Dissertation/Thesis Abstract

Functional analysis of KAP1 genomic recruitment and investigation of its transcriptional regulatory effects on the genome
by Iyengar, Sushma S., Ph.D., University of California, Davis, 2011, 231; 3482231
Abstract (Summary)

In mammalian cells, multiple cellular processes such as gene silencing, cell growth, cell differentiation, maintenance of pluripotency, neoplastic transformation, DNA repair and apoptosis converge on the evolutionarily conserved protein KAP1 (KRAB-associated protein1). Despite the advances made through biochemical and other in vitro studies on KAP1, not much was known about (1) its in vivo binding sites, (2) its mechanism of recruitment to the genome or (3) its effect on the transcriptional output of its target genes. Presented within is a summary of my dissertation research which attempts to answer these three questions about KAP1 and ultimately to understand how KAP1 mediates diverse effects on cell biology.

As a first step towards identifying the in vivo genomic binding sites of KAP1, I performed ChIP-chip experiments in Ntera2D1 cells using an antibody targeted against KAP1 in combination with tiling arrays that spanned the entire human genome, leading to the identification of ∼7000 KAP1 binding sites. Subsequently, using a combination of ChIP-chip and ChIP-seq techniques, I identified KAP1 targets in numerous other cell types, including both normal and tumor cells.

I hypothesized that there are two mechanisms of KAP1 recruitment to DNA. To test this hypothesis, I created a series of FLAG-tagged KAP1 mutants and stably introduced them into HEK293 cells. Each mutant inactivated a different functional domain of the KAP1 protein; specifically I mutated the N-terminal RBCC domain (required for interaction with KRAB-ZNFs), the C-terminal PB domain (required for interaction with chromatin modifying enzymes), and the HP1 binding domain (required for interaction with HP1). I performed ChIP-seq experiments using an antibody that recognizes the FLAG tag to test the requirement of each domain of KAP1 for genomic recruitment. My experiments (i) prove that interaction of KAP1 with KRAB-ZNFs is required for a subset of targets, (ii) suggest the presence of a hitherto uncharacterized mechanism of KAP1 recruitment to its promoter bound targets, and (iii) reveal a previously unknown role for HP1 in tethering KAP1 to DNA.

I performed ChIP-seq experiments using an antibody that recognizes the FLAG tag to test the possible role of each ZNF in recruiting KAP1 to its genomic binding sites. Surprisingly, although the KRAB-ZNFs interacted with endogenous KAP1, the ChIP-seq assay was not able to detect any genomic binding sites for these proteins. It is possible that the chosen KRAB-ZNF proteins have a role in mediating interaction between KAP1 and RNA or other non-chromatin proteins, and therefore their function cannot be elucidated using the ChIP-seq assay. It is also possible that the selected KRAB-ZNF proteins bind DNA only upon certain developmental or environmental triggers, and hence ChIP-seq could not identify any genomic targets for these proteins under the given conditions.

To address the question of how KAP1 was recruited to its promoter targets, I used a bioinformatics approach to identify highly enriched transcription factor binding motifs within the central 100 bp of the top 100 promoters bound by KAP1. This approach identified the CCGGAA motif; this motif is generally used by the ETS family of transcription factors, and ELK4 specifically binds to a very similar sequence as found in the KAP1 promoter peaks. Therefore I hypothesized that ELK4 may be involved in the recruitment of KAP1 to promoters. I then experimentally tested this prediction by performing ChIP-seq for ELK4 in HEK293 cells and showed that out of 1084 promoter-bound targets of KAP1, 358 (∼33%) co-localized with ELK4 bound sites, suggesting that ELK4 may play an important role in recruiting KAP1 to this category of genomic binding sites.

Finally, to address the role of KAP1 as a global regulator of transcription, I assessed the mRNA levels of its target genes in cells deficient for KAP1 using Illumina gene expression arrays and RNA-seq. Surprisingly, I showed that there is no correlation between the enrichment of KAP1 at its strongest binding sites (ZNF 3' ends) and the expression of the bound ZNF genes. Also there was no change in mRNA splicing or processing of KAP1-bound ZNF genes, which are enriched for the heterochromatic mark H3me3K9 even in KAP1-deficient cells. (Abstract shortened by UMI.)

Indexing (document details)
Advisor: Farnham, Peggy J.
Commitee: Chedin, Frederic, Korf, Ian, Segal, David J.
School: University of California, Davis
Department: Genetics
School Location: United States -- California
Source: DAI-B 73/03, Dissertation Abstracts International
Subjects: Genetics
Keywords: Epigenetic regulation, Gene silencing, Genomic recruitment, Heterochromatin
Publication Number: 3482231
ISBN: 9781267028785