Kevin R. Gillinder

Crim1KST264/KST264 mice display a disruption of the Crim1 gene resulting in perinatal lethality with defects in multiple organ systems.

Crim1 is a transmembrane protein, containing six vWF‐C type cysteine‐rich repeats, that tethers growth factors to the cell surface. A mouse line, KST264, generated in a LacZ insertion mutagenesis gene‐trap screen, was examined to elucidate Crim1 function in development. We showed that Crim1KST264/KST264 mice were not null for Crim1 due to the production of a shortened protein isoform. These mice are likely to represent an effective hypomorph or a dominant‐negative for Crim1. Transgene expression recapitulated known Crim1 expression in lens, brain, and limb, but also revealed expression in the smooth muscle cells of the developing heart and renal vasculature, developing cartilage, mature ovary and detrusor of the bladder. Transgene expression was also observed in glomerular epithelial cells, podocytes, mesangial cells, and urothelium in the kidney. Crim1KST264/KST264 mice displayed perinatal lethality, syndactyly, eye, and kidney abnormalities. The severe and complex phenotype observed in Crim1KST264/KST264 mice highlights the importance of Crim1 in numerous aspects of organogenesis. Developmental Dynamics 236:502–511, 2007.

A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing

BackgroundCRISPR-Cas9 is a revolutionary genome editing technique that allows for efficient and directed alterations of the eukaryotic genome. This relatively new technology has already been used in a large number of ‘loss of function’ experiments in cultured cells. Despite its simplicity and efficiency, screening for mutated clones remains time-consuming, laborious and/or expensive.ResultsHere we report a high-throughput screening strategy that allows parallel screening of up to 96 clones, using next-generation sequencing. As a proof of principle, we used CRISPR-Cas9 to disrupt the coding sequence of the homeobox gene, Evx1 in mouse embryonic stem cells. We screened 67 CRISPR-Cas9 transfected clones simultaneously by next-generation sequencing on the Ion Torrent PGM. We were able to identify both homozygous and heterozygous Evx1 mutants, as well as mixed clones, which must be identified to maintain the integrity of subsequent experiments.ConclusionsOur CRISPR-Cas9 screening strategy could be widely applied to screen for CRISPR-Cas9 mutants in a variety of contexts including the generation of mutant cell lines for in vitro research, the generation of transgenic organisms and for assessing the veracity of CRISPR-Cas9 homology directed repair. This technique is cost and time-effective, provides information on clonal heterogeneity and is adaptable for use on various sequencing platforms.

KLF1 Null Neonates Display Hydrops Fetalis and a Deranged Erythroid Transcriptome

We describe a case of severe neonatal anemia with kernicterus caused by compound heterozygosity for null mutations in KLF1, each inherited from asymptomatic parents. One of the mutations is novel. This is the first described case of a KLF1-null human. The phenotype of severe nonspherocytic hemolytic anemia, jaundice, hepatosplenomegaly, and marked erythroblastosis is more severe than that present in congenital dyserythropoietic anemia type IV as a result of dominant mutations in the second zinc-finger of KLF1. There was a very high level of HbF expression into childhood (>70%), consistent with a key role for KLF1 in human hemoglobin switching. We performed RNA-seq on circulating erythroblasts and found that human KLF1 acts like mouse Klf1 to coordinate expression of many genes required to build a red cell including those encoding globins, cytoskeletal components, AHSP, heme synthesis enzymes, cell-cycle regulators, and blood group antigens. We identify novel KLF1 target genes including KIF23 and KIF11 which are required for proper cytokinesis. We also identify new roles for KLF1 in autophagy, global transcriptional control, and RNA splicing. We suggest loss of KLF1 should be considered in otherwise unexplained cases of severe neonatal NSHA or hydrops fetalis.

Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viability

Abstract The rules of engagement between zinc finger transcription factors and DNA have been partly defined by in vitro DNA-binding and structural studies, but less is known about how these rules apply in vivo. Here, we demonstrate how a missense mutation in the second zinc finger of Krüppel-like factor-1 (KLF1) leads to degenerate DNA-binding specificity in vivo, resulting in ectopic transcription and anemia in the Nan mouse model. We employed ChIP-seq and 4sU-RNA-seq to identify aberrant DNA-binding events genome wide and ectopic transcriptional consequences of this binding. We confirmed novel sequence specificity of the mutant recombinant zinc finger domain by performing biophysical measurements of in vitro DNA-binding affinity. Together, these results shed new light on the mechanisms by which missense mutations in DNA-binding domains of transcription factors can lead to autosomal dominant diseases.

Direct targets of pSTAT5 signalling in erythropoiesis.

Erythropoietin (EPO) acts through the dimeric erythropoietin receptor to stimulate proliferation, survival, differentiation and enucleation of erythroid progenitor cells. We undertook two complimentary approaches to find EPO-dependent pSTAT5 target genes in murine erythroid cells: RNA-seq of newly transcribed (4sU-labelled) RNA, and ChIP-seq for pSTAT5 30 minutes after EPO stimulation. We found 302 pSTAT5-occupied sites: ~15% of these reside in promoters while the rest reside within intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of pSTAT5 peaks contain a central palindromic GAS element, TTCYXRGAA. There was significant enrichment for GATA motifs and CACCC-box motifs within the neighbourhood of pSTAT5-bound peaks, and GATA1 and/or KLF1 co-occupancy at many sites. Using 4sU-RNA-seq we determined the EPO-induced transcriptome and validated differentially expressed genes using dynamic CAGE data and qRT-PCR. We identified known direct pSTAT5 target genes such as Bcl2l1, Pim1 and Cish, and many new targets likely to be involved in driving erythroid cell differentiation including those involved in mRNA splicing (Rbm25), epigenetic regulation (Suv420h2), and EpoR turnover (Clint1/EpsinR). Some of these new EpoR-JAK2-pSTAT5 target genes could be used as biomarkers for monitoring disease activity in polycythaemia vera, and for monitoring responses to JAK inhibitors.

Krüppel-like factors compete for promoters and enhancers to fine-tune transcription

Abstract 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 transcriptional activators via recruitment of co-activators such as EP300, whereas KLF3 and related members act as transcriptional repressors via recruitment of C-terminal Binding Proteins. KLF1 directly activates the Klf3 gene via an erythroid-specific promoter. Herein, we show KLF1 and KLF3 bind common as well as unique sites within the erythroid cell genome by ChIP-seq. We show KLF3 can displace KLF1 from key erythroid gene promoters and enhancers in vivo. Using 4sU RNA labelling and RNA-seq, we show this competition results in reciprocal transcriptional outputs for >50 important genes. Furthermore, Klf3−/− mice displayed exaggerated recovery from anemic stress and persistent cell cycling consistent with a role for KLF3 in dampening KLF1-driven proliferation. We suggest this study provides a paradigm for how KLFs work in incoherent feed-forward loops or networks to fine-tune transcription and thereby control diverse biological processes such as cell proliferation.

The Evx1/Evx1as gene locus regulates anterior-posterior patterning during gastrulation

Thousands of sense-antisense mRNA-lncRNA gene pairs occur in the mammalian genome. While there is usually little doubt about the function of the coding transcript, the function of the lncRNA partner is mostly untested. Here we examine the function of the homeotic Evx1-Evx1as gene locus. Expression is tightly co-regulated in posterior mesoderm of mouse embryos and in embryoid bodies. Expression of both genes is enhanced by BMP4 and WNT3A, and reduced by Activin. We generated a suite of deletions in the locus by CRISPR-Cas9 editing. We show EVX1 is a critical downstream effector of BMP4 and WNT3A with respect to patterning of posterior mesoderm. The lncRNA, Evx1as arises from alternative promoters and is difficult to fully abrogate by gene editing or siRNA approaches. Nevertheless, we were able to generate a large 2.6 kb deletion encompassing the shared promoter with Evx1 and multiple additional exons of Evx1as. This led to an identical dorsal-ventral patterning defect to that generated by micro-deletion in the DNA-binding domain of EVX1. Thus, Evx1as has no function independent of EVX1, and is therefore unlikely to act in trans. We predict many antisense lncRNAs have no specific trans function, possibly only regulating the linked coding genes in cis.

