Developing a pill to treat sickle cell disease.

A newly identified epigenetic modifier increases fetal hemoglobin in preclinical studies.

Super-enhancers include classical enhancers and facilitators to fully activate gene expression.

Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.

Ancient genomic linkage couples metabolism with erythroid development.

Generation of mature cells from progenitors requires tight coupling of differentiation and metabolism. During erythropoiesis, erythroblasts are required to massively upregulate globin synthesis then clear extraneous material and enucleate to produce erythrocytes1-3. Nprl3 has remained in synteny with the α-globin genes for >500 million years4, and harbours the majority of the α-globin enhancers5. Nprl3 is a highly conserved inhibitor of mTORC1, which controls cellular metabolism. However, whether Nprl3 itself serves an erythroid role is unknown. Here, we show that Nprl3 is a key regulator of erythroid metabolism. Using Nprl3-deficient fetal liver and adult competitive bone marrow - fetal liver chimeras, we show that NprI3 is required for sufficient erythropoiesis. Loss of Nprl3 elevates mTORC1 signalling, suppresses autophagy and disrupts erythroblast glycolysis and redox control. Human CD34+ progenitors lacking NPRL3 produce fewer enucleated cells and demonstrate dysregulated mTORC1 signalling in response to nutrient availability and erythropoietin. Finally, we show that the α-globin enhancers upregulate NprI3 expression, and that this activity is necessary for optimal erythropoiesis. Therefore, the anciently conserved linkage of NprI3, α-globin and their associated enhancers has enabled coupling of metabolic and developmental control in erythroid cells. This may enable erythropoiesis to adapt to fluctuating nutritional and environmental conditions.

Understanding fundamental principles of enhancer biology at a model locus: Analysing the structure and function of an enhancer cluster at the α-globin locus.

Despite ever-increasing accumulation of genomic data, the fundamental question of how individual genes are switched on during development, lineage-specification and differentiation is not fully answered. It is widely accepted that this involves the interaction between at least three fundamental regulatory elements: enhancers, promoters and insulators. Enhancers contain transcription factor binding sites which are bound by transcription factors (TFs) and co-factors expressed during cell fate decisions and maintain imposed patterns of activation, at least in part, via their epigenetic modification. This information is transferred from enhancers to their cognate promoters often by coming into close physical proximity to form a 'transcriptional hub' containing a high concentration of TFs and co-factors. The mechanisms underlying these stages of transcriptional activation are not fully explained. This review focuses on how enhancers and promoters are activated during differentiation and how multiple enhancers work together to regulate gene expression. We illustrate the currently understood principles of how mammalian enhancers work and how they may be perturbed in enhanceropathies using expression of the α-globin gene cluster during erythropoiesis, as a model.

Scalable in vitro production of defined mouse erythroblasts.

Mouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios.

Reactivation of a developmentally silenced embryonic globin gene.

The α- and ß-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues.

The mouse alpha-globin cluster: a paradigm for studying genome regulation and organization.

The mammalian globin gene clusters provide a paradigm for studying the relationship between genome structure and function. As blood stem cells undergo lineage specification and differentiation to form red blood cells, the chromatin structure and expression of the α-globin cluster change. The gradual activation of the α-globin genes in well-defined cell populations has enabled investigation of the structural and functional roles of its enhancers, promoters and boundary elements. Recent studies of gene regulatory processes involving these elements at the mouse α-globin cluster have brought new insights into the general principles underlying the three-dimensional structure of the genome and its relationship to gene expression throughout time.

A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions.

Self-interacting chromatin domains encompass genes and their cis-regulatory elements; however, the three-dimensional form a domain takes, whether this relies on enhancer-promoter interactions, and the processes necessary to mediate the formation and maintenance of such domains, remain unclear. To examine these questions, here we use a combination of high-resolution chromosome conformation capture, a non-denaturing form of fluorescence in situ hybridisation and super-resolution imaging to study a 70 kb domain encompassing the mouse α-globin regulatory locus. We show that this region forms an erythroid-specific, decompacted, self-interacting domain, delimited by frequently apposed CTCF/cohesin binding sites early in terminal erythroid differentiation, and does not require transcriptional elongation for maintenance of the domain structure. Formation of this domain does not rely on interactions between the α-globin genes and their major enhancers, suggesting a transcription-independent mechanism for establishment of the domain. However, absence of the major enhancers does alter internal domain interactions. Formation of a loop domain therefore appears to be a mechanistic process that occurs irrespective of the specific interactions within.

