Eya protein phosphatase activity regulates Six1DachEya transcriptional effects in mammalian organoge

1
Eya protein phosphatase activity regulates
Six1-Dach-Eya transcriptional effects in
mammalian organogenesisXue Li, Kenneth A. Oghi,
Jie Zhang, Anna Krones, Kevin T. Bush,
Christopher K. Glass, Sanjay K. Nigam, Aneel K.
Aggarwal, Richard Maas, David W. Rose, and
Michael G. Rosenfeld. (Nature, Vol 426, November
20, 2003, 247-254)

• Babak Pedram and Jenna McNeill
• March 29, 2004

2
Outline

• Overview of Six, Eya, and Dach proteins.
• Experiments on cell proliferation/survival and
  results.
• Discussion.
• Summary.

3
Overview

• Understanding the relationship between gene
  activation and repression is imperative in
  explaining mammalian organogenesis.
• Three genetically interacting proteins
• Sine oculis (Six)
• Eyes absent (Eya)
• Dachshund (Dach)
• Six1 is required for the development of
• Murine kidney
• Muscle
• Inner ear

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Overview

• Sine oculis (Six) ? DNA-binding homeodomain
  factor
• Eyes absent (Eya) ? Nuclear cofactor
• Dachshund (Dach) ? Nuclear cofactor
• Mutations leads to the failure of eye formation
  in Drosophila.
• Six1-6, Eya1-4, and Dach1-2/Ski/Sno are
  co-expressed in multiple organs
• Eyes
• Inner ear
• Pituitary gland
• Muscle
• Kidneys

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Overview

• Eya and Dach function as cofactors for Six.
• In Drosophila model, there is a synergistic
  interactions between so, eya and dac during eye
  development.
• Eya serves as co-activator of Six in the
  regulation of downstream genes.
• Eya has no apparent DNA-binding activity.
• Eya is translocated from the cytoplasm to the
  nucleus by Six proteins.

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Overview

• Dach is closely related to Ski/Sno (transcription
  co-repressors).
• They form repression complexes with N-CoR, Sin3A
  and members of histone deacetylases in order to
  functionally repress downstream gene expression.
• Example
• Six6 and Dach together repress the
  cyclin-dependent
• protein kinase inhibitor p27kip1, controlling
  retinal and
• pituitary precursor cell proliferation.

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Eyes absent (Eya) Overview

• Transcriptional co-activator.
• Promotes eye specification and development.
• Member of a evolutionary conserved set of nuclear
  transcription factors and cofactors collectively
  termed retinal determination (RD) gene network.
• Binds to other RD members
• Sine oculis (so)
• Dachshund (dac)
• Belongs to phosphatase subgroup, haloacid
  dehalogenase (HAD)
• (edited by ICS - original presentation had
  dehydrogenase) .
• (Drosophila model)

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Eya Overview

• Eya is also a protein tyrosine phosphatase.
• Classical tyrosine phosphatases use cysteine as a
  nucleophile.
• Eya in contrast uses a nucleophilic aspartic acid
  in a metal-dependent reaction.
• N-terminal motif ? Phosphatase activity.
• C-terminal motif (Eya domain) ? Where Mg2 binds
  Six and Dach.
• Phosphatase activity of Eya contributes to its
  ability to induce eye formation in Drosophila.

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Objective

• Demonstrate genetic interactions between Six and
  Eya factors that regulate precursor cell
  proliferation and survival during mammalian
  organogenesis.

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Six1 regulates precursor cell proliferation and
survival

• Six1 is highly expressed in the kidneys, otic
  placode, skeletal muscle, pituitary and
  developing nasal structures.
• Role of Six1 was investigated using
• Generated mice null for Six1 genomic locus using
  homologous recombination in embryonic stem cells.
• Mice null was produced by replacing the
  functionally conserved Six domain (SD) and DNA
  binding homeodomain (HD) with IRES-LacZ and
  PGK-Neo sequences respectively.
• 5-bromo-4-chloro-3-indolyl-ß-D-galactoside
  (X-gal) staining was used.

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• Effects
• All organs expressing Six1 were affected
• Failure of renal organogenesis.
• Asymmetrical reduction or complete absence of
  kidneys.
• Reduced muscle development.
• Absence of diaphragm, tongue, and migratory
  hypaxial muscles of the forelimbs.
• Impaired nasal development.
• Pituitary showed minimal decrease in size (
• Rib-cage significantly effected (deformation and
  fusion of distal rib cartilage).
• No development of inner ear structures.