Neomorphic effects of the neonatal anemia (Nan-Eklf) mutation contribute to deficits throughout development

ABSTRACT Transcription factor control of cell-specific downstream targets can be significantly altered when the controlling factor is mutated. We show that the semi-dominant neonatal anemia (Nan) mutation in the EKLF/KLF1 transcription factor leads to ectopic expression of proteins that are not normally expressed in the red blood cell, leading to systemic effects that exacerbate the intrinsic anemia in the adult and alter correct development in the early embryo. Even when expressed as a heterozygote, the Nan-EKLF protein accomplishes this by direct binding and aberrant activation of genes encoding secreted factors that exert a negative effect on erythropoiesis and iron use. Our data form the basis for a novel mechanism of physiological deficiency that is relevant to human dyserythropoietic anemia and likely other disease states. Highlighted article: The Nan mouse contains a heterozygous mutation in Eklf/Klf1 that ectopically activates misexpressed genes, leading to intrinsic erythroid and systemic developmental deficits.

Variable serologic and other phenotypes due to KLF1 mutations: LETTERS TO THE EDITOR

Variable serologic and other phenotypes due to KLF1 mutations We note with interest the recent online prepublication (now published) in TRANSFUSION by Keller and colleagues entitled “Novel mutations in KLF1 encoding the In(Lu) phenotype reflect a diversity of clinical presentations.” This article describes seven cases of the In(Lu) blood serology phenotype resulting from mutations in KLF1. It details important findings with respect to new human KLF1 mutations and serologic phenotypes, but some aspects of KLF1 function require clarification with respect to the literature. We provide a schematic of genotype–phenotype correlations to help blood service providers make appropriate recommendations for patients and clients. In many cases, there are likely to be important genetic counseling issues that are like those which are pertinent for counseling families at risk of thalassemia major. These issues are currently underappreciated. Case 2 in the article by Keller and colleagues is a 49-year-old Caucasian with the Lu(a–b–) serotype due to a mutation in Exon 2 of KLF1, c.90aG>A, leading to an early stop codon, p.Trp30Ter. This mutation was previously reported in a family who had a child with unexpected hydrops fetalis. The father of this child had an isolated p.Trp30Ter mutation, elevated HbF (2.4%), and reduced CD44 (Indian) surface expression on red blood cells (RBCs). The mother carried a common frameshift mutation. Serology was not performed in the family reported by Magor and coworkers, but the In(Lu) phenotype was presumably present in the father as it is in Case 2 of the report in TRANSFUSION. So, this new case confirms p.Trp30Ter is a loss-of-function mutation, and thus the hydrops case reported by Magor and coworkers must have resulted from absence of KLF1 function. Case 3 describes a new mutation in a highly conserved threonine (Thr-304) within linker 1 (TGEKP), between zinc fingers 1 and 2. Although this amino acid does not contact DNA directly, the TGEKP sequence is highly conserved and likely to play a critical role in overall DNA-binding affinity, although this needs to be tested further in biochemical and/or genetic studies. This is the first report of a mutation in linker 1 of KLF1 but there are a few reports of human and mouse mutations in linker 2. These are interesting because many retain partial KLF1 function in binding assays (i.e., weak alleles (w) in Fig. 1) and genetically. This is evidenced by the fact that homozygosity for the H350R mutation in murine linker 2 leads to a mild hemolytic anemia, and many of the human mutations are also clinically mild. Interestingly, when coinherited with a KLF1 lossof-function mutation on the other allele, these linker 2 mutations result in an intermediate hemolytic phenotype (known as congenital nonspherocytic hemolytic anemia [CNSHA]) between