Minor Loops in Major Folds: Enhancer-Promoter Looping, Chromatin Restructuring, and Their Association with Transcriptional Regulation and Disease.

The organization and folding of chromatin within the nucleus can determine the outcome of gene expression. Recent technological advancements have enabled us to study chromatin interactions in a genome-wide manner at high resolution. These studies have increased our understanding of the hierarchy and dynamics of chromatin domains that facilitate cognate enhancer-promoter looping, defining the transcriptional program of different cell types. In this review, we focus on vertebrate chromatin long-range interactions as they relate to transcriptional regulation. In addition, we describe how the alteration of boundaries that mark discrete regions in the genome with high interaction frequencies within them, called topological associated domains (TADs), could lead to various phenotypes, including human diseases, which we term as "TADopathies.

The human ARF tumor suppressor senses blastema activity and suppresses epimorphic tissue regeneration.

The control of proliferation and differentiation by tumor suppressor genes suggests that evolution of divergent tumor suppressor repertoires could influence species regenerative capacity. To directly test that premise, we humanized the zebrafish p53 pathway by introducing regulatory and coding sequences of the human tumor suppressor ARF into the zebrafish genome. ARF was dormant during development, in uninjured adult fins, and during wound healing, but was highly expressed in the blastema during epimorphic fin regeneration after amputation. Regenerative, but not developmental signals resulted in binding of zebrafish E2f to the human ARF promoter and activated conserved ARF-dependent Tp53 functions. The context-dependent activation of ARF did not affect growth and development but inhibited regeneration, an unexpected distinct tumor suppressor response to regenerative versus developmental environments. The antagonistic pleiotropic characteristics of ARF as both tumor and regeneration suppressor imply that inducing epimorphic regeneration clinically would require modulation of ARF -p53 axis activation

Meta-analysis of massively parallel reporter assays enables prediction of regulatory function across cell types.

Deciphering the potential of noncoding loci to influence gene regulation has been the subject of intense research, with important implications in understanding genetic underpinnings of human diseases. Massively parallel reporter assays (MPRAs) can measure regulatory activity of thousands of DNA sequences and their variants in a single experiment. With increasing number of publically available MPRA data sets, one can now develop data-driven models which, given a DNA sequence, predict its regulatory activity. Here, we performed a comprehensive meta-analysis of several MPRA data sets in a variety of cellular contexts. We first applied an ensemble of methods to predict MPRA output in each context and observed that the most predictive features are consistent across data sets. We then demonstrate that predictive models trained in one cellular context can be used to predict MPRA output in another, with loss of accuracy attributed to cell-type-specific features. Finally, we show that our approach achieves top performance in the Fifth Critical Assessment of Genome Interpretation "Regulation Saturation" Challenge for predicting effects of single-nucleotide variants. Overall, our analysis provides insights into how MPRA data can be leveraged to highlight functional regulatory regions throughout the genome and can guide effective design of future experiments by better prioritizing regions of interest

MPRAnalyze: statistical framework for massively parallel reporter assays.

Massively parallel reporter assays (MPRAs) can measure the regulatory function of thousands of DNA sequences in a single experiment. Despite growing popularity, MPRA studies are limited by a lack of a unified framework for analyzing the resulting data. Here we present MPRAnalyze: a statistical framework for analyzing MPRA count data. Our model leverages the unique structure of MPRA data to quantify the function of regulatory sequences, compare sequences' activity across different conditions, and provide necessary flexibility in an evolving field. We demonstrate the accuracy and applicability of MPRAnalyze on simulated and published data and compare it with existing methods

CRISPR-Cas9-mediated functional dissection of 3'-UTRs.

Many studies using reporter assays have demonstrated that 3' untranslated regions (3'-UTRs) regulate gene expression by controlling mRNA stability and translation. Due to intrinsic limitations of heterologous reporter assays, we sought to develop a gene editing approach to investigate the regulatory activity of 3'-UTRs in their native context. We initially used dual-CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 targeting to delete DNA regions corresponding to nine chemokine 3'-UTRs that destabilized mRNA in a reporter assay. Targeting six chemokine 3'-UTRs increased chemokine mRNA levels as expected. However, targeting CXCL1, CXCL6 and CXCL8 3'-UTRs unexpectedly led to substantial mRNA decreases. Metabolic labeling assays showed that targeting these three 3'-UTRs increased mRNA stability, as predicted by the reporter assay, while also markedly decreasing transcription, demonstrating an unexpected role for 3'-UTR sequences in transcriptional regulation. We further show that CRISPR-Cas9 targeting of specific 3'-UTR elements can be used for modulating gene expression and for highly parallel localization of active 3'-UTR elements in the native context. Our work demonstrates the duality and complexity of 3'-UTR sequences in regulation of gene expression and provides a useful approach for modulating gene expression and for functional annotation of 3'-UTRs in the native context

Genomic and epigenomic mapping of leptin-responsive neuronal populations involved in body weight regulation.

Genome wide association studies (GWAS) in obesity have identified a large number of noncoding loci located near genes expressed in the central nervous system. However, due to the difficulties in isolating and characterizing specific neuronal subpopulations, few obesity-associated SNPs have been functionally characterized. Leptin responsive neurons in the hypothalamus are essential in controlling energy homeostasis and body weight. Here, we combine FACS-sorting of leptin-responsive hypothalamic neuron nuclei with genomic and epigenomic approaches (RNA-seq, ChIP-seq, ATAC-seq) to generate a comprehensive map of leptin-response specific regulatory elements, several of which overlap obesity-associated GWAS variants. We demonstrate the usefulness of our leptin-response neuron regulome, by functionally characterizing a novel enhancer near Socs3, a leptin response-associated transcription factor. We envision our data to serve as a useful resource and a blueprint for functionally characterizing obesity-associated SNPs in the hypothalamus

A genome-wide association study identifies four novel susceptibility loci underlying inguinal hernia.

Inguinal hernia repair is one of the most commonly performed operations in the world, yet little is known about the genetic mechanisms that predispose individuals to develop inguinal hernias. We perform a genome-wide association analysis of surgically confirmed inguinal hernias in 72,805 subjects (5,295 cases and 67,510 controls) and confirm top associations in an independent cohort of 92,444 subjects with self-reported hernia repair surgeries (9,701 cases and 82,743 controls). We identify four novel inguinal hernia susceptibility loci in the regions of EFEMP1, WT1, EBF2 and ADAMTS6. Moreover, we observe expression of all four genes in mouse connective tissue and network analyses show an important role for two of these genes (EFEMP1 and WT1) in connective tissue maintenance/homoeostasis. Our findings provide insight into the aetiology of hernia development and highlight genetic pathways for studies of hernia development and its treatment

Functionally conserved enhancers with divergent sequences in distant vertebrates

Conserved transcription factor binding motifs in the five zebrafish/mouse syntenic enhancers. Identical n-mers (n ≥ 7) identified in the zebrafish, mouse, and human sequences of the five syntenic CNS were examined for the presence of transcription factor binding motifs; only motifs with E-value E ≤ 0.1 are shown. (XLSX 15 kb