Erythroid Kruppel-like factor directly activates the basic Kruppel-like factor gene in erythroid cells

The Sp/Kriippel-like factor (Sp/KIf) family is comprised of around 25 zinc finger transcription factors that recognize CACCC boxes and GC-rich elements. We have investigated basic Kruppel-like factor (Bklf/Klf3) and show that in erythroid tissues its expression is highly dependent on another family member, erythroid Kruppel-like factor (Eklf/Kif1). 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, la, 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

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-bl, 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

A Large Gene Network in Immature Erythroid Cells Is Controlled by the Myeloid and B Cell Transcriptional Regulator PU.1

PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages

Diel Variations in Survey Catch Rates and Survey Catchability of Spiny Dogfish and their Pelagic Prey in the Northeast US Continental Shelf Large Marine Ecosystem

This study examines the potential uncertainty in survey biomass estimates of Spiny Dogfish Squalus acanthias in the Northeast U.S. Continental Shelf Large Marine Ecosystem (NES LME). Diel catch-per-unit-effort (CPUE) estimates are examined from the Northeast Fisheries Science Center bottom trawl surveys conducted during autumn (1963-2009) and spring (1968-2009). Influential environmental variables on survey catchability are identified for Spiny Dogfish life history stages and five pelagic prey species: Butterfish Peprilus triacanthus, Atlantic Herring Clupea harengus, shortfin squid Illex spp., longfin squid Doryteuthis spp., and Atlantic Mackerel Scomber scombrus. Daytime survey catchability was significantly higher than nighttime catchability for most species during autumn and for mature male Spiny Dogfish, shortfin squid, and longfin squid during spring in the NES LME. For most stages and species examined, breakpoint analyses identified significant increases in CPUE in the morning, peak CPUE during the day, and significant declines in CPUE in the late afternoon. Seasonal probabilities of daytime catch were largely driven by solar zenith angle for most species, with stronger trends identified during autumn. Unadjusted CPUE estimates appear to overestimate absolute abundance, with adjustments resulting in reductions in absolute abundance ranging from 41% for Spiny Dogfish to 91% for shortfin and longfin squids. These findings have important implications for Spiny Dogfish regarding estimates of population consumption of key pelagic prey species and their ecological footprint within the NES LME

Erythroid Kruppel-Like Factor and the Cell Cycle: A Role beyond Globin Gene Regulation

