FY2011 Annual Report

Developmental Signalling Unit

Professor Mary Ann Price

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Abstract

My lab is interested in the medically and developmentally important Hedgehog (Hh) signal transduction pathway, which is conserved in metazoans. In many of its roles in cell-fate specification, in cancer, and in the regulation of stem cells, the pathway acts to change expression of genes in target cells, via the Gli/Ci family of zinc finger transcription factors. Ci, the sole family member in Drosophila, can act as a repressor in the absence of Hh signalling and an activator in the presence of signalling. This switch in Ci activity is mediated by partial degradation of the full-length molecule by the proteasome to a shorter repressor form. Hh signalling inhibits this processing reaction and further converts full-length Ci to an activator form. My lab has been studying the regulation of Ci using molecular genetic, biochemical, and cell biological approaches. Additionally, we are studying the role of Hh signalling in lymphocyte differentiation and proliferation.

Our aims are:

  • to better understand the cis requirements for Ci-75 formation using structure/function  analysis and model substrate experiments,
  • to determine novel components involved in Ci processing by doing a cell-based genome-wide double-stranded RNA interference (RNAi) screen,
  • to gain insight into Ci regulation by examining its subcellular localization, and
  • to understand the role of Hh in lymphocyte differentiation and proliferation.

1. Staff

  • Chika Azama – research administrator/secretary
  • Olga Elisseeva – post-doc
  • Marco Tsui – post-doc
  • Umesh Gangishetti – post-doc
  • Yasuji Kido – shared post-doc with Samatey unit
  • Satoshi Hasegawa – technician/post-doc
  • Anri Itoh  - technician (started September 2011)
  • Michiko Arai – technician (moved to Sugiyama unit in October 2011)
  • Spencer Spratt – post-doc (left in December 2011)

2. Collaborations

Nothing to report.

3. Activities and Findings

1. cis requirements for Ci-75 formation. Previously, we determined which regions of Ci are required for Ci-75 formation (Wang and Price (2008) Mol. Cell Biol. 28: 5555-5568). In that work, we showed that a region in the C-terminus of Ci-155 (amino acids 1235 to 1397) is required for Ci-75 formation because it is necessary for initiation of proteasomal processing. Anri Itoh, Marco Tsui, and Satoshi Hasegawa have narrowed this region down to amino acids 1319 to 1337 by showing that a Ci mutant lacking these 18 amino acids does not form Ci-75 (Figure 1), while deletion of other regions within the larger domain have no effect on Ci processing. Like the larger domain we started with, this smaller region is required for the initiation of degradation by the proteasome (Figure 2). We determine this by measuring the half-life of the Ci mutants. Wild-type Ci has a half-life of 1.34h, while mutants such as Ci3m, which are not ubiquitylated, and therefore, do not initiate proteasomal degradation, have a longer half life (4.2h). CiD1319-1337 similarly has a longer half-life (4.68h) than wild-type Ci.

 

Fig. 1. Amino acids 1319-1337 are required for Ci-75 formation. A. A schematic of the C-terminal region of Ci-155 showing the region deleted in our Ci mutant constructs. All constructs are tagged at the N-terminus with three copies of the hemagglutinin (HA) tag. B. Results of immunoprecipitation (IP) Western Blot (WB) assay. We transiently transfect the indicated constructs into Kc cells, make extracts, IP with an anti-HA antibody (12CA5) and WB with a second anti-HA antibody (3F10).

            

Figure 2. Amino acids 1319 to 1370 are required for initiation of degradation. A. Cycloheximide (CHX)-chase experiment. Samples of Kc cells transfected with the indicated construct are taken at 0, 2, 4, or 6 hours after CHX treatment. The WB is probed with an anti-HA antibody (3F10). B. Quantitation is shown in the graph. C. Half-life is shown in the table.

We are trying to determine how this region is involved in initiation of degradation. Our preliminary results suggest that it is required for ubiquitylation of Ci-155. We are now determining whether this region is involved in binding to the E3 Ubiquitin ligase SCFslimb.