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Figure 1a. X-gal staining of Six1
expression. Source Nature. Vol. 426. November
20, 2003
13
Figure 1b,c. (b) Decreased size or absence of
kidneys (K) (c) X-gal staining of forelimb muscle
precursor cells in embryos.
Source Nature. Vol. 426. November 20, 2003
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Figure 1d,e. (d) Decreased histological staining
of hypaxial muscle (e) Staining showing fused
ribs and absent inner ear structures. Source
Nature. Vol. 426. November 20, 2003
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Immunohistochemical Analyses

• To define the molecular basis of Six1-induced
  alterations in target tissue development,
  expression of Pax3, Lbx1, c-Met and Hgf were
  analyzed.
• Normal expression of all four markers were
  observed.
• Severe reduction of muscle precursor cells was
  observed (consistent with X-gal staining
  pattern).
• Expression level of Gdnf and Six2 per cell was
  reduced.

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Figure 2a. Eya1, Lbx1 and c-Met normal expression
and reduction in Gdnf and Six2 (asterisk) and
muscle precursor cells. Source Nature. Vol.
426. November 20, 2003
17

• Marker genes Eya1, Pax2, c-Ret, Wt1 and Wnt genes
  also had normal cellular expression during kidney
  organogenesis.
• Gdnf and Six2 expression per cell was depleted
  compared to a heterozygous littermate.
• Gdnf is a glial cell neurotrophic factor involved
  in neuron formation.
• The level of Lbx2 expression was lower.

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Figure 2b. Eya1, c-Met and Pax2 normal cellular
expression. Reduced expression of Gdnf and Six2
in the kidney (asterisk).
Figure 2c. Shows no Lbx2 expression in the mutant
kidney and normal Wt1 and Wnt gene expression.
19
Conclusion

• Therefore, results suggest a proliferation/surviva
  l defect during both muscle and kidney
  development in mutant mice (Six1-/-).

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Cell Proliferation Test

• Assessed rate of cell proliferation using
  5-bromodeoxyuridine labelling (BrdU) in vivo.
• Test was used on mice at embryonic day (E) 9.5,
  10.5 and 11.5.
• In mutant mice (Six1-/-) cells that express BrdU
  exhibited a significantly lower rate of cell
  proliferation (21.5).
• Six/- mice cell proliferation rate was 60.5.

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Cell Survival Test (TUNEL)

• Used ATdT-mediated dUTP nick end labelling
  (TUNEL) assay.
• Mutant mice (Six1-/-) showed 4 times the amount
  of apoptosis in hypaxial muscle precursors and
  3.5 times more apoptosis in the developing
  kidney.
• Similar results were found in the eye imaginal
  disk of Sine oculis mutated Drosophila.

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Figure 3. BrdU (red) test displaying decreased
cell proliferation (decreased BrdU
incorporation). Tunnel assay displaying increased
cell apoptosis (increased BrdU incorporation)
Source Nature. Vol. 426. November 20, 2003.

(edited by ICS)
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Is Six1 Cell-autonomous?

• Needed to determine if the Six1 dependent
  proliferation events were cell autonomous.
• Single-cell nuclear microinjection assay was
  implemented.
• Used IgG antibodies against Six1 or Eya3.
• Injected into C2C12 cells.
• Cell line found in the murine myoblast that
  express Six1 and Eya3, as well as other family
  members.
• BrdU was not incorporated.

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Figure 4. The effect of Six1 and Eya3 in cell
proliferation. Source Nature. Vol. 426.
November 20, 2003
25
Confirmation of Cell Autonomy

• Used small interfering RNAs (siRNA).
• Targeted murine Six1 and Eya3 sequences.
• Injected the siRNA in to the nucleus.
• Within 24hrs there was a significant knockdown of
  RNA transcripts.
• Polymerase chain reaction with reverse
  transcripts (RT-PCR) did not display the targeted
  transcripts for specific siRNAs.
• This was not the case for control siRNAs.

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Figure 5. Inhibition of of cell proliferation
(C2C12) by Six1 and Eya1 siRNAs. Source Nature.
Vol. 426. November 20, 2003
27
Cell Survival Test (AIF-1 Staining)

• Used AIF-1 staining as a marker of apoptosis.
• Found that apoptosis of C2C12 cells was not
  associated with anti-Six1 nor anti-Eya3 IgGs nor
  specific siRNAs.

