Systems-level understanding of signaling regulation on the cell fate decision between proliferation and differentiation [electronic resource]

Differentiation versus proliferation decisions have to be tightly regulated since excessive proliferation or premature differentiation can be harmful to multicellular organisms. While both growth factor-activated PI3K/AKT and Ras/ERK signaling have been shown to play crucial roles in proliferation and differentiation, it is not understood if and how these two pathways work in concert to make balanced cell fate decisions. Here, in the first part of my thesis, using single cell analysis of nerve growth factor (NGF)-induced PC12 cell differentiation, we uncovered a two dimensional (2D) phospho-ERK (pERK), phospho-AKT (pAKT) signaling code that reveals a sharp boundary that specifies cell fate. The boundary position remains invariant when different stimuli are used or upstream signaling components perturbed. In addition, we found that a marked cell-to-cell signal variation broadens the signal distribution in the pERK and pAKT plane so that the population spreads across the boundary, inhabiting both the differentiation and proliferation regions and creating two subpopulations with defined outcomes. We further screened for regulators of cell fate with a rat signaling siRNA library, and identified the RasGAP Rasa2, which actively maintains the stochastically distributed pERK-pAKT signals close to the decision boundary. We show that Rasa2 RasGAP activity is regulated by PI3K and acts as a negative feedback regulator between the Ras and PI3K pathways. Finally, we demonstrate that Rasa2-dependent positioning at the boundary allows for uniform NGF stimulation to create a subpopulation of cells that differentiates with each cycle of proliferation. Thus, by linking a complex signaling system to a simpler intermediate response map, cells gain unique integration and control capabilities to balance cell number expansion with differentiation. In the second part of the thesis, we further studied how proliferation decisions are made in G1 phase of the cell cycle. We focused on the role of dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (Dyrk1a), the strongest hit identified from our siRNA screen which markedly promotes proliferation when knocked down. Combining long-term time-lapse microscopy, single-cell image analysis and biochemical experiments, we found that Dyrk1a exhibits a sensitive dosage-dependent effect on G1 duration through direct regulation of cyclin D1stability. Knockdown of Dyrk1a greatly increases cyclin D1 and shortens G1 duration. Surprisingly, it also creates a second distinct population with cells exiting the cell cycle. This bifurcation is due to the upregulation of p21 at high cyclin D1 levels. This led to an unexpected finding that there exists a two-dimensional p21-cyclin D1 response map that encodes G1 cell fates. We further show that different doses of Dyrk1a function to shift the relative balance between cyclin D1 and p21, thereby allowing an sensitive control of G1 cell fates. The Dyrk1a gene is localized at chromosome 21, which has three copies in patients with Down syndrome (DS). Therefore, we extended our observation to the study of the cell cycle in DS-derived fibroblasts. We showed that with a 1.5-fold increase in Dyrk1a, DS cells have extended G1 duration, reduced proliferation and lower cyclin D1-to-p21 ratio relative to normal cells. Strikingly, all of these phenotypes could be partially rescued by inhibiting Dyrk1a kinase activity or knockdown of Dyrk1a. Our observation likely explains the reduced risk of solid tumors in adults with Down syndrome and provides potential therapeutic insight in terms of treating the DS-associated neurogenesis and proliferation defects during development. Altogether, this thesis has established two important concepts for cell fate decisions. First, single-cell fate could be more accurately predicted by considering two dimensional inputs, emphasizing the existence of hubs that function as information integration points where critical decisions are made. Second, natural variations could be advantageous in maintaining population balance and provide cells with decision plasticity. We envision that these findings will point out new directions for future studies on cell fate decisions and lead to a greater understanding of stem cell regulation and cancer development.

Keywords: Chemical and Systems Biology.
Advisor: Meyer Tobias primary advisor. Thesis advisor
Chen James
Dolmetsch Ricardo E. advisor. Thesis advisor
Ferrell James Ellsworth advisor. Thesis advisor