Characterization and mutation of sumoylation sites within ZBP-89

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Characterization and mutation of sumoylation
sites within ZBP-89

• M. Bruce, M. Salmon and Z. Zehner
• HHMI Presentations 2007

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Vimentin
An intermediate filament protein Expressed early
in development and turned off in the
differentiation of many cell types such as muscle
neural cells Found expressed in most
metastatic tumor cells!
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Regulatory Elements for the Vimentin Gene
STAT3
Sp1/Sp3
c-Jun/Fos
ZBP -89
ZBP-89
ZBP -89
ASE AP-1 PS
PS PS GC1
RESULT GC-box 1 binds Sp1 is required to
activate vimentin gene expression. ZBP-89
binds the PS elements and represses vimentin
expression. STAT3 and c-Jun bind upstream
elements and overrides ZBP-89 repression

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ZBP-89

• Known Bifunctional Transcription Factor
• transcriptional activator p21
• transcriptional repressor Vimentin
• Zinc Finger Motif binds DNA
• Propose ZBP-89 functions as a repressor when
  one or more of its four sumoylation sites are
  sumoylated

Sumo site 1
Sumo site 2
Sumo sites 3 4
Acidic Region
Zinc Finger
Serine Rich region
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ZBP-89 As an Activator

• ZBP-89 activates P21 by binding to it promoter
  site and complexes to SP1

-Recruits p300, a histone acetyltransferase -Pr
omotes acetylation -Opens chromatin so genes
are transcribed
Bai and Merchant , JBC, 1999.
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ZBP-89 As a repressor
Binds to Vimentin promoter (3 known
sites) Recruits HDAC 1 to the vimentin promoter-
Represses vimentin gene expression via Histone
H3 and H4 de-acetylation
TSA
HDAC 1
ZBP-89
Sp1
Vimentin proximal promoter
Wu, Zhang, Salmon, Zehner, Genes to Cell 2007.
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Sumoylation

• A post-translational modification of proteins
  involving the covalent attachment of SUMO (small
  ubiquitin-related modifier) to proteins and
  affecting biological processes
• Four different sumoylation sites ?KXD/E
• Sites 1, 2, 4 are conserved human to rat
• Site 3 not conserved TKKD vs. IKKD

K Lysine Site where sumo peptide added
Sumo site 1
Sumo site 2
Sumo sites 3 4
Acidic Region
Zinc Finger
Serine Rich region
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My Experiment

• Mutate the Sumoylation sites in ZBP-89
• This wipes out sumoylation sites allowing us to
  determine if this changes the function of ZBP-89
• Express ZBP-89 with mutated sites in Drosophila
  S2 Cells lack ZBP-89
• Measure expression of p21 and Vimentin
• Want to see if wiping out sumoylation sites
  affects ZBP- 89 promoting aspects as well as
  repressing

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My Experiment

• PCR
• Transformations
• Plasmid Preps
• Sequencing
• Transfection of Cells
• Assays

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My Experiment
Is to Mutate Lys to Arg in sumoylation sites
Lys

• gaggagacagtgaaaaatgatgaagagcag
• ..
• GAGGAGACAGTGCGAAATGATGAAGAGCAG

• Site 1 mutation

Obtained Several plasmid with this mutation
Arg
tgtccctataagcgtaaagcaggaaattac
. TGTCCCTATAAGCGT-ACGCAGGAAATTAC
? Site 2 potential mutation ?
Agtactaaagtaaaagatgagtatatggttgcag ..
AGTACTAAAGTACGAGATGAGTATATGGTT
GCAG
? Site 4 mutation
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Transfections Assays

• Transient Calcium Phosphate Transfection
• Used S2 Drosophila Cells
• Why? Lack Sp1 and ZBP-89
• B-gal Assay
• Serves as an internal control for differences in
  transfection frequency
• CAT Assay
• -Vimentin promoter fused to CAT Chloramphenical
  AcetylTansferase
• -Purpose is to determine how sumoylation affects
  ZBP activity
• -Used to measure P21 and Vimentin expression

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Results?????

• Coming soon to a theatre near you

WHO WILL COME OUT ON TOP? VIMENTIN OR P21 ??