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Rescue

• Confirmation of the specificty of the effect was
  confirmed using rescue.
• Simultaneous microinjection of purified
  bacterially expressed holoproteins.

Figure 6. Rescue by bacterial holoproteins
(wild-type Eya3). Source Nature. Vol. 426.
November 20, 2003
29
Six1 gene targets

• 50 of the most statistically significant gene
  candidates, including Gdnf, Six2 and c-Myc were
  analyzed by in situ hybridization of E11.0 and
  E13.5 Six1-/- mutant and wild-type littermate
  controls.
• Results
• In both limb-muscle precursor cells and
  developing kidney precursor cells, c-Myc and Gdnf
  expression were absent (E11.0, E13.5).

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• To evaluate the potential roles of the Six1 gene,
  single-cell nuclear microinjection assay was
  used.
• Anti-Six1 IgG was injected.
• Results
• Expression of both c-Myc and Gdnf reporters was
  dependent on the expression of Six1.

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Function of Eya

• Wanted to determine the roles of Eya, Dach in
  Six1mediated proliferation events.
• Co-transfection analysis using Six1-responsive
  myogenin promoter was performed.
• Reporter gene expression was repressed by Six1 in
  heterologous 293 cells.
• Eya3 reversed repression and showed a small
  amount of activation above baseline levels.
• Six1 and Eya3 interact to activate genes
  downstream.

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Figure 7. Reversal of Six1 repression by
Eya3. Source Nature. Vol. 426. November 20, 2003
33
Tested Enzymatic Activity

• Aspartate mutation eliminates phosphatase
  activity.
• Eya1 and Eya3 holoproteins displayed phosphatase
  activity.
• Mutated the first aspartate to alanine in the
  catalytic motif.
• Enzymatic activity was lost.

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Eya3 and Gene Transcription

• Needed to find out if aforementioned enzymatic
  activity was a requirement for the effects of
  Eya3 on gene transcription.
• Used single cell microinjection assay.
• To assess if Eya3 could overcome the inhibitory
  actions of Gal4-Six1 fusion protein on a
  UAS-dependent reporter.

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Eya3 and Gene Transcription

• Wild type Eya3 completely reversed effects of
  Gal4-Six1.
• Mutant Eya3 did not reverse the Gal4-Six1.
• Six1 repressed a reporter driven by
  Six-responsive elements.
• This was also reversed by wild type Eya3 and not
  mutant Eya3.

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Is Eya Phosphatase Activity a Requirement for
Cell Proliferation?

• Effect of phosphatase activity, using anti-Eya3
  IgG or Eya3 siRNA, was evaluated using C2C12
  cells as a model.
• Anti-Eya3 IgG and Eya3 siRNA both inhibited cell
  proliferation in C2C12 cells as assessed by Brdu
  incorporation.
• Co-injection of Eya3 wild-type protein completely
  negated the effects of anti-Eya3 IgG and Eya3
  siRNA.
• However, Eya3 mutant protein failed to rescue.

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Conclusion

• Therefore, Eya phosphatase activity is essential
  for regulating precursor cell proliferation.

38
Interaction Between Six1 and Dach1

• Use two-hybrid assay with myogenin Six1-regulated
  reporter.
• Found Six1-dependent repression was enhanced when
  expressed with Dach1.
• However, VP16-Dach1 acted as a strong activator.
• Therefore, Six1 and Dach interact with one
  another.

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Figure 11. Interaction between Dach1 and VP16 as
found in the two-hybrid assay. Source Nature.
Vol. 426. November 20, 2003
40
What is the Role of Eya in Six-Dach Mediated
Repression?

• Used single nuclear microinjection.
• Gal4-Dach fusion protein and Six1 inhibited the
  UAS-tk reporter gene expression.
• Again wild-type Eya3 was able to rescue this
  repression but the mutant Eya was not able to do
  so.
• Suggesting that Eya phosphatase function is
  required to reverse Dach-Six-mediated repression.

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Conclusion

• Therefore in order to activate the repressed
  Six-Dach complex, Eya is required.

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Role of Dach1 in C2C12 cells

• Nuclear microinjection of anti-Dach1 IgG and
  Dach1 siRNA were used.
• In both cases it was found that Dach1 is required
  for effective serum-stimulated proliferation.
• Question
• Are both Dach and Eya required for Six1-mediated
  gene activation?