2. Novel genes required for Hh to block Ci processing. The lab previously conducted a cell-based genome wide RNAi screen to determine which Drosophila genes are required for Ci processing in the absence of Hh. This screen involved using a stable Kc cell line (a Drosophila hemocyte line which recapitulates in vivo Ci processing) expressing a reporter consisting of Ci tagged at the N-terminus with firefly luciferase and at the C-terminus with Renilla luciferase (Figure 3).

Figure 3. Schematic of our Ci processing reporter. F stands for firefly luciferase; R stands for Renilla luciferase.

Because the Renilla tag is degraded when Ci is processed, measurement of the Renilla/firefly luciferase (R/F) ratio reflects the amount of processing. We obtained funding (Kakenhi grant) to do a second screen, this time in the presence of Hh. Hh blocks Ci processing and thus increases the R/F ratio. Removal of genes required for this outcome of Hh signalling by RNAi will prevent this increase in R/F ratio (Figure 4). For example, pre-treatment of the cells with

Figure 4. Schematic of the screen. The screen uses Kc cells expressing the  F-Ci-R reporter. Addition of Hh blocks processing leading to a higher R/F luciferase (R/F) ratio (upper right panel). The reporter line will be treated with dsRNAs corresponding to each gene in the Drosophila genome in the presence of Hh. Most dsRNAs will have no effect on Hh blocking Ci processing, leaving the R/F ratio unchanged (lower left panel). Genes required for Hh to block processing, however, will give a reduced R/F ratio (lower right panel).

smoothened (smo) double-stranded RNA prevents this increase, since Smo is required to transduce Hh signalling to the intracellular components of the pathway (Figure 5). Umesh Gangishetti will conduct the screen on our reporter cell line, in the presence of Hh, to determine which genes are required for Hh to block Ci processing. First we are determining how best to introduce Hh. In the past, we have transfected a Hh expression construct into the Kc cells, but this is inappropriate for a screen because many genes will affect modification and secretion of Hh, and will give us false-positives.

Figure 5. Pilot experiment for the screen. Kc cells expressing the F-Ci-R reporter are treated with no dsRNA, GFP dsRNA, or smo dsRNA, either with (gray bar)or without  (white bar) added Hh. With no RNAi or an unrelated dsRNA, addition of Hh inreases the R/F ratio. However, with smo RNAi, Hh has no effect.

3. Subcellular localization of Ci-155. Marco Tsui finds that Ci is localized in the cytoplasm in a punctate pattern on top of diffuse Ci staining in wing imaginal discs, whether it is endogenous Ci or Ci expressed from a transgene (Figure 6A and 6B). Ci constructs in various Drosophila cell lines transfected show a punctate pattern with less diffuse staining in the cytoplasm (Figure 6C). A punctate pattern in the cytoplasm often reflects localization to intracellular membrane vesicles, such as endosomes. So far, we have not found any vesicle markers that significantly co-localizes with Ci. Furthermore, we do not find that activation of Hh or knocking out Cos2, a kinesin related protein and Ci-interacting protein that also localizes to cytoplasmic puncta, significantly change the punctate localization of Ci, though both increase the amount of full-length Ci. We have isolated a short (~200 amino acid) domain, which we call the PFR (puncta forming region) that is sufficient to localize an exogenous protein (EGFP) to cytoplamsmic puncta (Figure 6D). We are currently determining whether deletion of this region from Ci-155 ablates the puntate localization, and whether its deletion affects the activity or regulation of Ci (processing, transcription, or regulation by Hh).

Figure 6. Punctate pattern of Ci. A. Endogenous Ci in the wing disc, stained with 2A1 antibody which recognizes only Ci-155. B. Ci-EGFP expressed in the wing disc C. Ci-EGFP expressed in clone 8 cells. D. CiPFR-EGFP in S2R+ cells (PFR stands for puncta forming region, a ~200 amino acid region of Ci).

4. Hh in the immune system. It is well established that Hh plays a role in the early development and maturation of T cells in the thymus, but less is known about the role of Hh in circulating T cells. Olga Elisseeva finds by RT-PCR that the components of Hh signalling are found in circulating T cells and that the cells produce Hh. Rather than treating mixtures of either CD4+ or CD8+ cells with Hh, we have separated them into naïve, memory, and effector subclasses. The effect of adding or blocking Hh in these cells depends on the subtype. For example, treatment of naïve CD8+ cells with Shh leads to increased differentiation to effector cells. We are also looking at the localization of Hh signalling components in T cells as they engage with antigen presenting cells.

