SUMMARY While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ~1000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retro-translocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.

Endoplasmic reticulum (ER) stress activates a set of signaling pathways, collectively termed the unfolded protein response (UPR). The three UPR branches (IRE1, PERK, and ATF6) promote cell survival by reducing misfolded protein levels. UPR signaling also promotes apoptotic cell death if ER stress is not alleviated. How the UPR integrates its cytoprotective and proapoptotic outputs to select between life or death cell fates is unknown. We found that IRE1 and ATF6 activities were attenuated by persistent ER stress in human cells. By contrast, PERK signaling, including translational inhibition and proapoptotic transcription regulator Chop induction, was maintained. When IRE1 activity was sustained artificially, cell survival was enhanced, suggesting a causal link between the duration of UPR branch signaling and life or death cell fate after ER stress. Key findings from our studies in cell culture were recapitulated in photoreceptors expressing mutant rhodopsin in animal models of retinitis pigmentosa.

The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. Here we demonstrate that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, our results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi.

The X-linked Xist gene encodes a large untranslated RNA that has been implicated in mammalian dosage compensation and in spermatogenesis. To investigate the function of the Xist gene product, we have generated male and female mice that carry a deletion in the structural gene but maintain a functional Xist promoter. Mutant males were healthy and fertile. Females that inherited the mutation from their mothers were also normal and had the wild-type paternal X chromosome inactive in every cell. In contrast to maternal transmission, females that carry the mutation on the paternal X chromosome were severely growth-retarded and died early in embryogenesis. The wild-type maternal X chromosome was inactive in every cell of the growth-retarded embryo proper, whereas both X chromosomes were expressed in the mutant female trophoblast where X inactivation is imprinted. However, an XO mouse with a paternally inherited Xist mutation was healthy and appeared normal. The imprinted lethal phenotype of the mutant females is therefore due to the inability of extraembryonic tissue with two active X chromosomes to sustain the embryo. Our results indicate that the Xist RNA is required for female dosage compensation but plays no role in spermatogenesis.

Histone lysine residues can be mono-, di-, or trimethylated. These posttranslational modifications regulate the affinity of effector proteins and may also impact chromatin structure independent of their role as adaptors. In order to study histone lysine methylation, particularly in the context of chromatin, we have developed a chemical approach to install analogs of methyl lysine into recombinant proteins. This approach allows for the rapid generation of large quantities of histones in which the site and degree of methylation can be specified. We demonstrate that these methyl-lysine analogs (MLAs) are functionally similar to their natural counterparts. These methylated histones were used to examine the influence of specific lysine methylation on the binding of effecter proteins and the rates of nucleosome remodeling. This simple method of introducing site-specific and degree-specific methylation into recombinant histones provides a powerful tool to investigate the biochemical mechanisms by which lysine methylation influences chromatin structure and function.

SUMMARY Proper regulation of chromatin structure is necessary for the maintenance of cell type-specific gene expression patterns. The embryonic stem cell (ESC) expression pattern governs self-renewal and pluripotency. Here, we present an RNAi screen in mouse ESCs of 1008 loci encoding chromatin proteins. We identified 68 proteins that exhibit diverse phenotypes upon knockdown (KD), including seven subunits of the Tip60-p400 complex. Phenotypic analyses revealed that Tip60-p400 is necessary to maintain characteristic features of ESCs. We show that p400 localization to the promoters of both silent and active genes is dependent upon histone H3 lysine 4 trimethylation (H3K4me3). Furthermore, the Tip60-p400 KD gene expression profile is enriched for developmental regulators and significantly overlaps with that of the transcription factor Nanog. Depletion of Nanog reduces p400 binding to target promoters without affecting H3K4me3 levels. Together, these data indicate that Tip60-p400 integrates signals from Nanog and H3K4me3 to regulate gene expression in ESCs.

Dosage compensation in mammals is achieved by the transcriptional inactivation of one X chromosome in female cells. From the time X chromosome inactivation was initially described, it was clear that several mechanisms must be precisely integrated to achieve correct regulation of this complex process. X-inactivation appears to be triggered upon differentiation, suggesting its regulation by developmental cues. Whereas any number of X chromosomes greater than one is silenced, only one X chromosome remains active. Silencing on the inactive X chromosome coincides with the acquisition of a multitude of chromatin modifications, resulting in the formation of extraordinarily stable facultative heterochromatin that is faithfully propagated through subsequent cell divisions. The integration of all these processes requires a region of the X chromosome known as the X-inactivation center, which contains the Xist gene and its cis-regulatory elements. Xist encodes an RNA molecule that plays critical roles in the choice of which X chromosome remains active, and in the initial spread and establishment of silencing on the inactive X chromosome. We are now on the threshold of discovering the factors that regulate and interact with Xist to control X-inactivation, and closer to an understanding of the molecular mechanisms that underlie this complex process.

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