Michael R. Tallack

A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells

KLF1 regulates a diverse suite of genes to direct erythroid cell differentiation from bipotent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which KLF1 operates, we performed KLF1 ChIP-seq in the mouse. We found at least 945 sites in the genome of E14.5 fetal liver erythroid cells which are occupied by endogenous KLF1. Many of these recovered sites reside in erythroid gene promoters such as Hbb-b1, but the majority are distant to any known gene. Our data suggests KLF1 directly regulates most aspects of terminal erythroid differentiation including production of alpha- and beta-globin protein chains, heme biosynthesis, coordination of proliferation and anti-apoptotic pathways, and construction of the red cell membrane and cytoskeleton by functioning primarily as a transcriptional activator. Additionally, we suggest new mechanisms for KLF1 cooperation with other transcription factors, in particular the erythroid transcription factor GATA1, to maintain homeostasis in the erythroid compartment.

Erythroid Krüppel-Like Factor Directly Activates the Basic Krüppel-Like Factor Gene in Erythroid Cells

ABSTRACT The Sp/Krüppel-like factor (Sp/Klf) family is comprised of around 25 zinc finger transcription factors that recognize CACCC boxes and GC-rich elements. We have investigated basic Krüppel-like factor (Bklf/Klf3) and show that in erythroid tissues its expression is highly dependent on another family member, erythroid Krüppel-like factor (Eklf/Klf1). We observe that Bklf mRNA is significantly reduced in erythroid tissues from Eklf-null murine embryos. We find that Bklf is driven primarily by two promoters, a ubiquitously active GC-rich upstream promoter, 1a, and an erythroid downstream promoter, 1b. Transcripts from the two promoters encode identical proteins. Interestingly, both the ubiquitous and the erythroid promoter are dependent on Eklf in erythroid cells. Eklf also activates both promoters in transient assays. Experiments utilizing an inducible form of Eklf demonstrate activation of the endogenous Bklf gene in the presence of an inhibitor of protein synthesis. The kinetics of activation are also consistent with Bklf being a direct Eklf target. Chromatin immunoprecipitation assays confirm that Eklf associates with both Bklf promoters. Eklf is typically an activator of transcription, whereas Bklf is noted as a repressor. Our results support the hypothesis that feedback cross-regulation occurs within the Sp/Klf family in vivo.

Prediction of novel long non-coding RNAs based on RNA-Seq data of mouse Klf1 knockout study

BackgroundStudy on long non-coding RNAs (lncRNAs) has been promoted by high-throughput RNA sequencing (RNA-Seq). However, it is still not trivial to identify lncRNAs from the RNA-Seq data and it remains a challenge to uncover their functions.ResultsWe present a computational pipeline for detecting novel lncRNAs from the RNA-Seq data. First, the genome-guided transcriptome reconstruction is used to generate initially assembled transcripts. The possible partial transcripts and artefacts are filtered according to the quantified expression level. After that, novel lncRNAs are detected by further filtering known transcripts and those with high protein coding potential, using a newly developed program called lncRScan. We applied our pipeline to a mouse Klf1 knockout dataset, and discussed the plausible functions of the novel lncRNAs we detected by differential expression analysis. We identified 308 novel lncRNA candidates, which have shorter transcript length, fewer exons, shorter putative open reading frame, compared with known protein-coding transcripts. Of the lncRNAs, 52 large intergenic ncRNAs (lincRNAs) show lower expression level than the protein-coding ones and 13 lncRNAs represent significant differential expression between the wild-type and Klf1 knockout conditions.ConclusionsOur method can predict a set of novel lncRNAs from the RNA-Seq data. Some of the lncRNAs are showed differentially expressed between the wild-type and Klf1 knockout strains, suggested that those novel lncRNAs can be given high priority in further functional studies.

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.

EKLF/KLF1 controls cell cycle entry via direct regulation of E2f2.

Differentiation of erythroid cells requires precise control over the cell cycle to regulate the balance between cell proliferation and differentiation. The zinc finger transcription factor, erythroid Krüppel-like factor (EKLF/KLF1), is essential for proper erythroid cell differentiation and regulates many erythroid genes. Here we show that loss of EKLF leads to aberrant entry into S-phase of the cell cycle during both primitive and definitive erythropoiesis. This cell cycle defect was associated with a significant reduction in the expression levels of E2f2 and E2f4, key factors necessary for the induction of S-phase gene expression and erythropoiesis. We found and validated novel intronic enhancers in both the E2f2 and E2f4 genes, which contain conserved CACC, GATA, and E-BOX elements. The E2f2 enhancer was occupied by EKLF in vivo. Furthermore, we were able to partially restore cell cycle dynamics in EKLF−/− fetal liver upon additional genetic depletion of Rb, establishing a genetic causal link between reduced E2f2 and the EKLF cell cycle defect. Finally, we propose direct regulation of the E2f2 enhancer is a generic mechanism by which many KLFs regulate proliferation and differentiation.

