O-Glycosylation of
Transcription Factor BCL11b:
Truth or Myth?
Elizabeth Pendergrass
Dr. Theresa Filtz
Dept Of Pharmaceutical Sciences
College Of Pharmacy
Oregon State University
T-cell Leukemia
 T-cell Leukemia: Cancer of the white blood cells, or
T-cells.


Affects approximately 50,000 people per year
About 4,000 people die from it annually
 Recovery Rate: ~90% in children
 Relapse Rate: ~50% in adults
 Looking for potential drug
targets
“Leukemia - Chronic T-Cell Lymphocytic - Statistics.” Cancer.Net. N.p., n.d. Web. 20 Aug. 2015.
BCL11b: a haploinsufficient tumor suppressor
 Loss or mutation of BCL11b results in T-cell
leukemia
 BCL11b mutation is associated with ~15% of T-cell
leukemia cases
Bcl11b is upregulated at DN2 stage
DN1
Specification
DN2
Commitment
DN3

-selection
DN4
Thymus
B
m
DC
NK
BM
Progenitor
ISP
NKT
Treg
Bcl11b needed for proliferation of
immature myeloid T cell
progenitors
Bcl11b needed for progression
into CD4 or CD8 SP cells
DP
Positive
selection
CD4
CD4
CD8
CD8
CTL
iTregTh1 Th2 Th17
Periphery
TCR-dependent TCR-independent
BCL11b: Essential for Thymocyte Development
Bcl11b known to be modified by
phosphorylation and sumoylation
Scheme c/o Ellen Rothenberg, Cal Tech
Genetic Regulation
Transcription Factors – Control gene expression by
activating or inhibiting transcription of DNA sequences
Regulated by localization,
synthesis/degradation, and
post-translational
modifications.
Post Translational Modification
PTM
Major Targeted
Amino Acids
Modifies BCL11b
Phosphorylation
Ser, Thr, Tyr
Yes
Sumoylation
Lys
Yes
O-Glycosylation
Ser, Thr
?
Methods for Detecting O-Glycosylation
 Antibodies targeted to O-glycosylation residues
 Beta-N-Acetylhexosaminidase to remove
glycosylated residues
 Wheat germ agglutinin to precipitate glycosylated
proteins
 Mass Spectrometry to characterize location on
protein
PUGNAc
 Inhibits O-GlcNAc-β-N-acetylglucosaminidase
(the enzyme for deglycosylation) in the cells
 Hypothesis: If BCL11b is O-glycosylated, PUGNAc
treatment will cause a significant increase in
O-glycosylation signal.
 Can easily be transfected to
HEK293T Cells
Human Embryonic
Kidney Cells
Transformed
express BCL11b
 Can also easily be treated with
PUGNAc
Experimental Methods
 Cell Culture
 Immunoprecipitation
 Western Blot
http://www.activemotif.com/images/products/coip_flowchart_big.jpg
Changes in O-Glycosylation caused by PugNAc
In BCL11b-Transfected HEK cells
HEK Cells Treated with PUGNAc
IB: BCL11b
IB:O-Glycosylation
Overlay
400
Ratio of O-Gly:Total Protein
130 kDa
350
300
250
200
150
100
50
0
0 min
15 min
30 min
Treatment Duration
45 min
Beta-N-acetylhexosaminidase
 An enzyme that removes an O-linked oligosaccharide
from a glycoprotein
 Designing a negative control for the O-glycosylation
antibodies
Beta-N-acetylhexosaminidase
HEK 203T cells Treated with
Beta-N-acetylhexosaminidase over time
+
IB: BCL11b
+
+
+
IB: O-Glycosylation
+
+
Ratio of Glycosylation to Total BCL11b
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Beta0
Overlay
Beta5-
Beta5+
Beta10Beta10+
Treatment Type (min)
Beta30-
Beta30+
Mouse
Thymocytes
Why are they a
better model?
Natively express BCL11b
Phosphorylation and sumoylation
change dynamically in thymocytes upon
stimulation
Is BCL11b glycosylated?
Does it also change?
P/A
(Phorbal Ester and A23187)
 Mimics T-cell receptor activated signaling and
phosphorylation cascades


MAPK pathway stimulated by Phorbol ester
Ca2+ pathway stimulated by A23187
Image from Walter Vogel;
Data from JBC vol 287:26971
Changes in Glycosylation (P/A)
On BCL11b in Thymocytes
Thymocytes Stimulated with P/A
IB:BCL11b
IB:O-Glycosylation
Ratio of Glycosylation to Total BCL11b
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
no drug
Overlay
-Non-specific binding of the antibody
7 min
30 min
60 min
Treatment Type and Duration
3 hr
Wheat Germ Agglutinin (WGA)
 Binds to N-acetyl-D-glucosamine
 Procedure is identical to the immunoprecipitation
described previously
 Pulls down all glycosylated proteins
 Gel is then scanned for specific proteins
 AKT1 is used as a positive control, as it is known to
be glycosylated in mouse thymocytes
Wheat Germ Agglutinin Blot
130 kDa
Red = IB:BCL11b
Green = IB:O-Gly
72 kDa
Green = IB:AKT1
Result: WGA did not bind BCL11b
AKT1 was present in the WGA lane
Conclusion: BCL11b is not directly glycosylated
Overlay*
Thymocytes Scanned for Phosphorylation
130 kDa
30 min
Treatments
Result: Phosphorylation levels of BCL11b decreased when PUGNAc was added
Conclusion: Glycosylation levels affect the activity of BCL11b, but not directly.
Changes in Phosphorylation caused by PUGNAc
In BCL11b-Transfected HEK293T cells
IB: BCL11b Signal
Overlay
Ratio of Phosphorylation to Total BCL11b
Phosphorylation Signal
Phosphorylation of HEK cells
Treated with PUGNAc
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Series1
0 min
0.338500714
20 min
0.302533413
45 min
0.437811187
Next Up:
 Determining the effects
of glycosylation on other
proteins in the NuRD
complex and along the
MAPK and Ca2+
dependent pathways.
Special Thanks
Funding Sources
 College of Pharmacy
 URISC
In-Lab Assistance
 Dr. Theresa Filtz
 Wisam Selman
 Heather Wisner
 Xuemei Wan
 Dr. Chrissa Kioussi
 Arun Singh
 Vera Chang
 Elahe Esfandiari