Jelena Telenius

Genetic dissection of the α-globin super-enhancer in vivo

Many genes determining cell identity are regulated by clusters of Mediator-bound enhancer elements collectively referred to as super-enhancers. These super-enhancers have been proposed to manifest higher-order properties important in development and disease. Here we report a comprehensive functional dissection of one of the strongest putative super-enhancers in erythroid cells. By generating a series of mouse models, deleting each of the five regulatory elements of the α-globin super-enhancer individually and in informative combinations, we demonstrate that each constituent enhancer seems to act independently and in an additive fashion with respect to hematological phenotype, gene expression, chromatin structure and chromosome conformation, without clear evidence of synergistic or higher-order effects. Our study highlights the importance of functional genetic analyses for the identification of new concepts in transcriptional regulation.

Multiplexed analysis of chromosome conformation at vastly improved sensitivity

Methods for analyzing chromosome conformation in mammalian cells are either low resolution or low throughput and are technically challenging. In next-generation (NG) Capture-C, we have redesigned the Capture-C method to achieve unprecedented levels of sensitivity and reproducibility. NG Capture-C can be used to analyze many genetic loci and samples simultaneously. High-resolution data can be produced with as few as 100,000 cells, and single-nucleotide polymorphisms can be used to generate allele-specific tracks. The method is straightforward to perform and should greatly facilitate the investigation of many questions related to gene regulation as well as the functional dissection of traits examined in genome-wide association studies.

Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia

Abstractβ-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with β-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for β-thalassemia.β-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.

Functional characterisation of cis-regulatory elements governing dynamic Eomes expression in the early mouse embryo

The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. The cis-acting regulatory elements that direct expression in the anterior visceral endoderm (AVE), primitive streak (PS) and definitive endoderm (DE) have yet to be defined. Here, we identified three gene-proximal enhancer-like sequences (PSE_a, PSE_b and VPE) that faithfully activate tissue-specific expression in transgenic embryos. However, targeted deletion experiments demonstrate that PSE_a and PSE_b are dispensable, and only VPE is required for optimal Eomes expression in vivo. Embryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions in embryonic stem cells (ESCs), prior to gene activation. The locus resides in a large (500 kb) pre-formed compartment in ESCs and activation during DE differentiation occurs in the absence of 3D structural changes. ATAC-seq analysis reveals that VPE, PSE_a and four additional putative enhancers display increased chromatin accessibility in DE that is associated with Smad2/3 binding coincident with transcriptional activation. By contrast, activation of the Eomes target genes Foxa2 and Lhx1 is associated with higher order chromatin reorganisation. Thus, diverse regulatory mechanisms govern activation of lineage specifying TFs during early development. Summary: Expression of the mouse T-box factor Eomes is controlled by a key gene-proximal enhancer-like element, with changes in chromatin accessibility influencing its activity in definitive endoderm.

High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates

The emergence in recent years of DNA editing technologies—Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors—have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis.

NGseqBasic - a single-command UNIX tool for ATAC-seq, DNaseI-seq, Cut-and-Run, and ChIP-seq data mapping, high-resolution visualisation, and quality control

With decreasing cost of next-generation sequencing (NGS), we are observing a rapid rise in the volume of ‘big data’ in academic research, healthcare and drug discovery sectors. The present bottleneck for extracting value from these ‘big data’ sets is data processing and analysis. Considering this, there is still a lack of reliable, automated and easy to use tools that will allow experimentalists to assess the quality of the sequenced libraries and explore the data first hand, without the need of investing a lot of time of computational core analysts in the early stages of analysis. NGseqBasic is an easy-to-use single-command analysis tool for chromatin accessibility (ATAC, DNaseI) and ChIP sequencing data, providing support to also new techniques such as low cell number sequencing and Cut-and-Run. It takes in fastq, fastq.gz or bam files, conducts all quality control, trimming and mapping steps, along with quality control and data processing statistics, and combines all this to a single-click loadable UCSC data hub, with integral statistics html page providing detailed reports from the analysis tools and quality control metrics. The tool is easy to set up, and no installation is needed. A wide variety of parameters are provided to fine-tune the analysis, with optional setting to generate DNase footprint or high resolution ChIP-seq tracks. A tester script is provided to help in the setup, along with a test data set and downloadable example user cases. NGseqBasic has been used in the routine analysis of next generation sequencing (NGS) data in high-impact publications 1,2. The code is actively developed, and accompanied with Git version control and Github code repository. Here we demonstrate NGseqBasic analysis and features using DNaseI-seq data from GSM689849, and CTCF-ChIP-seq data from GSM2579421, as well as a Cut-and-Run CTCF data set GSM2433142, and provide the one-click loadable UCSC data hubs generated by the tool, allowing for the ready exploration of the run results and quality control files generated by the tool. Availability Download, setup and help instructions are available on the NGseqBasic web site http://userweb.molbiol.ox.ac.uk/public/telenius/NGseqBasicManual/external/ Bioconda users can load the tool as library “ngseqbasic”. The source code with Git version control is available in https://github.com/Hughes-Genome-Group/NGseqBasic/releases. Contact jelena.telenius@imm.ox.ac.uk

