SISTEMAS INTERNOS DE CONTROL Y GESTIÓN DE RIESGOS EN RELACIÓN CON EL PROCESO DE

The large differences in PRC1 protein levels among hematopoietic cell types beg the question of how they might impact PRC1 function. Beyond the general observation that, in primary cells, levels of PRC1 subunits decreased to a different extent compared to those in primitive progenitors contained in the Lin- population, no clear pattern could be established. The exception, for canonical subunits, was the population of B-cell precursors (CD19+ cells). In particular, it was unexpected to determine the magnitude of the fall in RING1A and RING1B levels with differentiation. The lowest levels of canonical subunits in neutrophils may not need of an interpretation considering their short lifespan but only if PRC1 function is considered under the usual perspective of "memory maintenance". Indeed, most functions in differentiated cell types, unveiled through loss-of-function mutations, are associated to longer lived, lymphoid cells (Beguelin et al., 2016; Ikawa et al., 2016). A switch of CBX subunits, from CBX7 to CBX8 has been shown in primitive hematopoietic progenitors (Klauke et al., 2013) so that CBX7 expression in the most undifferentiated type is silenced to turn on that of its paralog CBX8. The process parallels that previously shown in ES cells and its differentiated descendents (Morey et al., 2012). Also, during the progress of this thesis, a systematic comparison between the contents of PcG proteins in ES cells and neural progenitors shows that the differences were reflected in the nature of the complexes formed (Kloet et al., 2016). Here, is worth insisting in that the differences in content reported refer

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only to individual subunits and that quantitatively are independent of each other, due to limitations in the methodology used.

Looking at the expression of mRNAs encoding these subunits (Fig. D1) it is plainly evident the lack of correlation with protein levels. The comparison shown in Fig. D1 includes only primary cells since we have no data for immortalized progenitors (de Graaf et al., 2016). As it has been mentioned above, protein levels, whether of canonical or non-canonical PRC1 subunits are lower in every cell type tested when compared with those in primitive, Lin- progenitors. And yet, mRNA levels in the two groups of progenitors that make that pool are not consistently higher than those measured in more differentiated cell types which, in contrast contain much reduced levels of protein. Taking more differentiated cell types the conclusion is similar even though in some cases the correlation between mRNA and protein levels may hold, at least in part.

Whichever regulatory pathway is involved we think it is affected by the proliferative status of the cell. Our comparison of spleen cells between the quiescent population and that of cells taken into proliferation by LPS, however, is affected by the very different conditions the cells are: in the first case they were simply taken from the mouse whereas, in the second, the cells had grown in enriched media under non-physiological dosages of oxygen. The latter conditions are bound to altermetabolic/energetic rates. However, the comparison of immortal progenitors growing in the same rich medium but differing extremely in their proliferative rates also sustains the conclusion that, on average, dividing cells tend to be equipped with higher levels of PRC1 subunits. Of note, among the primary cells studied, even if not actively dividing, the Lin- population enriched in PRC1 subunits contains the larger proportion of cells undergoing proliferation.

Lastly, it was surprising the realization of how the content of PRC1 subunits was altered by the depletion of RING1B, and how the deficiency in RING1A, instead, turned out to produce very mild effects. Thus, although the levels of most subunits in RING1B-deficient cells decreased/increased by half/double compared to control cells, in some cases variations were far more extreme with opposing alterations depending on the cell type.

The minor effects seen in RING1A-deficient cells could be argued that is the consequence of adaptations associated to the nature of the mutation: tissues depleted of RING1A throughout development where some adjustment may have taken place to ameliorate negative effects of the mutation.

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Compared to such a constitutive depletion, our conditional deletion of RING1B may appear more as an acute inactivation. However, our in vivo depletion of RING1B differs from an abrupt inactivation as that seen after 4'-OHT-induced depletion in cultured cells. The reason is that in our protocol, we waited for a substantial period, 8-10 weeks, of time pass after RING1B depletion, in order to ensure complete loss of RING1B in the longer-lived lymphoid compartment. Not having examined mRNA levels encoding PRC1 subunits in mutant cells we cannot elaborate on the nature of the mechanism involved. It is worth noting, however, that

Figure D1. mRNA expression of chosen Polycomb products in hematopoietic cells.(A) RING proteins and canonical subunits. (B) non-canonical subunits. MPP- multipotent progenitor, RPP - restricted potential progenitor

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whereas RING1A protein levels are unregulated in all cell types studied, other subunits showed changes towards accumulation or reduction. Therefore, a lineal relationship with transcriptional activity is difficult, more so considering the residence, in differentiated cells, of PRC1 complexes on both, silent and active loci (Frangini et al., 2013; Kloet et al., 2016; Loubiere et al., 2016; Morey et al., 2015; Schaaf et al., 2013).