IDENTIFICATION OF THE MOLECULAR PATHWAYS LINKING H2A.Z (H2AZ.1 and H2AZ.2) TO CHROMOSOME SEGREGATION FIDELITY

Cell division is a fundamental process that is at the basis of our existence. From the single cell, about 37.2 trillion cells are generated to make up an adult human being. Divisions need to occur in an extremely accurate manner in order to produce daughter cells that are healthy and viable. Defects in cell division are at the basis of several human pathologies ranging from Down syndrome to cancer. Understanding how cell division occurs faithfully and how mistakes are avoided would lead us to better diagnostic tools and intervention opportunity to either prevent or cure some of these diseases. The key machinery for segregating the chromosomes during cell division is the centromere/kinetochore. This structure is composed of DNA and proteins that are directly linked to DNA and forms a special DNA structure at the centromere, which is essential for chromosome segregation. I recently identified some of these proteins that, when altered, compromise the fidelity of segregation. The project aims at identifying how these proteins work, how they contribute to maintain healthy cells for generations and what are the direct consequences when they mal-function. The outcome of this study would not only further our knowledge on the chromosome segregation machinery but also provide new possible targets for pharmacological interventions.

The centromere is key for the faithful segregation of chromosomes during cell division as it represents the only point where a mutli-protein complex called kinetochore assembles on mitotic chromosomes. Kinetochores bind the spindle microtubules and allow the equal distribution of sister chromatids. Understanding the establishment and maintenance of centromeric chromatin is fundamental to comprehend the assembly and function of the centromere and kinetochore structures. Centromeric chromatin is very specialized and contains a H3 histone variant called CENP-A that defines the position of the centromere. Failure of incorporating CENP-A into chromatin leads to kinetochore defects and major chromosome segregation errors.
We have discovered that one of the H2A.Z variant (H2A.Z.2 but not H2A.Z.1) is also a key regulator of this process. We revealed that the two variants that only differ by 3 amino acids have distinct functions in cell cycle regulation. Loss of H2A.Z.2 leads to increased chromosome instability and premature loss of sister chromatid cohesion; concomitantly, it also causes a reduction in CENP-A at the centromere. Here we will investigate how H2A.Z.2 influences centromeric function by identifying the protein complexes that are specifically recruited to chromatin by the H2A.Z variants (AIM1) and identifying the link between H2A.Z and CENP-A deposition/stability (AIM2).
To this purpose, we will use CRISR-Cas9-mediated cell engineering to modify the endogenous proteins either with a degron module (that will allow the quick degradation of the protein at a specific time), a purification tag (that will allow to conduct purification and mass spectrometry analyses) and a visualization tag (that will allow to monitor in vivo the dynamics, incorporation and turn over). Combining these cell lines with an additional tag for visualizing CENP-A, we will be able to monitor and investigate the link between H2A.Z dynamics and CENP-A maintenance at the centromere.