Christopher M. Vockley

Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers

Technologies that enable targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we describe a programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. The fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers. Gene activation by the targeted acetyltransferase was highly specific across the genome. In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with an individual guide RNA. We also show that the core p300 domain can be fused to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a robust tool for manipulating gene regulation.

RNA-guided gene activation by CRISPR-Cas9–based transcription factors

Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators

Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function.

Regulation of chromatin accessibility and Zic binding at enhancers in the developing cerebellum

To identify chromatin mechanisms of neuronal differentiation, we characterized chromatin accessibility and gene expression in cerebellar granule neurons (CGNs) of the developing mouse. We used DNase-seq to map accessibility of cis-regulatory elements and RNA-seq to profile transcript abundance across postnatal stages of neuronal differentiation in vivo and in culture. We observed thousands of chromatin accessibility changes as CGNs differentiated, and verified, using H3K27ac ChIP-seq, reporter gene assays and CRISPR-mediated activation, that many of these regions function as neuronal enhancers. Motif discovery in differentially accessible chromatin regions suggested a previously unknown role for the Zic family of transcription factors in CGN maturation. We confirmed the association of Zic with these elements by ChIP-seq and found, using knockdown, that Zic1 and Zic2 are required for coordinating mature neuronal gene expression patterns. Together, our data reveal chromatin dynamics at thousands of gene regulatory elements that facilitate the gene expression patterns necessary for neuronal differentiation and function.

Evidence for multiple roles for grainyheadlike 2 in the establishment and maintenance of human mucociliary airway epithelium

Most of the airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated basal progenitor cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia, there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in coordinating multiple cellular processes required for epithelial morphogenesis, differentiation, remodeling, and repair. However, only a few target genes have been identified, and little is known about GRHL function in the adult lung. Here we focus on the role of GRHL2 in primary human bronchial epithelial cells, both as undifferentiated progenitors and as they differentiate in air–liquid interface culture into an organized mucociliary epithelium with transepithelial resistance. Using a dominant-negative protein or shRNA to inhibit GRHL2, we follow changes in epithelial phenotype and gene transcription using RNA sequencing or microarray analysis. We identify several hundreds of genes that are directly or indirectly regulated by GRHL2 in both undifferentiated cells and air–liquid interface cultures. Using ChIP sequencing to map sites of GRHL2 binding in the basal cells, we identify 7,687 potential primary targets and confirm that GRHL2 binding is strongly enriched near GRHL2-regulated genes. Taken together, the results support the hypothesis that GRHL2 plays a key role in regulating many physiological functions of human airway epithelium, including those involving cell morphogenesis, adhesion, and motility.

Massively parallel quantification of the regulatory effects of noncoding genetic variation in a human cohort

We report a novel high-throughput method to empirically quantify individual-specific regulatory element activity at the population scale. The approach combines targeted DNA capture with a high-throughput reporter gene expression assay. As demonstration, we measured the activity of more than 100 putative regulatory elements from 95 individuals in a single experiment. In agreement with previous reports, we found that most genetic variants have weak effects on distal regulatory element activity. Because haplotypes are typically maintained within but not between assayed regulatory elements, the approach can be used to identify causal regulatory haplotypes that likely contribute to human phenotypes. Finally, we demonstrate the utility of the method to functionally fine map causal regulatory variants in regions of high linkage disequilibrium identified by expression quantitative trait loci (eQTL) analyses.

