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Studying the Evolution of gene expression regulation in the hypoxia-adapted rodent Spalax

Supervisor: Thomas Hankeln

Co-Supervisor: Miguel Andrade


Scientific Background:
Changes in regulatory elements of gene expression, i.e. cis-acting sequences (promotors, enhancers) and trans-acting proteins (transcription factors), are considered having a prime role in the divergent evolution and adaptation of species. Little is known specifically on how cis- and trans-regulatory sequence divergence is responsible for complex phenotypes. The blind mole rat Spalax is a subterranean rodent, which has adapted to survive severe lack of oxygen. Hypoxia tolerance is accompanied by longevity and cancer resistance, and it is largely unclear how these phenotypes are inter-connected on the molecular level. Hence, we started investigating the contribution of gene regulatory changes to the evolution of Spalax. In GenEvo phase 1, we used ‘omics methods to systematically identify differential gene expression (mRNA, small RNA, proteome) in hypoxic and normoxic Spalax tissues as compared to other hypoxia-sensitive and -tolerant rodents. We could identify gene categories (e.g. DNA repair, tumor and hypoxia genes) which are differentially regulated in all Spalax organs and may explain the mole rat’s adaptive phenotype. Selected Spalax candidate genes are now expressed in mammalian cell cultures to investigate their phenotypic consequences. Via bioinformatics, we identified cis-regulatory regions of differentially regulated candidate genes which display conspicuous insertion/deletions in Spalax, but not other taxa. Such regions are now tested by luciferase reporter assays to study the functional relevance of Spalax-specific cis-regulatory sequence changes. To investigate the potential significance of changes in trans-acting regulatory proteins, a bioinformatic pipeline is conceptualized to detect and evaluate Spalax-specific amino acid changes in candidate master transcription factors, as evidence for positive Darwinian selection on genes.


PhD Project: Studying the Evolution of gene expression regulation in the hypoxia-adapted rodent Spalax
In GenEvo phase 2, we ask the following questions:
(1) Where are the functional cis-regulatory elements in the Spalax genome and which genes do they regulate? The landscape of active enhancers/promoters will be identified in normoxic and hypoxic Spalax fibroblast cell culture (using ATAC-Seq to measure chromatin accessibility and Hi-C to identify promoter-enhancer interactions). These epigenetic data will link regulatory sites to their genes and expression data, thereby forming the basis for genome-wide studies of evolutionary patterns.
(2) Which mode of evolutionary change dominates the cis-acting regulatory sequences in Spalax versus hypoxia–sensitive mammals? We will study signatures of selective regimes in cis-regulatory sequences by defining conserved regions (evolving under selective constraint) and regions evolving faster than 4-fold degenerate synonymous codon positions of neighbouring genes, possibly reflecting positive selection for adaptive change.
(3) Do conspicuous changes in cis-regulatory regions confer an adaptive molecular phenotype, i.e. differential gene regulation, in vitro? Cis-regulatory regions of candidate genes showing the strongest differences in Spalax gene regulation will be tested experimentally in vitro by reporter gene assays.
(4) Can conspicuous amino acid changes in trans-acting regulators explain Spalax’ phenotypes?

For this, candidates will be ectopically expressed in mammalian cell cultures to investigate if Spalax’ phenotypic traits, i.e. hypoxia resistance, tumor cell features and senescence can be recovered. In a longer-term perspective, we seek to explain how the different aspects of the Spalax organismal phenotype evolved in parallel and to determine which evolutionary mechanisms predominated. 

Prof. Andrade’s group will contribute essential bioinformatics expertise in developing pipelines for detection of positive and negative selection in (non-)coding DNA, using a combination of different algorithmic approaches. We also cooperate with Core Facilities at IMB (proteomics, ATAC-Seq, HiC).


Publications relevant to this project
Schmidt H, Malik A, Bicker A, Poetzsch G, Avivi A, Shams I, Hankeln T (2017) Hypoxia tolerance, longevity and cancer-resistance in the mole rat Spalax – a liver transcriptomics approach. Sci Rep. 7:14348.
Schmidt H, Hangmann J, Shams I, Avivi A and Hankeln T (2016). Molecular evolution of antioxidant and hypoxia response in long-lived, cancer-resistant blind mole rats: The Nrf2-Keap1 pathway. Gene, 577, 293-298.
Fang X, Nevo E, ..... Hankeln T, ...., Brodsky L and Wang J (2014). Genome-wide adaptive complexes to underground stresses in blind mole rats Spalax. Nat Commun, 5, 3966.