Multimodal perturbation analyses of cyclin-dependent kinases reveal a network of synthetic lethalities associated with cell-cycle regulation and transcriptional regulation.

Cell-cycle control is accomplished by cyclin-dependent kinases (CDKs), motivating extensive research into CDK targeting small-molecule drugs as cancer therapeutics. Here we use combinatorial CRISPR/Cas9 perturbations to uncover an extensive network of functional interdependencies among CDKs and related factors, identifying 43 synthetic-lethal and 12 synergistic interactions. We dissect CDK perturbations using single-cell RNAseq, for which we develop a novel computational framework to precisely quantify cell-cycle effects and diverse cell states orchestrated by specific CDKs. While pairwise disruption of CDK4/6 is synthetic-lethal, only CDK6 is required for normal cell-cycle progression and transcriptional activation. Multiple CDKs (CDK1/7/9/12) are synthetic-lethal in combination with PRMT5, independent of cell-cycle control. In-depth analysis of mRNA expression and splicing patterns provides multiple lines of evidence that the CDK-PRMT5 dependency is due to aberrant transcriptional regulation resulting in premature termination. These inter-dependencies translate to drug-drug synergies, with therapeutic implications in cancer and other diseases.

Long-distance association of topological boundaries through nuclear condensates.

The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of "housekeeping" genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates.

Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs.

Recruiting endogenous adenosine deaminases using exogenous guide RNAs to edit cellular RNAs is a promising therapeutic strategy, but editing efficiency and durability remain low using current guide RNA designs. In this study, we engineered circular ADAR-recruiting guide RNAs (cadRNAs) to enable more efficient programmable adenosine-to-inosine RNA editing without requiring co-delivery of any exogenous proteins. Using these cadRNAs, we observed robust and durable RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, and high transcriptome-wide specificity. Additionally, we increased transcript-level specificity for the target adenosine by incorporating interspersed loops in the antisense domains, reducing bystander editing. In vivo delivery of cadRNAs via adeno-associated viruses enabled 53% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler syndrome. cadRNAs enable efficient programmable RNA editing in vivo with diverse protein modulation and gene therapeutic applications.

Comprehensive interrogation of the ADAR2 deaminase domain for engineering enhanced RNA editing activity and specificity.

Adenosine deaminases acting on RNA (ADARs) can be repurposed to enable programmable RNA editing, however their exogenous delivery leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, especially those flanked by a 5' guanosine is very low thus limiting their utility as a transcriptome engineering toolset. Towards addressing these issues, we first performed a novel deep mutational scan of the ADAR2 deaminase domain, directly measuring the impact of every amino acid substitution across 261 residues, on RNA editing. This enabled us to create a domain-wide mutagenesis map while also revealing a novel hyperactive variant with improved enzymatic activity at 5'-GAN-3' motifs. As overexpression of ADAR enzymes, especially hyperactive variants, can lead to significant transcriptome-wide off-targeting, we next engineered a split-ADAR2 deaminase which resulted in >100-fold more specific RNA editing as compared to full-length deaminase overexpression. Taken together, we anticipate this systematic engineering of the ADAR2 deaminase domain will enable broader utility of the ADAR toolset for RNA biotechnology applications.

Computational approaches for inferring 3D conformations of chromatin from chromosome conformation capture data.

Chromosome conformation capture (3C) and its variants are powerful experimental techniques for probing intra- and inter-chromosomal interactions within cell nuclei at high resolution and in a high-throughput, quantitative manner. The contact maps derived from such experiments provide an avenue for inferring the 3D spatial organization of the genome. This review provides an overview of the various computational methods developed in the past decade for addressing the very important but challenging problem of deducing the detailed 3D structure or structure population of chromosomal domains, chromosomes, and even entire genomes from 3C contact maps.

Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly.

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17ß-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA-dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.

In vivo RNA editing of point mutations via RNA-guided adenosine deaminases.

We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.

Quantification of DNA cleavage specificity in Hi-C experiments.

Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered.

Efficient estimation of contact probabilities from inter-bead distance distributions in simulated polymer chains.

