Molecular and functional analyses of the plant specific 3xHMG-box proteins expressed during mitosis/meiosis

The plant specific family of 3xHMG-box proteins contains three HMG-box domains and an N-terminal basic domain that synergistically contribute to its DNA binding and bending properties. So far, they resemble the first group of HMG-box containing proteins which association with chromatin is restricted to mitosis and meiosis. In this work, a reporter system that allows in vivo studies of 3xHMG-box proteins was successfully developed and tested. Through the usage of the endogenous promoters of 3xHMG-box1 and 3xHMG-box2, expression of the reporter constructs should correspond to the expression of the endogenous genes with respect to transcript level and cell cycle dependency. The reporter system was used to monitor occurance and distribution of 3xHMG-box proteins during G2/M phase in root cells and to test the effect of a putative D-box motif in degradation of 3xHMG-box2. In contrast to overexpression of 3xHMG-box1-GFP-fusion proteins, overexpression of fusion proteins that consist of 3xHMG-box1 and a GFP with an attached nuclear localization signal leads to strong developmental defects in A. thaliana plants. The 3xHMG-box1-GFP-NLS derived signal was observed to be accumulated as distinct speckles within in the nucleolus, in which rDNA is transcribed and processed. Growth defects in 3xHMG-box1-GFP-NLS overexpression lines could not be connected to decreased transcript levels of 45 rDNA or altered compaction state of 45S rDNA gene regions. Construction of truncated and chimeric proteins in which the N-terminal domains of 3xHMG-box1 and 3xHMG-box2 were exchanged, suggested a function of the N-terminal domain for the specificity of 3xHMG-box1 for certain rDNA regions. EMSA experiments with 45S rDNA fragments and recombinant N-terminal domains of 3xHMG-box1 and 3xHMG-box2 did not support a sequence specific binding of 3xHMG-box1 N-terminal domain for 45S rDNA. Association of 3xHMG-box proteins with NORs in allotetraploid A. suecica was tested in subsequent Immunostain and FISH experiments. 3xHMG-box proteins were found to associate with silenced A. thaliana NORs but also with some of the A. arenosa NORs. Furthermore, plants that simultaneously express 3xHMG-box proteins fused to GFP and linker histones fused to RFP were analyzed. No evidences for antagonistic binding could be obtained