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. 2010 Mar 2;107(9):4087-92.
doi: 10.1073/pnas.0914595107. Epub 2010 Feb 10.

Function and specificity of synthetic Hox transcription factors in vivo

Dimitrios K Papadopoulos  1 , Vladana Vukojevic, Yoshitsugu Adachi, Lars Terenius, Rudolf Rigler, Walter J Gehring
Affiliations

Function and specificity of synthetic Hox transcription factors in vivo

Dimitrios K Papadopoulos et al. Proc Natl Acad Sci U S A. .

Abstract

Homeotic (Hox) genes encode transcription factors that confer segmental identity along the anteroposterior axis of the embryo. However the molecular mechanisms underlying Hox-mediated transcription and the differential requirements for specificity in the regulation of the vast number of Hox-target genes remain ill-defined. Here we show that synthetic Sex combs reduced (Scr) genes that encode the Scr C terminus containing the homedomain (HD) and YPWM motif (Scr-HD) are functional in vivo. Synthetic Scr-HD peptides can induce ectopic salivary glands in the embryo and homeotic transformations in the adult fly, act as transcriptional activators and repressors during development, and participate in protein-protein interactions. Their transformation capacity was found to be enhanced over their full-length counterpart and mutations known to transform the full-length protein into constitutively active or inactive variants behaved accordingly in the synthetic peptides. Our results show that synthetic Scr-HD genes are sufficient for homeotic function in Drosophila and suggest that the N terminus of Scr has a role in transcriptional potency, rather than specificity. We also demonstrate that synthetic peptides behave largely in a predictable way, by exhibiting Scr-specific phenotypes throughout development, which makes them an important tool for synthetic biology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Synthetic Scr peptides induce homeotic transformations in the fly (A–C). Expression of the synthetic genes throughout the embryo results in the formation of additional salivary glands in the cephalic region for Scr-HDwt (A) and Scr-HDAA (B), but not Scr-HDDD (C). (D) Wild type embryo also treated with heat shock allows the development of one normal pair of salivary glands. Arrowheads show the ectopic—and asterisks, the normal—salivary glands. All constructs were induced using Heat-Shock-Gal4. Stainings are on stage 16 embryos for dCREB-A (19), a salivary gland luminal marker. Scale bar 100 μm. (E–I) Expression of Scr-HDwt (E) and Scr-HDAA (F) in the antennal disc results in complete antenna-to-tarsus transformations. (G) Magnification of the outlined area in (E) shows pigmented cells (Arrowhead) at the distal part of the transformed A3. (H) Scr-HDDD only confers a small reduction in the size of the arista. (I) Wild type antenna. (J–K) Sex comb teeth on antennal tarsi generated by ectopic expression of Scr-HDwt (J) and Scr-HDAA (K) (Arrowheads). All transformations were generated using a Dll-Gal4 driver.
Fig. 2.
Fig. 2.
Synthetic Scr peptides interact with Pax transcription factors in vivo (A–D). Eye-reduction phenotypes exhibited by ectopic expression of Scr-HDwt (A, B, and D) and Scr-HDAA (C). According to the strength of expression, different lines exhibited phenotypes ranging from eye-reduction (A–C) to eye-absence (D). (E) Ectopic expression of Scr-HDDD resulted in no detectable phenotype. (F) Wild type head. (G–I) Ectopic expression of Scr-HDwt in the eye-disc (G) does not repress ey (H). Dashed lines show the domain of colocalization of Scr-HDwt and Ey (I). The dppblink-Gal4 driver has been used throughout.
Fig. 3.
Fig. 3.
Synthetic Scr-HD peptides act as transcriptional activators and repressors in the antennal primordium. (A–F) Repression of Spalt major (Salm) in the antennal disc by ectopic expression of Scr-HDwt (A and D) and Scr-HDAA (B and E). No repression was observed with Scr-HDDD (C and F). (G–L) Repression of distal antenna (dan) in the antennal disc is complete with Scr-HDAA (H and K) and incomplete with Scr-HDwt (G and J), leaving a patch of cells that retain Dan activity (Arrowhead in G). These cells do not express ectopic Scr-HDwt. (I and L) Scr-HDDD does not repress dan. (M–R) Repression of Hth results in a shift of the Hth-Dll boundary. Partial repression of Hth by Scr-HDwt (M and P) and Scr-HDAA (N and Q), as compared to Scr-HDDD (O and R), where no repression is observed. (S–U) X-gal stainings showing the activation of grain (grn) by Scr-HDwt (S) and Scr-HDAA (T) (Arrowheads). Scr-HDDD (U) fails to activate ectopic grn expression. (V–Y) Normal expression of Salm (V), dan (W), Hth-Dll (X), and grn (Y) in eye-antennal discs. Dll-Gal4 was used to drive expression of all constructs (A-Y).
