Evolution of gene expression between closely related taxa of Mus I n a u g u r a l – D i s s e r t a t i o n zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Christian Voolstra aus Offenbach am Main Köln, 2006 Berichterstatter: Prof. Dr. Diethard Tautz Prof. Dr. Thomas Wiehe Tag der letzten mündlichen Prüfung: 29.05.2006 Table of Contents Table of Contents Table of Contents ........................................................................................................................i Danksagung................................................................................................................................ v Zusammenfassung.....................................................................................................................vi Abstract ...................................................................................................................................viii 1 Introduction ........................................................................................................................ 1 1.1 Microarrays as a tool to study evolution of gene expression ..................................... 1 1.2 Intra-specific transcriptome variation ........................................................................ 2 1.3 Inter-specific transcriptome variation - tempo and mode of transcriptome evolution3 1.4 Transcriptome divergence and speciation.................................................................. 4 1.5 The genetic basis of gene expression differences ...................................................... 5 1.6 Model/study system – the Mus species complex ....................................................... 6 1.7 Project description...................................................................................................... 8 2 Material & Methods........................................................................................................... 9 2.1 Animals ...................................................................................................................... 9 2.2 Microarrays .............................................................................................................. 11 2.3 Sample preparation................................................................................................... 11 2.4 Hybridization............................................................................................................ 11 2.5 Data Acquistion........................................................................................................ 12 2.6 Experimental design................................................................................................. 12 2.7 Genomic DNA hybridizations.................................................................................. 13 2.8 Data processing and statistical analyses................................................................... 13 2.9 Identification of differentially expressed genes with SAM (Significance Analysis of Microarrays) ......................................................................................................................... 13 i Table of Contents 2.10 Identification of lineage-specific genes of subspecies of Mus musculus................. 14 2.11 Mitochondrial D-loop sequencing............................................................................ 14 2.12 Confirmation of differentially expressed genes from the SAM analyses with quantitative real-time PCR (qRT-PCR) ............................................................................... 15 2.13 Identification of functional categories within lists of differentially expressed genes with DAVID (Database for Annotation, Visualization and Integrated Discovery).............. 16 2.14 Identification of biological processes within lists of differentially expressed genes with PANTHER (Protein ANalysis THrough Evolutionary Relationships)......................... 17 2.15 Scaled divergence analysis....................................................................................... 17 2.16 dN/dS analysis.......................................................................................................... 18 3 Results .............................................................................................................................. 19 3.1 Differentially expressed genes between species and subspecies of Mus ................. 19 3.1.1 Genomic DNA hybridizations (CGHs)............................................................ 19 3.1.2 SAM (Significance Analysis of Microarrays) analysis – number of differentially expressed genes between species/subspecies for the different tissues....... 21 3.1.2.1 Gene expression differences between Mus musculus and Mus spretus ....... 21 3.1.2.2 Gene expression differences between subspecies of Mus musculus............ 22 3.1.2.3 Comparison of gene expression differences between species and subspecies of Mus ...................................................................................................................... 22 3.1.2.4 Distribution of fold-changes (FC) among significantly differentially expressed genes............................................................................................................ 26 3.1.2.5 Lineage-specific genes in Mus musculus ..................................................... 29 3.2 Confirmation of SAM candidate genes with quantitative real-time PCR (qRT-PCR) .................................................................................................................................. 32 3.2.1 Genes differentially expressed between Mus spretus and Mus musculus........ 32 3.2.2 Genes differentially expressed between subspecies of Mus musculus............. 36 3.3 Identification of functional categories within lists of differentially expressed genes with DAVID (Database for Annotation, Visualization and Integrated Discovery).............. 37 3.3.1 Categories of functional classification within the list of differentially expressed genes identified in the brain ............................................................................................. 38 ii Table of Contents 3.3.2 Categories of functional classification within the list of differentially expressed genes identified in the liver/kidney .................................................................................. 40 3.3.3 Categories of functional classification within the list of differentially expressed genes identified in the testis ............................................................................................. 42 3.4 Identification of biological processes within lists of differentially expressed genes with PANTHER (Protein ANalysis THrough Evolutionary Relationships)......................... 44 3.5 Genome-wide patterns of expression divergence..................................................... 47 3.5.1 Scaled divergence analysis............................................................................... 47 3.5.2 dN/dS analysis.................................................................................................. 53 3.5.2.1 Distribution of dN/dS ratios ......................................................................... 55 3.5.2.2 Patterns of correlation of evolution of gene expression and sequence evolution ...................................................................................................................... 55 4 DISCUSSION .................................................................................................................. 61 4.1 Gene expression differences between species and subspecies of Mus..................... 61 4.2 Taxonomic status of Mus musculus ssp. .................................................................. 62 4.3 Confirmation of differentially expressed genes with qRT-PCR .............................. 63 4.4 Functional annotation of candidate genes ................................................................ 64 4.5 Correlations of expression divergence and nucleotide sequence divergence .......... 66 4.6 Towards evolutionary patterns of species divergence/speciation regarding gene expression divergence .......................................................................................................... 67 5 Literature .......................................................................................................................... 71 6 Appendix .......................................................................................................................... 77 6.1 Differentially expressed genes between Mus spretus and Mus musculus (SAM 2- class unpaired analysis, 200 permutations, FDR < 5%)....................................................... 77 6.1.1 brain – over-expressed genes in Mus spretus................................................... 77 6.1.2 brain – over-expressed genes in Mus musculus ............................................... 78 6.1.3 liver/kidney – over-expressed genes in Mus spretus........................................ 78 6.1.4 liver/kidney – over-expressed genes in Mus musculus .................................... 81 6.1.5 testis – over-expressed genes in Mus spretus................................................... 85 iii Table of Contents 6.1.6 testis – over-expressed genes in Mus musculus ............................................... 89 6.2 Differentially expressed genes across subspecies of Mus