Search

Regulation of neuronal metabolism during early brain development is crucial for directing synaptic plasticity and proper circuit formation. Alterations in neuronal glycolysis or mitochondrial function are associated with several neuropsychiatric disorders, including schizophrenia. Recently, loss-of-function mutations in SETD1A, a histone methyltransferase, have been linked to increased schizophrenia risk and global developmental delay. Here, we show that heterozygous disruption of SETD1A in human induced pluripotent stem cell (hiPSC)-derived neurons results in reduced neurite outgrowth and spontaneous activity, two phenotypes commonly associated with schizophrenia, as well as alterations in metabolic capacity. Furthermore, supplementing culture media with metabolic intermediates ameliorated changes in neurite outgrowth and spontaneous activity, suggesting that metabolic dysfunction contributes to neuronal phenotypes caused by SETD1A haploinsufficiency. These findings highlight a previously unknown connection between SETD1A function, metabolic regulation, and neuron development, and identifies alternative avenues for therapeutic development.

The human immune system is composed of a distributed network of cells circulating throughout the body, which must dynamically form physical associations and communicate using interactions between their cell-surface proteomes. Despite their therapeutic potential, our map of these surface interactions remains incomplete. Here, using a high-throughput surface receptor screening method, we systematically mapped the direct protein interactions across a recombinant library that encompasses most of the surface proteins that are detectable on human leukocytes. We independently validated and determined the biophysical parameters of each novel interaction, resulting in a high-confidence and quantitative view of the receptor wiring that connects human immune cells. By integrating our interactome with expression data, we identified trends in the dynamics of immune interactions and constructed a reductionist mathematical model that predicts cellular connectivity from basic principles. We also developed an interactive multi-tissue single-cell atlas that infers immune interactions throughout the body, revealing potential functional contexts for new interactions and hubs in multicellular networks. Finally, we combined targeted protein stimulation of human leukocytes with multiplex high-content microscopy to link our receptor interactions to functional roles, in terms of both modulating immune responses and maintaining normal patterns of intercellular associations. Together, our work provides a systematic perspective on the intercellular wiring of the human immune system that extends from systems-level principles of immune cell connectivity down to mechanistic characterization of individual receptors, which could offer opportunities for therapeutic intervention., Nature, 608 (7922), ISSN:0028-0836, ISSN:1476-4687

Embarking on a PhD provides many opportunities for personal and professional development beyond scientific research. This instalment of the Words of Advice series aims to provide guidance and tips on harnessing these resources to build a well-rounded CV and increase your chances of getting hired after your PhD. We provide two perspectives on developing your CV to optimise career opportunities in academia and beyond. The first perspective is by Dr Zheng-Shan Chong, a post-doctoral researcher in Singapore, and focuses on the acquisition of a wide range of skills and experience that could open doors to a career outside of academia. Beyond her day job, Shan manages an article series on bioentrepreneurship and career development for Biotech Connection Singapore, which has allowed her to speak to several researchers who have successfully transitioned to non-research roles. Here, she summarises the insights gained from these conversations. This is followed by advice and tips from Dr Sara Clohisey, a post-doctoral researcher in Edinburgh who changed fields after her PhD, from Drosophila cell biology to human genetics and virology. Although not quite as dramatic as leaving academia completely, this shift prompted her to rethink her approach to writing an academic CV so that it would appeal to an employer from a different field. Sara's perspective is particularly geared towards careers in research. We hope that these unique perspectives from experienced individuals who have successfully navigated the path from graduate student to working scientist will prove useful to those who are planning their next moves after completing a PhD.

Neighbouring cells can recognise and communicate with each other by direct binding between cell surface receptor and ligand pairs. Examples of cellular recognition events include pathogen entry into a host cell, sperm-egg fusion, and self/nonself discrimination by the immune system. Despite growing appreciation of cell surface recognition molecules as potential therapeutic targets, identifying key factors contributing to cellular recognition remains technically challenging to perform on a genome-wide scale. Recently, genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) knockout or activation (CRISPR-KO/CRISPRa) screens have been applied to identify the molecular determinants of cellular recognition. In this review, we discuss how CRISPR-KO/CRISPRa screening has contributed to our understanding of cellular recognition processes, and how it can be applied to investigate these important interactions in a range of biological contexts. Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.

Extracellular interactions between cell surface receptors are necessary for signaling and adhesion but identifying them remains technically challenging. We describe a cell-based genome-wide approach employing CRISPR activation to identify receptors for a defined ligand. We show receptors for high-affinity antibodies and low-affinity ligands can be unambiguously identified when used in pools or as individual binding probes. We apply this technique to identify ligands for the adhesion G-protein-coupled receptors and show that the Nogo myelin-associated inhibitory proteins are ligands for ADGRB1. This method will enable extracellular receptor-ligand identification on a genome-wide scale. Electronic supplementary material The online version of this article (10.1186/s13059-018-1581-3) contains supplementary material, which is available to authorized users.

