Publications

Author: 

LC Ly, Y Yang, F Li, M Crossley, Y Shi, KGR Quinlan

Key Summary: 

Here, we describe protocols to interrogate the binding of the zinc fingers of the transcription factor ZBTB7A to the fetal γ-globin (HBG) promoter. We detail the steps for performing electrophoretic mobility shift assays (EMSAs), X-ray crystallography, and isothermal titration calorimetry (ITC) to explore this interaction. These techniques could readily be applied to the structural studies of other zinc finger transcription factors and cognate DNA sequences.

For complete details on the use and execution of this protocol, please refer to Yang et al. (2021).

Author: 

Crossley M, Christakopoulos GE, Weiss MJ

Key Summary: 

Sickle cell disease (SCD) is a common genetic blood disorder associated with acute and chronic pain, progressive multiorgan damage, and early mortality. Recent advances in technologies to manipulate the human genome, a century of research and the development of techniques enabling the isolation, efficient genetic modification, and reimplantation of autologous patient hematopoietic stem cells (HSCs), mean that curing most patients with SCD could soon be a reality in wealthy countries. In parallel, ongoing research is pursuing more facile treatments, such as in-vivo-delivered genetic therapies and new drugs that can eventually be administered in low- and middle-income countries where most SCD patients reside.

Author: 

Topfer SK, Feng R, Huang P, Ly LC, Martyn GE, Blobel GA, Weiss MJ, Quinlan KG, Crossley M

Key Summary: 

The benign condition hereditary persistence of fetal hemoglobin (HPFH) is known to ameliorate symptoms of co-inherited β-hemoglobinopathies, such as sickle cell disease and β-thalassemia. The condition is sometimes associated with point mutations in the fetal globin promoters that disrupt the binding of the repressors BCL11A or ZBTB7A/LRF, which have been extensively studied. HPFH is also associated with a range of deletions within the β-globin locus that all reside downstream of the fetal HBG2 gene. These deletional forms of HPFH are poorly understood and are the focus of this study. Numerous different mechanisms have been proposed to explain how downstream deletions can boost the expression of the fetal globin genes, including the deletion of silencer elements, of genes encoding noncoding RNA, and bringing downstream enhancer elements into proximity with the fetal globin gene promoters. Here we systematically analyze the deletions associated with both HPFH and a related condition known as δβ-thalassemia and propose a unifying mechanism. In all cases where fetal globin is upregulated, the proximal adult β-globin (HBB) promoter is deleted. We use clustered regularly interspaced short palindromic repeats-mediated gene editing to delete or disrupt elements within the promoter and find that virtually all mutations that reduce ΗΒΒ promoter activity result in elevated fetal globin expression. These results fit with previous models where the fetal and adult globin genes compete for the distal locus control region and suggest that targeting the ΗΒΒ promoter might be explored to elevate fetal globin and reduce sickle globin expression as a treatment of β-hemoglobinopathies.

Author: 

Yang Y, Ren R, Ly LC, Horton JR, Li F, Quinlan KG, Crossley M, Shi Y, Cheng X.

Key Summary: 

Elevated levels of fetal globin(link is external) protect against β-hemoglobinopathies, such as sickle cell(link is external) disease and β-thalassemia. Two zinc-finger (ZF) repressors(link is external), BCL11A(link is external) and ZBTB7A/LRF, bind directly to the fetal globin promoter elements(link is external) positioned at −115 and −200, respectively. Here, we describe X-ray structures of the ZBTB7A DNA-binding domain, consisting of four adjacent ZFs, in complex with the −200 sequence element, which contains two copies of four consecutive C:G base pairs. ZF1 and ZF2 recognize the 5′ C:G quadruple, and ZF4 contacts the 3′ C:G quadruple. Natural non-coding DNA mutations(link is external) associated with hereditary persistence of fetal hemoglobin(link is external) (HPFH) impair ZBTB7A DNA binding(link is external), with the most severe disruptions resulting from mutations in the base pairs recognized by ZF1 and ZF2. Our results firmly establish ZBTB7A/LRF as a key molecular regulator of fetal globin expression and inform genome-editing strategies that inhibit repressor binding and boost fetal globin expression to treat hemoglobinopathies.

