A Three-Way Combinatorial CRISPR Screen for Analyzing Interactions among Druggable Targets.

We present a CRISPR-based multi-gene knockout screening system and toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations and successfully validate double- and triple-combination regimens for suppression of cancer cell growth and protection against Parkinson's disease-associated toxicity. This system overcomes the practical challenges of experimenting on a large number of high-order genetic and drug combinations and can be applied to uncover the rare synergistic interactions between druggable targets.

Probing flecainide block of INa using human pluripotent stem cell-derived ventricular cardiomyocytes adapted to automated patch-clamping and 2D monolayers.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are emerging tools for applications such as drug discovery and screening for pro-arrhythmogenicity and cardiotoxicity as leading causes for drug attrition. Understanding the electrophysiology (EP) of hPSC-CMs is essential but conventional manual patch-clamping is highly laborious and low-throughput. Here we adapted hPSC-CMs derived from two human embryonic stem cell (hESC) lines, HES2 and H7, for a 16-channel automated planar-recording approach for single-cell EP characterization. Automated current- and voltage-clamping, with an overall success rate of 55.0 ±â€¯11.3%, indicated that 90% of hPSC-CMs displayed ventricular-like action potential (AP) and the ventricular cardiomyocytes (VCMs) derived from the two hESC lines expressed similar levels of INa, ICaL, Ikr and If and similarly lacked Ito and IK1. These well-characterized hPSC-VCMs could also be readily adapted for automated assays of pro-arrhythmic drug screening. As an example, we showed that flecainide (FLE) induced INa blockade, leftward steady-state inactivation shift, slowed recovery from inactivation in our hPSC-VCMs. Since single-cell EP assay is insufficient to predict drug-induced reentrant arrhythmias, hPSC-VCMs were further reassembled into 2D human ventricular cardiac monolayers (hvCMLs) for multi-cellular electrophysiological assessments. Indeed, FLE significantly slowed the conduction velocity while causing AP prolongation. Our RNA-seq data suggested that cell-cell interaction enhanced the maturity of hPSC-VCMs. Taken collectively, a combinatorial approach using single-cell EP and hvCMLs is needed to comprehensively assess drug-induced arrhythmogenicity.

Effect of valproic acid on radiation-induced DNA damage in euchromatic and heterochromatic compartments.

The distinction between heterochromatin and euchromatin in the double-strand break (DSB) damage pathway is of interest, recent reports indicate that chromatin is not created equally nor is it acquiescent to DSBs. Using the classical histone deacetylase inhibitor, Trichostatin A, we have previously demonstrated that chromatin represents a heterogeneous substrate with respect to histone tail modification by histone deacetylase inhibitors and consequent responses to DNA damage and repair. Here, we extended the initial findings by investigating the radiation sensitizing properties of the widely used antiepileptic, valproic acid. Clonogenic survival assays confirm that valproic acid is an efficient sensitizer of radiation-induced cell death. The radiosensitizing effect is correlated with valproic acid-mediated histone hyperacetylation, chromatin decondensation and enhanced formation of radiation-induced gammaH2AX preferentially on euchromatic alleles. Heterochromatin was much more resistant to histone tail modification, changes in chromatin architecture and DNA damage. These findings are consolidated by studies with the structurally related analogue, valpromide, which does not inhibit histone deacetylase enzymes. At a relatively low concentration (1 mM) valpromide did not cause chromatin modifications and radiation sensitivity, providing further evidence that the radiation sensitizing properties of valproic acid are at least in part, due to histone modification-dependent effects on euchromatin. When higher concentrations (5 mM) were used, both compounds resulted in significant radiation sensitivity, albeit, with differing efficacy (dose modifying factors of 1.5 and 1.2 for valproic acid and valpromide, respectively). The findings imply that histone-modification independent mechanisms also contribute to the radiation sensitizing properties of valproic acid. Overall, our findings are consistent with the emerging interest in the use histone deacetylase inhibitors in combination with radiotherapy for the treatment of cancer.

Brahma links the SWI/SNF chromatin-remodeling complex with MeCP2-dependent transcriptional silencing.

Transcriptional repression of methylated genes can be mediated by the methyl-CpG binding protein MeCP2. Here we show that human Brahma (Brm), a catalytic component of the SWI/SNF-related chromatin-remodeling complex, associates with MeCP2 in vivo and is functionally linked with repression. We used a number of different molecular approaches and chromatin immunoprecipitation strategies to show a unique cooperation between Brm, BAF57 and MeCP2. We show that Brm and MeCP2 assembly on chromatin occurs on methylated genes in cancer and the gene FMR1 in fragile X syndrome. These experimental findings identify a new role for SWI/SNF in gene repression by MeCP2.