Detection of gene cis-regulatory element perturbations in single-cell transcriptomes.

We introduce poly-adenine CRISPR gRNA-based single-cell RNA-sequencing (pAC-Seq), a method that enables the direct observation of guide RNAs (gRNAs) in scRNA-seq. We use pAC-Seq to assess the phenotypic consequences of CRISPR/Cas9 based alterations of gene cis-regulatory regions. We show that pAC-Seq is able to detect cis-regulatory-induced alteration of target gene expression even when biallelic loss of target gene expression occurs in only ~5% of cells. This low rate of biallelic loss significantly increases the number of cells required to detect the consequences of changes to the regulatory genome, but can be ameliorated by transcript-targeted sequencing. Based on our experimental results we model the power to detect regulatory genome induced transcriptomic effects based on the rate of mono/biallelic loss, baseline gene expression, and the number of cells per target gRNA.

Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay.

A key mechanism in cellular regulation is the ability of the transcriptional machinery to physically access DNA. Transcription factors interact with DNA to alter the accessibility of chromatin, which enables changes to gene expression during development or disease or as a response to environmental stimuli. However, the regulation of DNA accessibility via the recruitment of transcription factors is difficult to study in the context of the native genome because every genomic site is distinct in multiple ways. Here we introduce the multiplexed integrated accessibility assay (MIAA), an assay that measures chromatin accessibility of synthetic oligonucleotide sequence libraries integrated into a controlled genomic context with low native accessibility. We apply MIAA to measure the effects of sequence motifs on cell type-specific accessibility between mouse embryonic stem cells and embryonic stem cell-derived definitive endoderm cells, screening 7905 distinct DNA sequences. MIAA recapitulates differential accessibility patterns of 100-nt sequences derived from natively differential genomic regions, identifying E-box motifs common to epithelial-mesenchymal transition driver transcription factors in stem cell-specific accessible regions that become repressed in endoderm. We show that a single binding motif for a key regulatory transcription factor is sufficient to open chromatin, and classify sets of stem cell-specific, endoderm-specific, and shared accessibility-modifying transcription factor motifs. We also show that overexpression of two definitive endoderm transcription factors, T and Foxa2, results in changes to accessibility in DNA sequences containing their respective DNA-binding motifs and identify preferential motif arrangements that influence accessibility.

A CRISPR view of gene regulation.

Due to plummeting costs, whole genome sequencing of patients and cancers will soon become routine medical practice; however, we cannot currently predict how non-coding genotype affects cellular gene expression. Gene regulation research has recently been dominated by observational approaches that correlate chromatin state with regulatory function. These approaches are limited to the available genotypes and cannot scratch the surface of possible sequence combinations, and thus there is a need for perturbation-based approaches to better understand how DNA encodes gene regulatory functions. CRISPR/Cas9 genome editing has revolutionized our ability to alter genome sequence, and CRISPR/Cas9-based assays have already begun to contribute to new paradigms of gene regulation. We discuss the variety of arenas in which current and future CRISPR-based technologies will aid in developing predictive understanding of how genome sequence leads to gene regulatory function.

High-throughput mapping of regulatory DNA.

Quantifying the effects of cis-regulatory DNA on gene expression is a major challenge. Here, we present the multiplexed editing regulatory assay (MERA), a high-throughput CRISPR-Cas9-based approach that analyzes the functional impact of the regulatory genome in its native context. MERA tiles thousands of mutations across ∼40 kb of cis-regulatory genomic space and uses knock-in green fluorescent protein (GFP) reporters to read out gene activity. Using this approach, we obtain quantitative information on the contribution of cis-regulatory regions to gene expression. We identify proximal and distal regulatory elements necessary for expression of four embryonic stem cell-specific genes. We show a consistent contribution of neighboring gene promoters to gene expression and identify unmarked regulatory elements (UREs) that control gene expression but do not have typical enhancer epigenetic or chromatin features. We compare thousands of functional and nonfunctional genotypes at a genomic location and identify the base pair-resolution functional motifs of regulatory elements.

EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B.

The Aurora B kinase coordinates kinetochore-microtubule attachments with spindle checkpoint signaling on each mitotic chromosome. We find that EB1, a microtubule plus end-tracking protein, is required to enrich Aurora B at inner centromeres in a microtubule-dependent manner. This regulates phosphorylation of both kinetochore and chromatin substrates. EB1 regulates the histone phosphorylation marks (histone H2A phospho-Thr120 and histone H3 phospho-Thr3) that localize Aurora B. The chromosomal passenger complex containing Aurora B can be found on a subset of spindle microtubules that exist near prometaphase kinetochores, known as preformed K-fibers (kinetochore fibers). Our data suggest that EB1 enables the spindle microtubules to regulate the phosphorylation of kinetochores through recruitment of the Aurora B kinase.

Insights into the inhibition of the p90 ribosomal S6 kinase (RSK) by the flavonol glycoside SL0101 from the 1.5 Å crystal structure of the N-terminal domain of RSK2 with bound inhibitor.

The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-α-L-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 Å resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the αD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.

Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis.

Aurora B is a component of the chromosomal passenger complex (CPC) required for correct spindle-kinetochore attachments during chromosome segregation and for cytokinesis. The chromatin factors that recruit the CPC to centromeres are unknown, however. Here we show that phosphorylation of histone H3 threonine 3 (H3T3ph) by Haspin is necessary for CPC accumulation at centromeres and that the CPC subunit Survivin binds directly to H3T3ph. A nonbinding Survivin-D70A/D71A mutant does not support centromeric CPC concentration, and both Haspin depletion and Survivin-D70A/D71A mutation diminish centromere localization of the kinesin MCAK and the mitotic checkpoint response to taxol. Survivin-D70A/D71A mutation and microinjection of H3T3ph-specific antibody both compromise centromeric Aurora B functions but do not prevent cytokinesis. Therefore, H3T3ph generated by Haspin positions the CPC at centromeres to regulate selected targets of Aurora B during mitosis.