Sequence and functional differences in the ATPase domains of CHD3 and SNF2H promise potential for selective regulability and drugability.

Chromatin remodelers use the energy of ATP hydrolysis to regulate chromatin dynamics. Their impact for development and disease requires strict enzymatic control. Here, we address the differential regulability of the ATPase domain of hSNF2H and hCHD3, exhibiting similar substrate affinities and enzymatic activities. Both enzymes are comparably strongly inhibited in their ATP hydrolysis activity by the competitive ATPase inhibitor ADP. However, the nucleosome remodeling activity of SNF2H is more strongly affected than that of CHD3. Beside ADP, also IP6 inhibits the nucleosome translocation of both enzymes to varying degrees, following a competitive inhibition mode at CHD3, but not at SNF2H. Our observations are further substantiated by mutating conserved Q- and K-residues of ATPase domain motifs. The variants still bind both substrates and exhibit a wild-type similar, basal ATP hydrolysis. Apart from three CHD3 variants, none of the variants can translocate nucleosomes, suggesting for the first time that the basal ATPase activity of CHD3 is sufficient for nucleosome remodeling. Together with the ADP data, our results propose a more efficient coupling of ATP hydrolysis and remodeling in CHD3. This aspect correlates with findings that CHD3 nucleosome translocation is visible at much lower ATP concentrations than SNF2H. We propose sequence differences between the ATPase domains of both enzymes as an explanation for the functional differences and suggest that aa interactions, including the conserved Q- and K-residues distinctly regulate ATPase-dependent functions of both proteins. Our data emphasize the benefits of remodeler ATPase domains for selective drugability and/or regulability of chromatin dynamics.

Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii.

For the model organism Chlamydomonas reinhardtii, a codon-adapted gene variant of the extracellular luciferase of Gaussia princeps was generated as a sensitive molecular tool to study gene expression from the nuclear genome. In the past, monitoring promoter activity in Chlamydomonas employing the commonly used luciferase encoded by Renilla reniformis was hampered due to the detection limit of the reporter assay, especially if analyzing weak promoters. In this work, the expression of Gaussia-luciferase from such promoters resulted in an average luminescent activity at least 500 times higher than that detected for the Renilla enzyme. The wildtype signal peptide of Gaussia princeps efficiently mediated the export of the luciferase into the culture medium of Chlamydomonas strain cw15arg ( - ), and the characterization of the secreted protein showed an unexpected temperature instability, probably arising from post-translational modifications made by the algae. To further test the utility of Gaussia-luciferase, promoter sequences originating from different viral genomes were analyzed for their ability to drive transgene expression in Chlamydomonas. Solely, the 35S-promoter of the Cauliflower mosaic virus (CaMV) displayed a significant transcriptional activity and this happened only when the shunting region of the 5'-untranslated region of the 35S-sequence was omitted from the luciferase expression cassette. Gaussia-luciferase proved to be a superior quantifiable reporter gene for the analysis of constitutive promoter sequences in Chlamydomonas reinhardtii.