Author: 

Knights, A. J., Vohralik, E. J., Houweling, P. J., Stout, E. S., Norton, L. J., Alexopoulos, S. J., ... & Quinlan, K. G.

Key Summary: 

The conversion of white adipocytes to thermogenic beige adipocytes represents a potential mechanism to treat obesity and related metabolic disorders. However, the mechanisms involved in converting white to beige adipose tissue remain incompletely understood. Here we show profound beiging in a genetic mouse model lacking the transcriptional repressor Krüppel-like factor 3 (KLF3). Bone marrow transplants from these animals confer the beige phenotype on wild type recipients. Analysis of the cellular and molecular changes reveal an accumulation of eosinophils in adipose tissue. We examine the transcriptomic profile of adipose-resident eosinophils and posit that KLF3 regulates adipose tissue function via transcriptional control of secreted molecules linked to beiging. Furthermore, we provide evidence that eosinophils may directly act on adipocytes to drive beiging and highlight the critical role of these little-understood immune cells in thermogenesis.

Author: 

Yang L, Chen Z, Stout ES, Delerue F, Ittner LM, Wilkins MR, Quinlan KG, Crossley M.

Author: 

Shah, M., Funnell, A.P., Quinlan, K.G. and Crossley, M.

Key Summary: 

Transcriptional silencing may not necessarily depend on the continuous residence of a sequence‐specific repressor at a control element and may act via a “hit and run” mechanism. Due to limitations in assays that detect transcription factor (TF) binding, such as chromatin immunoprecipitation followed by high‐throughput sequencing (ChIP‐seq), this phenomenon may be challenging to detect and therefore its prevalence may be underappreciated. To explore this possibility, erythroid gene promoters that are regulated directly by GATA1 in an inducible system are analyzed. It is found that many regulated genes are bound immediately after induction of GATA1 but the residency of GATA1 decreases over time, particularly at repressed genes. Furthermore, it is shown that the repressive mark H3K27me3 is seldom associated with bound repressors, whereas, in contrast, the active (H3K4me3) histone mark is overwhelmingly associated with TF binding. It is hypothesized that during cellular differentiation and development, certain genes are silenced by repressive TFs that subsequently vacate the region. Catching such repressor TFs in the act of silencing via assays such as ChIP‐seq is thus a temporally challenging prospect. The use of inducible systems, epitope tags, and alternative techniques may provide opportunities for detecting elusive “hit and run” transcriptional silencing. Also see the video abstract here https://youtu.be/vgrsoP_HF3g(link is external).

Link: PubMed

Author: 

Martyn, G. E., Wienert, B., Kurita, R., Nakamura, Y., Quinlan, K. G., & Crossley, M.

Key Summary: 

β-hemoglobinopathies, such as sickle cell disease and β-thalassemia result from mutations in the adult β-globin gene. Reactivating the developmentally silenced fetal γ-globin gene elevates fetal hemoglobin levels and ameliorates symptoms of β-hemoglobinopathies. The continued expression of fetal γ-globin into adulthood occurs naturally in a genetic condition termed Hereditary Persistence of Fetal Hemoglobin (HPFH). Point mutations in the fetal γ-globin proximal promoter can cause HPFH. The -113A>G HPFH mutation falls within the -115 cluster of HPFH mutations, a binding site for the fetal globin repressor BCL11A. We demonstrate that the -113A>G HPFH mutation, unlike other mutations in the cluster, does not disrupt BCL11A binding but rather creates a de novo binding site for the transcriptional activator GATA1. Introduction of the -113A>G HPFH mutation into erythroid cells using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system increases GATA1 binding and elevates fetal globin levels. These results reveal the mechanism by which the -113A>G HPFH mutation elevates fetal globin and demonstrate the sensitivity of the fetal globin promoter to point mutations that often disrupt repressor binding sites but here create a de novo site for an erythroid activator.

