Merlin Crossley

• BSc Hons (Melb), DPhil (Oxon)
• Deputy Vice-Chancellor Education at UNSW Sydney

Editing Combinations Of Transcription Factor Binding Sites To Maximise Fetal Hemoglobin Production Natural mutations in the HBG1/2 promoters increase HbF production, by either creating new binding sites for activators or impairing the binding of transcriptional repressors. For example, a variant at -175 facilitates TAL1 activator binding, and mutat...

Objectives Hydroxyurea (HU) is the most widely used therapy for adults and children with sickle cell disease (SCD). It is believed to act largely by inducing the transcription of fetal γ-globin genes to generate fetal hemoglobin (HbF), which inhibits the pathological polymerization of sickle hemoglobin (HbS). The mechanisms by which hydroxyurea ele...

Transcription factors often regulate numerous target genes. However, ZNF410 controls only a single gene, CHD4, in human erythroid cells by its highly restricted chromatin occupancy to the CHD4 locus via two clusters of ZNF410 binding motifs. Here, we uncover that ZNF410 controls chromatin accessibility and activity of the two CHD4 enhancer regions....

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for β-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and er...

Inducing fetal hemoglobin (HbF) in red blood cells can alleviate β-thalassemia and sickle cell disease. We compared five strategies in CD34⁺ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin –175A>G. Homozygous –175A>G edited eryth...

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 re...

The fetal-to-adult switch in hemoglobin production is a model of developmental gene control with relevance to the treatment of hemoglobinopathies. The expression of transcription factor BCL11A, which represses fetal β-type globin (HBG) genes in adult erythroid cells, is predominantly controlled at the transcriptional level but the underlying mechan...

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...

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle-cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters usi...

The benign condition Hereditary Persistence of Foetal Haemoglobin (HPFH) is known to ameliorate symptoms when co-inherited with b-haemoglobinopathies, such as sickle cell disease and b-thalassaemia. The condition is sometimes associated with point mutations in the foetal globin promoters that disrupt the binding of the repressors BCL11A or ZBTB7A/L...

Elevated levels of fetal globin protect against β-hemoglobinopathies, such as sickle cell disease and β-thalassemia. Two zinc-finger (ZF) repressors, BCL11A and ZBTB7A/LRF, bind directly to the fetal globin promoter elements positioned at −115 and −200, respectively. Here, we describe X-ray structures of the ZBTB7A DNA-binding domain, consisting of...

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates β-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to β-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcript...

Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding...

Transcription factors typically regulate a large number of genes. Here we found that transcription factor ZNF410 binds and activates the expression of a single direct target gene, CHD4, to enforce the silencing of the fetal hemoglobin genes (HBG1 and HBG2) in adult erythroid cells. ZNF410 is a pentadactyl DNA binding protein that emerged from a DNA...

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Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA binding protein that in human erythroid cells directly and measurably activates only one gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF41...

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 represso...

Alterations in DNA methylation occur during development, but the mechanisms by which they influence gene expression remain uncertain. There are few examples where modification of a single CpG dinucleotide directly affects transcription factor binding and regulation of a target gene in vivo. Here, we show that the erythroid transcription factor GATA...

Bacterial products such as lipopolysaccharide (or endotoxin) cause systemic inflammation, resulting in a substantial global health burden. The onset, progression, and resolution of the inflammatory response to endotoxin are usually tightly controlled to avoid chronic inflammation. Members of the NF-κB family of transcription factors are key drivers...

Hereditary persistence of fetal hemoglobin (HPFH) mutations are rare, naturally-occurring variations which reduce the severity of beta-hemoglobinopathies by increasing postnatal expression of the gamma-globin genes (HBG1/2). Genome editing to inhibit erythroid transcriptional silencers or alter cis-elements in the HBG1/2 promoters recapitulates HPF...

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 ph...

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 geneti...

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,...

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-speci...

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 again...

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-thalassemia1. Some forms of hereditary persistence of fetal hemoglobin (HPFH), a rare benign cond...

Genome editing to introduce specific mutations or to knock out genes in model cell systems has become an efficient platform for research in the fields of molecular biology, genetics, and cell biology. With recent rapid improvements in genome editing techniques, bench-top manipulation of the genome in cell culture has become progressively easier. Th...

Editorial summary Genome editing using clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated proteins offers the potential to facilitate safe and effective treatment of genetic diseases refractory to other types of intervention. Here, we identify some of the major challenges for clinicians, regulators, and human...

Key Points Introduction of the British HPFH mutation into the fetal globin promoter in a human cell model causes elevated fetal globin expression. The British HPFH mutation creates a de novo binding site both in vitro and in vivo for the potent erythroid activator KLF1.

