Inhibition of retinoic acid signaling impairs cranial and spinal neural tube closure in mice lacking the Grainyhead-like 3 transcription factor.

Neural tube closure is a dynamic morphogenic event in early embryonic development. Perturbations of this process through either environmental or genetic factors induce the severe congenital malformations known collectively as neural tube defects (NTDs). Deficiencies in maternal folate intake have long been associated with NTDs, as have mutations in critical neurulation genes that include the Grainyhead-like 3 (Grhl3) gene. Mice lacking this gene exhibit fully penetrant thoraco-lumbo-sacral spina bifida and a low incidence of exencephaly. Previous studies have shown that exposure of pregnant mice carrying hypomorphic Grhl3 alleles to exogenous retinoic acid (RA) increases the incidence and severity of NTDs in their offspring. Here, we demonstrate that inhibition of RA signaling using a high affinity pan-RA receptor antagonist administered to pregnant mice at E7.5 induces fully penetrant exencephaly and more severe spina bifida in Grhl3-null mice. Later administration, although prior to neural tube closure has no effect. Similarly, blockade of RA in the context of reduced expression of Grhl2, a related gene known to induce NTDs, has no effect. Taken together, these findings provide new insights into the complexities of the interplay between RA signaling and Grhl3-induced neurulation.

Corrupted DNA-binding specificity and ectopic transcription underpin dominant neomorphic mutations in KLF/SP transcription factors.

BACKGROUND: Mutations in the transcription factor, KLF1, are common within certain populations of the world. Heterozygous missense mutations in KLF1 mostly lead to benign phenotypes, but a heterozygous mutation in a DNA-binding residue (E325K in human) results in severe Congenital Dyserythropoietic Anemia type IV (CDA IV); i.e. an autosomal-dominant disorder characterized by neonatal hemolysis. RESULTS: To investigate the biochemical and genetic mechanism of CDA IV, we generated murine erythroid cell lines that harbor tamoxifen-inducible (ER™) versions of wild type and mutant KLF1 on a Klf1-/- genetic background. Nuclear translocation of wild type KLF1 results in terminal erythroid differentiation, whereas mutant KLF1 results in hemolysis without differentiation. The E to K variant binds poorly to the canonical 9 bp recognition motif (NGG-GYG-KGG) genome-wide but binds at high affinity to a corrupted motif (NGG-GRG-KGG). We confirmed altered DNA-binding specificity by quantitative in vitro binding assays of recombinant zinc-finger domains. Our results are consistent with previously reported structural data of KLF-DNA interactions. We employed 4sU-RNA-seq to show that a corrupted transcriptome is a direct consequence of aberrant DNA binding. CONCLUSIONS: Since all KLF/SP family proteins bind DNA in an identical fashion, these results are likely to be generally applicable to mutations in all family members. Importantly, they explain how certain mutations in the DNA-binding domain of transcription factors can generate neomorphic functions that result in autosomal dominant disease.

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

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.

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

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.

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.

Fibroblast growth factor-1 (FGF-1) promotes adipogenesis by downregulation of carboxypeptidase A4 (CPA4) - a negative regulator of adipogenesis implicated in the modulation of local and systemic insulin sensitivity.

Fibroblast growth factor-1 (FGF-1) promotes differentiation of human preadipocytes into mature adipocytes via modulation of a BMP and Activin Membrane-Bound Inhibitor (BAMBI)/Peroxisome proliferator-activated receptor (PPARγ)-dependent network. Here, we combined transcriptomic and functional investigations to identify novel downstream effectors aligned with complementary analyses of gene expression in human adipose tissue to explore relationships with insulin sensitivity. RNA-Seq and qRT-PCR analysis revealed significant down-regulation of carboxypeptidase A4 (CPA4) following FGF-1 treatment or induction of differentiation of human preadipocytes in a BAMBI/PPARγ-independent manner. siRNA-mediated knockdown of CPA4 resulted in enhanced differentiation of human preadipocytes. Furthermore, expression of CPA4 in subcutaneous adipose tissue correlated negatively with indices of local and systemic (liver and muscle) insulin sensitivity. These results identify CPA4 as a negative regulator of adipogenesis that is down-regulated by FGF-1 and a putative deleterious modulator of local and systemic insulin sensitivity. Further investigations are required to define the molecular mechanism(s) involved and potential therapeutic opportunities.

Identification of novel hypomorphic and null mutations in Klf1 derived from a genetic screen for modifiers of α-globin transgene variegation.

Position-effect variegation of transgene expression is sensitive to the chromatin state. We previously reported a forward genetic screen in mice carrying a variegated α-globin GFP transgene to find novel genes encoding epigenetic regulators. We named the phenovariant strains "Mommes" for modifiers of murine metastable epialleles. Here we report positional cloning of mutations in two Momme strains which result in suppression of variegation. Both strains harbour point mutations in the erythroid transcription factor, Klf1. One (D11) generates a stop codon in the zinc finger domain and a homozygous null phenotype. The other (D45) generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three. Homozygous MommeD45 mice have chronic microcytic anaemia which models the phenotype in a recently described family. This is the first genetic evidence that the linkers between the zinc fingers of transcription factors have a function beyond that of a simple spacer.