Link: Blood

Author: 

Artuz CM, Knights AJ, Funnell APW, Gonda TJ, Ravid K, Pearson RCM, Quinlan KGR, Crossley M.

Key Summary: 

The ability of transcriptional regulators to drive lineage conversion of somatic cells offers great potential for the treatment of human disease. To explore the concept of switching on specific target genes in heterologous cells, we developed a model system to screen candidate factors for their ability to activate the archetypal megakaryocyte-specific chemokine platelet factor 4 (PF4) in fibroblasts. We found that co-expression of the transcriptional regulators GATA1 and FLI1 resulted in a significant increase in levels of PF4, which became magnified over time. This finding demonstrates that such combinations can be used to produce potentially beneficial chemokines in readily available heterologous cell types.

Link: Biotechnology Reports

Author: 

Wienert B, Martyn GE, Funnell APW, Quinlan KGR, Crossley M.

Key Summary: 

Disorders in hemoglobin (hemoglobinopathies) were the first monogenic diseases to be characterized and remain among the most common and best understood genetic conditions. Moreover, the study of the β-globin locus provides a textbook example of developmental gene regulation. The fetal γ-globin genes (HBG1/HBG2) are ordinarily silenced around birth, whereupon their expression is replaced by the adult β-globin genes (HBB primarily and HBD). Over 50 years ago it was recognized that mutations that cause lifelong persistence of fetal γ-globin expression ameliorate the debilitating effects of mutations in β-globin. Since then, research has focused on therapeutically reactivating the fetal γ-globin genes. Here, we summarize recent discoveries, focusing on the influence of genome editing technologies, including CRISPR-Cas9, and emerging gene therapy approaches.

Link: PubMed

Author: 

Knights AJ, Vohralik EJ, Hoehn KL, Crossley M, Quinlan KGR

Key Summary: 

Despite promising early work into the role of immune cells such as eosinophils in adipose tissue (AT) homeostasis, recent findings revealed that elevating the number of eosinophils in AT alone is insufficient for improving metabolic impairments in obese mice. Eosinophils are primarily recognized for their role in allergic immunity and defence against parasitic worms. They have also been detected in AT and appear to contribute to adipose homeostasis and drive energy expenditure, but the underlying mechanisms remain elusive. It has long been recognized that immune cells such as macrophages respond to external signals to regulate adipose homeostasis and energy balance, however, less is known about the relevance of eosinophil activity in AT. As the authors propose in this review, given recent debate over the relative importance of their tissue-specific abundance, the stage is now set for exploring the functionality and activation states of AT eosinophils.

Link: PubMed

Author: 

Martyn, G.E., Wienert, B., Yang, L., Shah, M., Norton, L.J., Burdach, J., Kurita, R., Nakamura, Y., Pearson, R.C.M., Funnell, A.P.W., Quinlan, K.G.R. and Crossley, M.

Key Summary: 

β-hemoglobinopathies such as sickle cell disease (SCD) and β-thalassemia result from mutations in the adult HBB (β-globin) gene. Reactivating the developmentally silenced fetal HBG1 and HBG2 (γ-globin) genes is a therapeutic goal for treating SCD and β-thalassemia 1 . Some forms of hereditary persistence of fetal hemoglobin (HPFH), a rare benign condition in which individuals express the γ-globin gene throughout adulthood, are caused by point mutations in the γ-globin gene promoter at regions residing ~115 and 200 bp upstream of the transcription start site. We found that the major fetal globin gene repressors BCL11A and ZBTB7A (also known as LRF) directly bound to the sites at –115 and –200 bp, respectively. Furthermore, introduction of naturally occurring HPFH-associated mutations into erythroid cells by CRISPR–Cas9 disrupted repressor binding and raised γ-globin gene expression. These findings clarify how these HPFH-associated mutations operate and demonstrate that BCL11A and ZBTB7A are major direct repressors of the fetal globin gene.

Link: Nature Genetics