Control of bacterial anti-phage signaling by a WYL domain transcription factor

Bacteria use diverse immune systems to defend themselves from ubiquitous viruses termed bacteriophages (phages). Many anti-phage systems function by abortive infection to kill a phage-infected cell, raising the question of how these systems are regulated to avoid activation and cell killing outside the context of infection. Here, we identify a transcription factor associated with the widespread CBASS bacterial immune system, that we term CapW. CapW forms a homodimer and binds a palindromic DNA sequence in the CBASS promoter region. Two crystal structures of CapW reveal how the protein switches from a DNA binding-competent state to a ligand-bound state that cannot bind DNA due to misalignment of dimer-related DNA binding domains. We show that CapW strongly represses CBASS gene expression in uninfected cells, and that CapW disruption likely results in toxicity due to uncontrolled CBASS expression. Our results parallel recent findings with BrxR, a transcription factor associated with the BREX anti-phage system, and suggest that CapW and BrxR are the founding members of a family of universal anti-phage signaling proteins.

Transcription factor lineages in plant-pathogenic fungi, connecting diversity with fungal virulence

Plant-pathogenic fungi span diverse taxonomic lineages. Their host-infection strategies are often specialised and require the coordinated regulation of molecular virulence factors. Transcription factors (TFs) are fundamental regulators of gene expression, controlling development and virulence in plant pathogenic fungi. Recent research has established regulatory roles for several taxonomically conserved fungal TFs, but the evolution of specific virulence regulators is not well understood. This study sought to explore the representation of TFs across a taxonomically-diverse range of fungi, with a focus on plant pathogens. A significant trend was observed among the obligate, host-associated pathogens, which possess a reduced overall TF repertoire, alluding to a lack of pressure for maintaining diversity. A novel orthology-based analysis is then presented that refined TF classifications, traditionally based on the nature of the DNA-binding domains. Using this analysis, cases of TF over/underrepresentation across fungal pathogen lineages are systematically highlighted. Specific examples are then explored and discussed that included the TF orthologues of Ste12, Pf2 and EBR1, plus phytotoxic secondary-metabolite cluster regulators, which all presented novel and distinct evolutionary insights. Ultimately, as the examples presented demonstrate, this resource can be interrogated to guide functional studies that seek to characterise virulence-specific regulators and shed light on the factors underpinning plant pathogenicity.

The Impact of the HydroxyMethylCytosine epigenetic signature on DNA structure and function

We present a comprehensive, experimental and theoretical study of the impact of 5-hydroxymethylation of DNA cytosine. Using molecular dynamics, biophysical experiments and NMR spectroscopy, we found that Ten-Eleven translocation (TET) dioxygenases generate an epigenetic variant with structural and physical properties similar to those of 5-methylcytosine. Experiments and simulations demonstrate that 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) generally lead to stiffer DNA than normal cytosine, with poorer circularization efficiencies and lower ability to form nucleosomes. In particular, we can rule out the hypothesis that hydroxymethylation reverts to unmodified cytosine physical properties, as hmC is even more rigid than mC. Thus, we do not expect dramatic changes in the chromatin structure induced by differences in physical properties between d(mCpG) and d(hmCpG). Conversely, our simulations suggest that methylated-DNA binding domains (MBDs), associated with repression activities, are sensitive to the substitution d(mCpG) ➔ d(hmCpG), while MBD3 which has a dual activation/repression activity is not sensitive to the d(mCpG) d(hmCpG) change. Overall, while gene activity changes due to cytosine methylation are the result of the combination of stiffness-related chromatin reorganization and MBD binding, those associated to 5-hydroxylation of methylcytosine could be explained by a change in the balance of repression/activation pathways related to differential MBD binding.

Characterization of the Function of Two S1Fa-Like Family Genes From Populus trichocarpa

S1Fa-like transcription factors (TFs) are small molecular weight proteins that contain both nuclear localization and DNA binding domains. However, the functions of S1Fa-like TFs are poorly understood. In the present study, we identified the S1Fa-like TFs from the Populus trichocarpa genome, which revealed two S1Fa-like TF genes, PtS1Fa1 and PtS1Fa2. PtS1Fa1 and PtS1Fa2 expression was suppressed by drought and salt stress, and was also significantly altered by ABA, MeJA, or SA treatment. Both PtS1Fa1 and PtS1Fa2 are nuclear proteins. Transgenic P. trichocarpa plants overexpressing PtS1Fa1 and PtS1Fa2, respectively, were generated. The plants overexpressing PtS1Fa2 showed increased fresh weight, chlorophyll content, and root length and weight compared with those in wild-type (WT) P. trichocarpa under drought conditions. Meanwhile, these phenotype traits of plants overexpressing PtS1Fa1 were similar to those of WT plants. Furthermore, overexpression of PtS1Fa2 reduced the malondialdehyde (MDA) content, electrolyte leakage, H2O2 and O2- contents, and increased superoxide dismutase (SOD) and peroxidase (POD) activities. The expression of SOD and POD was also induced by PtS1Fa2. However, overexpression of PtS1Fa1 failed to affect any of these physiological parameters or SOD and POD gene expression. These results suggested that PtS1Fa2 plays a role in drought tolerance, and confers drought tolerance by increase antioxidant activity to reduce reactive oxygen species (ROS) accumulation.

