Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM

A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites. A wide spectrum of transitions, transversions and single base deletions are observed. We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ. We show that HR is error-prone in this context because of DNA damage checkpoint activation and base pair lesions and use this knowledge to shift the primary mutagenic outcome of targeted endonuclease breaks from HR-independent rearrangements to HR-dependent point mutations. The ability to switch repair in this way opens up the possibility of using targeted endonucleases in diverse organisms for in vivo-targeted mutagenesis.National Institute of Environmental Health Sciences (Pilot P30-ES002109

A regulatory role for repeated decoy transcription factor binding sites in target gene expression

Repetitive stretches of DNA that contain transcription factor (TF) binding sites can act as decoys that sequester TFs. This study shows that these decoy sites can have important indirect effects on transcriptional regulation by altering the dose–response between a TF and its target promoter

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

Cell-Cycle Dependence of Transcription Dominates Noise in Gene Expression

The large variability in mRNA and protein levels found from both static and dynamic measurements in single cells has been largely attributed to random periods of transcription, often occurring in bursts. The cell cycle has a pronounced global role in affecting transcriptional and translational output, but how this influences transcriptional statistics from noisy promoters is unknown and generally ignored by current stochastic models. Here we show that variable transcription from the synthetic tetO promoter in S. cerevisiae is dominated by its dependence on the cell cycle. Real-time measurements of fluorescent protein at high expression levels indicate tetO promoters increase transcription rate ~2-fold in S/G2/M similar to constitutive genes. At low expression levels, where tetO promoters are thought to generate infrequent bursts of transcription, we observe random pulses of expression restricted to S/G2/M, which are correlated between homologous promoters present in the same cell. The analysis of static, single-cell mRNA measurements at different points along the cell cycle corroborates these findings. Our results demonstrate that highly variable mRNA distributions in yeast are not solely the result of randomly switching between periods of active and inactive gene expression, but instead largely driven by differences in transcriptional activity between G1 and S/G2/M.GM095733BBBE 103316MIT Startup Fun

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.Massachusetts Institute of Technology (Startup funds)National Institutes of Health (U.S.) (grant GM95733

Transcriptional bursts from homologous loci are cell-cycle dependent and partially correlated.

<p>The 3-color diploid strain was grown in microfluidics with 50 ng/mL dox, reducing expression from P_7xtetO. (A) The probability that each P_7xtetO's transcription rate is above background (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003161#pcbi.1003161.s022" target="_blank">Text S1</a>), computed by averaging binarized individual cell responses (ON = 1, or OFF = 0) in each cell-cycle progression bin, increases after G1. (B) A 2D histogram of activation time for each promoter when both activate (t = 0 at budding). Most activation occurs near budding and is correlated. (C) Classifying all single-cell S/G2/M periods (from (A) plus those following an unobserved G1) by whether each P_7xtetO activates reveals correlations in sporadic expression. Error bars represent SEM from bootstrapping.</p

An instantaneous transcription rate is calculated from single-cell fluorescence time series.

<p>(A) In the 3-color diploid strain, tet-Trans-Activator (tTA) expressed from P<i>_MYO2</i> activates P_7xtetO and is repressed by doxycycline. Homologous copies of P_7xtetO drive expression of either <i>CFP</i> or <i>YFP</i> while P<i>_PGK1</i> drives <i>RFP</i> expression constitutively. (B) Monitoring growth of single cells in microfluidic culture. The segmented mother cell, daughter, and contiguous whole cell regions are outlined in blue, green, and red, respectively. For the mother and its daughters, (C) raw volume and (D) CFP concentration time series were smoothed to remove measurement noise; (E) integrated CFP fluorescence was calculated as their product (corresponding regions shown in B). The sum of bud and mother values until division constitutes a whole cell trace, which when extended past division, yields a running total trace that is fit to a differentiable smoothing spline. (F) Calculated relative mRNA levels and (G) instantaneous transcription rate.</p