טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentIsraitel Elena
SubjectDelineating Perturbation(s) of Differentiated Cell Identity
During Aging in the Drosophila Midgut
DepartmentDepartment of Medicine
Supervisor Professor Amir )Oryan )Orian


Abstract

Aging is characterized by a progressive functional decline at the molecular, cellular, tissue, and organismal levels. Cell identity is only loosely maintained during aging and is associated with the emergence of age-related diseases including cancer, neurodegeneration, cardiovascular disease, and increased susceptibility to infection. However, our knowledge regarding mechanisms that supervise the differentiated cell identity remain rudimentary.

Drosophila is a powerful model for aging and cell identity studies, including age-associated changes. Drosophila is characterized by a relatively short lifespan and is excellent for genetic manipulation. Importantly, the molecular mechanisms and genes that regulate cell identity and longevity in the fly are highly similar to that of humans. A specific tissue of interest is the midgut and its differentiated cell; the enterocyte (EC). Mechanisms that regulate and maintain ECs identity in the context of aging are unknown. We previously performed a transgenic RNA-i screen and identified the isopeptidase Non-stop (Not/dUSP22) as a key identity supervisor. Not is a homolog of the human deubiquitinating enzyme, ubiquitin-specific protease 22 (USP22) that was originally identified as part of the SAGA complex. However, EC-specific RNA-i knockdown of the SAGA complex members such as Sgf11or the GCN5 had no observed phenotype. This suggested that Not acts independently of the SAGA complex to maintain EC identity.

The broad aim of my study is to delineate the changes in cell identity of the Drosophila adult midgut ECs during aging by focusing on the isopeptidase Not as a key identity regulator.  In this study and via proteomic and genetic approaches I discovered a novel Not-related protein complex termed - Not identity complex (NIC) - that maintains EC identity. The complex contains known members of a boundary complex; Cp190, Eny2, Nup98, and Mdg4. In this study, I determined the physiological roles of Not that are relevant for ECs identity in the context of aging and elucidated its function as part of a NIC complex.

Using EC-specific knockdown of different subunits of the complex, I discovered a protective role of NIC in differentiated ECs that is highly relevant for aging. The loss of each of NIC subunits compromised EC identity. I found that loss of Not in ECs affects the levels and localization of NIC proteins. I determined that one of the NIC subunits that directly interacts with Not is the insulator protein Cp190. I performed RNA-sequencing to delineate Cp190 regulated gene targets in EC. Moreover, I found that NIC subunits maintain EC identity by regulating the large-scale nuclear organization and gene expression. Additionally, the expression of Not in aging EC protects NIC proteins from degradation and suppresses aging phenotypes. 

In conclusion, in ECs Not is part of the NIC that supervises EC identity by promoting a nuclear organization that is unique to ECs, establishing and maintaining chromatin environment that protects from premature aging.