Erythropoiesis, the process of producing mature erythrocytes from the haematopoietic stem cells (HSCs) that reside in the bone marrow, is tightly regulated at both the cell and molecular level by a well defined set of extracellular cytokine signals and intracellular transcription factors. Diseases affecting erythropoiesis are among the most commonly inherited conditions and result from disturbances to the cellular and molecular events that normally regulate this process. Erythroid Kruppel-like factor (EKLF/KLF1) is a transcription factor that is essential for erythropoiesis. EKLF is the founding member of the Kruppel-like factor family of transcription factors that bind to GC rich CACC-Box elements within gene promoters and activate transcription. The β-like globin genes are critical targets of EKLF through its binding at sites within the proximal promoters and the upstream locus control region (LCR) enhancer. Mice lacking EKLF die prior to birth by E16 with a phenotype that closely resembles the human disease thalassaemia. Thalassaemia is due to mutations in the α or β-globin genes, leading to globin chain imbalance, red cell destruction and ineffective erythropoiesis. However, restoration of expression of γ-globin (a β-like gene) failed to prevent embryonic lethality in EKLF knockout mice and suggested that additional target genes were critical to erythropoiesis. This thesis describes investigation into the transcriptional network of EKLF and an in depth analysis of previously uncharacterised phenotypes present in the EKLF knockout mouse. I have identified a suite of target genes for EKLF that include critical components of the cells cycle. I have also tested the hypothesis that EKLF is able to function in vivo as a tumour suppressor gene. Additionally, I report a role for EKLF in the determination of cell fate within the haematopoietic system and describe the development of a new approach to globally understanding erythroid transcription factor function. A previously performed microarray transcriptional profiling study provided a set of potential target genes for EKLF. I have expanded on this study by identifying that the cell cycle genes p18INK4c, and E2f2 are direct transcriptional targets of EKLF, where binding of EKLF occurs at the promoter and a novel intronic enhancer region, respectively. I have also described a previously undiscovered cell cycle phenotype of aberrant entry into S-phase in EKLF -/- erythroid cells that is directly related to abrogated expression of E2f2. The Kruppel-like factor family of genes have been implicated as players in the tumour process. By constructing a model for the loss of EKLF within HSCs in vivo, I have tested whether EKLF is functional as a tumour suppressor. The loss of EKLF in vivo was found to be insufficient to generate erythroleukaemia, however did result in erythroid hyperplasia, extramedullary haematopoieis and a mild macrocytic anaemia. In addition to regulation of erythropoiesis, EKLF performs a critical role in the lineage choice for a megakaryocyte-erythroid progenitor (MEP) between the megakaryocytic and erythroid lineages. This thesis describes that in the absence of EKLF, MEPs fail to commit properly to either lineage and proceed along a promiscuous pathway sharing the hallmarks of both megakaryocytes and erythroid cells. A detailed molecular mechanism for this phenotype remains undetermined, but is likely to involve interactions with the megakaryocyte transcription factor Fli1 and other members of the Kruppel-like factor family, such as BKLF (KLF3). While the transcriptional mechanisms that drive erythropoiesis have been slowly discovered, the development of chromatin immunoprecipitation (ChIP) assays and next generation DNA sequencing technology has presented the potential to rapidly enhance the progression of these studies. In this thesis I describe the development of ChIP-seq using Applied Biosystems SOLiD technology, an approach to rapidly identify binding sites for erythroid transcription factors in an unbiased genome wide approach. The work described in this thesis has expanded the transcriptional network of EKLF to include critical components of the cell cycle and has suggested many additional target genes from ChIP-seq requiring validation. As one of the major transcription factor players during erythropoiesis, EKLF performs many critical functions that include the regulation of the cell cycle, lineage selection and erythroid development. I suggest that current and future studies of EKLF function will influence our understanding of erythropoiesis and refine our understanding of human conditions such as thalassaemia and erythroleukaemia

Three fingers on the switch: Kruppel-like factor 1 regulation of gamma-globin to beta-globin gene switching

Purpose of revie

Erythroid Kruppel-Like factor regulates E2F4 and the G1 Cdk inhibitor, p18

Erythroid Kruppel-Like Factor (EKLF) is a zinc finger transcription factor which is essential for ß-globin gene expression. Knockout mice die from anemia at E15, but restoration of globin chain imbalance does not rescue anemia or increase survival. Cell lines derived from EKLF null mice undergo proliferation arrest upon reactivation of a conditional EKLF-ER fusion protein, suggesting a role in cell cycle control. A transcriptional profiling experiment comparing the global gene expression in EKLF null and wild type fetal liver identified many differentially expressed genes, a number of which function in G1 and at the G1/S checkpoint of the cell cycle. The Cyclin dependent kinase (Cdk) inhibitor, p18, and the S phase transcription factor E2F4 were both found to be significantly down regulated in EKLF null mice and this result was confirmed by real-time PCR. Interestingly, E2F4 knockout mice have a similar phenotype to EKLF knockout mice. Bioinformatic searches of the p18 and E2F4 genes shows that each contains phylogenetically conserved CACC box motifs capable of binding EKLF within longer regions of conservation in promoter and intron regions. The p18 gene contains two conserved CACCC sites upstream of the start of transcription, which are required for EKLF dependent promoter activity in luciferase reporter assays. The transcription factor E2F4 contains a conserved EKLF-binding CACC site within an intron that is closely associated with two conserved GATA1 binding sites. We show by a chromatin immunoprecipitation (ChIP) assays that the E2F4 intron and p18 promoter are occupied by EKLF in vivo. Together, these results suggest that EKLF is likely to directly regulate expression of key cell cycle genes in vivo to drive the switch from proliferation to differentiation of erythrocytes. The loss of EKLF is likely to result in aberrant proliferation and predisposition to leukemia