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Figure 12. Dach1 requirement in serum stimulated
proliferation. Source Nature. Vol. 426.
November 20, 2003
44
Dach and Eya requirement in Six1-mediated gene
activation

• IgGs that recognized both Eya1 and Eya3 or Dach1
  and Dach2 were used (ChIP assay).
• It was found that Six1 along with Eya1/3 and
  Dach1/2 were present on both c-Myc and Gdnf
  regulatory regions.
• Role of Dach in gene activation was also tested
  using single-cell nuclear microinjection assays
  in C2C12 cells.
• It was found that a specific Dach1 siRNA
  inhibited reporter gene expression driven by
  Six1-responsive elements comparable to that
  observed with anti-Six1 IgG.
• Indication of Dach2 requirement for activation of
  Six1 target genes.

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Figure 13. Dach role in gene activation in C2C12
cells. Source Nature. Vol. 426. November 20,
2003
46
Six1 and Eya1 Synergistically Regulate
Organogenesis

• Evaluate the interaction between Six1 and Eya1.
• Eya1-/- is best known mutant mice model.
• Phenotype is similar to Six1-/- mice in regards
  to renal and otic development.
• Therefore crossed Six1/- and Eya1/- double
  heterozygous mice.
• Produced Eya1-/- Six1-/- mice.

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Discoveries

• Kidney development impaired in double
  heterogygotes.
• Impairment not found in single heterzygotes for
  either gene deletion separately.
• Therefore Six1 and Eya1 may function in the same
  genetic pathway.
• Six1-/- Eya1-/- double gene deleted mice had no
  hypaxial muscle and minimal epaxial muscle.

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Figure 14. Double heterozygote mice
(Six/-Eya/-) showing kidney hypoplasia and
unilateral ablation (asterisk). Source Nature.
Vol. 426. November 20, 2003
49
Figure 15. Staining of hypaxial muscle, expaxial
muscle and the pituitary. Source Nature. Vol.
426. November 20, 2003
50
Discoveries

• This phenotype is found in Myog-/-, MyoD-/-,
  Myf5-/- and Pax3-/- Myf5-/- mutants.
• Six1 and Eya1 do not affect pituitary development
  unless both genes are absent in which the
  pituitary has a volume that is 5-10 time less
  than the wildtype pituitary.
• However, also demonstrates that Six and Eya
  interact during vertebrate organogenesis.

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Discussion

• There are multiple DNA-binding transcription
  factors that are tissue specific.
• As well, there are many co-activator and
  repressor complexes which act together to
  regulate transcription in a tissue and promoter
  specific fashion.
• Six, Eya and Dach have a genetic relationship
  with one another.

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Discussion

• Six has the ability to function both to activate
  or repress depending on the co-factors present
• Dach co-represses
• Eya co-activates
• Eya has phosphatase activity.
• Therefore, it can alleviate the effects of the
  Dach co-repressor inducing it to act as a
  required activator to recruit other co-activators.

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Discussion

• Six-Eya-Dach network is a molecular mechanism in
  which a recruited co-activator with phosphatase
  function can serve to activate target genes.
• Many phosphates act to regulate cellular
  processes.
• However, they may also act as a promoter specific
  transcriptional co-activator.

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Discussion

• The above experiments and results aid in the
  understanding of the Six-Eya-Dach genetic
  interactions.
• These may be further applied to other pathways
  involved in co-activator or co-repressor
  activities during mammalian organogenesis.

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Summary

• Three genetically interacting proteins
• Sine oculis (Six)- DNA binding homeodomain factor
• Eyes absent (Eya)- nuclear cofactor
• Dachshund (Dach)- nuclear cofactor
• Eya serves as co-activator of Six in the
  regulation of downstream genes.
• Mice null for Six1 have cell proliferation and
  survival impairments in all organs expressing
  Six1.
• Six1 and Dach interact enhancing repression of
  genes downstream.

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Summary

• Eya phosphatase activity is required for cell
  proliferation and releases repression of the Six1
  and Dach interaction.
• Six1 and Eya1 may function in the same pathway.
• Six1-/- Eya1-/- double gene deleted mice have no
  hypaxial muscle, minimal epaxial muscle.
• Development of the kidney is impaired in double
  heterozygotes.
• Six-Eya-Dach network is a molecular mechanism
  that acts both to repress and activate genes
  downstream that are essential to the development
  of various tissues during mammalian
  organogenesis.