KLF1 directly coordinates almost all aspects of terminal erythroid differentiation

The molecular events and transcriptional mechanisms that underlie erythropoiesis are of great interest to biologists and hematologists since disorders of erythrocytes are common and remain relatively poorly understood. Kruppel‐like factor 1 (KLF1) is a critical transcription factor for erythropoiesis in mice and man. Recently the use of chromatin immunoprecipitation (ChIP) coupled to next‐generation DNA sequencing (ChIP‐seq) has led to an updated understanding of how KLF1 functions in vivo. The full extent of KLF1 target genes have provided new insights into erythropoiesis, and have established that KLF1 controls almost all aspects of erythroid cell development and maturation.

Three fingers on the switch: Krüppel-like factor 1 regulation of γ-globin to β-globin gene switching.

Purpose of reviewKrüppel-like factor 1 (KLF1) regulates most aspects of erythropoiesis. Many years ago, transgenic mouse studies implicated KLF1 in the control of the human &ggr;-globin to &bgr;-globin switch. In this review, we will integrate these initial studies with recent developments in human genetics to discuss our present understanding of how KLF1 and its target genes direct the switch. Recent findingsRecent studies have shown that human mutations in KLF1 are common and mostly asymptomatic, but lead to significant increases in levels of fetal hemoglobin (HbF) (&agr;2&ggr;2) and adult HbA2 (&agr;2&dgr;2). Genome-wide association studies (GWAS) have demonstrated that three primary loci are associated with increased HbF levels in the population: the &bgr;-globin locus itself, the BCL11A locus, and a site between MYB and HBS1L. We discuss evidence that KLF1 directly regulates BCL11A, MYB and other genes, which are involved directly or indirectly in &ggr;-globin silencing, thus providing a link between GWAS and KLF1 in hemoglobin switching. SummaryKLF1 regulates the &ggr;-globin to &bgr;-globin genetic switch by many mechanisms. Firstly, it facilitates formation of an active chromatin hub (ACH) at the &bgr;-globin gene cluster. Specifically, KLF1 conscripts the adult-stage &bgr;-globin gene to replace the &ggr;-globin gene within the ACH in a stage-specific manner. Secondly, KLF1 acts as a direct activator of genes that encode repressors of &ggr;-globin gene expression. Finally, KLF1 is a regulator of many components of the cell cycle machinery. We suggest that dysregulation of these genes leads to cell cycle perturbation and ‘erythropoietic stress’ leading to indirect upregulation of HbF.

A mechanism for Ikaros regulation of human globin gene switching

The human β globin locus consists of an upstream LCR and functional genes arranged sequentially in the order of their expression during development: 5′‐HBE1, HBG2, HBG1, HBD, HBB‐3′. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Here we show that the transcription factor Ikaros plays a major role in the formation of the β‐globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA‐binding region of Ikaros is disrupted by a point mutation, there is concomitant marked down‐regulation of HBB, and up‐regulation of HBG expression. We show for the first time Ikaros and its family member Eos, bind to critical cis elements implicated in haemoglobin switching and deletional hereditary persistence of fetal haemoglobin (HPFH). Chromatin conformation capture (3C) data demonstrated that Ikaros facilitates long‐distance DNA looping between the LCR and a region upstream of HBD. This study provides new insights into the mechanism of stage‐specific assembly of the β‐globin ACH, and HPFH.

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.

Megakaryocyte-erythroid lineage promiscuity in EKLF null mouse blood

Commitment towards megakaryocyte versus erythroid blood cell lineages occurs in the megakaryocyte-erythroid progenitor, where mutually exclusive expression of either EKLF (Klf1) or Fli1 defines alternative outcomes. Here we show there is a marked increase in the number of circulating platelets in mice lacking the erythroid transcription factor EKLF. In addition, committed erythroid cells retain key signatures of megakaryocytes both on the cell surface and at the mRNA level. We also show that the effect of EKLF on megakaryocyte-erythroid progenitor lineage decision and commitment is cell autonomous in bone marrow reconstitution assays where stem cells lacking EKLF favor the megakaryocyte differentiation pathway. We conclude the megakaryocyte program is aberrantly activated in EKLF null erythroid cells.