ESR1: “Uncovering functional regulations of the SMC5/6 complex implicated in human disease”

IFOM (D. Branzei group): Uncovering the chromosome dynamics functions of the SMC5/6 complex implicated in human disease

i. Objective of research: To identify SMC5/6 functions in chromosome dynamics that promote genome stability

ii. Current state of the art: The Structural Maintenance of Chromosome (SMC) family of proteins, involving cohesin, condensin, and SMC5/6, are critical regulators of genome stability. Structurally, these complexes form molecular rings that can entrap and promote higher-order organization of the genomic DNA. The SMC5/6 complex ring, whose function is the least understood, is composed of two coiled-coil SMC heterodimers (SMC5/SMC6), which associate with a kleisin component (Nse4/NSMCE4) and one or more peripheral subunits (NSMCE1-NSMCE3 heterodimers interacting with NSMCE4 and SMC6, and NSMCE2 interacting with SMC5). SMC5/6 is critical for DNA repair and for supporting replication of difficult to replicate regions in the genome. Mutations in SMC5/6 components predispose to various cancers and result in debilitating diseases associated with severe developmental defects (NSMCE2-associated syndrome featuring primordial dwarfism and deregulation of glucose metabolism), or increased chromosome breakage and defective T and B cell function (NSMCE3-associated disorder also known as LICS). At the cellular level, mutations in SMC5/6 components cause impaired DNA repair, accumulation of unresolved topological and recombination structures, and chromosome segregation defects. It is likely that SMC5/6 is essential for the architectural organization of interphase DNA in coordination with cohesin, and these functions affect genome stability and DNA damage response.

iii. Research methodology and approach: Using in vivo genetics, imaging techniques and genomic approaches, we will identify the roles of the SMC5/6 complex in interphase and chromatin structure organization and its effects on DNA damage response and genome stability. We will use tools recently established in the lab, including an AID degron-based inducible proteolysis system of SMC5 in human TK6 cells and DT40 cells, and establish other necessary tools with mutations in cohesin, SMC5/6 and regulators. We will use Hi-C and NET-CAGE techniques to detect alterations in topologically associated domains (TADs) and map promoter-enhancer interactions affected by loss of SMC5, in spontaneous and topologically stressed conditions, comparing these changes with the ones induced by loss of cohesin, a main organizer of interphase chromatin in loops. These studies will be combined with BLISS studies of DSB mapping and analysis of transcriptional changes induced by SMC5 loss, for which preliminary data point to a function of SMC5/6 at specific structural elements and in regulating metabolic pathways responding to replication stress. We will further examine the hypothesis that SMC5/6 and cohesin are co-regulated by acetylation, and the effects of such deregulation on DNA damage response and chromosome structure.

iv. Originality and innovative aspects of the ESR project: The project will identify functions of SMC5/6 in interphase chromatin organization and its effects on genome stability and DNA damage response. The insights may allow development of rationalized intervention strategies for SMC5/6-related disorders and identify vulnerabilities in SMC5/6-mutated cancers.

v. Integration of the ESR project to the overall research programme: Our ESR will collaborate with the Crosetto group in mapping the DSBs arising upon SMC5 depletion in human cells, with the Mailand group on the effects on DDR associated with changes in interphase chromatin structure, and with Norgenotech and LXRepair on the development of novel DNA repair assays for DSB detection.