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On the Evolution of the Avian Transcriptome
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology.ORCID iD: 0000-0003-2439-6946
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Change in gene expression is a powerful tool for evolution, because seemingly small expression changes can contribute important steps towards adaptation without necessarily affecting the whole organism. There is still much to learn about how gene expression evolves on genome- and population-wide levels, especially in non-model organisms. This thesis addresses some important questions in gene expression evolution via the quantitative measurement of RNA and protein levels in birds.

First, I confirmed the state of incomplete dosage compensation in birds by sequencing the transcriptome of collared flycatchers (Ficedula albicollis). I showed that pleiotropy governs the evolution of expression male-bias from the Z chromosome. Sex-linked genes in females were more highly expressed than half the male expression level, indicative of a partial up-regulation. A comparison with data from ostrich (Struthio camelus), a bird with non-degenerated sex chromosomes, showed that sex-linked expression male-bias evolved following sex chromosome degradation.

Second, using a combination of RNA sequencing and proteome mass spectrometry in chicken (Gallus gallus), I asked whether complete dosage compensation was achieved through regulation at translation. I showed that this was not the case and that incomplete dosage compensation extends to the protein level in birds. In addition, sex-linked genes showed more often an increased amount of regulation at translational level than autosomal genes.

Third, I investigated gene expression divergence between collared and pied flycatchers (Ficedula hypoleuca) using RNA sequencing in multiple tissues and individuals. Tissues differed in the degree of expression variance and in the number of divergent genes, which I identified using expression QST. Variance within species was negatively correlated with expression breadth and protein interactivity, indicating that evolutionary constraints act predominantly within interbreeding populations. Among genes unique to one of the species, I identified one gene, DPP7, falling into a large genomic deletion fixed in pied flycatchers.

Fourth, I investigated allele-specific expression (ASE) in the two flycatcher populations. ASE was identified from genetic variants within transcripts using RNA sequencing reads. We developed a Bayesian negative binomial approach that gained statistical power by estimating expression variance from combined SNPs within a transcript and overdispersion from the whole dataset.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 42 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1270
Keyword [en]
evolution, gene expression, regulation, RNA-seq, transcriptomics, proteomics, sex chromosome, dosage compensation, divergence, ASE, birds, Ficedula, flycatcher, chicken
National Category
Biological Sciences Evolutionary Biology Genetics
Research subject
Biology with specialization in Evolutionary Genetics
Identifiers
URN: urn:nbn:se:uu:diva-259487ISBN: 978-91-554-9292-2 (print)OAI: oai:DiVA.org:uu-259487DiVA: diva2:844382
Public defence
2015-09-24, Ekmansalen, Norbyvägen 14, Uppsala, 13:15 (English)
Supervisors
Available from: 2015-09-02 Created: 2015-08-05 Last updated: 2015-10-01
List of papers
1. Transcriptome Sequencing Reveals the Character of Incomplete Dosage Compensation across Multiple Tissues in Flycatchers
Open this publication in new window or tab >>Transcriptome Sequencing Reveals the Character of Incomplete Dosage Compensation across Multiple Tissues in Flycatchers
2013 (English)In: Genome Biology and Evolution, ISSN 1759-6653, E-ISSN 1759-6653, Vol. 5, no 8, 1555-1566 p.Article in journal (Refereed) Published
Abstract [en]

Sex chromosome divergence, which follows the cessation of recombination and degeneration of the sex-limited chromosome, can cause a reduction in expression level for sex-linked genes in the heterozygous sex, unless some mechanisms of dosage compensation develops to counter the reduction in gene dose. Because large-scale perturbations in expression levels arising from changes in gene dose might have strong deleterious effects, the evolutionary response should be strong. However, in birds and in at least some other female heterogametic organisms, wholesale sex chromosome dosage compensation does not seem to occur. Using RNA-seq of multiple tissues and individuals, we investigated male and female expression levels of Z-linked and autosomal genes in the collared flycatcher, a bird for which a draft genome sequence recently has been reported. We found that male expression of Z-linked genes was on average 50% higher than female expression, although there was considerable variation in the male-to-female ratio among genes. The ratio for individual genes was well correlated among tissues and there was also a correlation in the extent of compensation between flycatcher and chicken orthologs. The relative excess of male expression was positively correlated with expression breadth, expression level, and number of interacting proteins (protein connectivity), and negatively correlated with variance in expression. These observations lead to a model of compensation occurring on a gene-by-gene basis, supported by an absence of clustering of genes on the Z chromosome with respect to the extent of compensation. Equal mean expression level of autosomal and Z-linked genes in males, and 50% higher expression of autosomal than Z-linked genes in females, is compatible with that partial compensation is achieved by hypertranscription from females' single Z chromosome. A comparison with male-to-female expression ratios in orthologous Z-linked genes of ostriches, where Z-W recombination still occurs, suggests that male-biased expression of Z-linked genes is a derived trait after avian sex chromosome divergence.

