High-throughput single-cell functional elucidation of neurodevelopmental disease-associated genes reveals convergent mechanisms altering neuronal differentiation

The overwhelming success of exome- and genome-wide association studies in discovering thousands of disease-associated genes necessitates developing novel high-throughput functional genomics approaches to elucidate the molecular mechanisms of these genes. Here, we have coupled multiplexed repression of neurodevelopmental disease-associated genes to single-cell transcriptional profiling in differentiating human neurons to rapidly assay the functions of multiple genes in a disease-relevant context, assess potentially convergent mechanisms, and prioritize genes for specific functional assays. For a set of 13 autism spectrum disorder (ASD)-associated genes, we show that this approach generated important mechanistic insights, revealing two functionally convergent modules of ASD genes: one that delays neuron differentiation and one that accelerates it. Five genes that delay neuron differentiation

Changes in nucleosome occupancy occur in a chromosome specific manner

In the eukaryotic nucleus, DNA is packaged into chromatin. The fundamental subunit of chromatin is the nucleosome, DNA is wrapped 1.6 times around a histone octamer core. Nuclear processes in eukaryotes are impacted by whether regulatory DNA is occupied by nucleosomes. We used microarrays to measure nucleosome occupancy in human cells post-Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation at hundreds of immunity-related loci. The detailed analysis of these technologies can be found in recent publications from our lab [1,3]. We found that nucleosome redistributions displayed chromosome specific nucleosome occupancy. This resource can be used to map nucleosome distributions in a variety of biological contexts

Single-Cell RNA-Seq Uncovers a Robust Transcriptional Response to Morphine by Glia

Summary: Molecular and behavioral responses to opioids are thought to be primarily mediated by neurons, although there is accumulating evidence that other cell types play a prominent role in drug addiction. To investigate cell-type-specific opioid responses, we performed single-cell RNA sequencing (scRNA-seq) of the nucleus accumbens of mice following acute morphine treatment. Differential expression analysis uncovered unique morphine-dependent transcriptional responses by oligodendrocytes and astrocytes. We examined the expression of selected genes, including Cdkn1a and Sgk1, by FISH, confirming their induction by morphine in oligodendrocytes. Further analysis using RNA-seq of FACS-purified oligodendrocytes revealed a large cohort of morphine-regulated genes. The affected genes are enriched for roles in cellular pathways intimately linked to oligodendrocyte maturation and myelination, including the unfolded protein response. Altogether, our data illuminate the morphine-dependent transcriptional response by oligodendrocytes and offer mechanistic insights into myelination defects associated with opioid abuse. : Avey et al. use single-cell RNA-seq to uncover cell-type-specific responses to morphine in the brains of mice, revealing a unique and robust response by oligodendrocytes, which they further validate by multiple approaches. These analyses offer insights into the molecular mechanisms underlying oligodendrocyte dysfunction in the context of opioid abuse. Keywords: opioid, morphine, addiction, single-cell, RNA-seq, oligodendrocyte, UPR, glucocorticoid, nucleus accumbens, myeli

Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication

<div><p>Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5’ UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates components of the host cell machinery via modulation of RSK activity. Thus, this study has important implications for the pathobiology of KSHV and other diseases in which RSK activity is dysregulated.</p></div

Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response

Nucleosome occupancy is critically important in regulating access to the eukaryotic genome. Few studies in human cells have measured genome-wide nucleosome distributions at high temporal resolution during a response to a common stimulus. We measured nucleosome distributions at high temporal resolution following Kaposi's-sarcoma-associated herpesvirus (KSHV) reactivation using our newly developed mTSS-seq technology, which maps nucleosome distribution at the transcription start sites (TSS) of all human genes. Nucleosomes underwent widespread changes in organization 24 hours after KSHV reactivation and returned to their basal nucleosomal architecture 48 hours after KSHV reactivation. The widespread changes consisted of an indiscriminate remodeling event resulting in the loss of nucleosome rotational phasing signals. Additionally, one in six TSSs in the human genome possessed nucleosomes that are translationally remodeled. 72% of the loci with translationally remodeled nucleosomes have nucleosomes that moved to positions encoded by the underlying DNA sequence. Finally we demonstrated that these widespread alterations in nucleosomal architecture potentiated regulatory factor binding. These descriptions of nucleosomal architecture changes provide a new framework for understanding the role of chromatin in the genomic response, and have allowed us to propose a hierarchical model for chromatin-based regulation of genome response

DAVID bioinformatic analysis of the phosphoproteomic screening results.

<p>The enrichment of Gene Ontology (GO) terms associated with the putative substrates of ORF45-activated RSK (121 unique phosphopeptides with ≥2.5-fold change in phosphorylation between A66F and F66A) was assessed in the background of the human proteome. The x-axes values denote the percentage of the 98 unique proteins that were enriched (black bars) or the fold enrichment (gray bars) for the indicated GO term, functional category, or protein domain. P-values were all <0.05. The exact p-values and additional information can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004993#ppat.1004993.s002" target="_blank">S2 File</a>.</p

Diagram of the upstream signaling pathways that converge on activation of AGC kinases.

<p>In dark blue are the KSHV proteins that have been shown to affect various nodes of the PI3K/Akt/mTOR and/or Raf/MEK/ERK signaling pathways. In red are the kinase inhibitors used in this study. AGC kinases, including Akt, p70S6K (S6K), and p90RSK (RSK), are shown in light blue, and phosphorylate their substrates at the RxRxxS*/T* motif, leading to regulation of key biological processes.</p

CRISPR/Cas9-mediated knockout of RSK1/2 results in the reduction of RSK substrate phosphorylation, viral late lytic gene expression, and virion production.

<p>(A and B) SLK-iBAC cells transduced with empty lentiCRISPR or single cell clones with RSK1/2 knockout (#1 and #15) were uninduced (U) or induced (I) with dox/butyrate, and cells were harvested at the indicated dpi. Lysates were analyzed by western blot with the indicated antibodies. (C) The indicated cell lines were induced as in (A and B). Twenty-four hpi, cells were harvested and RNAs were extracted, reverse transcribed, and subjected to qRT-PCR analysis using the indicated primers. Relative mRNA level is shown normalized to β-Actin. (D) KSHV progeny virion production is compromised by RSK knockout. SLK-iBAC cells were induced as described in (A and B). At the indicated hpi, media were collected from cells, extracellular virion DNA was extracted, and viral genome copy number was measured by qPCR.</p

The increased phosphorylation of putative RSK substrates induced by KSHV lytic reactivation is dramatically reduced by ORF45 mutation or deletion.

<p>Stable iSLK.219 or iSLK.BAC16 cells carrying the indicated BAC (F66A, Stop45, or the F66A revertant (A66F)), were uninduced (U) or induced (I) with doxycycline and butyrate. Cell lysates were harvested at 0, 6, 12, 24, 48, and 72 hours post-induction (hpi) and analyzed by western blot using the indicated antibodies.</p

Phosphoproteomic analysis allows for the identification of putative substrates of ORF45-activated RSK during KSHV lytic replication.

<p>(A) Flowchart of the experimental design. (B) Phosphorylation motif for RSK, the 110B7E antibody used for the PhosphoScan, and the experimental data. (C) Scatterplot representation of the unique phosphopeptides identified by the screening that met the cutoff for deltaCN, intensity, and spectral counts. (D) Pie chart of the protein type/function distribution among the unique phosphopeptides with at least a 2.5-fold change (A66F:F66A).</p