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Thalassaemia is one of the most common genetic diseases worldwide, with at least 60 000 severely affected individuals born every year. Individuals originating from tropical and subtropical regions are most at risk. Disorders of haemoglobin synthesis (thalassaemia) and structure (eg, sickle-cell disease) were among the first molecular diseases to be identified, and have been investigated and characterised in detail over the past 40 years. Nevertheless, treatment of thalassaemia is still largely dependent on supportive care with blood transfusion and iron chelation. Since 1978, scientists and clinicians in this specialty have met regularly in an international effort to improve the management of thalassaemia, with the aim of increasing the expression of unaffected fetal genes to improve the deficiency in adult β-globin synthesis. In this Seminar we discuss important advances in the understanding of the molecular and cellular basis of normal and abnormal expression of globin genes. We will summarise new approaches to the development of tailored pharmacological agents to alter regulation of globin genes, the first trial of gene therapy for thalassaemia, and future prospects of cell therapy.

A genome-wide analysis of CTCF binding sites yields compact sequence elements that function as chromatin insulators. Insertional mutagenesis and genotoxicity, which usually manifest as hematopoietic malignancy, represent major barriers to realizing the promise of gene therapy. Although insulator sequences that block transcriptional enhancers could mitigate or eliminate these risks, so far no human insulators with high functional potency have been identified. Here we describe a genomic approach for the identification of compact sequence elements that function as insulators. These elements are highly occupied by the insulator protein CTCF, are DNase I hypersensitive and represent only a small minority of the CTCF recognition sequences in the human genome. We show that the elements identified acted as potent enhancer blockers and substantially decreased the risk of tumor formation in a cancer-prone animal model. The elements are small, can be efficiently accommodated by viral vectors and have no detrimental effects on viral titers. The insulators we describe here are expected to increase the safety of gene therapy for genetic diseases.

Gene expression during cell development and differentiation is orchestrated by distal regulatory elements that precisely modulate cell selective gene activity. Gene therapy vectors leverage these elements for precise spatiotemporal transgene expression. Here, we develop a one-shot approach to screen candidate regulatory sequences from large-scale epigenomics data for programmable transgene expression within gene therapy viral vectors. We assess a library of 15,000 short sequences derived from developmentally active elements during erythropoiesis using a clinically relevant reporter vector. These elements display a gradient of transcriptional enhancer activity in erythroid cells, with high cell type restriction and developmental stage specificity. Finally, replacing the canonical β-globin μLCR with a compact enhancer in a β-thalassemia lentiviral vector successfully corrects the thalassemic phenotype in patient-derived hematopoietic and stem and progenitor cells (HSPCs), while increasing viral titers and cell transducibility. Our approach provides further insights into enhancer biology with wider implications for human gene therapy. This study presents a large-scale enhancer screening approach to optimize gene therapy vectors. A compact, potent, erythroid-specific enhancer used in a therapeutic vector, improved viral titers, transducibility, and restored hemoglobin levels in β-thalassemia patient-derived cells.

An obituary for Arno Motulsky, one of the founders of medical genetics, who died on Jan 17, 2018 at the age of 94 is presented. Motulsky started his postdoctoral medical genetics training program in the early 1960s, and during the 1960s, he trained several medical geneticists who subsequently became leaders in the field.

Sequences of the genome that are capable of silencing gene expression are thought to play a key role in gene regulation. However, very few silencer elements capable of functioning in mammalian cells have been described, and only a fraction of these have been tested for the ability to function in an autonomous fashion. We report here the characterization and functional validation of a constitutive autonomous silencer element from the human genome called T39, and the comparison of T39 to three other putative silencer elements previously described by others. Functional analysis included one assay for enhancer-blocking insulator activity and two independent assays for silencer activity, all based on stable transfection and comparison to a neutral spacer control. In erythroid K562 cells, T39 exhibited potent silencer activity, the previously described element PRE2-S5 exhibited modest silencer activity, and the two other previously described elements exhibited no silencer activity. T39 was further found to be capable of silencing three disparate promoters, of silencing gene expression in three disparate cell lines, and of functioning as a single copy in a topology-independent manner. Of the four elements analyzed, only T39 exhibits a constitutive pattern of DNase hypersensitivity and binding by CTCF. In its native location the T39 element also exhibits a unique interaction profile with a subset of distal putative regulatory elements. Taken together, these studies validate T39 as a constitutive autonomous silencer, identify T39 as a defined control for future studies of other regulatory elements such as insulators, and provide a basic chromatin profile for one highly potent silencer element.