The Krüppel-like factor (KLF) family of transcription factors play critical roles in haematopoiesis. KLF1, the founding member of the family, has been implicated in the control of both erythropoiesis and megakaryopoiesis. Here we describe a novel system using an artificial dominant negative isoform of KLF1 to investigate the role of KLF1 in the ery...

Krüppel-like factors (KLFs) are a family of 17 transcription factors characterized by a conserved DNA-binding domain of three zinc fingers and a variable N-terminal domain responsible for recruiting cofactors. KLFs have diverse functions in stem cell biology, embryo patterning, and tissue homoeostasis. KLF1 and related family members function as tr...

Key Points KLF1 directly drives expression of ZBTB7A, a key repressor of fetal γ-globin gene expression, in erythroid cells. An erythroid-specific regulation mechanism allows upregulation of a novel ZBTB7A transcript in erythroid cells.

Krüppel-like factors (KLF) are a group of 17 transcription factors with highly conserved DNA-binding domains that contain three C-terminal C2H2-type zinc fingers and a variable N-terminal domain responsible for recruiting cofactors 1. KLFs participate in diverse roles in stem cell renewal, early patterning, organogenesis and tissue homeostasis. Krü...

CCAAT boxes are motifs found within the proximal promoter of many genes, including the human globin genes. The highly conserved nature of CCAAT box motifs within the promoter region of both α-like and β-like globin genes emphasises the functional importance of the CCAAT sequence in globin gene regulation. Mutations within the β-globin CCAAT box res...

The Lgals3 gene encodes a multifunctional β-galactoside-binding protein, galectin-3. Galectin-3 has been implicated in a broad range of biological processes, from chemotaxis and inflammation to fibrosis and apoptosis. The role of galectin-3 as a modulator of inflammation has been studied intensively and recent evidence suggests that it may serve as...

Until recently our approach to the analysis of human genetic diseases has been to accurately phenotype patients and sequence the genes known to be associated with those phenotypes; for example, analysing the globin loci in cases of thalassemia. As sequencing has become increasingly accessible, a larger panel of genes is now analysed and whole exome...

Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that...

Reactivating the fetal globin gene Mutation of adult-type globin genes causes sickle cell disease and thalassemia. Although treating these hemoglobinopathies with gene therapy is possible, there is a pressing need for pharmacologic approaches to treat general patient populations. One promising approach is to reactivate repressed expression of fetal...

Transcription factors are often regarded as having two separable components: a DNA-binding domain (DBD) and a functional domain (FD), with the DBD thought to determine target gene recognition. While this holds true for DNA binding in vitro, it appears that in vivo FDs can also influence genomic targeting. We fused the FD from the well-characterized...

Induction of fetal-type hemoglobin (HbF: α2γ2) is a promising means to treat hemoglobinopathies; however, precisely how HbF expression is silenced in adult erythroid cells is not fully understood. Such knowledge is essential to develop mechanism-based, targeted approaches to reactivate HbF production. Here, we show that Leukemia/lymphoma Related Fa...

Genetic disorders resulting from defects in the adult globin genes are among the most common inherited diseases. Symptoms worsen from birth as fetal γ-globin expression is silenced. Genome editing could permit the introduction of beneficial single-nucleotide variants to ameliorate symptoms. Here, as proof of concept, we introduce the naturally occu...

Kruppel-like Factor 3 (KLF3/BKLF), a member of the Kruppel-like Factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. While there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal...

C-terminal binding proteins (CtBPs) are recruited by a variety of transcription factors to mediate gene repression. Nematode CTBP-1 has previously been shown to play a role in the regulation of lifespan; Caenorhabditis elegans strains carrying a deletion in the ctbp-1 gene showed a 10-20% increase in mean and maximal lifespan compared with wild-typ...

Table of DAVID enrichments used to annotate Figure 8 and the HGMD genes that overlapped our CRMs and singletons in the different phylogenetic categories.DOI: http://dx.doi.org/10.7554/eLife.02626.025

Quality control for ChIP-seq, CRM construction, and multi-species comparisons.DOI: http://dx.doi.org/10.7554/eLife.02626.004

Functional enrichment results obtained for CRMs and singletons using GREAT.DOI: http://dx.doi.org/10.7554/eLife.02626.011

Tally of genes with HGMD regulatory mutations that overlap liver CRMs or singletons. DOI: http://dx.doi.org/10.7554/eLife.02626.029

Table of CRMs and singletons along with the phylogenetic categories they were assigned.File coordinates are for the hg18 assembly.DOI: http://dx.doi.org/10.7554/eLife.02626.009

Motif enrichments of CRMs and singletons as well as overlaps of CRMs and Singletons with ENCODE data. DOI: http://dx.doi.org/10.7554/eLife.02626.027

Comparison of motif matches between CRMs and singletons. Chi-square test for differences between the number of peaks associated with CRMs and singletons, for each TF, that contained at least one predicted motif using three different p-value thresholds for motif scanning: stringent (10−4), moderate (10−3) and lenient (10−2). Blue shadows highlight s...