Between form and function: the complexity of genome folding.

It has been known for over a century that chromatin is not randomly distributed within the nucleus. However, the question of how DNA is folded and the influence of such folding on nuclear processes remain topics of intensive current research. A longstanding, unanswered question is whether nuclear organization is simply a reflection of nuclear processes such as transcription and replication, or whether chromatin is folded by independent mechanisms and this per se encodes function? Evidence is emerging that both may be true. Here, using the α-globin gene cluster as an illustrative model, we provide an overview of the most recent insights into the layers of genome organization across different scales and how this relates to gene activity.

Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo.

The genome is organized via CTCF-cohesin-binding sites, which partition chromosomes into 1-5 megabase (Mb) topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ∼80 kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ∼1 Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF-cohesin sites that are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF-cohesin boundary extends the sub-TAD to adjacent upstream CTCF-cohesin-binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF-cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.

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.

SCL-mediated regulation of the cell-cycle regulator p21 is critical for murine megakaryopoiesis.

Megakaryopoiesis is a complex process that involves major cellular and nuclear changes and relies on controlled coordination of cellular proliferation and differentiation. These mechanisms are orchestrated in part by transcriptional regulators. The key hematopoietic transcription factor stem cell leukemia (SCL)/TAL1 is required in early hematopoietic progenitors for specification of the megakaryocytic lineage. These early functions have, so far, prevented full investigation of its role in megakaryocyte development in loss-of-function studies. Here, we report that SCL critically controls terminal megakaryocyte maturation. In vivo deletion of Scl specifically in the megakaryocytic lineage affects all key attributes of megakaryocyte progenitors (MkPs), namely, proliferation, ploidization, cytoplasmic maturation, and platelet release. Genome-wide expression analysis reveals increased expression of the cell-cycle regulator p21 in Scl-deleted MkPs. Importantly, p21 knockdown-mediated rescue of Scl-mutant MkPs shows full restoration of cell-cycle progression and partial rescue of the nuclear and cytoplasmic maturation defects. Therefore, SCL-mediated transcriptional control of p21 is essential for terminal maturation of MkPs. Our study provides a mechanistic link between a major hematopoietic transcriptional regulator, cell-cycle progression, and megakaryocytic differentiation.

Genome-wide identification of TAL1's functional targets: insights into its mechanisms of action in primary erythroid cells.

Coordination of cellular processes through the establishment of tissue-specific gene expression programs is essential for lineage maturation. The basic helix-loop-helix hemopoietic transcriptional regulator TAL1 (formerly SCL) is required for terminal differentiation of red blood cells. To gain insight into TAL1 function and mechanisms of action in erythropoiesis, we performed ChIP-sequencing and gene expression analyses from primary fetal liver erythroid cells. We show that TAL1 coordinates expression of genes in most known red cell-specific processes. The majority of TAL1's genomic targets require direct DNA-binding activity. However, one-fifth of TAL1's target sequences, mainly among those showing high affinity for TAL1, can recruit the factor independently of its DNA binding activity. An unbiased DNA motif search of sequences bound by TAL1 identified CAGNTG as TAL1-preferred E-box motif in erythroid cells. Novel motifs were also characterized that may help distinguish activated from repressed genes and suggest a new mechanism by which TAL1 may be recruited to DNA. Finally, analysis of recruitment of GATA1, a protein partner of TAL1, to sequences occupied by TAL1 suggests that TAL1's binding is necessary prior or simultaneous to that of GATA1. This work provides the framework to study regulatory networks leading to erythroid terminal maturation and to model mechanisms of action of tissue-specific transcription factors.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis.

Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding-independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.