Minimal requirements for the epigenetic inheritance of engineered silent chromatin domains.

Mechanisms enabling genetically identical cells to differentially regulate gene expression are complex and central to organismal development and evolution. While gene silencing pathways involving DNA sequence-specific recruitment of histone-modifying enzymes are prevalent in nature, examples of sequence-independent heritable gene silencing are scarce. Studies of the fission yeast Schizosaccharomyces pombe indicate that sequence-independent propagation of heterochromatin can occur but requires numerous multisubunit protein complexes and their diverse activities. Such complexity has so far precluded a coherent articulation of the minimal requirements for heritable gene silencing by conventional in vitro reconstitution approaches. Here, we take an unconventional approach to defining these requirements by engineering sequence-independent silent chromatin inheritance in budding yeast Saccharomyces cerevisiae cells. The mechanism conferring memory upon these cells is remarkably simple and requires only two proteins, one that recognizes histone H3 lysine 9 methylation (H3K9me) and catalyzes the deacetylation of histone H4 lysine 16 (H4K16), and another that recognizes deacetylated H4K16 and catalyzes H3K9me. Together, these bilingual "read-write" proteins form an interdependent positive feedback loop that is sufficient for the transmission of DNA sequence-independent silent information over multiple generations.

Measuring prion propagation in single bacteria elucidates a mechanism of loss.

Prions are self-propagating protein aggregates formed by specific proteins that can adopt alternative folds. Prions were discovered as the cause of the fatal transmissible spongiform encephalopathies in mammals, but prions can also constitute nontoxic protein-based elements of inheritance in fungi and other species. Prion propagation has recently been shown to occur in bacteria for more than a hundred cell divisions, yet a fraction of cells in these lineages lost the prion through an unknown mechanism. Here, we investigate prion propagation in single bacterial cells as they divide using microfluidics and fluorescence microscopy. We show that the propagation occurs in two distinct modes. In a fraction of the population, cells had multiple small visible aggregates and lost the prion through random partitioning of aggregates to one of the two daughter cells at division. In the other subpopulation, cells had a stable large aggregate localized to the pole; upon division the mother cell retained this polar aggregate and a daughter cell was generated that contained small aggregates. Extending our findings to prion domains from two orthologous proteins, we observe similar propagation and loss properties. Our findings also provide support for the suggestion that bacterial prions can form more than one self-propagating state. We implement a stochastic version of the molecular model of prion propagation from yeast and mammals that recapitulates all the observed single-cell properties. This model highlights challenges for prion propagation that are unique to prokaryotes and illustrates the conservation of fundamental characteristics of prion propagation.

A bacteria-based genetic assay detects prion formation.

Prions are infectious, self-propagating protein aggregates that are notorious for causing devastating neurodegenerative diseases in mammals. Recent evidence supports the existence of prions in bacteria. However, the evaluation of candidate bacterial prion-forming proteins has been hampered by the lack of genetic assays for detecting their conversion to an aggregated prion conformation. Here we describe a bacteria-based genetic assay that distinguishes cells carrying a model yeast prion protein in its nonprion and prion forms. We then use this assay to investigate the prion-forming potential of single-stranded DNA-binding protein (SSB) of Campylobacter hominis Our findings indicate that SSB possesses a prion-forming domain that can transition between nonprion and prion conformations. Furthermore, we show that bacterial cells can propagate the prion form over 100 generations in a manner that depends on the disaggregase ClpB. The bacteria-based genetic tool we present may facilitate the investigation of prion-like phenomena in all domains of life.

A bacterial global regulator forms a prion.

Prions are self-propagating protein aggregates that act as protein-based elements of inheritance in fungi. Although prevalent in eukaryotes, prions have not been identified in bacteria. Here we found that a bacterial protein, transcription terminator Rho of Clostridium botulinum (Cb-Rho), could form a prion. We identified a candidate prion-forming domain (cPrD) in Cb-Rho and showed that it conferred amyloidogenicity on Cb-Rho and could functionally replace the PrD of a yeast prion-forming protein. Furthermore, its cPrD enabled Cb-Rho to access alternative conformations in Escherichia coli-a soluble form that terminated transcription efficiently and an aggregated, self-propagating prion form that was functionally compromised. The prion form caused genome-wide changes in the transcriptome. Thus, Cb-Rho functions as a protein-based element of inheritance in bacteria, suggesting that the emergence of prions predates the evolutionary split between eukaryotes and bacteria.

The bacterial cell cycle regulator GcrA is a σ70 cofactor that drives gene expression from a subset of methylated promoters.

Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we found that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active σ(70)-dependent promoters in vivo but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact σ(70) interact with domain 4, GcrA interfaces with domain 2, the region that binds the -10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrated that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identified a regulon of ∼ 200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than recruitment exclusively.

Prion propagation can occur in a prokaryote and requires the ClpB chaperone.

