Repeated evolution of heat responsiveness among Brassicaceae COPIA transposable elements

Eukaryotic genomes contain repetitive DNA sequences. This includes simple repeats and more complex transposable elements (TEs). Many TEs reach high copy numbers in the host genome, owing to their amplification abilities by specific mechanisms. There is growing evidence that TEs contribute to gene transcriptional regulation. However, excess of TE activity may lead to reduced genome stability. Therefore, TEs are suppressed by the transcriptional gene silencing machinery via specific chromatin modifications. In contrary, effectiveness of the epigenetic silencing mechanisms imposes risk for TE survival in the host genome. Therefore, TEs may have evolved specific strategies for bypassing epigenetic control and allowing the emergence of new TE copies. Recent studies suggested that the epigenetic silencing can be, at least transiently, attenuated by heat stress in A. thaliana. Heat stress induced strong transcriptional activation of COPIA78 family LTR-retrotransposons named ONSEN, and even their transposition in mutants deficient in siRNA-biogenesis. ONSEN transcriptional activation was facilitated by the presence of heat responsive elements (HREs) within the long terminal repeats, which serve as a binding platform for the HEAT SHOCK FACTORs (HSFs). This thesis focused on the evolution of ONSEN heat responsiveness in Brassicaceae. By using whole-transcriptome sequencing approach, multiple Arabidopsis lyrata ONSENs with conserved heat response were found and together with ONSENs from other Brassicaceae were used to reconstruct the evolution of ONSEN HREs. This indicated ancestral situation with two, in palindrome organized, HSF binding motifs. In the genera Arabidopsis and Ballantinia, a local duplication of this locus increased number of HSF binding motifs to four, forming a high-efficiency HRE. In addition, whole transcriptome analysis revealed novel heat-responsive TE families COPIA20, COPIA37 and HATE. Notably, HATE represents so far unknown COPIA family which occurs in several Brassicaceae species but is absent in A. thaliana. Putative HREs were identified within the LTRs of COPIA20, COPIA37 and HATE of A. lyrata, and could be preliminarily validated by transcriptional analysis upon heat induction in subsequent survey of Brassicaeae species. Subsequent phylogenetic analysis indicated a repeated evolution of heat responsiveness within Brassicaceae COPIA LTR-retrotransposons. This indicates that acquisition of heat responsiveness may represent a successful strategy for survival of TEs within the host genome.

Post-translational lysine-acetylation of Ran and its regulation by Sirtuin deacetylases

Through recent advances in high-throughput mass spectrometry it has become evident that post-translational N-(epsilon)-lysine-acetylation is a modification found on thousands of proteins of all cellular compartments and all essential physiological processes. Many aspects in the biology of lysine-acetylation are poorly understood, including its regulation by lysine-acetyltransferases and lysine-deacetylases (KDACs). Here, the role of this modification was investigated for the small GTP-binding protein Ran, which, inter alia, is essential for the regulation of nucleocytoplasmic transport. To this end, site-specifically acetylated Ran was produced in E. coli by genetic code expansion. For five previously identified sites, Ran acetylation was tested regarding its impact on the intrinsic GTP hydrolysis rate, the assembly of export complexes (modeled in vitro with the export receptor CRM1 and the export substrate Spn1) and the interaction of Ran with its GTPase activation protein RanGAP and RanBP1. Overall, mild effects of Ran acetylation were observed for intrinsic and RanGAP-stimulated GTP hydrolysis rates. The interaction of active Ran with RanBP1 was negatively influenced by Ran acetylation at K159. Moreover, CRM1 bound to Ran acetylated at K37, K99 or K159 interacted more strongly with Spn1. Thus, lysine-acetylation interferes with essential aspects of Ran function. An in vitro screen was performed to identify potential Ran KDACs. The NAD+-dependent KDACs of the Sirtuin class showed activity towards two acetylation sites of Ran, K37 and K71. The specificity of Sirtuins was further analyzed based on an additional Ran acetylation site, K38. Since deacetylation of RanAcK38 was much slower compared to RanAcK37, di-acetylated RanAcK37/38 was tested next. The deacetylation rate of di-acetylated Ran was comparable to that of RanAcK37. Deacetylation experiments under single turnover conditions revealed that deacetylation occurs first at the K38 site in the di-acetylated RanAcK37/38 background. The ability of Sirtuins to deacetylate two adjacent AcKs was further investigated based on two proteins, which had previously been found to be di-acetylated and targeted by Sirtuins, namely the tumor suppressor protein p53 and phosphoenolpyruvate carboxykinase 1 (PEPCK1). p53 was readily deacetylated at two di-acetylation sites (K372/372 and K381/382), whereas PEPCK1 was not deacetylated in vitro. Taken together, these results have important implications for the substrate specificity of Sirtuins.