Keyword
sex chromosomes, dosage compensation, sex-biased gene expression, expression variance, collared flycatcher
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-209498 (URN)10.1093/gbe/evt114 (DOI)000324595000009 ()
Available from: 2013-10-21 Created: 2013-10-21 Last updated: 2017-12-06Bibliographically approved
2. Quantitative Mass Spectrometry Reveals Partial Translational Regulation for Dosage Compensation in Chicken
Open this publication in new window or tab >>Quantitative Mass Spectrometry Reveals Partial Translational Regulation for Dosage Compensation in Chicken
Show others...
2015 (English)In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 32, no 10, 2716-2725 p.Article in journal (Refereed) Published
Abstract [en]

There is increasing evidence that dosage compensation is not a ubiquitous feature following sex chromosome evolution, especially not in organisms where females are the heterogametic sex, like in birds. Even when it occurs, compensation can be incomplete and limited to dosage-sensitive genes. However, previous work has mainly studied transcriptional regulation of sex-linked genes, which may not reflect expression at the protein level. Here, we used liquid chromatography–tandem mass spectrometry to detect and quantify expressed levels of more than 2,400 proteins in ten different tissues of male and female chicken embryos. For comparison, transcriptome sequencing was performed in the same individuals, five of each sex. The proteomic analysis revealed that dosage compensation was incomplete, with a mean male-to-female (M:F) expression ratio of Z-linked genes of 1.32 across tissues, similar to that at the RNA level (1.29). The mean Z chromosome-to-autosome expression ratio was close to 1 in males and lower than 1 in females, consistent with partly reduced Z chromosome expression in females. Although our results exclude a general mechanism for chromosome-wide dosage compensation at translation, 30% of all proteins encoded from Z-linked genes showed a significant change in the M:F ratio compared with the corresponding ratio at the RNA level. This resulted in a pattern where some genes showed balanced expression between sexes and some close to 2-fold higher expression in males. This suggests that proteomic analyses will be necessary to reveal a more complete picture of gene regulation and sex chromosome evolution.

Keyword
sex chromosome evolution, dosage compensation, chicken, proteomics, mass spectrometry
National Category
Evolutionary Biology Genetics
Research subject
Biology with specialization in Evolutionary Genetics
Identifiers
urn:nbn:se:uu:diva-258796 (URN)10.1093/molbev/msv147 (DOI)000361987100019 ()26108680 (PubMedID)
Funder
Swedish Research Council, 2010-5650, 2013-8271, 2011-4423EU, European Research Council, AdG 249976Knut and Alice Wallenberg Foundation
Available from: 2015-07-20 Created: 2015-07-20 Last updated: 2017-12-04Bibliographically approved
3. Divergence in gene expression within and between two closely related flycatcher species
Open this publication in new window or tab >>Divergence in gene expression within and between two closely related flycatcher species
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Compared to DNA sequence evolution, relatively little is known about the character of gene expression evolution as species diverge. For example, it is unclear if gene expression generally evolves in a clock-like manner (by stabilizing selection or from neutral evolution) or if there are frequent episodes of directional selection. To gain insights into the evolutionary divergence of gene expression patterns, we sequenced and compared the transcriptomes of multiple tissues from population samples of collared (Ficedula albicollis) and pied flycatchers (F. hypoleuca), a species pair which diverged less than one million years ago. Tissues resolved into separate clusters in non-metric multidimensional scaling ordination analysis and samples from the two species generally clustered by tissue rather than by species. Tissues differed in the degrees of expression variance within species and divergence between species. Variance was positively correlated with expression breadth and negatively correlated with protein interactivity, suggesting that pleiotropic constraints reduce gene expression variance within species. Variance was correlated with between-species divergence, consistent with a pattern expected from stabilizing selection and neutral evolution. Using an expression QST approach, we identified genes differentially expressed between species. We also identified 10 genes uniquely expressed in one of the species. For one such gene (DPP7, uniquely expressed in collared flycatcher), the absence of expression in pied flycatchers could be associated with a fixed ≈ 20 kb deletion including 11 out of 13 exons in this species. This study conducted in a young vertebrate speciation model system expands our knowledge of how gene expression evolves in natural populations.

Keyword
Gene regulation, speciation, transcriptomics, collared flycatcher, pied flycatcher, Ficedula
National Category
Evolutionary Biology Genetics
Research subject
Biology with specialization in Evolutionary Genetics
Identifiers
urn:nbn:se:uu:diva-258798 (URN)
Available from: 2015-08-04 Created: 2015-07-20 Last updated: 2015-10-01
4. Allele-specific gene expression inferred by a Bayesian negative binomial approach indicates abundant cis-regulatory variation in natural flycatcher populations
Open this publication in new window or tab >>Allele-specific gene expression inferred by a Bayesian negative binomial approach indicates abundant cis-regulatory variation in natural flycatcher populations
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Polymorphisms in cis-regulatory sequences can make the two alleles of a gene be expressed at different levels, providing a starting point for the evolution of divergence in gene expression. However, little is known about the genome-wide abundance of regulatory variation in natural populations. We performed RNA-seq of multiple tissues from population samples of two closely related flycatcher species and developed a Bayesian algorithm that maximises data usage by borrowing overdispersion estimates over the whole dataset and combines several SNPs per transcript to detect allele-specific expression (ASE). Of 2,576 transcripts analysed in collared flycatcher, ASE was detected in 185 (7.2%). A similar frequency was seen in the pied flycatcher but these frequencies are likely to be underestimates for several reasons. The proportion of ASE transcripts varied among tissues, being lowest in testis and highest in muscle. Often, ASE was seen in several tissues for particular gene-individual combinations. This is one of only very few large-scale studies investigating the incidence of ASE in the wild. The results suggest that genetic variation in regulatory sequences commonly affect gene expression in natural populations and that it provides a seedbed for phenotypic evolution via divergence in gene expression.

Keyword
regulatory sequences, gene expression evolution, ASE, RNA-seq
National Category
Evolutionary Biology Genetics
Research subject
Biology with specialization in Evolutionary Genetics
Identifiers
urn:nbn:se:uu:diva-258797 (URN)
Available from: 2015-08-05 Created: 2015-07-20 Last updated: 2015-10-01

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