Fig. 4.
Fig. 4.
Cells with no ectopic Scr-HDwt activity fail to repress dan but activate Elav and thus differentiate into compound eyes. (A) Elav gain-of-function, (B) Dan (Arrowheads), and (C) merge of (A) and (B) with Scr-HDwt. (D) Magnification of outlined area in (C). Arrow points at cells that express both dan and Elav but not ectopic Scr-HDwt. (E) Adult antenna transformed into a tarsus, which bears a small ectopic eye in the A3. Note the presence of ommatidia and interommatidial bristles (Red Asterisk). Scale bars in (C) 100 μm and in (D, E) 50 μm.
Fig. 5.
Fig. 5.
High-resolution APD imaging of DNA-Scr-HD interactions in live cells. (A) Third instar salivary gland polytene nuclei expressing Scr-HDwt, Scr-HDAA, Scr-HDDD, and Scr-formula image under the control of dppblink-Gal4. Ubiquitously expressed mRFP1-tagged histone H2B was used to visualize chromatin. Scr-HDwt and Scr-HDAA readily associate with the chromosomes (as shown in the Green Channel) but also show sites of accumulation along the chromosome where loose chromatin compaction is shown as a low histone signal (Arrows). Arrowheads point at sites of high accumulation observed for Scr-HDwt and Scr-HDAA. The nucleus expressing the inactive Scr-HDDD shows some association of the transcription factor with the DNA, but it is also dispersed in the nucleoplasm. There is no pronounced banding pattern observed in this case, which suggests absence of specific binding. Scr-formula image appears almost completely excluded from the chromosomes, mainly residing in the nucleoplasm. Scale bars in all cases are 20 μm. (B) Electrophoretic Mobility Shift Assay (EMSA) shows that only Scr-HDwt and Scr-HDAA bind DNA specifically in vitro. Both variants bound more strongly to BS2 than fkh250 (Left). Titration of peptide concentration (Right) revealed that even at high concentrations of transcription factor, Scr-HDDD and Scr-formula image do not bind DNA (BS2) specifically.
Fig. 6.
Fig. 6.
The full-length Scr peptide exhibits weaker homeotic function than its synthetic counterparts in vivo. (A) Partial antenna-to-tarsus transformation mediated by Scr full-length using the dppblink enhancer. (B) The same gain-of-function results in eye-reduction in the head. (C) Transformation using the Dll enhancer is also incomplete. (D) Adult head of a fly expressing Scr full-length using Dll-Gal4. (E–F) Repression of Salm by the full-length Scr in the antennal disc (Arrowheads in E) is complete. (G–H) The same applies in the repression of dan. (I–M) Repression of Dll and Hth is incomplete (I, K, and M) and considerable overlap of Hth and Dll is observed in the antennal disc (Dashed White Lines in M). Scr represses Hth also outside the Dll expression domain (Solid Green Line in K and M). (N–P) Cells that maintain Dan activity (Arrowhead in O) in the antennal disc express ectopic Elav (Arrowhead in N). The full-length Scr has been induced by dppblink-Gal4 (E–P).
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References

    1. Gehring WJ. Homeo boxes in the study of development. Science. 1987;236(4806):1245–1252. - PubMed
    1. Lewis EB. A gene complex controlling segmentation in Drosophila. Nature. 1978;276(5688):565–570. - PubMed
    1. Mann RS, Morata G. The developmental and molecular biology of genes that subdivide the body of Drosophila. Annu Rev Cell Dev Biol. 2000;16:243–271. - PubMed
    1. McGinnis W, Krumlauf R. Homeobox genes and axial patterning. Cell. 1992;68(2):283–302. - PubMed
    1. Kappen C, Ruddle FH. Evolution of a regulatory gene family: HOM/HOX genes. Curr Opin Genet Dev. 1993;3(6):931–938. - PubMed

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