Proximity sequencing for the detection of mRNA, extracellular proteins and extracellular protein complexes in single cells.

Complex cellular functions occur via the coordinated formation and dissociation of protein complexes. Functions such as the response to a signaling ligand can incorporate dozens of proteins and hundreds of complexes. Until recently, it has been difficult to measure multiple protein complexes at the single-cell level. Here, we present a step-by-step procedure for proximity sequencing, which enables the simultaneous measurement of proteins, mRNA and hundreds of protein complexes located on the outer membrane of cells. We guide the user through probe creation, sample preparation, staining, sequencing and computational quantification of protein complexes. This protocol empowers researchers to study, for example, the interplay between transcriptional states and cellular functions by coupling measurements of transcription to measurements of linked effector molecules, yet could be generalizable to other paired events. The protocol requires roughly 16 h spread over several days to complete by users with expertise in basic molecular biology and single-cell sequencing.

Computational prediction of protein interactions in single cells by proximity sequencing.

Proximity sequencing (Prox-seq) simultaneously measures gene expression, protein expression and protein complexes on single cells. Using information from dual-antibody binding events, Prox-seq infers surface protein dimers at the single-cell level. Prox-seq provides multi-dimensional phenotyping of single cells in high throughput, and was recently used to track the formation of receptor complexes during cell signaling and discovered a novel interaction between CD9 and CD8 in naïve T cells. The distribution of protein abundance can affect identification of protein complexes in a complicated manner in dual-binding assays like Prox-seq. These effects are difficult to explore with experiments, yet important for accurate quantification of protein complexes. Here, we introduce a physical model of Prox-seq and computationally evaluate several different methods for reducing background noise when quantifying protein complexes. Furthermore, we developed an improved method for analysis of Prox-seq data, which resulted in more accurate and robust quantification of protein complexes. Finally, our Prox-seq model offers a simple way to investigate the behavior of Prox-seq data under various biological conditions and guide users toward selecting the best analysis method for their data.

Herpesviruses mimic zygotic genome activation to promote viral replication.

DUX4 is a germline transcription factor and a master regulator of zygotic genome activation. During early embryogenesis, DUX4 is crucial for maternal to zygotic transition at the 2-8-cell stage in order to overcome silencing of genes and enable transcription from the zygotic genome. In adult somatic cells, DUX4 expression is silenced and its activation in adult muscle cells causes the genetic disorder Facioscapulohumeral Muscular Dystrophy (FSHD). Here we show that herpesviruses from alpha-, beta- and gamma-herpesvirus subfamilies as well as papillomaviruses actively induce DUX4 expression to promote viral transcription and replication. We demonstrate that HSV-1 immediate early proteins directly induce expression of DUX4 and its target genes including endogenous retroelements, which mimics zygotic genome activation. We further show that DUX4 directly binds to the viral genome and promotes viral transcription. DUX4 is functionally required for herpesvirus infection, since genetic depletion of DUX4 by CRISPR/Cas9 abrogates viral replication. Our results show that herpesviruses induce DUX4 expression and its downstream germline-specific genes and retroelements, thus mimicking an early embryonic-like transcriptional program that prevents epigenetic silencing of the viral genome and facilitates herpesviral gene expression.