Full tables of 2.5 kb and LD GWAS enrichments performed in Figure 7 and Table 1.The file also includes the categories used to annotate the NHGRI catalog.DOI: http://dx.doi.org/10.7554/eLife.02626.019

Deeply conserved CRMs within 2.5 kb of a lead GWAS SNP were overlapped with RegulomeDB annotated SNPs. SNPs with a high confidence evidence of regulatory potenital are shown (evidence levels 1 and 2 from Regulome DB). DOI: http://dx.doi.org/10.7554/eLife.02626.028

FOG1 is a transcriptional regulator that acts in concert with the hematopoietic master regulator GATA1 to coordinate the differentiation of platelets and erythrocytes. Despite considerable effort, however, the mechanisms through which FOG1 regulates gene expression are only partially understood. Here we report the discovery of a previously unrecogn...

Chromatin regulators contribute to the developmental control of gene expression. In the nematode Caenorhabditis elegans, the roles of chromatin regulation in development have been explored in several contexts, including vulval differentiation. The synthetic multivulva (synMuv) genes are regulators of vulval development in C. elegans and the protein...

Background Krüppel-like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor with diverse biological roles. During erythropoiesis, KLF3 acts as a feedback repressor of a set of genes that are activated by Krüppel-like Factor 1 (KLF1). Noting that KLF1 binds α-globin gene regulatory sequences during erythroid maturation, we s...

Background Retroviral elements are pervasively transcribed and dynamically regulated during development. While multiple histone- and DNA-modifying enzymes have broadly been associated with their global silencing, little is known about how the many diverse retroviral families are each selectively recognized. Results Here we show that the zinc finge...

Obesity is a major public health concern and a strong risk factor for insulin resistance, type 2 diabetes mellitus (T2DM), and cardiovascular disease. The last two decades have seen a reconsideration of the role of white adipose tissue (WAT) in whole body metabolism and insulin action. Adipose tissue-derived cytokines and hormones, or adipokines, a...

Background: Tightly regulated pathways maintain the balance between proliferation and differentiation within stem cell populations. In Caenorhabditis elegans, the germline is the only tissue that is maintained by stem-like cells into adulthood. In the current study, we investigated the role played by a member of the Homeodomain interacting protein...

Hemophilia B is a classic, monogenic blood clotting disease caused by mutations in the coagulation factor IX (F9) locus. Although interpreting mutations within the gene itself has been relatively straightforward, ascribing molecular mechanisms to the complete suite of mutations within the promoter region has proven somewhat difficult and has only r...

Transcription factors (TFs) are often regarded as being composed of a DNA-binding domain (DBD) and a functional domain. The two domains are considered separable and autonomous, with the DBD directing the factor to its target genes and the functional domain imparting transcriptional regulation. We examined an archetypal zinc finger (ZF) TF, Krüppel-...

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Histology of the pulmonary valve in adult Klf3 mutants. Pulmonary valve histology at low power (A,C) for wild type (WT) mice (left) and (A) for 2 different Klf3H275R/+ mutants (middle and right) and (C) for homozygous XS (middle) and CH mutants (right). (B,D) Higher power images of the pulmonary valves located in the boxed regions in (A,C). Pulmona...

Mapping mutation in ENU mutant line. (A) Genome scan of mice with high aortic blood velocity trait. Microsatellite markers polymorphic between parental strains (H) localized mutation between markers D5Mit346 and D5Mit201. (B) LOD score of trait on chromosome 5. Vertical dashed lines show fine mapped interval, which contained 35 genes. Vertical soli...

Enlarged heart of an adult Klf3H275R heterozygote that was found moribund. Heart image at 47 wk of a moribund Klf3H275R heterozygote (right) in comparison to a littermate control (left). Images show the dramatic cardiac enlargement typical of Klf3H275R heterozygotes that became acutely ill and were found moribund, likely due to high output heart fa...

Light and electron microscopy of the adult ventricular myocardium. In general, the structure of the mutant myocardium appeared normal when examined (A) by light microscopy, and (B) by electron microscopy. However, focal regions of contraction band necrosis (arrows) were sometimes observed in the Klf3H275R/+ myocardium (right) whereas this was rare...

Activator and repressor functions of Klf3H275R in mutant embryos. (A) Activation of Hsd3b6 was significantly diminished in heterozygous (Het) and homozygous (Homo) Klf3H275R embryos at E12.5 relative to wild type littermate controls (WT). (B) A trend towards a similar diminishment in Hsd3b6 expression in homozygous CH gene trap mutants was not stat...

Motion of the inter-ventricular septum during the cardiac cycle in adult wild type mice. Short axis view of the inter-ventricular septum, and right and left ventricles of a wild type adult mouse (58 wk) obtained using micro-ultrasound. The septum is near the middle of the image with the right ventricle on the left side, and the left ventricle on th...

