Molecular characterization of Mst77F and implication in Drosophila spermatogenesis
by Nils Kost
Date of Examination:2012-08-03
Date of issue:2013-04-19
Advisor:Dr. Wolfgang Fischle
Referee:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Steven Johnsen
Persistent Address:
http://dx.doi.org/10.53846/goediss-3806
Files in this item
Name:Thesis Nils Kost.pdf
Size:3.90Mb
Format:PDF
The following license files are associated with this item:
Abstract
English
The mechanisms of spermatogenesis, in particular the process of postmeiotic spermatid maturation, are little understood. During this developmental stage massive molecular and morphological rearrangements occur and the major events are conserved across higher eukaryots. This involves the condensation of the DNA after histone removal by two major classes of proteins: (a) transition proteins and (b) protamines. Beyond these proteins nothing is known about additional factors that directly impact DNA condensation. In Drosophila Mst77F has been implicated in the DNA condensation process on the basis of immunocytology experiments and its distant sequence homology towards histone H1 family proteins and protamines. Even though Mst77F clearly localizes inside the nucleus and is a component of the condensed DNA complex in differentiated sperm cells, a direct interaction with DNA, the prerequisite for condensation, could not be demonstrated. On the basis of these findings my hypothesis was that Mst77F is a structural component of spermatid DNA that contributes to the condensation process. To address this hypothesis I conducted bioinformatic analysis of the Mst77F protein sequence to identify a putative DNA interaction domain. I found a highly charged, intrinsically unstructured C-terminal domain. This domain shows similarity towards the C-terminal domain of linker histone H1 that could be shown to bind DNA/chromatin. Qualitative and quantitativ binding experiments with recombinant Mst77F mutants and short DNAs corroborated the in silico predicted DNA interaction interface. The Mst77F DNA interaction was further characterized in detail by equilibrium binding experiments that identified an ionic interaction mode and provided the thermodynamic parameters. Besides the binding to DNA the proposed Mst77F function in DNA condensation processes was investigated. Therefore, Mst77F complexes with short DNAs were analysed by different biochemical assay systems and compared to a proposed homolog, histone hH1.4. I found Mst77F specific DNA aggregation properties that rely on the N-terminal domain of the protein. These observed differences were subsequently further characterized by Atomic Force Microscopy with long DNAs. Based on the recorded structural differences protein multimerization experiments were conducted and the following model of the Mst77F-DNA interaction was proposed:The Mst77F C-terminal domain constitutes a flexible, extended domain in solution. In this conformation protein-protein interactions via the N-terminus are rare. Upon recognition of DNA by the C-terminal domain, Mst77F undergoes a conformational change that increases its affinity for self-interactions dramatically. The C-terminal domain on its own can aggregate multiple individual DNA molecules in a one-dimensional manner. However, spreading of aggregation towards highly condensed structures is mediated by cooperative protein multimerization. Additionally, the functional consequences of the formed Mst77F DNA complexes were tested in an in vitro transcription experiment. Lastly, since Mst77F arises when the genome is still in histone-based configuration my aim was to investigate binding-, structural- and functional effects also in the context of recombinant chromatin.The present work presents the first comprehensive in vitro study on a DNA architecture protein presumably involved in DNA condensation during postmeiotic spermatid maturation in Drosophila. The insights obtained by this study could help to improve the understanding of spermatogenesis, in particular observed differences between mice and flies in DNA condensation.
Keywords: Mst77F; Spermatogenesis; DNA condensation
