Activity-by-contact model of enhancer–promoter regulation from thousands of CRISPR perturbations

Fulco, Charles P.; Nasser, Joseph; Jones, Thouis R.; Munson, Glen; Bergman, Drew T.; Subramanian, Vidya; Grossman, Sharon R.; Anyoha, Rockwell; Doughty, Benjamin R.; Patwardhan, Tejal A.; Nguyen, Tung H.; Kane, Michael F.; Perez, Elizabeth M.; Durand, Neva C.; Lareau, Caleb A.; Stamenova, Elena K.; Aiden, E; Lander, Eric S.; Engreitz, Jesse M.

doi:10.1038/s41588-019-0538-0

Cited by 780 publications

(1,213 citation statements)

References 53 publications

Supporting

Mentioning

1,175

Contrasting

Order By: Relevance

“…Strikingly, replacing the sequence of this single regulatory element is sufficient to cause a more than two-fold increase in expression, which reveals that this repressor element has a major effect on Tdrd7 expression. This result contrasts previous findings that perturbations of individual regulatory elements generally have no measurable or only a small effect on gene expression (47)(48)(49)(50)(51)(52)(53). Previously-observed robustness at the transcriptional level can be attributed to functional redundancy, whereby multiple regulatory elements control the expression of a gene and a substantial change in gene expression can only be achieved by perturbations of multiple elements (50).…”

Section: Discussioncontrasting

confidence: 94%

Recapitulating evolutionary divergence in a singlecis-regulatory element is sufficient to cause expression changes of the lens geneTdrd7

Roscito

Subramanian

Naumann

et al. 2020

Preprint

View full text Add to dashboard Cite

Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved non-coding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis. We first show that this conserved element is a transcriptional repressor in lens cells and that the BMR sequence partially lost repressor activity. Next, we recapitulated the evolutionary changes by precisely replacing the endogenous regulatory element in a mouse line by the orthologous BMR sequence with CRISPR-Cas9. Strikingly, this repressor element has a large effect, causing a more than two-fold up-regulation of Tdrd7 in developing lens. Interestingly, the increased mRNA level does not result in a corresponding increase in TDRD7 protein nor an obvious lens phenotype, likely explained by buffering at the posttranscriptional level. Our results are consistent with eye degeneration in subterranean mammals having a polygenic basis where many small-effect mutations in different eyeregulatory elements collectively contribute to phenotypic differences.3

show abstract

Section: Discussioncontrasting

confidence: 94%

Recapitulating evolutionary divergence in a singlecis-regulatory element is sufficient to cause expression changes of the lens geneTdrd7

Roscito

Subramanian

Naumann

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…4k). Finally, we predicted likely enhancers using the recently developed activity-by-contact (ABC) model 36 , which integrates H3K27ac ChIP-seq, Hi-C, and gene expression data with chromatin accessibility to predict enhancers and link them to their target genes (Methods). Using this method, we were able to identify sets of high-confidence putative enhancers for each cell type and their likely target genes ( Fig.…”

Section: Single Cell Chromatin State Profiling Reveals Candidate Cellmentioning

confidence: 99%

“…The Activity-by-Contact (ABC) model 36 (https://github.com/broadinstitute/ABC-Enhancer-Gene-Prediction) was used for prediction of enhancer-gene interactions from scATACseq data. Cluster-specific ATAC-seq signal and peak outputs from MACS2 were used as inputs.…”

