1. Congenic mice reveal effect of SNP, genomic rearrangements and expression variation on genome wide gene expression
Introduction
There is still no well-defined strategy for discovering the Quantitive Trait Genes (QTG) amongst the hundreds of candidates underlying Quantitive Trait Loci (QTL). We have created three congenic mouse lines that have C57BL/6 regions covering QTL for response to infection with Trypanosoma congolense introgressed into an A/J background. Data is presented here from the two parental strains and one of the congenic lines that carries a 16Mb region of C57BL/6 DNA spanning the murine MHC on chromosme 17. We have undertaken a systematic analysis of the effect of SNP, gene expression and genomic rearrangements within the congenic regions on genome wide gene expression. Gene expression data was collected using Affymetrix 430_2 mouse expression arrays and SNP data was from the Perlegen 8m SNP set This data has been used to identify the genes and pathways regulated by the congenic region and at the same time to identify genetic polymorphisms within the region that could plausibly regulate the differences in genome wide gene expression that we have observed. All classes of polymorphism within the QTL had detectable effects on both cis and trans gene expression. A genomic duplication within the QTL was associated with altered expression of Glo1 within the duplicated region. A Taverna workflow was used to identify pathways that had altered gene expression and that also contained genes within the QTL, the workflow identified Daxx as a candidate for regulating apoptosis pathways and resequencing identified an amino acid indel in the p53 interacting domain of Daxx. A GeneGo network analysis showed that the p53 pathway was the one that differed most between congenic mice and controls in the spleen and is regulated by Daxx.
Noyes HA1
Agaba M2
Ogugo M2
Brass A3
Anderson S4
Archibald A4
Fisher P3
Hulme H3
Rennie K3
Kemp SJ1
1School of Biological Sciences
University of Liverpool
Crown Street
Liverpool
L69 7ZB
2International Livestock Research
Institute (ILRI)
P O Box 30709
Nairobi 00100
Kenya
3Department of Computer Science
University of Manchester
Oxford Road
Manchester
M13 9PL
4Roslin Institute
Roslin BioCentre
Midlothian
EH25 9PS
Correlation of SNP and Expression in Inbred mice
Trans regulation of gene expression in congenic mice
The number of probes in each 50 probe bin that were differentially expressed between C57BL/6 and A/J is shown in blue above the line. The number of SNP in the 1kb upstream of each gene in each 50 probe bin is shown in red below the line. The red section of the central bar for chromosome 17 indicates the position of a QTL controlling resistance to Trypanosoma congolense infection.
For each chromosome the absolute ratio of expression in the parental mice (green) is contrasted with the absolute ratio in the congenic mice (black). Each solid bar shows regions carrying C57BL/6 DNA in red on the A/J background (blue). Regions that have an excess (p<0.05) of differentially expressed genes in the congenic mice are marked with a star. These regions may contain binding sites for transcription factors from the inserted C57BL/6 regions that regulate multiple genes.
Relative Risk
Alignment of acidic region of Daxx
C57BL/6 ETDDDDDDDDDDDDEDNEESEEEEEEEEEEKEATEDEDED
129/J ETDDDDDDDDDDDDEDNEESEEEEEEEEEEKEATEDEDED
BALB/c ETDDDDD-DDDDDDEDNEESEEEEEEEEEEKEATEDEDED
A/J ETDDDDD-DDDDDDEDNEESEEEEEEEEEEKEATEDEDED
Networks of differentially expressed genes were identified using GeneGo. The largest network of genes that were differentially expressed in the spleen of mice carrying the C57BL/6 MHC region on an A/J background was centered around p53. The congenic region was scanned for genes that might regulate p53; 3 genes were identified of which one (Daxx) had an aspartate deletion in a highly acidic region of the gene that has been implicated in the interaction of Daxx with p53 (see inset)(J Biol Chem 2004, 279: ).
Pathways in the spleen regulated by congenic region
Relative Risk of a gene being differentially expressed between C57BL/6 and A/J with different numbers of SNP in the 1Kb upstream of each gene. There was only a significant association between SNP and expression for genes that were significantly differentially expressed at the p<0.005 level. The low p value associated with differentially expressed genes that appeared to be differentially regulated by upstream SNP implies that these genes are more precisely controlled than ones that are differentially regulated as a consequence of polymorphisms elsewhere in the genome.
Copy number variants (CNV) between C57BL/6 and A/J mice were detected using Agilent 240k whole genome CGH arrays. An amplifcation of the Glo1 gene was identified which correlated with differences in expression in Glo1 at five time points after infection with T. congolense indicating that the effect of polymorphism was not sensitive to the inflammation induced by the infection.
Effect of gene copy number on expression
Acknowledgements: We thank John Wambugu, Leanne Wardlesworth, Leo Zeef and Laurence Hall for technical assistance. These studies were funded by the Wellcome Trust