Transcription Factor Activity Profiling Reveals New Insights Into Megakaryocyte and Erythrocyte Differentiation

Transcription 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.

 

References

1          Doré, L. C. & Crispino, J. D. Transcription factor networks in erythroid cell and megakaryocyte development. Blood 118, 231-239, doi:10.1182/blood-2011-04-285981 (2011).

2          Bellis, A. D. et al. Cellular arrays for large-scale analysis of transcription factor activity. Biotechnology and Bioengineering 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 Journal of Haematology 135, 554-566, doi:10.1111/j.1365-2141.2006.06341.x (2006).