Novel roles for KLF1 in erythropoiesis revealed by mRNA-seq.

KLF1 (formerly known as EKLF) regulates the development of erythroid cells from bi-potent progenitor cells via the transcriptional activation of a diverse set of genes. Mice lacking Klf1 die in utero prior to E15 from severe anemia due to the inadequate expression of genes controlling hemoglobin production, cell membrane and cytoskeletal integrity, and the cell cycle. We have recently described the full repertoire of KLF1 binding sites in vivo by performing KLF1 ChIP-seq in primary erythroid tissue (E14.5 fetal liver). Here we describe the KLF1-dependent erythroid transcriptome by comparing mRNA-seq from Klf1(+/+) and Klf1(-/-) erythroid tissue. This has revealed novel target genes not previously obtainable by traditional microarray technology, and provided novel insights into the function of KLF1 as a transcriptional activator. We define a cis-regulatory module bound by KLF1, GATA1, TAL1, and EP300 that coordinates a core set of erythroid genes. We also describe a novel set of erythroid-specific promoters that drive high-level expression of otherwise ubiquitously expressed genes in erythroid cells. Our study has identified two novel lncRNAs that are dynamically expressed during erythroid differentiation, and discovered a role for KLF1 in directing apoptotic gene expression to drive the terminal stages of erythroid maturation.

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

BACKGROUND: CRISPR-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. RESULTS: Here 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. CONCLUSIONS: Our 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.

Single-cell transcriptomics reveals the cellular identity of a novel progenitor population crucial for murine neural tube closure.

Neural tube closure in vertebrates is achieved through a highly dynamic and coordinated series of morphogenic events involving neuroepithelium, surface ectoderm, and neural plate border. Failure of this process in the caudal region causes spina bifida. Grainyhead-like 3 (GRHL3) is an indispensable transcription factor for neural tube closure as constitutive inactivation of the Grhl3 gene in mice leads to fully penetrant spina bifida. Here, through single-cell transcriptomics we show that at E8.5, the time-point preceding mouse neural tube closure, co-expression of Grhl3, Tfap2a, and Tfap2c defines a previously unrecognised progenitor population of surface ectoderm integral for neural tube closure. Deletion of Grhl3 expression in this cell population using a Tfap2a-Cre transgene recapitulates the spina bifida observed in Grhl3-null animals. Moreover, conditional inactivation of Tfap2c expression in Grhl3-expressing neural plate border cells also induces spina bifida. These findings indicate that a specific neural plate border cellular cohort is required for the early-stage neurulation.

Fragmentation of tissue-resident macrophages during isolation confounds analysis of single-cell preparations from mouse hematopoietic tissues.

Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations. The macrophage remnant binding profile reflects interactions between macrophages and other cell types in vivo. Depletion of CD169+ macrophages in vivo eliminates F4/80+ remnant attachment. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters, and mRNA are all detected in non-macrophage cells including isolated stem and progenitor cells. Analysis of RNA sequencing (RNA-seq) data, including publicly available datasets, indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single-cell analysis of disaggregated hematopoietic tissues. Hematopoietic tissue macrophage fragmentation undermines the accuracy of macrophage ex vivo molecular profiling and creates opportunity for misattribution of macrophage-expressed genes to non-macrophage cells.

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.

Rapid Molecular Profiling of Myeloproliferative Neoplasms Using Targeted Exon Resequencing of 86 Genes Involved in JAK-STAT Signaling and Epigenetic Regulation.

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells and an increased risk of late transformation to acute myeloid leukemia or primary myelofibrosis. Approximately 15% of MPN cases do not carry mutations in JAK2, CALR, or MPL and are thus often referred to as triple-negative cases. These are caused by a diverse set of rare mutations in cytokine receptors, JAK-STAT signaling pathway components, or epigenetic modifiers. In addition, some cases diagnosed as MPN are reactive rather than clonal disorders, so a negative result from a genetic screen can be informative. To obtain a comprehensive rapid molecular diagnosis for most MPNs, we developed an assay to detect genetic mutations (single nucleotide variants and/or small insertions/deletions) in 86 genes using targeted exon resequencing (AmpliSeq) and a bench-top semiconductor machine (Ion Torrent Personal Genome Machine). Our assay reliably detects well characterized mutations in JAK2, CALR, and MPL, but also rarer mutations in ASXL1, TET2, SH2B3, and other genes. Some of these mutations are novel. We find multiple mutations in advanced cases, suggesting co-operation between Janus kinase-STAT pathway mutations and epigenetic mutations in disease progression. This assay can be used to follow molecular progression, clonal heterogeneity, and drug resistance in MPNs.

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.