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Characterization and
mutation of sumoylation
sites within ZBP-89
M. Bruce, M. Salmon and Z. Zehner
HHMI Presentations 2007
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!
Regulatory Elements for the Vimentin
Gene
STAT3
ASE
c-Jun/Fos
AP-1
ZBP89
ZBP
-89
ZBP
-89
PS
PS
PS
Sp1/
Sp3
GC#1
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
ZBP-89
Known Bifunctional Transcription Factor

transcriptional activator
p21
 transcriptional repressor
Vimentin
 Zinc Finger Motif binds DNA
Sumo
site 1
Acidic
Region
Sumo
site 2
Sumo sites
3&4
Zinc
Finger
Serine
Rich region
Propose: ZBP-89 functions as a repressor when one or more
of its four sumoylation sites are sumoylated
ZBP-89: As an Activator
ZBP-89 activates P21 by binding to it promoter
site and complexes to SP1
-Recruits p300, a histone
acetyltransferase
-Promotes acetylation
-Opens chromatin so genes
are transcribed
Bai and Merchant , JBC, 1999.
ZBP-89: As a repressor
Binds to Vimentin promoter
(3 known sites)
TSA
Recruits HDAC 1 to the
vimentin promoterRepresses vimentin gene
expression via Histone H3
and H4 de-acetylation
HDAC 1
ZBP-89
Vimentin proximal promoter
Wu, Zhang, Salmon, Zehner, Genes to Cell 2007.
Sp1
Sumoylation
A post-translational modification of proteins involving
the covalent attachment of SUMO (small ubiquitinrelated modifier) to proteins and affecting biological
processes
Four different sumoylation sites ΨKXD/E
Sites 1, 2, 4 are conserved human to rat
Site 3 not conserved TKKD vs. IKKD
Sumo
site 1
Acidic
Region
Sumo
site 2
K = Lysine: Site where
sumo peptide added
Sumo sites
3&4
Zinc
Finger
Serine
Rich region
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 ZBP89 promoting aspects as well as repressing
My Experiment
•PCR
•Transformations
•Plasmid Preps
•Sequencing
•Transfection of
Cells
•Assays
My Experiment
Is to Mutate Lys to Arg in sumoylation sites
Lys
gaggagacagtgaaaaatgatgaagagcag
||||||||||||..||||||||||||||||
GAGGAGACAGTGCGAAATGATGAAGAGCAG
Arg
tgtccctataagcgtaaagcaggaaattac
||||||||||||||| |.||||||||||||
TGTCCCTATAAGCGT-ACGCAGGAAATTAC
Agtactaaagtaaaagatgagtatatggttgcag
||||||||||||..||||||||||||||||||||
AGTACTAAAGTACGAGATGAGTATATGGTTGCAG
Site 1 mutation
Obtained Several
plasmid with this
mutation
 Site 2 potential
mutation ?
 Site 4 mutation
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
Results?????

Coming soon to a theatre near you…
WHO WILL COME OUT ON TOP?
VIMENTIN OR P21 ??