Body and organ weights of XS and CH homozygous adult mice at 18–82 wk. (DOCX)

Increased mRNA expression of Lgals3 in Klf3H275R mutant embryos. Lgals3 mRNA was significantly increased in (A) homozygous (Homo) Klf3H275R embryos at E12.5 relative to wild type littermate controls (WT). (B) Lgals3 was significantly increased in livers from homozygous (Homo) and heterozygous (Het) Klf3H275R embryos at E12.5. These results show tha...

Abnormal motion of the inter-ventricular septum during the cardiac cycle in adult CH homozygous mice. Short axis view of the inter-ventricular septum, and right and left ventricles of a homozygous Klf3 gene trap mutant of the CH line (age 58 wk) obtained using micro-ultrasound. The septum is near the middle of the image with the visibly enlarged ri...

Stage of perinatal or postnatal lethality in Klf3H272R mutants. (DOCX)

Lethality at weaning of XS and CH gene trap mutants. (DOCX)

Differential gene expression of embryos at E12.5 by microarray with False Discovery Rate (FDR) <0.1. A. WT (n = 4) versus CH homozygotes (n = 4). B. Klf3H275R homozygotes (n = 3) versus CH homozygotes (n = 4). C. Klf3H275R heterozygotes (n = 4) versus CH homozygotes (n = 4). (DOCX)

PCR primers for genomic DNA used to genotype mouse lines. (DOCX)

Hematological parameters in adult Klf3 mutants. Blood of Klf3H275R and of XS and CH gene trap lines was sampled at 9–19 wk. RBC = red blood cell counts; MCV = red blood cell volume; RDW = red blood cell distribution width; WBC = white blood cell count; WT = wild-type; Het = heterozygotes; Homo = homozygotes. No homozygote Klf3H275R mice survived to...

KLF3H275R protein does not interfere with the ability of WT KLF3 to bind to DNA. No reduction in binding of WT KLF3 to KLF3's canonical CACCC binding region of the β-globin gene promoter was observed when (A) the recombinant WT protein was combined with recombinant bacterial GST-Klf3H275R zinc finger 1–3 protein in ratios of 1∶1, 1∶2, and 1∶4 or (B...

LacZ staining in the adult heart and aorta showing Klf3 gene expression. (A) LacZ-staining (blue) in homozygous XS mice shows Klf3 gene expression in the atrial myocardium, left atrioventricular valve, left ventricular myocardium, and aorta. Lower magnification images are shown on left (20×) and higher magnification images on right (100×). (B) Imag...

List of 35 genes in mapping interval on Chromosome 5. (DOCX)

Heat map showing relative microarray gene expression of Klf3H275R and CH mutants versus wild type. Genes with a greater than 1.5-fold difference in expression in Klf3H275R/H275R embryos relative to wild type are shown. These differences were not statistically significant at a false discovery rate of 0.1. Lilra6 and Hsd3b6 mRNA was measured by qRT-P...

Body and organ weights of Klf3H275R heterozygous mice at 18–25 wk. (DOCX)

qRT-PCR primer sequences for mRNA. (DOCX)

KLF3 is a Krüppel family zinc finger transcription factor with widespread tissue expression and no previously known role in heart development. In a screen for dominant mutations affecting cardiovascular function in N-ethyl-N-nitrosourea (ENU) mutagenized mice, we identified a missense mutation in the Klf3 gene that caused aortic valvular stenosis a...

Krüppel-like factors 3 and 8 (KLF3 and KLF8) are highly related transcriptional regulators that bind to similar sequences of DNA. We have previously shown that in erythroid cells there is a regulatory hierarchy within the KLF family, whereby KLF1 drives the expression of both the Klf3 and Klf8 genes and KLF3 in turn represses Klf8 expression. While...

Krüppel-like Factor 3 (KLF3) is a transcriptional regulator that we have shown to be involved in the regulation of adipogenesis in vitro. Here we report that KLF3 null mice are lean and protected from diet-induced obesity and glucose intolerance. On a chow diet, plasma levels of leptin are decreased, and adiponectin is increased. Despite significan...

Hemophilia B, or the "royal disease," arises from mutations in coagulation factor IX (F9). Mutations within the F9 promoter are associated with a remarkable hemophilia B subtype, termed hemophilia B Leyden, in which symptoms ameliorate after puberty. Mutations at the -5/-6 site (nucleotides -5 and -6 relative to the transcription start site, design...

Classical zinc fingers (ZFs) are one of the most abundant and best characterized DNA-binding domains. Typically, tandem arrays of three or more ZFs bind DNA target sequences with high affinity and specificity, and the mode of DNA recognition is sufficiently well understood that tailor-made ZF-based DNA-binding proteins can be engineered. We have sh...