Funciones de las subunidades específicas de complejos Polycomb vPRC1, RYBP y YAF2, en el establecimiento y mantenimiento de estados inmortalizados leucémicos

Abstract

The products of the Polycomb group of genes are a heterogeneous set of proteins that assemble in a variety of multiprotein complexes acting as chromatin regulators. The best-known outcome of such an activity, the transcriptional repression of targets with functions in developmental and differentiation events, is critical to tissue homeostasis and their alteration in oncogenic processes. One of the two major groups of Polycomb complexes are the repressive type I complexes (PRC1), which are responsible for a specific monoubiquitylation at the C-terminal tail of the histone H2A. All PRC1 complexes share a core heterodimeric E3 ligase made of two RING finger proteins, of which one of them, the RING1A or RING1B paralogs, are always present. Of the non-core - accessory - subunits, the paralogs RYBP and YAF2 identify the subset of variant PRC1 complexes, endowed with the highest and pioneering activity modifying H2A. In general, the different subunits in multiprotein chromatin regulators contribute both general and specific functions to the global activity of the complexes. Despite their widespread presence in variant PRC1, little is known on the functions of RYBP and YAF2. Here, I have focused my study on their involvement in the initiation and maintenance of aberrant hematopoiesis as in acute myeloid leukemia (AML). I have used a genetic approach with murine hematopoietic progenitors in an ex vivo immortalization model as a proxy of AML. RYBP deletion and YAF2 down-regulation alone or combined show that, despite their similarity, it is YAF2, but not RYBP that is critical in the establishment of the transformed state. I also show that YAF2 acts stabilizing RING1A and RING1B protein levels and that the dosage of RING1B in particular is essential for the oncogenic activity of MLL1-fusion proteins such as those identified in leukemic patients. The acute contribution of YAF2 to hematopoietic immortalization, however, is less decisive in the establishment of pre-leukemic states. I also find that at high expression levels, RYBP can compensate for the absence of YAF2, and that the lower RING1 stabilization function is determined by its longer linker unique and disordered sequence that separates the N- and Cterminal domains conserved in YAF2. I also provide evidence for the importance of the direct contact between RYBP/YAF2 and RING1 paralogs as part of the mechanisms underlying protein stabilization. In the maintenance of the leukemic state, YAF2 again acts in an essential manner, although RING1 stabilization processes are of less importance compared to their function supporting cell survival. These results characterize functional differences between paralogs, illustrate functions little studied on the dynamics of multiprotein complexes and call for attention to epistasis in the interpretation of results from genetic analysis.