Understanding the relationship between IRF-1 and the transcriptional repressor ZNF350
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Date
04/07/2015Author
Mallin, Lucy Janet
Metadata
Abstract
Interferon regulatory factor-1 (IRF-1) is a transcription factor and tumour suppressor,
involved in many diverse cellular processes including immune responses and growth
regulation. An interesting feature of IRF-1 is that it can both activate and repress
gene expression, possibly by acting with co-activator or co-repressor proteins. In a
previous phage display assay, a homologous peptide to the known repressor protein,
zinc finger 350 (ZNF350), was found to bind to the C-terminus of IRF-1. ZNF350,
also known as ZBRK1 (Zinc finger and BRCA1-interacting protein with KRAB
domain-1), is a member of the Krüppel-associated box (KRAB)-containing zinc
finger (KZF) proteins, which is a group of the widely distributed transcriptional
repression proteins in mammals. ZNF350 has previously been shown to repress the
expression of a number of genes including ANG1 and GADD45A, often in complex
with other proteins. This study confirms the direct interaction between IRF-1 and
ZNF350 and identifies key residues, including the LXXLL repression motif within
the C-terminus of IRF-1, necessary for the binding interface. The two proteins have
additionally been shown to interact within a cellular environment, shown by using
techniques including immunoprecipitation and a proximity ligation assay. In
addition, the ZNF350/IRF-1 complex formation appears to occur in the basal state of
the cell, as opposed to in response to cellular stress such as viral infection or DNA
damage. On the basis of ZNF350 being a negative regulator of transcription, a novel
technique was developed to identify putative targets of both ZNF350 and IRF-1.
This involved an initial bioinformatics screen using candidate IRF-1 binding site data
obtained from CENTIPEDE, an algorithm that combines genome sequence
information, with cell-specific experimental data to map bound TF binding sites.
This allowed for the identification of novel target genes that contained the ZNF350
consensus binding site, GGGxxCAGxxxTTT, within close proximity to an IRF-1
consensus site, such as the immune response gene IL-12A. Lastly, a peptide phage
display screen was combined with high-throughput sequencing to identify other
potential binding partners of ZNF350 and perhaps help to understand the mechanism
by which transcriptional repression is controlled by complex formation.