Significance Retroviruses can integrate their DNA into the host-cell genome. Inherited retroviral DNA and other transposable elements account for more than half of the human genome. Transposable elements must be tightly regulated to restrict their proliferation and prevent toxic gene expression. KAP1/TRIM28 is an essential regulator of transposable element transcription. We determined the crystal structure of the KAP1 TRIM. The structure identifies a protein–protein interaction site required for recruitment of KAP1 to transposable elements. An epigenetic gene silencing assay confirms the importance of this site for KAP1-dependent silencing. We also show that KAP1 self-assembles in solution, but this self-assembly is not required for silencing. Our work provides insights into KAP1-dependent silencing and tools for expanding our mechanistic understanding of this process., Transcription of transposable elements is tightly regulated to prevent genome damage. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, inducing the assembly of an epigenetic silencing complex, with chromatin remodeling activities that repress transcription of the targeted retrotransposon and adjacent genes. Our biophysical and structural data show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevent higher-order oligomerization without significant loss of retrotransposon silencing activity, indicating that, in contrast to other TRIM-family proteins, self-assembly is not essential for KAP1 function. The crystal structure of the KAP1 TRIM dimer identifies the KRAB domain binding site in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 TRIM responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.

Transcription of transposable elements is tightly regulated to prevent genome damage. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, inducing the assembly of an epigenetic silencing complex, with chromatin remodeling activities that repress transcription of the targeted retrotransposon and adjacent genes. Our biophysical and structural data show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevent higher-order oligomerization without significant loss of retrotransposon silencing activity, indicating that, in contrast to other TRIM-family proteins, self-assembly is not essential for KAP1 function. The crystal structure of the KAP1 TRIM dimer identifies the KRAB domain binding site, in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 TRIM responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing. Significance Retroviruses can integrate their DNA into the host-cell genome. Inherited retroviral DNA and other transposable elements account for over half of the human genome. T ransposable elements must be tightly regulated to restrict their proliferation and prevent toxic gene expression. KAP1/TRIM28 is an essential regulator of transposable element transcription. We determined the crystal structure of the KAP1 TRIM. The structure identifies a protein-protein interaction site required for recruitment of KAP1 to transposable elements. An epigenetic gene silencing assay confirms the importance of this site for KAP1-dependent silencing. We also show that KAP1 self-assembles in solution, but this self-assembly is not required for silencing. Our work provides insights into KAP1-dependent silencing, and tools for expanding our mechanistic understanding of this process.

Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process. Repressors and effectors form close interacting networks in pathways, including RNA processing, G protein signaling, protein ubiquitination, and chromatin modification. Combinatorial knockdown of five repressors (SMAD3, ZMYM2, SFRS11, SAE1, and ESET) synergistically resulted in ∼85% TRA-1-60-positive cells. Removal of the novel splicing factor SFRS11 during reprogramming is accompanied by rapid acquisition of pluripotency-specific spliced forms. Mechanistically, SFRS11 regulates exon skipping and mutually exclusive splicing of transcripts in genes involved in cell differentiation, mRNA splicing, and chromatin modification. Our study provides insights into the reprogramming process, which comprises comprehensive and multi-layered transcriptional, splicing, and epigenetic machineries., Singapore. Agency for Science, Technology and Research (grant JCO R09138), Singapore. Agency for Science, Technology and Research (grant JCO R09125), Singapore. Agency for Science, Technology and Research (Investigatorship research award), National Institutes of Health (U.S.) (NIH grant CA196631-01A1), National Institutes of Health (U.S.) (NIH grant 1U54GM114838-01)

Table S1. A table listing the gRNAs sequences targeting the promoter regions for the named genes. The gene symbol, accession number of the target transcript and chromosomal location are provided. Table S2. A table detailing the sequences of the synthesized DNA fragments and PCR primers used for plasmid construction and sequencing, primers used for q-RT-PCR, and primers for gRNA library preparation and amplification. Table S3. A table providing the sources, and where appropriate, clone names of the primary monoclonal and conjugated secondary antibodies used in this study. (PDF 553 kb)

Figure S1. CRISPR activation enables rapid and stable upregulation of cell surface proteins. Figure S2. A CRISPR activation gRNA library targeting membrane-associated proteins. Figure S3. Enrichment of gRNAs targeting known receptors in cells selected using their corresponding ligand. Figure S4. ADGRB1 directly interacts with all three members of the RTN4R family. (PDF 489 kb)

Table S1. A table listing the gRNAs sequences targeting the promoter regions for the named genes. The gene symbol, accession number of the target transcript and chromosomal location are provided. Table S2. A table detailing the sequences of the synthesized DNA fragments and PCR primers used for plasmid construction and sequencing, primers used for q-RT-PCR, and primers for gRNA library preparation and amplification. Table S3. A table providing the sources, and where appropriate, clone names of the primary monoclonal and conjugated secondary antibodies used in this study. (PDF 553 kb)

Figure S1. CRISPR activation enables rapid and stable upregulation of cell surface proteins. Figure S2. A CRISPR activation gRNA library targeting membrane-associated proteins. Figure S3. Enrichment of gRNAs targeting known receptors in cells selected using their corresponding ligand. Figure S4. ADGRB1 directly interacts with all three members of the RTN4R family. (PDF 489 kb)