Background Mapping of DNase I hypersensitive sites (DHSs) is a powerful tool to experimentally identify cis -regulatory elements (CREs). Among CREs, enhancers are abundant and predominantly act in driving cell-specific gene expression. Krüppel-like factors (KLFs) are a family of eukaryotic transcription factors. Several KLFs have been demonstrated to play important roles in hematopoiesis. However, transcriptional regulation of KLFs via CREs, particularly enhancers, in erythroid cells has been poorly understood. Results In this study, 23 erythroid-specific or putative erythroid-specific DHSs were identified by DNase-seq in the genomic regions of 17 human KLFs, and their enhancer activities were evaluated using dual-luciferase reporter (DLR) assay. Of the 23 erythroid-specific DHSs, the enhancer activities of 15 DHSs were comparable to that of the classical enhancer HS2 in driving minimal promoter (minP). Fifteen DHSs, some overlapping those that increased minP activities, acted as enhancers when driving the corresponding KLF promoters (KLF-Ps) in erythroid cells; of these, 10 DHSs were finally characterized as erythroid-specific KLF enhancers. These 10 erythroid-specific KLF enhancers were further confirmed using chromatin immunoprecipitation coupled to sequencing (ChIP-seq) data-based bioinformatic and biochemical analyses. Conclusion Our present findings provide a feasible strategy to extensively identify gene- and cell-specific enhancers from DHSs obtained by high-throughput sequencing, which will help reveal the transcriptional regulation and biological functions of genes in some specific cells.

Recombinant murine retroviruses are widely used as delivery vectors for gene therapy. However, once integrated into a chromosome, these vectors often suffer from profound position effects, with vector silencing observed in vitro and in vivo. To overcome this problem, we investigated whether the HS4 chromatin insulator from the chicken β -globin locus control region could protect a retrovirus vector from position effects. When used to flank a reporter vector, this element significantly increased the fraction of transduced cells that expressed the provirus in cultures and in mice transplanted with transduced marrow. These results demonstrate that a chromatin insulator can improve the expression performance of a widely used class of gene therapy vectors by protecting these vectors from chromosomal position effects.

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis -regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis -regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation. An extensive map of human DNase I hypersensitive sites, markers of regulatory DNA, in 125 diverse cell and tissue types is described; integration of this information with other ENCODE-generated data sets identifies new relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. ENCODE: a map of accessible chromatin This paper describes the first extensive map of human DNaseI hypersensitive sites — markers of regulatory DNA — in 125 diverse cell and tissue types. Integration of this information with other data sets generated by ENCODE (Encyclopedia of DNA Elements) identified new relationships between chromatin accessibility, transcription, DNA methylation and regulatory-factor occupancy patterns. Evolutionary-conservation analysis revealed signatures of recent functional constraint within DNaseI hypersensitive sites.

Tiling of regulatory DNA with mutations introduced by genome editing nucleases and linking the resulting alleles to a phenotypic readout allows the precise determination of functional sequence motifs within these regions. Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe an approach that exploits the error-prone nature of genome editing–induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.

The Neolithic populations, which colonized Europe approximately 9,000 y ago, presumably migrated from Near East to Anatolia and from there to Central Europe through Thrace and the Balkans. An alternative route would have been island hopping across the Southern European coast. To test this hypothesis, we analyzed genome-wide DNA polymorphisms on populations bordering the Mediterranean coast and from Anatolia and mainland Europe. We observe a striking structure correlating genes with geography around the Mediterranean Sea with characteristic east to west clines of gene flow. Using population network analysis, we also find that the gene flow from Anatolia to Europe was through Dodecanese, Crete, and the Southern European coast, compatible with the hypothesis that a maritime coastal route was mainly used for the migration of Neolithic farmers to Europe.