The Evolution of SlyA/RovA Transcription Factors from Repressors to Countersilencers in Enterobacteriaceae.

Gene duplication and subsequent evolutionary divergence have allowed conserved proteins to develop unique roles. The MarR family of transcription factors (TFs) has undergone extensive duplication and diversification in bacteria, where they act as environmentally responsive repressors of genes encoding efflux pumps that confer resistance to xenobiotics, including many antimicrobial agents. We have performed structural, functional, and genetic analyses of representative members of the SlyA/RovA lineage of MarR TFs, which retain some ancestral functions, including repression of their own expression and that of divergently transcribed multidrug efflux pumps, as well as allosteric inhibition by aromatic carboxylate compounds. However, SlyA and RovA have acquired the ability to countersilence horizontally acquired genes, which has greatly facilitated the evolution of Enterobacteriaceae by horizontal gene transfer. SlyA/RovA TFs in different species have independently evolved novel regulatory circuits to provide the enhanced levels of expression required for their new role. Moreover, in contrast to MarR, SlyA is not responsive to copper. These observations demonstrate the ability of TFs to acquire new functions as a result of evolutionary divergence of both cis-regulatory sequences and in trans interactions with modulatory ligands.IMPORTANCE Bacteria primarily evolve via horizontal gene transfer, acquiring new traits such as virulence and antibiotic resistance in single transfer events. However, newly acquired genes must be integrated into existing regulatory networks to allow appropriate expression in new hosts. This is accommodated in part by the opposing mechanisms of xenogeneic silencing and countersilencing. An understanding of these mechanisms is necessary to understand the relationship between gene regulation and bacterial evolution. Here we examine the functional evolution of an important lineage of countersilencers belonging to the ancient MarR family of classical transcriptional repressors. We show that although members of the SlyA lineage retain some ancestral features associated with the MarR family, their cis-regulatory sequences have evolved significantly to support their new function. Understanding the mechanistic requirements for countersilencing is critical to understanding the pathoadaptation of emerging pathogens and also has practical applications in synthetic biology.

Modulation of H-NS transcriptional silencing by magnesium.

The bacterial histone-like protein H-NS silences AT-rich DNA, binding DNA as 'stiffened' filaments or 'bridged' intrastrand loops. The switch between these modes has been suggested to depend on the concentration of divalent cations, in particular Mg2+, with elevated Mg2+ concentrations associated with a transition to bridging. Here we demonstrate that the observed binding mode is a function of the local concentration of H-NS and its cognate binding sites, as well as the affinity of the interactions between them. Mg2+ does not control a binary switch between these two modes but rather modulates the affinity of this interaction, inhibiting the DNA-binding and silencing activity of H-NS in a continuous linear fashion. The direct relationship between conditions that favor stiffening and transcriptional silencing activity suggests that although both modes can occur in the cell, stiffening is the predominant mode of binding at silenced genes.

Integrated circuits: how transcriptional silencing and counter-silencing facilitate bacterial evolution.

Horizontal gene transfer is a major contributor to bacterial evolution and diversity. For a bacterial cell to utilize newly-acquired traits such as virulence and antibiotic resistance, new genes must be integrated into the existing regulatory circuitry to allow appropriate expression. Xenogeneic silencing of horizontally-acquired genes by H-NS or other nucleoid-associated proteins avoids adventitious expression and can be relieved by other DNA-binding counter-silencing proteins in an environmentally-responsive and physiologically-responsive manner. Biochemical and genetic analyses have recently demonstrated that counter-silencing can occur at a variety of promoter architectures, in contrast to classical transcriptional activation. Disruption of H-NS nucleoprotein filaments by DNA bending is a suggested mechanism by which silencing can be relieved. This review discusses recent advances in our understanding of the mechanisms and importance of xenogeneic silencing and counter-silencing in the successful integration of horizontally-acquired genes into regulatory networks.

Evolutionary expansion of a regulatory network by counter-silencing.

Horizontal gene transfer plays a major role in bacterial evolution. Successful acquisition of new genes requires their incorporation into existing regulatory networks. This study compares the regulation of conserved genes in the PhoPQ regulon of Salmonella enterica serovar Typhimurium with that of PhoPQ-regulated horizontally acquired genes, which are silenced by the histone-like protein H-NS. We demonstrate that PhoP upregulates conserved and horizontally acquired genes by distinct mechanisms. Conserved genes are regulated by classical PhoP-mediated activation and are invariant in promoter architecture, whereas horizontally acquired genes exhibit variable promoter architecture and are regulated by PhoP-mediated counter-silencing. Biochemical analyses show that a horizontally acquired promoter adopts different structures in the silenced and counter-silenced states, implicating the remodelling of the H-NS nucleoprotein filament and the subsequent restoration of open-complex formation as the central mechanism of counter-silencing. Our results indicate that counter-silencing is favoured in the regulatory integration of newly acquired genes because it is able to accommodate multiple promoter architectures.

Maintenance of silent chromatin through replication requires SWI/SNF-like chromatin remodeler SMARCAD1.

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.

The SNF2-family member Fun30 promotes gene silencing in heterochromatic loci.

Chromatin regulates many key processes in the nucleus by controlling access to the underlying DNA. SNF2-like factors are ATP-driven enzymes that play key roles in the dynamics of chromatin by remodelling nucleosomes and other nucleoprotein complexes. Even simple eukaryotes such as yeast contain members of several subfamilies of SNF2-like factors. The FUN30/ETL1 subfamily of SNF2 remodellers is conserved from yeasts to humans, but is poorly characterized. We show that the deletion of FUN30 leads to sensitivity to the topoisomerase I poison camptothecin and to severe cell cycle progression defects when the Orc5 subunit is mutated. We demonstrate a role of FUN30 in promoting silencing in the heterochromatin-like mating type locus HMR, telomeres and the rDNA repeats. Chromatin immunoprecipitation experiments demonstrate that Fun30 binds at the boundary element of the silent HMR and within the silent HMR. Mapping of nucleosomes in vivo using micrococcal nuclease demonstrates that deletion of FUN30 leads to changes of the chromatin structure at the boundary element. A point mutation in the ATP-binding site abrogates the silencing function of Fun30 as well as its toxicity upon overexpression, indicating that the ATPase activity is essential for these roles of Fun30. We identify by amino acid sequence analysis a putative CUE motif as a feature of FUN30/ETL1 factors and show that this motif assists Fun30 activity. Our work suggests that Fun30 is directly involved in silencing by regulating the chromatin structure within or around silent loci.