Functional studies of the regulatory protein Ki-1/57 in cells and mice

Author(s)

Ângela Saito

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2015-08-13

Examining board members

Jörg Kobarg; Deborah Schechtman; Alisson Campos Cardoso; Claudio Chrysostomo Werneck; Juliana Helena Costa Smetana

• Advisor: José Xavier Neto; Jörg Kobarg

Abstract

The Ki-1/57 protein was discovered by cross-reactivity of the monoclonal antibody Ki-1 in cells of Hodgkin¿s lymphoma. The interaction of Ki-1/57 with proteins that participate in transcription, RNA processing and stability, and translation suggested its involvement in regulatory mechanisms of gene expression. A paralog protein, CGI-55, has high degree of sequence similarity with Ki-1/57 and was initially identified as a protein that regulates mRNA stability. Ki-1/57 and CGI-55 have some mutual interaction partners, they are methylated by PRMT1 and can locate in small nuclear and cytoplasmic structures under the action of different stimuli. In this study, functional assays were conducted in human cells for the better understanding of the Ki-1/57 and CGI-55 function to the regulation of gene expression and in the mechanisms of cellular stress response. Also, a knockout lineage for Ki-1/57 using the CRISPR/Cas9 system was generated and Ki-1/57 expression studies in mouse tissues were performed. Through global gene expression analysis on DNA microarray, it was observed a predominantly repressive role of Ki-1/57 and CGI-55 after the overexpression of these proteins in human cells. Many of the altered genes are related to proliferation pathways, apoptosis and cell cycle control, suggesting a possible functional relationship of both proteins with cellular stress response mechanisms. Overexpression of Ki-1/57 and CGI-55 in cells caused a reduction in proliferation, possibly due to an arrest in the G1 phase of the cell cycle. Moreover, when K-1/57 was overexpressed, there was a protective effect against apoptosis after treatment with the endoplasmic reticulum stress inductor, thapsigargin. In addition, it was observed that Ki-1/57 and CGI-55 are SUMOylated, wherein the modification of Ki-1/57 by SUMO was important for its right activity in the splicing site selection of the E1A gene. Immunofluorescence assays showed that overexpression of Ki-1/57 in HeLa cells affect the distribution of PML-NBs, an important subnuclear structure formed in response to a variety of stresses and functionally related SUMOylation and to transcriptional regulation. Also in this work, a knockout mice lineage for Ki-1/57 was generated through the CRISPR/Cas9 system. A plasmid, containing the information for expression of the hSpCas9 endonuclease and a guide RNA to the exon one of Habp4 gene, was injected into the pro-nucleus of mouse embryo and mosaic animals were generated. These animals were crossed with wild-type mice to segregate the mutant alleles and create the heterozygous lines. The intercrossing between two heterozygous generated viable and fertile homozygous knockouts. Luciferase reporter gene assays activated by the stress-responsive transcription factor MEF2C were performed in cell culture and initial characterization of mutant animals by qRT-PCR showed the potential role of Ki-1/57 on the MEF2C transcriptional activity modulation. Therefore, these findings together point out to the role of Ki-1/57 on the regulation of gene expression and cellular stress response mechanisms. The knockout mice for Ki-1/57, developed in this study, will contribute for future studies on its role at different stimuli and stress situations (AU)

• FAPESP's process: 10/15760-2 - Functional characterization of the human regulatory protein Ki-1/57: cellular and in vivo model studies
• Grantee: Ângela Saito
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)