A Single Heterochromatin Boundary Element Imposes Position-Independent Antisilencing Activity in Saccharomyces cerevisiae Minichromosomes

Chromatin boundary elements serve as cis-acting regulatory DNA signals required to protect genes from the effects of the neighboring heterochromatin. In the yeast genome, boundary elements act by establishing barriers for heterochromatin spreading and are sufficient to protect a reporter gene from transcriptional silencing when inserted between the silencer and the reporter gene. Here we dissected functional topography of silencers and boundary elements within circular minichromosomes in Saccharomyces cerevisiae. We found that both HML-E and HML-I silencers can efficiently repress the URA3 reporter on a multi-copy yeast minichromosome and we further showed that two distinct heterochromatin boundary elements STAR and TEF2-UASrpg are able to limit the heterochromatin spreading in circular minichromosomes. In surprising contrast to what had been observed in the yeast genome, we found that in minichromosomes the heterochromatin boundary elements inhibit silencing of the reporter gene even when just one boundary element is positioned at the distal end of the URA3 reporter or upstream of the silencer elements. Thus the STAR and TEF2-UASrpg boundary elements inhibit chromatin silencing through an antisilencing activity independently of their position or orientation in S. cerevisiae minichromosomes rather than by creating a position-specific barrier as seen in the genome. We propose that the circular DNA topology facilitates interactions between the boundary and silencing elements in the minichromosomes

End-joining long nucleic acid polymers

Many experiments involving nucleic acids require the hybridization and ligation of multiple DNA or RNA molecules to form a compound molecule. When one of the constituents is single stranded, however, the efficiency of ligation can be very low and requires significant individually tailored optimization. Also, when the molecules involved are very long (>10 kb), the reaction efficiency typically reduces dramatically. Here, we present a simple procedure to efficiently and specifically end-join two different nucleic acids using the well-known biotin–streptavidin linkage. We introduce a two-step approach, in which we initially bind only one molecule to streptavidin (STV). The second molecule is added only after complete removal of the unbound STV. This primarily forms heterodimers and nearly completely suppresses formation of unwanted homodimers. We demonstrate that the joining efficiency is 50 ± 25% and is insensitive to molecule length (up to at least 20 kb). Furthermore, our method eliminates the requirement for specific complementary overhangs and can therefore be applied to both DNA and RNA. Demonstrated examples of the method include the efficient end-joining of DNA to single-stranded and double-stranded RNA, and the joining of two double-stranded RNA molecules. End-joining of long nucleic acids using this procedure may find applications in bionanotechnology and in single-molecule experiments

The Set2/Rpd3S Pathway Suppresses Cryptic Transcription without Regard to Gene Length or Transcription Frequency

In cells lacking the histone methyltransferase Set2, initiation of RNA polymerase II transcription occurs inappropriately within the protein-coding regions of genes, rather than being restricted to the proximal promoter. It was previously reported that this “cryptic” transcription occurs preferentially in long genes, and in genes that are infrequently transcribed. Here, we mapped the transcripts produced in an S. cerevisiae strain lacking Set2, and applied rigorous statistical methods to identify sites of cryptic transcription at high resolution. We find that suppression of cryptic transcription occurs independent of gene length or transcriptional frequency. Our conclusions differ with those reported previously because we obtained a higher-resolution dataset, we accounted for the fact that gene length and transcriptional frequency are not independent variables, and we accounted for several ascertainment biases that make cryptic transcription easier to detect in long, infrequently transcribed genes. These new results and conclusions have implications for many commonly used genomic analysis approaches, and for the evolution of high-fidelity RNA polymerase II transcriptional initiation in eukaryotes

The role of multiple marks in epigenetic silencing and the emergence of a stable bivalent chromatin state

We introduce and analyze a minimal model of epigenetic silencing in budding yeast, built upon known biomolecular interactions in the system. Doing so, we identify the epigenetic marks essential for the bistability of epigenetic states. The model explicitly incorporates two key chromatin marks, namely H4K16 acetylation and H3K79 methylation, and explores whether the presence of multiple marks lead to a qualitatively different systems behavior. We find that having both modifications is important for the robustness of epigenetic silencing. Besides the silenced and transcriptionally active fate of chromatin, our model leads to a novel state with bivalent (i.e., both active and silencing) marks under certain perturbations (knock-out mutations, inhibition or enhancement of enzymatic activity). The bivalent state appears under several perturbations and is shown to result in patchy silencing. We also show that the titration effect, owing to a limited supply of silencing proteins, can result in counter-intuitive responses. The design principles of the silencing system is systematically investigated and disparate experimental observations are assessed within a single theoretical framework. Specifically, we discuss the behavior of Sir protein recruitment, spreading and stability of silenced regions in commonly-studied mutants (e.g., sas2, dot1) illuminating the controversial role of Dot1 in the systems biology of yeast silencing.Comment: Supplementary Material, 14 page

