probe. A phosphomimetic mutation in linker 1 (Sens-L1-S-E) reduces the Sens-DNA interaction. However, a phosphomimetic mutation in linker 2 (Sens-L2-T-E) does not show any DNA-binding activity under these conditions. The gel on the right shows 20% of the input for the mutant proteins and 20% of the wild-type Sens input is shown in Fig. 6E. (B,C) Ectopic expression of sens-L2-T-E using Eq-Gal4 driver induces formation of extra bristles at both 25°C and 28°C, as shown in B and C, respectively.

 

Figure 5
View larger version (25K):

[in a new window]
  		Fig. 5. Sens is recruited to the ac regulatory region via protein-protein interactions. (A) An S2 cell transcription assay using the ac-luc reporter with mutations in various combinations of E-boxes. wt, wild type; E1-3, mutant E-boxes 1-3 (see Fig. 1A). Blue bars represent the presence, and red bars represent the absence, of the S-box on the ac-luc reporter. The absence of the S-box does not affect the expression mediated by Ac and Da when no Sens protein is present. (B) Amplification of the expression induced by Ac and Da upon addition of Sens. The absence of the S-box significantly increases the ability of Sens to synergize with proneural proteins on the ac-luc reporter.

 

Figure 6
View larger version (26K):

[in a new window]
  		Fig. 6. Sens binds proneural proteins through its Zn-finger domains. (A) An IP assay shows that Sens interacts with Ac in COS-7 cells. (B) In vitro translated c-myc-tagged Sens interacts with bacterially expressed GST-tagged proneural proteins in a GST pull-down assay. Input lane shows 20% of the input used in the GST pull-down assay. (C) Sens binds to Sc through its Zn-finger domains. Bacterially expressed GST-tagged Sc protein can interact with in vitro translated full-length Sens (a), and Sens Zn-finger domains (c), but not with Sens N terminus (b), which lacks the Zn-finger domains. (D) Sens can still bind GST-tagged Sc if any of its Zn-finger domains are deleted (b,c,d,e). However, Sens loses its ability to bind Sc if Zn finger2, linker2 and Zn finger3 are deleted together (f). The bottom gel shows western blot analysis using anti c-myc antibody to detect 20% of the input. (E) Sens-L2-T-E binds Sc in a GST pull-down assay. The binding is weaker than wild-type Sens, in line with the weaker bristle phenotype (see Fig. 4B,C). Input lanes show 20% of the actual input.

 

Figure 7
View larger version (17K):

[in a new window]
  		Fig. 7. Sens can synergize with proneural proteins on the UAS-tk-luc reporter in S2 cell transcription assays. (A) Schematics of the UAS-tk-luc reporter construct. (B) S2 cell transcription assays using UAS-tk-luc reporter. Sens expression alone does not have a significant effect on the basal expression of the UAS-tk-luc reporter. However, Sens is able to synergize with Sc and Ac. (C) E(spl)m7 strongly inhibits the expression induced by the Sc-Gal4DBD and the synergism between Sens and Sc-Gal4DBD. (D) When fused to Gal4DBD, Sens acts as a strong repressor on the UAS-tk-luc reporter. Ac and Sc fail to synergize with DNA-bound Sens.

 

Figure 8
View larger version (16K):

[in a new window]
  		Fig. 8. A model for the dual role of Sens Zn fingers in the transcriptional regulation of proneural target genes. E represents E-box, S represents S-box. The four ovals in Sens depict Zn fingers. See Discussion for details.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006