Hormone-responsive enhancer-activity maps reveal predictive motifs, indirect repression, and targeting of closed chromatin.

Steroid hormones act as important developmental switches, and their nuclear receptors regulate many genes. However, few hormone-dependent enhancers have been characterized, and important aspects of their sequence architecture, cell-type-specific activating and repressing functions, or the regulatory roles of their chromatin structure have remained unclear. We used STARR-seq, a recently developed enhancer-screening assay, and ecdysone signaling in two different Drosophila cell types to derive genome-wide hormone-dependent enhancer-activity maps. We demonstrate that enhancer activation depends on cis-regulatory motif combinations that differ between cell types and can predict cell-type-specific ecdysone targeting. Activated enhancers are often not accessible prior to induction. Enhancer repression following hormone treatment seems independent of receptor motifs and receptor binding to the enhancer, as we show using ChIP-seq, but appears to rely on motifs for other factors, including Eip74. Our strategy is applicable to study signal-dependent enhancers for different pathways and across organisms.

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.

Transcriptional plasticity promotes primary and acquired resistance to BET inhibition.

Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL-AF9;Nras(G12D)-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.

Dissection of thousands of cell type-specific enhancers identifies dinucleotide repeat motifs as general enhancer features.

Gene expression is determined by genomic elements called enhancers, which contain short motifs bound by different transcription factors (TFs). However, how enhancer sequences and TF motifs relate to enhancer activity is unknown, and general sequence requirements for enhancers or comprehensive sets of important enhancer sequence elements have remained elusive. Here, we computationally dissect thousands of functional enhancer sequences from three different Drosophila cell lines. We find that the enhancers display distinct cis-regulatory sequence signatures, which are predictive of the enhancers' cell type-specific or broad activities. These signatures contain transcription factor motifs and a novel class of enhancer sequence elements, dinucleotide repeat motifs (DRMs). DRMs are highly enriched in enhancers, particularly in enhancers that are broadly active across different cell types. We experimentally validate the importance of the identified TF motifs and DRMs for enhancer function and show that they can be sufficient to create an active enhancer de novo from a nonfunctional sequence. The function of DRMs as a novel class of general enhancer features that are also enriched in human regulatory regions might explain their implication in several diseases and provides important insights into gene regulation.

STARR-seq - principles and applications.

Differential gene expression is the basis for cell type diversity in multicellular organisms and the driving force of development and differentiation. It is achieved by cell type-specific transcriptional enhancers, which are genomic DNA sequences that activate the transcription of their target genes. Their identification and characterization is fundamental to our understanding of gene regulation. Features that are associated with enhancer activity, such as regulatory factor binding or histone modifications can predict the location of enhancers. Nonetheless, enhancer activity can only be assessed by transcriptional reporter assays. Over the past years massively parallel reporter assays have been developed for large scale testing of enhancers. In this review we focus on the principles and applications of STARR-seq, a functional assay that quantifies enhancer strengths in complex candidate libraries and thus allows activity-based enhancer identification in entire genomes. We explain how STARR-seq works, discuss current uses and give an outlook to future applications.

An Intrinsic Oscillation of Gene Networks Inside Hair Follicle Stem Cells: An Additional Layer That Can Modulate Hair Stem Cell Activities.

This article explores and summarizes recent progress in and the characterization of main players in the regulation and cyclic regeneration of hair follicles. The review discusses current views and discoveries on the molecular mechanisms that allow hair follicle stem cells (hfSCs) to synergistically integrate homeostasis during quiescence and activation. Discussion elaborates on a model that shows how different populations of skin stem cells coalesce intrinsic and extrinsic mechanisms, resulting in the maintenance of stemness and hair regenerative potential during an organism's lifespan. Primarily, we focus on the question of how the intrinsic oscillation of gene networks in hfSCs sense and respond to the surrounding niche environment. The review also investigates the existence of a cell-autonomous mechanism and the reciprocal interactions between molecular signaling axes in hfSCs and niche components, which demonstrates its critical driving force in either the activation of whole mini-organ regeneration or quiescent homeostasis maintenance. These exciting novel discoveries in skin stem cells and the surrounding niche components propose a model of the intrinsic stem cell oscillator which is potentially instructive for translational regenerative medicine. Further studies, deciphering of the distribution of molecular signals coupled with the nature of their oscillation within the stem cells and niche environments, may impact the speed and efficiency of various approaches that could stimulate the development of self-renewal and cell-based therapies for hair follicle stem cell regeneration.

Genome-wide quantitative enhancer activity maps identified by STARR-seq.

Genomic enhancers are important regulators of gene expression, but their identification is a challenge, and methods depend on indirect measures of activity. We developed a method termed STARR-seq to directly and quantitatively assess enhancer activity for millions of candidates from arbitrary sources of DNA, which enables screens across entire genomes. When applied to the Drosophila genome, STARR-seq identifies thousands of cell type-specific enhancers across a broad continuum of strengths, links differential gene expression to differences in enhancer activity, and creates a genome-wide quantitative enhancer map. This map reveals the highly complex regulation of transcription, with several independent enhancers for both developmental regulators and ubiquitously expressed genes. STARR-seq can be used to identify and quantify enhancer activity in other eukaryotes, including humans.