Non-canonical Staphylococcus aureus pathogenicity island repression

ABSTRACTMobile genetic elements (MGEs) control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here we describe an unprecedented repression-derepression mechanism involved in the transfer of the Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation, never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterise a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.SignificanceWhile most repressors controlling the transfer of mobile genetic elements are dimers, we demonstrate here that the Staphylococcal pathogenicity island 1 (SaPI1) is repressed by two tetramers, which have a novel structural fold in their body that has never been seen before in other proteins. Moreover, by solving the structure of the SaPI1 repressor in complex with its inducing protein Sri, we have demonstrated that Sri forces the SaPI1 repressor to adopt a conformation that is incompatible with DNA binding, explaining how SaPI1 is induced. Finally, our results demonstrate that this repression system is not exclusive of the SaPIs but widespread in nature. Our studies provide important insights understanding how SaPIs spread in nature.

TEM1 combinatorially binds to FLOWERING LOCUS T and recruits a Polycomb factor to repress the floral transition in Arabidopsis

Arabidopsis TEMPRANILLO 1 (TEM1) is a transcriptional repressor that participates in multiple flowering pathways and negatively regulates the juvenile-to-adult transition and the flowering transition. To understand the molecular basis for the site-specific regulation of FLOWERING LOCUS T (FT) by TEM1, we determined the structures of the two plant-specific DNA-binding domains in TEM1, AP2 and B3, in complex with their target DNA sequences from the FT gene 5′-untranslated region (5′-UTR), revealing the molecular basis for TEM1 specificity for its DNA targets. In vitro binding assays revealed that the combination of the AP2 and B3 binding sites greatly enhanced the overall binding of TEM1 to the FT 5′-UTR, indicating TEM1 combinatorically recognizes the FT gene 5′-UTR. We further showed that TEM1 recruits the Polycomb repressive complex 2 (PRC2) to the FT 5′-UTR. The simultaneous binding of the TEM1 AP2 and B3 domains to FT is necessary for deposition of H3K27me3 at the FT 5′-UTR and for the flowering repressor function of TEM1. Overall, our data suggest that the combinatorial recognition of FT 5′-UTR by TEM1 ensures H3K27me3 deposition to precisely regulate the floral transition.

Evolution of binding preferences among whole-genome duplicated transcription factors

Throughout evolution, new transcription factors (TFs) emerge by gene duplication, promoting growth and rewiring of transcriptional networks. How TF duplicates diverge is known for only a few studied cases. To provide a genome-scale view, we considered the 35% of budding yeast TFs, classified as whole-genome duplication (WGD)-retained paralogs. Using high-resolution profiling, we find that ~60% of paralogs evolved differential binding preferences. We show that this divergence results primarily from variations outside the DNA binding domains (DBDs), while DBD preferences remain largely conserved. Analysis of non-WGD orthologs revealed that ancestral preferences are unevenly split between duplicates, while new targets are acquired preferentially by the least conserved paralog (biased sub/neo-functionalization). Dimer-forming paralogs evolved mostly one-sided dependency, while other paralogs interacted through low-magnitude DNA-binding competition that minimized paralog interference. We discuss the implications of our findings for the evolutionary design of transcriptional networks.

Targeted base editing in the plastid genome of Arabidopsis thaliana

Bacterial cytidine deaminase fused to the DNA binding domains of transcription activator-like effector nucleases was recently reported to transiently substitute a targeted C to a T in mitochondrial DNA of mammalian cultured cells1. We applied this system to targeted base editing in the Arabidopsis thaliana plastid genome. The targeted Cs were homoplasmically substituted to Ts in some plantlets of the T1 generation and the mutations were inherited by their offspring independently of their nuclear-introduced vectors.

On exploring effects of coevolving residues on DNA binding specificity of transcription factors

Transcription factors (TFs) regulate gene expression by specifically binding to DNA targets. Many factors have been revealed to influence TF-DNA binding specificity. Coevolution of residues in proteins occurs due to a common evolutionary history. However, it is unclear how coevolving residues in TFs contribute to DNA binding specificity. Here, we systematically analyzed TF-DNA interactions from high-throughput experiments for seven TF families, including Homeobox, HLH, bZIP_1, Ets, HMG_box, zf-C4 and Zn_clus TFs. Based on TF-DNA interactions, we detected TF subclass determining sites (TSDSs) defining the heterogeneity of DNA binding preference for each TF family. We showed that the TSDSs were more likely to be coevolving with TSDSs than with non-TSDSs, particularly for Homeobox, HLH, Ets, bZIP_1 and HMG_box TF families. Mutation of the highly coevolving residues could significantly reduce the stability of TF-DNA complex. The distant residues from the DNA interface also contributed to TF-DNA binding activity. Overall, our study gave evidence of the functional importance of coevolved residues in refining transcriptional regulation and provided clues to the application of engineered DNA-binding domains and protein.