Mapping chromatin modifications at the single cell level.

Understanding chromatin regulation holds enormous promise for controlling gene regulation, predicting cellular identity, and developing diagnostics and cellular therapies. However, the dynamic nature of chromatin, together with cell-to-cell heterogeneity in its structure, limits our ability to extract its governing principles. Single cell mapping of chromatin modifications, in conjunction with expression measurements, could help overcome these limitations. Here, we review recent advances in single cell-based measurements of chromatin modifications, including optimization to reduce DNA loss, improved DNA sequencing, barcoding, and antibody engineering. We also highlight several applications of these techniques that have provided insights into cell-type classification, mapping modification co-occurrence and heterogeneity, and monitoring chromatin dynamics.

High-throughput discovery and characterization of viral transcriptional effectors in human cells.

Viruses encode transcriptional regulatory proteins critical for controlling viral and host gene expression. Given their multifunctional nature and high sequence divergence, it is unclear which viral proteins can affect transcription and which specific sequences contribute to this function. Using a high-throughput assay, we measured the transcriptional regulatory potential of over 60,000 protein tiles across ∼1,500 proteins from 11 coronaviruses and all nine human herpesviruses. We discovered hundreds of transcriptional effector domains, including a conserved repression domain in all coronavirus Spike homologs, dual activation-repression domains in viral interferon regulatory factors (VIRFs), and an activation domain in six herpesvirus homologs of the single-stranded DNA-binding protein that we show is important for viral replication and late gene expression in Kaposi's sarcoma-associated herpesvirus (KSHV). For the effector domains we identified, we investigated their mechanisms via high-throughput sequence and chemical perturbations, pinpointing sequence motifs essential for function. This work massively expands viral protein annotations, serving as a springboard for studying their biological and health implications and providing new candidates for compact gene regulation tools.

The H3.3 K36M oncohistone disrupts the establishment of epigenetic memory through loss of DNA methylation.

Histone H3.3 is frequently mutated in cancers, with the lysine 36 to methionine mutation (K36M) being a hallmark of chondroblastomas. While it is known that H3.3K36M changes the cellular epigenetic landscape, it remains unclear how it affects the dynamics of gene expression. Here, we use a synthetic reporter to measure the effect of H3.3K36M on silencing and epigenetic memory after recruitment of KRAB: a member of the largest class of human repressors, commonly used in synthetic biology, and associated with H3K9me3. We find that H3.3K36M, which decreases H3K36 methylation, leads to a decrease in epigenetic memory and promoter methylation weeks after KRAB release. We propose a new model for establishment and maintenance of epigenetic memory, where H3K36 methylation is necessary to convert H3K9me3 domains into DNA methylation for stable epigenetic memory. Our quantitative model can inform oncogenic mechanisms and guide development of epigenetic editing tools.

High-throughput functional characterization of combinations of transcriptional activators and repressors.

Despite growing knowledge of the functions of individual human transcriptional effector domains, much less is understood about how multiple effector domains within the same protein combine to regulate gene expression. Here, we measure transcriptional activity for 8,400 effector domain combinations by recruiting them to reporter genes in human cells. In our assay, weak and moderate activation domains synergize to drive strong gene expression, whereas combining strong activators often results in weaker activation. In contrast, repressors combine linearly and produce full gene silencing, and repressor domains often overpower activation domains. We use this information to build a synthetic transcription factor whose function can be tuned between repression and activation independent of recruitment to target genes by using a small-molecule drug. Altogether, we outline the basic principles of how effector domains combine to regulate gene expression and demonstrate their value in building precise and flexible synthetic biology tools. A record of this paper's transparent peer review process is included in the supplemental information.

Dynamic spreading of chromatin-mediated gene silencing and reactivation between neighboring genes in single cells.

In mammalian cells genes that are in close proximity can be transcriptionally coupled: silencing or activating one gene can affect its neighbors. Understanding these dynamics is important for natural processes, such as heterochromatin spreading during development and aging, and when designing synthetic gene regulation circuits. Here, we systematically dissect this process in single cells by recruiting and releasing repressive chromatin regulators at dual-gene synthetic reporters, and measuring how fast gene silencing and reactivation spread as a function of intergenic distance and configuration of insulator elements. We find that silencing by KRAB, associated with histone methylation, spreads between two genes within hours, with a time delay that increases with distance. This fast KRAB-mediated spreading is not blocked by the classical cHS4 insulators. Silencing by histone deacetylase HDAC4 of the upstream gene can also facilitate background silencing of the downstream gene by PRC2, but with a days-long delay that does not change with distance. This slower silencing can sometimes be stopped by insulators. Gene reactivation of neighboring genes is also coupled, with strong promoters and insulators determining the order of reactivation. Our data can be described by a model of multi-gene regulation that builds upon previous knowledge of heterochromatin spreading, where both gene silencing and gene reactivation can act at a distance, allowing for coordinated dynamics via chromatin regulator recruitment.

CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons.

CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type-specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons. VIDEO ABSTRACT.

Memory regulatory T cells reside in human skin.

Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity. We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in inflammatory skin disease.