Section: Enhancer-gene Predicted Interactionsmentioning

confidence: 99%

Single cell epigenomic atlas of the developing human brain and organoids

Rs¹,

Wilfert

et al. 2019

Preprint

View full text Add to dashboard Cite

31Dynamic changes in chromatin accessibility coincide with important aspects of neuronal differentiation, such as 32 fate specification and arealization and confer cell type-specific associations to neurodevelopmental disorders. 33However, studies of the epigenomic landscape of the developing human brain have yet to be performed at single-34 cell resolution. Here, we profiled chromatin accessibility of >75,000 cells from eight distinct areas of developing 35human forebrain using single cell ATAC-seq (scATACseq). We identified thousands of loci that undergo 36 extensive cell type-specific changes in accessibility during corticogenesis. Chromatin state profiling also reveals 37 novel distinctions between neural progenitor cells from different cortical areas not seen in transcriptomic profiles 38 and suggests a role for retinoic acid signaling in cortical arealization. Comparison of the cell type-specific 39 chromatin landscape of cerebral organoids to primary developing cortex found that organoids establish broad 40 cell type-specific enhancer accessibility patterns similar to the developing cortex, but lack many putative 41 regulatory elements identified in homologous primary cell types. Together, our results reveal the important 42 contribution of chromatin state to the emerging patterns of cell type diversity and cell fate specification and 43 provide a blueprint for evaluating the fidelity and robustness of cerebral organoids as a model for cortical 44 development. 45 46Main text 47The diverse cell types of the human cerebral cortex (Fig. 1a) have been mostly classified based on a handful of 48 morphological, anatomical, and physiological features. Recent innovations in single cell genomics, such as single 49 cell mRNA sequencing (scRNA-seq), have enabled massively parallel profiling of thousands of molecular 50 features in every cell, uncovering the remarkable molecular diversity of cell types previously considered 51 homologous, such as excitatory neurons located in different areas of the cerebral cortex 1-6 . However, the 52 developmental mechanisms underlying the emergence of distinct cellular identities are largely unknown, as most 53 cortical neurons are generated at stages that are inaccessible to experimentation 5 . 54 55Over 60 years ago, Conrad Waddington introduced the concept of an epigenomic landscape to account for the 56 emergence of distinct cell fates 7 . In particular, chromatin state defines the functional architecture of the genome 57

show abstract

“…A recently introduced extension of CRISPRi targets intergenic regulators along with fluorescent in situ hybridization against genes in the same chromodomain to identify regulatortarget pairs. Fluorescence-intensity sorting into bins and subsequent RNA sequencing of stained cells then could be used to pair regulators (via guide RNA sequence) and target genes (altered fluorescent intensity associated to a guide RNA's presence) (88). While this assay was performed in K562 cells, it is not hard to envision its extension to neural cell types in vitro or in vivo.…”

Section: Mpras Enable Identification Of Functional Regulators and Varmentioning

confidence: 99%

The Oft-Overlooked Massively Parallel Reporter Assay: Where, When, and Which Psychiatric Genetic Variants are Functional?

Mulvey¹,

Lagunas²,

Dougherty³

2020

Preprint

View full text Add to dashboard Cite

Neuropsychiatric phenotypes have been long known to be influenced by heritable risk factors. The past decade of genetic studies have confirmed this directly, revealing specific common and rare genetic variants enriched in disease cohorts. However, the early hope for these studies-that only a small set of genes would be responsible for a given disorder-proved false. The picture that has emerged is far more complex: a given disorder may be influenced by myriad coding and noncoding variants of small effect size, and/or by rare but severe variants of large effect size, many de novo.Noncoding genomic sequences harbor a large portion of these variants, the molecular functions of which cannot usually be inferred from sequence alone. This creates a substantial barrier to understanding the higher-order molecular and biological systems underlying disease risk. Fortunately, a proliferation of genetic technologies-namely, scalable oligonucleotide synthesis, high-throughput RNA sequencing, CRISPR, and CRISPR derivatives-have opened novel avenues to experimentally identify biologically significant variants en masse. These advances have yielded an especially versatile technique adaptable to large-scale functional assays of variation in both untranscribed and untranslated regulatory features: Massively Parallel Reporter Assays (MPRAs). MPRAs are powerful molecular genetic tools that can be used to screen tens of thousands of predefined sequences for functional effects in a single experiment. This approach has several ideal features for psychiatric genetics, but remains underutilized in the field to date. To emphasize the opportunities MPRA holds for dissecting psychiatric polygenicity, we review here its applications in the literature, discuss its ability to test several biological variables implicated in psychiatric disorders, illustrate this flexibility with a proof-of-principle, in vivo cell-type specific implementation of the assay, and envision future outcomes of applying MPRA to both computational and experimental neurogenetics.

show abstract

Activity-by-contact model of enhancer–promoter regulation from thousands of CRISPR perturbations

Cited by 780 publications

References 53 publications

Recapitulating evolutionary divergence in a singlecis-regulatory element is sufficient to cause expression changes of the lens geneTdrd7

Recapitulating evolutionary divergence in a singlecis-regulatory element is sufficient to cause expression changes of the lens geneTdrd7

Single cell epigenomic atlas of the developing human brain and organoids

The Oft-Overlooked Massively Parallel Reporter Assay: Where, When, and Which Psychiatric Genetic Variants are Functional?

Contact Info

Product

Resources

About