the phenotypes of humans with homozygous loss-of-function mutations and heterozygous loss-of-function mutations (w/– alleles in Fig. 1). It remains to be seen whether the same is true for the Thr304Lys mutation described by Keller and colleagues in TRANSFUSION. Serologic testing for In(Lu) is likely to detect most of the weak alleles of KLF1 but not necessarily all of them (Fig. 1). Similarly, testing for elevated HbF levels is likely to detect many weak alleles but not all of them. Some hypomorphic KLF1 mutations may result in reduced DNA binding to some gene promoters or enhancers but not to others, as is the case for the E339D Klf1 mutation in mice. This could explain some of the phenotypic variation described with these alleles. Cases 5 and 6 are unrelated, each with the same two variants present in cis, c.304T>C (p.Ser102Pro) and c.318T>G (p.Tyr106Ter). Both cases have the Lu(a–b–) serotype. There has been some controversy about whether p.Ser102Pro is a pathogenic variant as it is present at approximately 50% minor allele frequencies in many populations. Keller and coworkers investigated the serology in an additional 91 cases of this variant and confirm none of them have In(Lu). So, this confirms the c.304T>C (p.Ser102Pro) SNP (rs2072597) is a common normal variant (1/1 in Fig. 1). The In(Lu) serotype in Cases 5 and 6 is simply explained by the presence of an additional stop codon (p.Tyr106Ter) in cis to the c.304T>C SNP. Case 4 describes a child with congenital dyserythropoietic anemia (CDA) due to a heterozygous variant c.973G>A that results in p.Glu325Lys. This rare condition was originally described by Arnaud and coworkers. Keller and colleagues suggest that the CDA phenotype may not be due to the heterozygous mutation in KLF1 because all other loss-of-function mutations simply result in a phenotype characterized by In(Lu), increased HbF and/or HbA2, and mildly reduced CD44 expression on the RBC surface. We disagree with this conclusion and suggest the p.Glu325Lys variant is likely to be an interesting dominant gain-of-function mutation in KLF1. The best evidence for this comes from detailed ChIP, RNA-seq, and biophysical studies on a mutation in Klf1 at the equivalent amino acid, p.Glu339Asp, in mice. This mutation results in an altered DNA-binding specificity in vivo and in vitro and consequent activation of genes not normally expressed in erythroid cells. These are toxic to the developing erythroid cell and result in hemolysis. Although the disease mechanism is doi: 10.1111/trf.14529

Mutant KLF1 in Adult Anemic Nan Mice Leads to Profound Transcriptome Changes and Disordered Erythropoiesis

Anemic Nan mice carry a mutation (E339D) in the second zinc finger of erythroid transcription factor KLF1. Nan-KLF1 fails to bind a subset of normal KLF1 targets and ectopically binds a large set of genes not normally engaged by KLF1, resulting in a corrupted fetal liver transcriptome. Here, we performed RNAseq using flow cytometric-sorted spleen erythroid precursors from adult Nan and WT littermates rendered anemic by phlebotomy to identify global transcriptome changes specific to the Nan Klf1 mutation as opposed to anemia generally. Mutant Nan-KLF1 leads to extensive and progressive transcriptome corruption in adult spleen erythroid precursors such that stress erythropoiesis is severely compromised. Terminal erythroid differentiation is defective in the bone marrow as well. Principle component analysis reveals two major patterns of differential gene expression predicting that defects in basic cellular processes including translation, cell cycle, and DNA repair could contribute to disordered erythropoiesis and anemia in Nan. Significant erythroid precursor stage specific changes were identified in some of these processes in Nan. Remarkably, however, despite expression changes in large numbers of associated genes, most basic cellular processes were intact in Nan indicating that developing red cells display significant physiological resiliency and establish new homeostatic set points in vivo.