Understanding The Molecular Mechanisms Of Transcriptional Repression Mediated By Krab Zinc Finger Proteins And Trim28

Abstract

KRAB domain Zinc Finger proteins make up the largest family of transcription factors in mammals. Previous studies on a handful of KRAB Zinc Finger proteins have demonstrated that KRAB domains possess the ability to repress transcription, and that this activity is mediated by an interaction with TRIM28. Depleting TRIM28 in mice zygotes results in pre gastrula stage embryonic lethality; however, little is known about how TRIM28 functions with individual KRAB domain proteins to control development. chatwo, an ENU-induced mutation, creates a hypomorphic allele of Trim28. Interestingly, the phenotype of chatwo mutants closely resembles the phenotype of KRAB Zinc Finger protein Zfp568 mutants. My results demonstrate at the molecular level that TRIM28 physically interacts with ZFP568, and is required for ZFP568 to mediate transcriptional repression. I characterized molecularly the Trim28chatwo allele and found that the chatwo mutation impairs the repressive activity of TRIM28, and affects the stability of TRIM28-ZFP568 protein complexes. My results also provide evidence that ZFP568 repressive activity is more severely affected than other KRAB Zinc Finger proteins by TRIM28 depletion, supporting a model in which KRAB Zinc Finger proteins differentially require TRIM28. By studying mutations in KRAB Zinc Finger protein ZFP568, I found that some KRAB motif amino acid residues are more critical than others to mediate transcriptional repression. My results also revealed that TRIM28 binding is not sufficient for KRAB domain repressive activity. By analyzing the repressive activity and ability to bind TRIM28 of a wide collection of KRAB domain proteins, I showed that repressive activities of KRAB domain proteins vary broadly amongst different family members, and importantly, the repressive activities of KRAB Zinc Finger proteins do not correlate with their ability to interact with TRIM28. Overall, this study provides novel contributions to the current understanding of the transcriptional roles of KRAB Zinc Finger proteins.