Prions are self-propagating protein aggregates that are characteristically transmissible. In mammals, the PrP protein can form a prion that causes the fatal transmissible spongiform encephalopathies. Prions have also been uncovered in fungi, where they act as heritable, protein-based genetic elements. We previously showed that the yeast prion protein Sup35 can access the prion conformation in Escherichia coli. Here, we demonstrate that E. coli can propagate the Sup35 prion under conditions that do not permit its de novo formation. Furthermore, we show that propagation requires the disaggregase activity of the ClpB chaperone. Prion propagation in yeast requires Hsp104 (a ClpB ortholog), and prior studies have come to conflicting conclusions about ClpB's ability to participate in this process. Our demonstration of ClpB-dependent prion propagation in E. coli suggests that the cytoplasmic milieu in general and a molecular machine in particular are poised to support protein-based heredity in the bacterial domain of life.

Key features of σS required for specific recognition by Crl, a transcription factor promoting assembly of RNA polymerase holoenzyme.

Bacteria use multiple sigma factors to coordinate gene expression in response to environmental perturbations. In Escherichia coli and other γ-proteobacteria, the transcription factor Crl stimulates σ(S)-dependent transcription during times of cellular stress by promoting the association of σ(S) with core RNA polymerase. The molecular basis for specific recognition of σ(S) by Crl, rather than the homologous and more abundant primary sigma factor σ(70), is unknown. Here we use bacterial two-hybrid analysis in vivo and p-benzoyl-phenylalanine cross-linking in vitro to define the features in σ(S) responsible for specific recognition by Crl. We identify residues in σ(S) conserved domain 2 (σ(S)2) that are necessary and sufficient to allow recognition of σ(70) conserved domain 2 by Crl, one near the promoter-melting region and the other at the position where a large nonconserved region interrupts the sequence of σ(70). We then use luminescence resonance energy transfer to demonstrate directly that Crl promotes holoenzyme assembly using these specificity determinants on σ(S). Our results explain how Crl distinguishes between sigma factors that are largely homologous and activates discrete sets of promoters even though it does not bind to promoter DNA.

Direct activator/co-activator interaction is essential for bacteriophage T4 middle gene expression.

The bacteriophage T4 AsiA protein is a bifunctional regulator that inhibits transcription from the major class of bacterial promoters and also serves as an essential co-activator of transcription from T4 middle promoters. AsiA binds the primary s factor in Escherichia coli, sigma(70), and modifies the promoter recognition properties of the sigma(70)-containing RNA polymerase(RNAP) holoenzyme. In its role as co-activator, AsiA directs RNAP to T4 middle promoters in the presence of the T4-encoded activator MotA. According to the current model for T4 middle promoter activation, AsiA plays an indirect role in stabilizing the activation complex by facilitating interaction between DNA-bound MotA and sigma(70). Here we show that AsiA also plays a direct role in T4 middle promoter activation by contacting the MotA activation domain. Furthermore,we show that interaction between AsiA and the beta-flap domain of RNAP is important for co-activation. Based on our findings, we propose a revised model for T4 middle promoter activation, with AsiA organizing the activation complex via three distinct protein-protein interactions.

The bacteriophage T4 AsiA protein contacts the beta-flap domain of RNA polymerase.

To initiate transcription from specific promoters, the bacterial RNA polymerase (RNAP) core enzyme must associate with the initiation factor sigma, which contains determinants that allow sequence-specific interactions with promoter DNA. Most bacteria contain several sigma factors, each of which directs recognition of a distinct set of promoters. A large and diverse family of proteins known as "anti-sigma factors" regulates promoter utilization by targeting specific sigma factors. The founding member of this family is the AsiA protein of bacteriophage T4. AsiA specifically targets the primary sigma factor in Escherichia coli, sigma(70), and inhibits transcription from the major class of sigma(70)-dependent promoters. AsiA-dependent transcription inhibition has been attributed to a well-documented interaction between AsiA and conserved region 4 of sigma(70). Here, we establish that efficient AsiA-dependent transcription inhibition also requires direct protein-protein contact between AsiA and the RNAP core. In particular, we demonstrate that AsiA contacts the flap domain of the RNAP beta-subunit (the beta-flap). Our findings support the emerging view that the beta-flap is a target site for regulatory proteins that affect RNAP function during all stages of the transcription cycle.

Rsd family proteins make simultaneous interactions with regions 2 and 4 of the primary sigma factor.

Bacterial anti-sigma factors typically regulate sigma factor function by restricting the access of their cognate sigma factors to the RNA polymerase (RNAP) core enzyme. The Escherichia coli Rsd protein forms a complex with the primary sigma factor, sigma(70), inhibits sigma(70)-dependent transcription in vitro, and has been proposed to function as a sigma(70)-specific anti-sigma factor, thereby facilitating the utilization of alternative sigma factors. In prior work, Rsd has been shown to interact with conserved region 4 of sigma(70), but it is not known whether this interaction suffices to account for the regulatory functions of Rsd. Here we show that Rsd and the Rsd orthologue AlgQ, a global regulator of gene expression in Pseudomonas aeruginosa, interact with conserved region 2 of sigma(70). We show further that Rsd and AlgQ can interact simultaneously with regions 2 and 4 of sigma(70). Our findings establish that the abilities of Rsd and AlgQ to interact with sigma(70) region 2 are important determinants of their in vitro and in vivo activities.