3 rd Annual Co-op Showcase

Cyclin E-CDK2 phosphorylates E2F4 to promote E2F1

binding to E2F promoter elements and to initiate transcription
of cell cycle genes
Rachael Sverdlove, Amy E. Schade, James A. DeCaprio (MD)
 Acknowledgments
3 rd Annual Co-op Showcase
I would like to thank the entire DeCaprio Lab at DFCI for their help and support:
Amy Schade, PhD Candidate
Mina Milad, CURE Student
Timothy Branigan, PhD Candidate
Roxana Tarnita, PhD Candidate
Esther Park, PhD Candidate
Camille Cushman, PhD Candidate
Sarah Clark, PhD Student
Brianna Lowey, PhD Student
Mona Ahmed, PhD Candidate
Tom Frost, PhD Student
Jason Nomburg, PhD Student
Jingwei Cheng, PhD
James A. DeCaprio, MD
This CaNCURE co-op research project was supported by National Cancer Institute grant #1CA174650-02.
Further gratitude is extended to Dr. Van De Ven (PhD) and Dr. Sridhar (PhD) for mentoring me and providing
This CaNCURE co-op research project was supported by National
Cancer Institute grant #1CA174650-02.
assistance.
 Introduction
3 rd Annual Co-op Showcase
Cell cycle gene expression

DREAM RB-DP1-E2F1
S-phase genes
DP1
p130 DP1 RB
MuvB
E2F promoter E2F promoter
elements elements

G0 G1 S
Cell cycle gene expression

P
P RB P
P
DP1
P
p130 P
S-phase genes
MuvB
DP1

E2F promoter E2F promoter

elements elements

G1 S
 Introduction
3 rd Annual Co-op Showcase

G0 Early G1 Late G1

DP1 DP1
p130
DREAM
MuvB

E2F promoter E2F promoter E2F promoter

elements elements elements

RB-DP1-E2F1 DP1
DP1 DP1
RB

E2F promoter E2F promoter E2F promoter

elements elements elements
 Introduction: Dynlacht et al
3 rd Annual Co-op Showcase

Cyclin A-
CDK2 P

E2F E2F E2F

promoter promoter promoter
elements elements elements

5’ 3’ 5’ 3’ 5’ 3’

• To find an S/T P phosphorylation site on E2F4, we used BLASTp and compared this protein
to E2F1.
• 44% identical
• 2 matching SP sites at bases: 384 E2F4 / 402 E2F1, 384 E2F4 / 332 E2F1
 3 rd Annual Co-op Showcase

Leading Question: Does RB-DP1-E2F1 promote DREAM disruption

at E2F promoter elements?

Hypothesis: Cyclin E-CDK2 phosphorylates E2F4 to promote

E2F1 out-competition for binding to E2F promoters to enable cell
cycle gene expression
Cyclin E-
CDK2 P

E2F promoter E2F promoter

elements elements
 Big Picture
3 rd Annual Co-op Showcase

Binding Affinity Assay: helps us infer the tendency of the E2F1/ E2F4 to bind to E2F
promoters.
Streptavidin
Protein Protein
Biotin DP1 Biotin
of DP1 of Biotin
interest interest
E2F
E2F E2F
promoter
promoter promoter
elements
elements elements
5’ 3’ 5’ 3’ 5’ 3’

This process will further be used to determine: Immunoblot for either E2F4, E2F1
1) if E2F1 and E2F4 compete for E2F promoters
2) if E2F4 pretreatment with Cyclin E-CDK2 changes its
binding affinity