A physical wiring diagram for the human immune system.

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 proteomes1. Despite their therapeutic potential2, our map of these surface interactions remains incomplete3,4. 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.

PHF2 regulates genome topology and DNA replication in neural stem cells via cohesin.

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs), which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here, we discover that the histone demethylase PHF2 associates with RAD21, a core subunit of cohesin, to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably, the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient, active replication origins, and can act as boundaries to separate adjacent domains. Accordingly, PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore, we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation, and that PHF2's histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops, where cohesin represses dormant replication origins directly or indirectly, to sustain DNA replication in NSC.

Structure of KAP1 tripartite motif identifies molecular interfaces required for retroelement 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.

Metabolic contributions to neuronal deficits caused by genomic disruption of schizophrenia risk gene SETD1A.

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.

How to build a well-rounded CV and get hired after your PhD.

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.

Investigating Cellular Recognition Using CRISPR/Cas9 Genetic Screening.

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.

Pooled extracellular receptor-ligand interaction screening using CRISPR activation.

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.

RNAi Reveals Phase-Specific Global Regulators of Human Somatic Cell Reprogramming.

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.