(247h) Transcription Factor Activity Profiling Reveals New Insights Into Megakaryocyte and Erythrocyte Differentiation

Duncan, M. T. - Presenter, Northwestern University
Shin, S., Northwestern University
Mays, Z., Northwestern University
Shea, L. D., Northwestern University
Miller, W. M., Northwestern University

factor activity profiling reveals new insights into megakaryocyte and
erythrocyte differentiation

Mark T. Duncan,1 Seungjin Shin,1 Zachary
Mays, Lonnie D. Shea,2 William M. Miller2

Department of Chemical and Biological Engineering,
Northwestern University, Evanston, IL, United States of America

1M.T.D. and S.S. contributed
equally to this work. 2Co-corresponding authors. 


The differentiation of
hematopoietic stem and progenitor cells (HSPCs) is a complex process that is
regulated by a multitude of transcription factors (TFs).  In particular, the
lineage-choice at the bifurcation between the megakaryocyte (MK) and erythrocyte
(E) lineages highly depends on the interactions within a network of TFs.1 
Post-lineage commitment, the maturation process of both red blood cells (RBCs)
and MKs continues to depend on activation of the appropriate TFs at the
appropriate time.  We applied our recently developed dynamic TF activity array
to study E/MK differentiation.2,3  The assay is based on the lentiviral
delivery of TF-specific binding elements that drive the expression of firefly
luciferase, which is subsequently monitored by daily bioluminescence imaging of
the same cells for 5-6 days.  In the initial studies, we assessed the dynamic
activity of a panel of 8 E/MK relevant TFs during hemin-mediated E and
PMA-mediated MK differentiation of the bipotent cell line K562.  We observed
numerous trends in TF activities that were consistent with results from the
literature.  In particular, we observed that PMA strongly induced the activity
of NF-E2 and FLI-1, both of which are required for terminal MK maturation,
while PMA repressed the activity of the myeloid-promoting TFs PU.1 and c-myb.  Consistent
with its known degradation in response to PMA, GATA-1 activity was also rapidly
reduced.  Conversely, during hemin-mediated E differentiation, we found that
NF-E2, GATA-2, and TAL1 activities were elevated.  To confirm the specificity
of the observed trends to the E/MK differentiation programs, we generated
GATA-1 silenced K562 cells, which we confirmed were unable to differentiate
towards either lineage.  Profiling the TF activities in these cells after hemin
or PMA treatment revealed that only activation of NF-E2 appeared to be
independent of either differentiation program.  This indicates that NF-E2
activation is not far downstream of the initial signaling events induced by the
addition of PMA or hemin.

We further utilized our assay to
explore terminal MK maturation and, in particular, polyploidization.  For that
purpose, we examined the effects of NIC, a potent enhancer of MK
polyploidization,4 on TF
activities in the MK-committed cell line CHRF-288-11.  Concomitant with
increasing ploidy, NIC greatly enhanced the PMA-mediated increases in activities
of NF-E2, FLI-1, and p53.  Moreover, a preliminary experiment in primary MKs
suggests that NIC does indeed significantly increase NF-E2 activity during
normal MK differentiation. 

Based on the results of these
studies, we conclude that dynamic profiling of TF activity is a novel, useful
tool that will complement more traditional, low-throughput assays (e.g. qPCR, microarray,
western blot, ChIP) in understanding stem and progenitor cell differentiation.  In
particular, the dynamic, non-invasive measurements of TF activity provide a rapid
means of identifying the critical TFs at different stages of the
differentiation process.  Additionally, the described technology can easily be
extended to simultaneously monitor the activity of a large numbers of TFs,
making it suitable for exploratory studies to identify TFs with previously
unknown involvement in the differentiation process.



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2          Bellis, A. D. et al. Cellular arrays for
large-scale analysis of transcription factor activity. Biotechnology and
108, 395-403, doi:10.1002/bit.22916 (2011).

3          Weiss, M. S. et al. Dynamic, Large-Scale Profiling
of Transcription Factor Activity from Live Cells in 3D Culture. PLoS ONE
5, e14026, doi:10.1371/journal.pone.0014026 (2010).

4          Giammona, L. M., Fuhrken, P. G., Papoutsakis, E. T.
& Miller, W. M. Nicotinamide (vitamin B3) increases the polyploidisation
and proplatelet formation of cultured primary human megakaryocytes. British
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