Zinc finger proteins: Unravelling Their Role from Bacteria to Humans

Introduction. Cys2His2 zinc-finger proteins represent one of the most abundant classes of eukaryotic proteins. These proteins contain a zinc-finger (ZF) motif that allows the interaction with nucleic acids or with other proteins and play a role in several processes. ZNF639 and ZBTB2 are two human ZF proteins, with respectively nine and four Cys2His2 ZFs at the C-terminus. They play a role in cancer, and interact with each other. ZBTB2 is a subunit of the NuRD complex having a role in chromatin remodelling. ZNF639 can bind viral DNA sequences. A single prokaryotic Cys2His2 ZF domain was also identified in the C-terminal domain (CTD) of the protein Ros from Agrobacterium tumefaciens. Ros homologs, such as MucR and Ml5, have been identified mainly in α-proteobacteria, giving rise to the Ros/MucR protein family. MucR/Ros proteins regulate the expression of genes involved in infection of and symbiosis with eukaryotic hosts. These proteins bind DNA by the ZF, form higher-order oligomers by the N-terminal domain (NTD) and are thought to be H-NS like proteins. Objectives. My project aims to unravel the biological role of ZNF639 and ZBTB2 and of some members of MucR/Ros protein family such as MucR from Brucella abortus, Ml5 from Misorhizobium loti, MucR (hereafter MucR1) and two putative MucRs (hereafter MucR2 and MucR3) from Sinorhizobium meliloti, a nitrogen-fixing bacterium. Methods. Expression of recombinant proteins and purification by cationic exchange chromatography, Electrophoretic Mobility Shift Assay, Light Scattering analysis, cryo-EM experiments, Mass Spectrometry analysis, NMR spectroscopy, AlphaFold, RT-qPCRs, RNA-sequencing, analysis of RNA-seq results, Plant-Bacteria interactions. Results and discussion (I). Interactome maps of ZNF639 and ZBTB2 in U87MG cell line confirm the interaction of these two proteins and show some common interactors: Sm-D3 and Sm-F. These molecules are part of the Sm complex, a core component of snRNPs essential for splicing. These results suggest a potential role of ZNF639 and ZBTB2 in the splicing process. Results and discussion (II). We demonstrated that Ros/MucR family is the first sub-family of Histone Nucleoid Structuring like proteins (H-NS like proteins) discovered in α-proteobacteria. We identified by mass spectrometry two new Ros/MucR family members, MucR2 and MucR3 from S. meliloti. MucR2 differs from MucR1 and MucR3 in that it possesses a prolonged random coil N-terminus. MucR2 and MucR3 NTDs show a substitution of a hydrophobic residue, key for oligomerization of MucR proteins, by a Thr. By LS analysis we demonstrate that MucR2 is a monomeric protein unable to oligomerize because of the simultaneous presence of the prolonged N-terminus, Arg9 and Thr64. MucR3 forms oligomers only at high concentrations, while MucR3-L9R mutant cannot oligomerize even reaching high concentrations. Neither MucR2 nor MucR3 can bridge DNA. RNA-seq under free living conditions reveals that MucR2 and MucR3 work mainly as activator of gene transcription differently from MucR. Knockout mutants of S. meliloti show that the absence of mucr1 gene strongly affects symbiosis with Medicago sativa plant. Functional and structural analyses lead us to define MucR2 and MucR3 as atypical members of MucR/Ros family, as they cannot oligomerize and structure DNA.