Detection and characterization of silencers and enhancer-blockers in the greater CFTR locus

Silencers and enhancer-blockers (EBs) are cis-acting, negative regulatory elements (NREs) that control interactions between promoters and enhancers. Although relatively uncharacterized in terms of biological mechanisms, these elements are likely to be abundant in the genome. We developed an experimental strategy to identify silencers and EBs using transient transfection assays. A known insulator and EB from the chicken beta-globin locus, cHS4, served as a control element for these assays. We examined 47 sequences from a 1.8-Mb region of human chromosome 7 for silencer and EB activities. The majority of functional elements displayed directional and promoter-specific activities. A limited number of sequences acted in a dual manner, as both silencers and EBs. We examined genomic data, epigenetic modifications, and sequence motifs within these regions. Strong silencer elements contained a novel CT-rich motif, often in multiple copies. Deletion of the motif from three regions caused a measurable loss of silencing ability in these sequences. Moreover, five duplicate occurrences of this motif were identified in the cHS4 insulator. These motifs provided an explanation for an uncharacterized silencing activity we measured in the insulator element. Overall, we identified 15 novel NREs, which contribute new insights into the prevalence and composition of sequences that negatively regulate gene expression.

Decoding the role of regulatory element polymorphisms in complex disease.

Genetic variation in gene regulatory elements contributes to diverse human diseases, ranging from rare and severe developmental defects to common and complex diseases such as obesity and diabetes. Early examples of regulatory mechanisms of human diseases involve large chromosomal rearrangements that change the regulatory connections within the genome. Single nucleotide variants in regulatory elements can also contribute to disease, potentially via demonstrated associations with changes in transcription factor binding, enhancer activity, post-translational histone modifications, long-range enhancer-promoter interactions, or RNA polymerase recruitment. Establishing causality between non-coding genetic variants, gene regulation, and disease has recently become more feasible with advances in genome-editing and epigenome-editing technologies. As establishing causal regulatory mechanisms of diseases becomes routine, functional annotation of target genes is likely to emerge as a major bottleneck for translation into patient benefits. In this review, we discuss the history and recent advances in understanding the regulatory mechanisms of human disease, and new challenges likely to be encountered once establishing those mechanisms becomes rote.

Enhanced MyoD-induced transdifferentiation to a myogenic lineage by fusion to a potent transactivation domain.

Genetic reprogramming holds great potential for disease modeling, drug screening, and regenerative medicine. Genetic reprogramming of mammalian cells is typically achieved by forced expression of natural transcription factors that control master gene networks and cell lineage specification. However, in many instances, the natural transcription factors do not induce a sufficiently robust response to completely reprogram cell phenotype. In this study, we demonstrate that protein engineering of the master transcription factor MyoD can enhance the conversion of human dermal fibroblasts and adult stem cells to a skeletal myocyte phenotype. Fusion of potent transcriptional activation domains to MyoD led to increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. This work supports a general strategy for synthetically enhancing the direct conversion between cell types that can be applied in both synthetic biology and regenerative medicine.

Many long intergenic non-coding RNAs distally regulate mRNA gene expression levels

Long intergenic non-coding RNAs (lincRNA) are members of a class of non-protein-coding RNA transcript that has recently been shown to contribute to gene regulatory processes and disease etiology. It has been hypothesized that lincRNAs influence disease risk through the regulation of mRNA transcription [88], possibly by interacting with regulatory proteins such as chromatin-modifying complexes [37, 50]. The hypothesis of the regulation of mRNA by lincRNAs is based on a small number of specific lincRNAs analyses; the cellular roles of lincRNAs regulation have not been catalogued genome-wide. Relative to mRNAs, lincRNAs tend to be expressed at lower levels and in more tissue-specific patterns, making genome-wide studies of their regulatory capabilities difficult [15]. Here we develop a method for Mendelian randomization leveraging expression quantitative trait loci (eQTLs) that regulate the expression levels of lincRNAs (linc-eQTLs) to perform such a study across four primary tissues. We find that linc-eQTLs are largely similar to protein-coding eQTLs (pc-eQTLs) in cis-regulatory element enrichment, which supports the hypothesis that lincRNAs are regulated by the same transcriptional machinery as protein-coding RNAs [15, 80] and validates our linc-eQTLs. We catalog 74 lincRNAs with linc-eQTLs that are in linkage disequilibrium with TASs and are in protein-coding gene deserts; the putative lincRNA-regulated traits are highly enriched for adipose-related traits relative to mRNA-regulated traits.