Preliminary data

We here present some preliminary results that show how cell cycle influences the expression distribution of the miRNA target.

We first show how different phases of the cell cycle can be discriminated by using the DNA marker. The distribution across the entire cell population of the fluorescence intensity (arbitrary units) in the Hoechst channel is reported in figure 6.2. Cells in phase G2 have approximately twice the amount of DNA of cells in phase G0/G1. Looking at the distribution of the Hoechst, we can indeed notice two peaks, one located at a value of intensity that is approximately two times bigger than the one of the other peak. These two maxima correspond to cells in phase G0/G1 and G2, while the region between them is populated by cells in phase S. The difference in the relative abundance of cells in the three regions is due to the different amount of time that the cells spend in each phase of the cycle. By sorting the cells according to their fluorescence intensity in the Hoechst channel, we can select sub-populations belonging to the desired phase. We will use this technique to study the effect of the variability induced by cell cycle in the regulation of the exogenous target of miR-20a.

As an additional observation, we mention that a rough distinction between cells in phase G0/G1 and cells in phase G2 can be obtained by discriminating them according to their size. Indeed, selecting cells with small sizes is approximately equivalent to select cells in G0/G1, while selecting bigger cells, is approximately equivalent to select cells in G2, see figure 6.3.

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Fig. 6.2 Hoechst DNA stain to identify the cell cycle phases.

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Fig. 6.4 No miRNA binding sites. Scatter plots of the fluorescence intensity in the eYFP and mChery channels, each dot is a cell. Cells are sorted according to their phase of the cycle.

The first experimental observation that can be made on the basis of our pre- liminary data, is that the cell cycle does not influence the transcriptional activity of the plasmid. The scatter plots in figure 6.4, where each dot is a cell, are the result of fluorescence measurements performed on cells transfected with the plasmid containing no miRNA binding sites on the mCherry gene. The y-axis indicates the fluorescence of the target mCherry, while the x-axis indicates the one of the control eYFP, proxy for the transcriptional activity of the construct. The left-most plot in figure is obtained from the entire population, while the others are the result of sub-populations homogeneous with respect to the phase of the cell cycle. As can be seen, there is a linear dependence between mCherry and eYFP. This is expected, since the miRNA does not regulate mCherry in this construct. In addition to that, we see that this linear dependence is the same, regardless of the phase of the cell cycle considered. This fact indicates that the expression of the construct is independent from the cell cycle. As a consequence, in case of mCherry regulated by the miRNA, differences among the phases should be attributed to the variability of the miRNA expression along the cell cycle.

The scatter plots corresponding to the experiments in which the transfected construct has 1, 4 or 7 miRNA binding sites on the mCherry gene, are shown in figure 6.5. From these results we observe that the expression of miR-20a changes with the cell cycle progression. For a given transcriptional activity (i.e. a given eYFP level) the intensity of mCherry changes both depending on the miRNA repression strenght (i.e. the number of miRNA binding sites present on the construct) and on the cell cycle phase. Since we showed that the transfected plasmids do not change expression with the cell cycle progression, the variability in mCherry expression

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Fig. 6.5 1, 4 and 7 miRNA binding sites. Scatter plots of the fluorescence intensity in the eYFP and mChery channels, each dot is a cell. Cells are sorted according to their phase of the cycle.

observed for the different cell cycle phases is the fingerprint of a variation in the miR-20a expression.

As can be seen in the scatter plots of the entire population, the regulation operated by the miRNA can induce bimodal expression distributions of the target, for a wide range of transcriptional activity. As expected, this effect is more evident in case of higher repression strength, i.e. 4 or 7 binding sites. In these conditions we can discriminate a repressed peak, whose mean value seems to follow a threshold-like behavior as a function of the transcriptional activity, and an unrepressed peak, whose mean value is essentially linear as a function of the transcriptional activity. The cell cycle influences the shape of the expression distributions. Indeed, in the scatter plots corresponding to sub-populations homogeneous with respect to the phase of the

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cycle, the distribution is clearly different from the one of the whole population. In particular, we see that by sorting according to the phase of the cycle, we can roughly select cells belonging mostly to one of the peaks of the population distribution. This seems more evident for cells in phase G0/G1.

The distribution observed in the sub-populations, could be the result of the noise reduction due to the homogeneity with respect to the phase of the cell cycle. Our model predicts the shrinking of the range of bimodality as extrinsic noise is decreased, eventually leading to the disappearance of the bimodal shape. Nonetheless, these results require further analysis and some open questions still remain. In the following section we will discuss these aspects.