The estimation of contact probabilities (CP) from conformations of simulated bead-chain polymer models is a key step in methods that aim to elucidate the spatial organization of chromatin from analysis of experimentally determined contacts between different genomic loci. Although CPs can be estimated simply by counting contacts between beads in a sample of simulated chain conformations, reliable estimation of small CPs through this approach requires a large number of conformations, which can be computationally expensive to obtain. Here we describe an alternative computational method for estimating relatively small CPs without requiring large samples of chain conformations. In particular, we estimate the CPs from functional approximations to the cumulative distribution function (cdf) of the inter-bead distance for each pair of beads. These cdf approximations are obtained by fitting the extended generalized lambda distribution (EGLD) to inter-bead distances determined from a sample of chain conformations, which are in turn generated by Monte Carlo simulations. We find that CPs estimated from fitted EGLD cdfs are significantly more accurate than CPs estimated using contact counts from samples of limited size, and are more precise with all sample sizes, permitting as much as a tenfold reduction in conformation sample size for chains of 200 beads and samples smaller than 10(5) conformations. This method of CP estimation thus has potential to accelerate computational efforts to elucidate the spatial organization of chromatin.

Recovering ensembles of chromatin conformations from contact probabilities.

The 3D higher order organization of chromatin within the nucleus of eukaryotic cells has so far remained elusive. A wealth of relevant information, however, is increasingly becoming available from chromosome conformation capture (3C) and related experimental techniques, which measure the probabilities of contact between large numbers of genomic sites in fixed cells. Such contact probabilities (CPs) can in principle be used to deduce the 3D spatial organization of chromatin. Here, we propose a computational method to recover an ensemble of chromatin conformations consistent with a set of given CPs. Compared with existing alternatives, this method does not require conversion of CPs to mean spatial distances. Instead, we estimate CPs by simulating a physically realistic, bead-chain polymer model of the 30-nm chromatin fiber. We then use an approach from adaptive filter theory to iteratively adjust the parameters of this polymer model until the estimated CPs match the given CPs. We have validated this method against reference data sets obtained from simulations of test systems with up to 45 beads and 4 loops. With additional testing against experiments and with further algorithmic refinements, our approach could become a valuable tool for researchers examining the higher order organization of chromatin.

Computational prediction of efficient splice sites for trans-splicing ribozymes.

Group I introns have been engineered into trans-splicing ribozymes capable of replacing the 3'-terminal portion of an external mRNA with their own 3'-exon. Although this design makes trans-splicing ribozymes potentially useful for therapeutic application, their trans-splicing efficiency is usually too low for medical use. One factor that strongly influences trans-splicing efficiency is the position of the target splice site on the mRNA substrate. Viable splice sites are currently determined using a biochemical trans-tagging assay. Here, we propose a rapid and inexpensive alternative approach to identify efficient splice sites. This approach involves the computation of the binding free energies between ribozyme and mRNA substrate. We found that the computed binding free energies correlate well with the trans-splicing efficiency experimentally determined at 18 different splice sites on the mRNA of chloramphenicol acetyl transferase. In contrast, our results from the trans-tagging assay correlate less well with measured trans-splicing efficiency. The computed free energy components suggest that splice site efficiency depends on the following secondary structure rearrangements: hybridization of the ribozyme's internal guide sequence (IGS) with mRNA substrate (most important), unfolding of substrate proximal to the splice site, and release of the IGS from the 3'-exon (least important). The proposed computational approach can also be extended to fulfill additional design requirements of efficient trans-splicing ribozymes, such as the optimization of 3'-exon and extended guide sequences.

Policing starter unit selection of the enterocin type II polyketide synthase by the type II thioesterase EncL.

Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete 'Streptomyces maritimus'. The enterocin biosynthesis gene cluster (enc) codes for proteins involved in the assembly and attachment of the rare benzoate primer that initiates polyketide assembly with the addition of seven malonate molecules and culminates in a Favorskii-like rearrangement of the linear poly-ß-ketone to give its distinctive non-aromatic, caged core structure. Fundamental to enterocin biosynthesis, which utilizes a single acyl carrier protein (ACP), EncC, for both priming with benzoate and elongating with malonate, involves maintaining the correct balance of acyl-EncC substrates for efficient polyketide assembly. Here, we report the characterization of EncL as a type II thioesterase that functions to edit starter unit (mis)priming of EncC. We performed a series of in vivo mutational studies, heterologous expression experiments, in vitro reconstitution studies, and Fourier-transform mass spectrometry-monitored competitive enzyme assays that together support the proposed selective hydrolase activity of EncL toward misprimed acetyl-ACP over benzoyl-ACP to facilitate benzoyl priming of the enterocin PKS complex. While this system resembles the R1128 PKS that also utilizes an editing thioesterase (ZhuC) to purge acetate molecules from its initiation module ACP in favor of alkylacyl groups, the enterocin system is distinct in its usage of a single ACP for both priming and elongating reactions with different substrates.

Biophysics of knotting.