Responsibility and laboratory animal research governance

The use of animals in experiments and research remains highly contentious. Laboratory animal research governance provides guidance and regulatory frameworks to oversee the use and welfare of laboratory animals and relies heavily on the replacement, reduction, and refinement (3Rs) principles to demonstrate responsibility. However, the application of the 3Rs is criticized for being too narrow in focus and closing down societal concerns and political questions about the purpose of animal laboratory research. These critiques challenge the legitimacy of responsibility in laboratory animal research governance and call for new approaches. With the advent of the "Responsible Research and Innovation" (RRI) agenda, we investigate whether the notion of responsibility in the controversial area of animal research governance could be enhanced by examining the 3Rs through RRI. Our analysis reveals RRI has the potential to helpfully augment the 3Rs in three key ways: recognizing the need to include a broader range of experts and publics in animal research governance; emphasizing the importance for animal research scientists of taking societal, and not just role, responsibilities into account; and acknowledging the political questions animal research raises

Functional and Structural Insights Revealed by Molecular Dynamics Simulations of an Essential RNA Editing Ligase in Trypanosoma brucei

RNA editing ligase 1 (TbREL1) is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD) simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme

Composition and Acidification of the Culture Medium Influences Chronological Aging Similarly in Vineyard and Laboratory Yeast

Chronological aging has been studied extensively in laboratory yeast by culturing cells into stationary phase in synthetic complete medium with 2% glucose as the carbon source. During this process, acidification of the culture medium occurs due to secretion of organic acids, including acetic acid, which limits survival of yeast cells. Dietary restriction or buffering the medium to pH 6 prevents acidification and increases chronological life span. Here we set out to determine whether these effects are specific to laboratory-derived yeast by testing the chronological aging properties of the vineyard yeast strain RM11. Similar to the laboratory strain BY4743 and its haploid derivatives, RM11 and its haploid derivatives displayed increased chronological life span from dietary restriction, buffering the pH of the culture medium, or aging in rich medium. RM11 and BY4743 also displayed generally similar aging and growth characteristics when cultured in a variety of different carbon sources. These data support the idea that mechanisms of chronological aging are similar in both the laboratory and vineyard strains

HNO Binding in a Heme Protein: Structures, Spectroscopic Properties, and Stabilities

HNO can interact with numerous heme proteins, but atomic level structures are largely unknown. In this work, various structural models for the first stable HNO heme protein complex, MbHNO (Mb, myoglobin), were examined by quantum chemical calculations. This investigation led to the discovery of two novel structural models that can excellently reproduce numerous experimental spectroscopic properties. They are also the first atomic level structures that can account for the experimentally observed high stabilities. These two models involve two distal His conformations as reported previously for MbCNR and MbNO. However, a unique dual hydrogen bonding feature of the HNO binding was not reported before in heme protein complexes with other small molecules such as CO, NO, and O2. These results shall facilitate investigations of HNO bindings in other heme proteins

Silent but Not Static: Accelerated Base-Pair Substitution in Silenced Chromatin of Budding Yeasts

Subtelomeric DNA in budding yeasts, like metazoan heterochromatin, is gene poor, repetitive, transiently silenced, and highly dynamic. The rapid evolution of subtelomeric regions is commonly thought to arise from transposon activity and increased recombination between repetitive elements. However, we found evidence of an additional factor in this diversification. We observed a surprising level of nucleotide divergence in transcriptionally silenced regions in inter-species comparisons of Saccharomyces yeasts. Likewise, intra-species analysis of polymorphisms also revealed increased SNP frequencies in both intergenic and synonymous coding positions of silenced DNA. This analysis suggested that silenced DNA in Saccharomyces cerevisiae and closely related species had increased single base-pair substitution that was likely due to the effects of the silencing machinery on DNA replication or repair