4. Publications

Oral presentations: 

  • “Invertebrate Embryology and Patterning,” OIST Developmental Neurobiology Course, Okinawa, Japan (July 2011)

Posters:

  • Hasegawa, S., Spratt, S., Arai, M., Wang, Y., Price, M.A. A genome-wide search for novel Hedgehog signalling regulators involved in Cubitus interruptus (Ci) processing, 44th Annual Meeting of the Japanese Society for Developmental Biology, Ginowan, Okinawa, May 18-21, 2011.
  • Hasegawa, S., Spratt, S., Wang, Y., Price, M.A. A genome-wide search for novel Hedgehog signalling regulators involved in Cubuitus interruptus (Ci) processing. 1st Asia-Pacific Drosophila Research Conference, Taipei, Taiwan, May 22-25, 2011.
  • Spratt, S., Hasegawa, S., Gangishetti, U., Arai, M., Wang, Y., Tsui, M., Price, M.A. A genome-wide search for novel Hedgehog signalling regulators involved in Cubitus interruptus (Ci) processing, Hh-Gli Signalling in Development, Regeneration and Cancer, Crete, Greece, June 23-25, 2011.
  • Tsui, M., Itoh, A., Hasegawa, S., Price, M.A. Structure-Function Study on the Sequence Requirements for Ci Processing, Hedgehog 2012, Biopolis, Singapore, March 18-21, 2012.

5. Intellectual Property Rights and Other Specific Achievements

Nothing to report.

6. Meetings and Events

Co-organizer, OIST Developmental Neurobiology Course, 17-31 July 2011

  • Co-organizers: Davie Van Vactor (Harvard/OIST), Akinao Nose (University of Tokyo), Ichiro Masai (OIST)
  • Speakers: James Briscoe (NIMR), Chris Doe (Univ. Oregon), David Feldheim (UC Santa Cruz), Josh Huang (Cold Spring Harbor), Yuh Nung Jan (UCSF), Kozo Kaibuchi (Nagoya), Masayuki Miura (Tokyo), Takeharu Nagai (Hokkaido), Hitoshi Okamoto (RIKEN BSI), Lee Rubin (Harvard), Hideyuki Okano (Keio Univ.), K. VijayRaghavan (NCBS), Bernardo Sabatini (Harvard), Hitoshi Sakano (Tokyo), Elke Stein (Yale)

Co-organizer, Hedgehog 2012, 18-21 March 2012

  • Main organizer: Philip Ingham (IMCB, Singapore); other co-organizers: Xinhua Lin (CAS, Beijing), Fred de Sauvage (Genentech)
  • Speakers: Dereck Amakye (Novartis), Phil Beachy (Stanford), James Briscoe (NIMR), Thomas Burglin (Karolinska), Tessa Crompton (UCL), Freek Van Eeden (Sheffield), Jonathan Eggenschwiler (Princeton), Michael Galko (Univ. Texas MD Anderson Cancer Center), Isabel Guerrero (Univ. Autonoma de Madrid), Masami Ishibashi (Chiba), Dan Kalderon (Columbia), Rolf Karlstrom (U Mass), Takehiro Kusakabe (Konan), Xinhua Lin (CAS, Beijing), Andy McMahon (Harvard), Stacey Ogden (St. Jude Children’s Research Hospital), Anne Plessis (Institut Jacques Monod), Margaret Read (Infinity), Rajat Rohatgi (Stanford), Ariel Ruiz I Altaba (Univ Geneva Medical School), Adrian Salic (Harvard), Fred de Sauvage (Genentech), Matthew Scott (Stanford), Toshihiko Shiroishi (NIG, Japan), Christian Siebold (Oxford), Marta Swierczynska (Max Planck ICBG), Cliff Tabin (Harvard), Pascal Therond (CNRS Nice), Rune Toftgard (Karolinska), Brandon Wainwritght (Univ. Queensland), Carol Wicking (Univ. Queensland), Gen Yamada (Wakayama Medical Univ.)

7. Other

Outreach activities:
Onna/OIST Children’s School of Science (course teacher for 3rd and 4th grade course”)