Knots appear in a wide variety of biophysical systems, ranging from biopolymers, such as DNA and proteins, to macroscopic objects, such as umbilical cords and catheters. Although significant advancements have been made in the mathematical theory of knots and some progress has been made in the statistical mechanics of knots in idealized chains, the mechanisms and dynamics of knotting in biophysical systems remain far from fully understood. We report on recent progress in the biophysics of knotting-the formation, characterization, and dynamics of knots in various biophysical contexts.

Interpretation of tandem mass spectra obtained from cyclic nonribosomal peptides.

Natural and non-natural cyclic peptides are a crucial component in drug discovery programs because of their considerable pharmaceutical properties. Cyclosporin, microcystins, and nodularins are all notable pharmacologically important cyclic peptides. Because these biologically active peptides are often biosynthesized nonribosomally, they often contain nonstandard amino acids, thus increasing the complexity of the resulting tandem mass spectrometry data. In addition, because of the cyclic nature, the fragmentation patterns of many of these peptides showed much higher complexity when compared to related counterparts. Therefore, at the present time it is still difficult to annotate cyclic peptides MS/MS spectra. In this current work, an annotation program was developed for the annotation and characterization of tandem mass spectra obtained from cyclic peptides. This program, which we call MS-CPA is available as a web tool (http://lol.ucsd.edu/ms-cpa_v1/Input.py). Using this program, we have successfully annotated the sequence of representative cyclic peptides, such as seglitide, tyrothricin, desmethoxymajusculamide C, dudawalamide A, and cyclomarins, in a rapid manner and also were able to provide the first-pass structure evidence of a newly discovered natural product based on predicted sequence. This compound is not available in sufficient quantities for structural elucidation by other means such as NMR. In addition to the development of this cyclic annotation program, it was observed that some cyclic peptides fragmented in unexpected ways resulting in the scrambling of sequences. In summary, MS-CPA not only provides a platform for rapid confirmation and annotation of tandem mass spectrometry data obtained with cyclic peptides but also enables quantitative analysis of the ion intensities. This program facilitates cyclic peptide analysis, sequencing, and also acts as a useful tool to investigate the uncommon fragmentation phenomena of cyclic peptides and aids the characterization of newly discovered cyclic peptides encountered in drug discovery programs.

Top-down mass spectrometry on low-resolution instruments: characterization of phosphopantetheinylated carrier domains in polyketide and non-ribosomal biosynthetic pathways.

Mass spectrometry (MS) is an important tool for studying non-ribosomal peptide, polyketide, and fatty acid biosynthesis. Here we describe a new approach using multi-stage tandem MS on a common ion trap instrument to obtain high-resolution measurements of the masses of substrates and intermediates bound to phosphopantetheinylated (holo) carrier proteins. In particular, we report the chemical formulas of 12 diagnostic MS(3) fragments of the phosphopantetheine moiety ejected from holo carrier proteins during MS(2). We demonstrate our method by observing the formation of holo-AcpC, a putative acyl carrier protein from Streptococcus agalactiae.

Enzymatic total synthesis of enterocin polyketides.

Polyketides are clinically important natural products that often require elaborate organic syntheses owing to their complex chemical structures. Here we report the multienzyme total synthesis of the Streptomyces maritimus enterocin and wailupemycin bacteriostatic agents in a single reaction vessel from simple benzoate and malonate substrates. To our knowledge, our results represent the first in vitro assembly of a complete type II polyketide synthase enzymatic pathway to natural products.

Low-temperature structural transitions: circumventing the broken-ergodicity problem.

We consider systems undergoing very-low-temperature solid-solid transitions, exhibiting the well-known "broken-ergodicity" problem that is often so severe that even the replica exchange method converges too slowly. We propose an improvement of the latter, which consists of coupling the lower-temperature random walks to analytically generated random walks corresponding to an auxiliary harmonic superposition system. Numerically accurate results are obtained for several Lennard-Jones clusters, which have so far been treated only by the harmonic superposition approximation.

Structural transformations and melting in neon clusters: quantum versus classical mechanics.

The extraordinary complexity of Lennard-Jones (LJ) clusters, which exhibit numerous structures and "phases" when their size or temperature is varied, presents a great challenge for accurate numerical simulations, even without accounting for quantum effects. To study the latter, we utilize the variational Gaussian wave packet method in conjunction with the exchange Monte Carlo sampling technique. We show that the quantum nature of neon clusters has a substantial effect on their size-temperature "phase diagrams," particularly the critical parameters of certain structural transformations. We also give a numerical confirmation that none of the nonicosahedral structures observed for some classical LJ clusters are favorable in the quantum case.