Single-cell chromatin interactions reveal regulatory hubs in dynamic compartmentalized domains

The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory elements within them interact within single cells is not understood. To address this we developed Tri-C, a new Chromosome Conformation Capture (3C) approach to identify concurrent chromatin interactions at individual alleles within single cells. The heterogeneity of interactions observed between such cells shows that CTCF-mediated formation of chromatin domains and interactions within them are dynamic processes. Importantly, our analyses reveal higher-order structures involving simultaneous interactions between multiple enhancers and promoters within individual cells. This provides a structural basis for understanding how multiple cis-elements act together to establish robust regulation of gene expression.

DOT1L inhibition reveals a distinct class of enhancers dependent on H3K79 methylation

Enhancer elements are a key regulatory feature of many important genes. Several general features including the presence of specific histone modifications are used to identify and subcategorize enhancers. Here we identify a distinct subset of enhancers in leukemia cells that are functionally dependent upon H3K79me3. Using the DOT1L inhibitor, EPZ-5676, we show that loss of H3K79me3 at these H3K79me3 enhancer elements (KEEs) leads to reduced chromatin accessibility, histone acetylation and transcription factor binding. We then use Capture-C, a high-resolution chromosome conformation capture technique, to show that H3K79me3 is required for KEE interactions with the promoter as well as transcription of the associated genes. Together these data implicate H3K79me3 in having a functional role at a subset of active enhancers where it helps maintain histone acetylation and chromatin accessibility, potentially by promoting phase-separated condensates.

Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains

The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory elements within them interact in single cells is not understood. To address this we developed Tri-C, a new chromosome conformation capture (3C) approach, to characterize concurrent chromatin interactions at individual alleles. Analysis by Tri-C identifies heterogeneous patterns of single-allele interactions between CTCF boundary elements, indicating that the formation of chromatin domains likely results from a dynamic process. Within these domains, we observe specific higher-order structures that involve simultaneous interactions between multiple enhancers and promoters. Such regulatory hubs provide a structural basis for understanding how multiple cis-regulatory elements act together to establish robust regulation of gene expression.Tri-C is a new 3C approach to identify concurrent chromatin interactions at individual alleles. The authors observe specific higher-order structures involving simultaneous interactions between multiple enhancers and promoters, called regulatory hubs.

How to Tackle Challenging ChIP-Seq, with Long-Range Cross-Linking, Using ATRX as an Example.

Chromatin immunoprecipitation coupled with high-throughput, next-generation DNA sequencing (ChIP-seq) has enabled researchers to establish the genome-wide patterns of chromatin modifications and binding of chromatin-associated proteins. Well-established protocols produce robust ChIP-seq data for many proteins by sequencing the DNA obtained following immunoprecipitation of fragmented chromatin using a wide range of specific antibodies. In general, the quality of these data mainly depends on the specificity and avidity of the antibody used. However, even using optimal antibodies, ChIP-seq can become more challenging when the protein associates with chromatin via protein-protein interactions rather than directly binding DNA. An example of such a protein is the alpha-thalassaemia mental retardation X-linked (ATRX) protein; a chromatin remodeler that associates with the histone chaperone DAXX, in the deposition of the replication-independent histone variant H3.3 and plays an important role in maintaining chromatin integrity. Inherited mutations of ATRX cause syndromal mental retardation (ATR-X Syndrome) whereas acquired mutations are associated with myelodysplasia, acute myeloid leukemia (ATMDS syndrome), and a range of solid tumors. Therefore, high quality ChIP-seq data have been needed to analyze the genome-wide distribution of ATRX, to advance our understanding of its normal role and to comprehend how mutations contribute to human disease. Here, we describe an optimized ChIP-seq protocol for ATRX which can also be used to produce high quality data sets for other challenging proteins which are indirectly associated with DNA and complement the ChIP-seq toolkit for genome-wide analyses of histone chaperon complexes and associated chromatin remodelers. Although not a focus of this chapter, we will also provide some insight for the analysis of the large dataset generated by ChIP-seq. Even though this protocol has been fully optimized for ATRX, it should also provide guidance for efficient ChIP-seq analysis, using the appropriate antibodies, for other proteins interacting indirectly with DNA.