Effective interpretation of genome function and genetic variation requires a shift from epigenetic mapping of cis-regulatory elements (CREs) to characterization of endogenous function. We developed HCR-FlowFISH, a broadly applicable approach to characterize CRISPR-perturbed CREs via accurate quantification of native transcripts, alongside CASA (CRISPR Activity Screen Analysis), a hierarchical Bayesian model to quantify CRE activity. Across >325,000 perturbations, we provide evidence that CREs can regulate multiple genes, skip over the nearest gene, and can display activating and/or silencing effects. At the cholesterol-level associated FADS locus, we combine endogenous screens with reporter assays to exhaustively characterize multiple genome-wide association signals, functionally nominating causal variants and importantly, identifying their target genes.

Genome-wide association studies have uncovered hundreds of autoimmune disease-associated loci; however, the causal genetic variant(s) within each locus are mostly unknown. Here, we perform high-throughput allele-specific reporter assays to prioritize disease-associated variants for five autoimmune diseases. By examining variants that both promote allele-specific reporter expression and are located in accessible chromatin, we identify 60 putatively causal variants that enrich for statistically fine-mapped variants by up to 57.8-fold. We introduced the risk allele of a prioritized variant (rs72928038) into a human T cell line and deleted the orthologous sequence in mice, both resulting in reduced BACH2 expression. Naïve CD8 T cells from mice containing the deletion had reduced expression of genes that suppress activation and maintain stemness. Our results represent an example of an effective approach for prioritizing variants and studying their physiologically relevant effects.

Cis-regulatory elements (CREs) control gene expression, orchestrating tissue identity, developmental timing, and stimulus responses, which collectively define the thousands of unique cell types in the body. While there is great potential for strategically incorporating CREs in therapeutic or biotechnology applications that require tissue specificity, there is no guarantee that an optimal CRE for an intended purpose has arisen naturally through evolution. Here, we present a platform to engineer and validate synthetic CREs capable of driving gene expression with programmed cell type specificity. We leverage innovations in deep neural network modeling of CRE activity across three cell types, efficient in silico optimization, and massively parallel reporter assays (MPRAs) to design and empirically test thousands of CREs. Through in vitro and in vivo validation, we show that synthetic sequences outperform natural sequences from the human genome in driving cell type-specific expression. Synthetic sequences leverage unique sequence syntax to promote activity in the on-target cell type and simultaneously reduce activity in off-target cells. Together, we provide a generalizable framework to prospectively engineer CREs and demonstrate the required literacy to write regulatory code that is fit-for-purpose in vivo across vertebrates.

Planar cell polarity (PCP) refers to the coordination of global organ axes and individual cell polarity in vertebrate and invertebrate epithelia. Mechanisms of PCP have been best studied in the Drosophila wing, in which each epidermal cell produces a single wing hair at the distal cell edge, and this spatial specification is mediated by redistribution of the core group proteins, including the seven-pass transmembrane cadherin Flamingo ⁄ Starry night (Fmi ⁄ Stan), to selective plasma membrane domains. Through genetic screening, we found that a mutation of the SMC3 gene caused dramatic misspecification of wing hair positions. SMC3 protein is one subunit of the cohesin complex, which regulates sister chromatid cohesion and also plays a role in transcriptional control of gene expression. In the SMC3 mutant cells, Fmi appeared to be upregulated by a posttranscriptional mechanism(s), and this elevation of Fmi was at least one cause of the PCP defect. In addition to the PCP phenotype, the loss of the cohesin function affected wing morphogenesis at multiple levels: one malformation was loss of the wing margin, and this was most likely a result of downregulation of the homeodomain protein Cut. At the cellular level, apical cell size and hexagonal packing were affected in the mutant wing. Dysfunction of cohesin in humans results in Cornelia de Lange syndrome (CdLS), which is characterized by various developmental abnormalities and mental retardation. Our analysis of cohesin in epithelia may provide new insight into cellular and molecular mechanisms of CdLS.

Human CD8 + T cells are functionally heterogeneous and can be divided into phenotypically and functionally distinct subsets according to CCR7 and CD45RA expression levels. Among these, CCR7 low CD45RA low effector memory CD8 + T cells (Tem) and CCR7 low CD45RA high CD8 + T cells, which are designated as Temra and considered to be terminally differentiated cells, are Ag-experienced T cells but show different functionalities. Here, we show that, while Tem proliferate vigorously and produce IFN-γ persistently and robustly, Temra proliferate poorly and lose the ability to produce IFN-γ over time after TCR stimulation. Temra showed impaired cell growth upon TCR stimulation, which was associated with defective activation of the mammalian target of rapamycin (mTOR) signaling. Furthermore, rapamycin, an inhibitor of mTOR signaling, interfered with the robust and continuous proliferation of and IFN-γ production by Tem at later time points after TCR stimulation. Thus, these data collectively indicate that activation of mTOR signaling is required for the robust functions of Tem cells in humans and suggest that defective mTOR signaling in Temra contributes to their functional impairment.Keywords: CCR7 r CD45RA r CD8 + T cells r IFN-γ r mTOR signaling Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionMemory CD8 + T cells develop and persist for a long period of time after the first encounter with exogenous antigen [1,2]. They are phenotypically and functionally heterogeneous and surface markers have been used to delineate different memory T-cell subsets. For example, in mice, CD62L low CD127 low cells are called effector memory cells and CD62L high CD127 high cells are called central memory cells [3,4]. It has been proposed that effector memory cells display a higher ability to produce effector cytokines than central memory cells, whereas the latter display a higher ability to proliferate in response to secondary Ag encounter than the former, and that they are in anatomically different locations [4,5].Correspondence: Dr. Ruka Setoguchi e-mail: ruka@ak.med.kyoto-u.ac.jp Human CD8 + T cells in peripheral blood are also heterogeneous and can be divided into four subpopulations according to their level of surface expression of CCR7 and CD45RA, CCR7 high CD45RA high naïve phenotype, CCR7 high CD45RA low central memory (Tcm) phenotype, CCR7 low CD45RA low effector memory (Tem) phenotype, and CCR7 low CD45RA high CD8 + T cells that are considered to be terminally differentiated cells (Temra) [6,7] The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr kinase that plays an important role in promoting mRNA translation and protein synthesis [16,17]. One of the key players operating downstream of mTOR signaling is ribosomal protein S6 kinase (S6K), and one of its major substrates, ribosomal protein S6 (rpS6), is essential for ribosomal biogenesis [18]. Activation of mTOR signaling in T cells occurs in response to nutrients, cytokines, an...