Differential cofactor dependencies define distinct types of human enhancers.

All multicellular organisms rely on differential gene transcription regulated by genomic enhancers, which function through cofactors that are recruited by transcription factors1,2. Emerging evidence suggests that not all cofactors are required at all enhancers3-5, yet whether these observations reflect more general principles or distinct types of enhancers remained unknown. Here we categorized human enhancers by their cofactor dependencies and show that these categories provide a framework to understand the sequence and chromatin diversity of enhancers and their roles in different gene-regulatory programmes. We quantified enhancer activities along the entire human genome using STARR-seq6 in HCT116 cells, following the rapid degradation of eight cofactors. This analysis identified different types of enhancers with distinct cofactor requirements, sequences and chromatin properties. Some enhancers were insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4 and regulated distinct transcriptional programmes. In particular, canonical Mediator7 seemed dispensable for P53-responsive enhancers, and MED14-depleted cells induced endogenous P53 target genes. Similarly, BRD4 was not required for the transcription of genes that bear CCAAT boxes and a TATA box (including histone genes and LTR12 retrotransposons) or for the induction of heat-shock genes. This categorization of enhancers through cofactor dependencies reveals distinct enhancer types that can bypass broadly utilized cofactors, which illustrates how alternative ways to activate transcription separate gene expression programmes and provide a conceptual framework to understand enhancer function and regulatory specificity.

Identification of ALK in Thinness.

There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.

STARR-seq and UMI-STARR-seq: Assessing Enhancer Activities for Genome-Wide-, High-, and Low-Complexity Candidate Libraries.

The identification of transcriptional enhancers and the quantitative assessment of enhancer activities is essential to understanding how regulatory information for gene expression is encoded in animal and human genomes. Further, it is key to understanding how sequence variants affect enhancer function. STARR-seq enables the direct and quantitative assessment of enhancer activity for millions of candidate sequences of arbitrary length and origin in parallel, allowing the screening of entire genomes and the establishment of genome-wide enhancer activity maps. In STARR-seq, the candidate sequences are cloned downstream of the core promoter into a reporter gene's transcription unit (i.e., the 3' UTR). Candidates that function as active enhancers lead to the transcription of reporter mRNAs that harbor the candidates' sequences. This direct coupling of enhancer sequence and enhancer activity in cis enables the straightforward and efficient cloning of complex candidate libraries and the assessment of enhancer activities of millions of candidates in parallel by quantifying the reporter mRNAs by deep sequencing. This article describes how to create focused and genome-wide human STARR-seq libraries and how to perform STARR-seq screens in mammalian cells, and also describes a novel STARR-seq variant (UMI-STARR-seq) that allows the accurate counting of reporter mRNAs for STARR-seq libraries of low complexity. © 2019 The Authors. Basic Protocol 1: STARR-seq plasmid library cloning Basic Protocol 2: Mammalian STARR-seq screening protocol Alternate Protocol: UMI-STARR-seq screening protocol-unique molecular identifier integration Support Protocol: Transfection of human cells using the MaxCyte STX scalable transfection system.

Resolving systematic errors in widely used enhancer activity assays in human cells.

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated. These cause confounding false positives and negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core promoter and by inhibiting two IFN-I-inducing kinases, enabling genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells and are key to the characterization of human enhancers.

Promoting transcription over long distances.

Promoters and enhancers have long been regarded as distinct elements, a notion that has been challenged more recently. Two new studies now identify promoters that function as long-range enhancers in vivo to regulate the transcription of distal genes.