Partial reprogramming of heterologous cells by defined factors to generate megakaryocyte lineage-restricted biomolecules.

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-specific chemokine platelet factor 4 (PF4) in fibroblasts. We found that co-expression of the transcriptional regulators GATA1 and FLI1 resulted in a significant increase in levels of PF4, which became magnified over time. This finding demonstrates that such combinations can be used to produce potentially beneficial chemokines in readily available heterologous cell types.

Natural regulatory mutations elevate the fetal globin gene via disruption of BCL11A or ZBTB7A binding.

ß-hemoglobinopathies such as sickle cell disease (SCD) and ß-thalassemia result from mutations in the adult HBB (ß-globin) gene. Reactivating the developmentally silenced fetal HBG1 and HBG2 (γ-globin) genes is a therapeutic goal for treating SCD and ß-thalassemia 1 . Some forms of hereditary persistence of fetal hemoglobin (HPFH), a rare benign condition in which individuals express the γ-globin gene throughout adulthood, are caused by point mutations in the γ-globin gene promoter at regions residing ~115 and 200 bp upstream of the transcription start site. We found that the major fetal globin gene repressors BCL11A and ZBTB7A (also known as LRF) directly bound to the sites at -115 and -200 bp, respectively. Furthermore, introduction of naturally occurring HPFH-associated mutations into erythroid cells by CRISPR-Cas9 disrupted repressor binding and raised γ-globin gene expression. These findings clarify how these HPFH-associated mutations operate and demonstrate that BCL11A and ZBTB7A are major direct repressors of the fetal globin gene.

Direct competition between DNA binding factors highlights the role of Krüppel-like Factor 1 in the erythroid/megakaryocyte switch.

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 erythroid/megakaryocytic switch in vivo. We developed murine cell lines stably overexpressing a GST-KLF1 DNA binding domain fusion protein (GST-KLF1 DBD), as well as lines expressing GST only as a control. Interestingly, overexpression of GST-KLF1 DBD led to an overall reduction in erythroid features and an increase in megakaryocytic features indicative of a reduced function of endogenous KLF1. We simultaneously compared in vivo DNA occupancy of both endogenous KLF1 and GST-KLF1 DBD by ChIP qPCR. Here we found that GST-KLF1 DBD physically displaces endogenous KLF1 at a number of loci, providing novel in vivo evidence of direct competition between DNA binding proteins. These results highlight the role of KLF1 in the erythroid/megakaryocyte switch and suggest that direct competition between transcription factors with similar consensus sequences is an important mechanism in transcriptional regulation.

KLF1 directly activates expression of the novel fetal globin repressor ZBTB7A/LRF in erythroid cells.

Genes encoding the human ß-like hemoglobin proteins undergo a developmental switch from fetal γ-globin to adult ß-globin expression around the time of birth. ß-hemoglobinopathies, such as sickle-cell disease and ß-thalassemia, result from mutations affecting the adult ß-globin gene. The only treatment options currently available carry significant adverse effects. Analyses of heritable variations in fetal hemoglobin (HbF) levels have provided evidence that reactivation of the silenced fetal γ-globin genes in adult erythroid cells is a promising therapy. The γ-globin repressor BCL11A has become the major focus, with several studies investigating its regulation and function as a first step to inhibiting its expression or activity. However, a second repression mechanism was recently shown to be mediated by the transcription factor ZBTB7A/LRF, suggesting that understanding the regulation of ZBTB7A may also be useful. Here we show that Krüppel-like factor 1 (KLF1) directly drives expression of ZBTB7A in erythroid cells by binding to its proximal promoter. We have also uncovered an erythroid-specific regulation mechanism, leading to the upregulation of a novel ZBTB7A transcript in the erythroid compartment. The demonstration that ZBTB7A, like BCL11A, is a KLF1 target gene also fits with the observation that reduced KLF1 expression or activity is associated with HbF derepression.

Krüppel-like Factor 3 (KLF3/BKLF) Is Required for Widespread Repression of the Inflammatory Modulator Galectin-3 (Lgals3).

The Lgals3 gene encodes a multifunctional ß-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 a protective factor in obesity and other metabolic disorders. Despite considerable interest in galectin-3, little is known about its physiological regulation at the transcriptional level. Here, using knockout mice, chromatin immunoprecipitations, and cellular and molecular analyses, we show that the zinc finger transcription factor Krüppel-like factor 3 (KLF3) directly represses galectin-3 transcription. We find that galectin-3 is broadly up-regulated in KLF3-deficient mouse tissues, that KLF3 occupies regulatory regions of the Lgals3 gene, and that KLF3 directly binds its cognate elements (CACCC boxes) in the galectin-3 promoter and represses its activation in cellular assays. We also provide mechanistic insights into the regulation of Lgals3, demonstrating that C-terminal binding protein (CtBP) is required to drive optimal KLF3-mediated silencing. These findings help to enhance our understanding of how expression of the inflammatory modulator galectin-3 is controlled, opening up avenues for potential therapeutic interventions in the future.

Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain.

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 bindingin vitro, it appears thatin vivoFDs can also influence genomic targeting. We fused the FD from the well-characterized transcription factor Krüppel-like Factor 3 (KLF3) to an artificial zinc finger (AZF) protein originally designed to target the Vascular Endothelial Growth Factor-A (VEGF-A) gene promoter. We compared genome-wide occupancy of the KLF3FD-AZF fusion to that observed with AZF. AZF bound to theVEGF-Apromoter as predicted, but was also found to occupy approximately 25,000 other sites, a large number of which contained the expected AZF recognition sequence, GCTGGGGGC. Interestingly, addition of the KLF3 FD re-distributes the fusion protein to new sites, with total DNA occupancy detected at around 50,000 sites. A portion of these sites correspond to known KLF3-bound regions, while others contained sequences similar but not identical to the expected AZF recognition sequence. These results show that FDs can influence and may be useful in directing AZF DNA-binding proteins to specific targets and provide insights into how natural transcription factors operate.

Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin.

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 occurring Hereditary Persistance of Fetal Haemoglobin (HPFH) -175T>C point mutation associated with elevated fetal γ-globin into erythroid cell lines. We show that this mutation increases fetal globin expression through de novo recruitment of the activator TAL1 to promote chromatin looping of distal enhancers to the modified γ-globin promoter.

Phosphorylation of Krüppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity.

Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although 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 transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3.

Repression of chimeric transcripts emanating from endogenous retrotransposons by a sequence-specific transcription factor.

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 finger protein Krüppel-like Factor 3 (KLF3) specifically silences transcription from the ORR1A0 long terminal repeat in murine fetal and adult erythroid cells. In the absence of KLF3, we detect widespread transcription from ORR1A0 elements driven by the master erythroid regulator KLF1. In several instances these aberrant transcripts are spliced to downstream genic exons. One such chimeric transcript produces a novel, dominant negative isoform of PU.1 that can induce erythroid differentiation. CONCLUSIONS: We propose that KLF3 ensures the integrity of the murine erythroid transcriptome through the selective repression of a particular retroelement and is likely one of multiple sequence-specific factors that cooperate to achieve global silencing.

Differential regulation of the α-globin locus by Krüppel-like Factor 3 in erythroid and non-erythroid cells.

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 sought to determine whether KLF3 also interacts with the α-globin locus to regulate transcription. RESULTS: We found that expression of a human transgenic α-globin reporter gene is markedly up-regulated in fetal and adult erythroid cells of Klf3-/- mice. Inspection of the mouse and human α-globin promoters revealed a number of canonical KLF-binding sites, and indeed, KLF3 was shown to bind to these regions both in vitro and in vivo. Despite these observations, we did not detect an increase in endogenous murine α-globin expression in Klf3-/- erythroid tissue. However, examination of murine embryonic fibroblasts lacking KLF3 revealed significant de-repression of α-globin gene expression. This suggests that KLF3 may contribute to the silencing of the α-globin locus in non-erythroid tissue. Moreover, ChIP-Seq analysis of murine fibroblasts demonstrated that across the locus, KLF3 does not occupy the promoter regions of the α-globin genes in these cells, but rather, binds to upstream, DNase hypersensitive regulatory regions. CONCLUSIONS: These findings reveal that the occupancy profile of KLF3 at the α-globin locus differs in erythroid and non-erythroid cells. In erythroid cells, KLF3 primarily binds to the promoters of the adult α-globin genes, but appears dispensable for normal transcriptional regulation. In non-erythroid cells, KLF3 distinctly binds to the HS-12 and HS-26 elements and plays a non-redundant, albeit modest, role in the silencing of α-globin expression.

Regions outside the DNA-binding domain are critical for proper in vivo specificity of an archetypal zinc finger transcription factor.

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-like factor 3 with an N-terminal domain that binds the corepressor CtBP and a DBD composed of three ZFs at its C-terminus. We established a system to compare the genomic occupancy profile of wild-type Krüppel-like factor 3 with two mutants affecting the N-terminal functional domain: a mutant unable to contact the cofactor CtBP and a mutant lacking the entire N-terminal domain, but retaining the ZFs intact. Chromatin immunoprecipitation followed by sequencing was used to assess binding across the genome in murine embryonic fibroblasts. Unexpectedly, we observe that mutations in the N-terminal domain generally reduced binding, but there were also instances where binding was retained or even increased. These results provide a clear demonstration that the correct localization of TFs to their target genes is not solely dependent on their DNA-contact domains. This informs our understanding of how TFs operate and is of relevance to the design of artificial ZF proteins.

Generation of mice deficient in both KLF3/BKLF and KLF8 reveals a genetic interaction and a role for these factors in embryonic globin gene silencing.

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 the erythroid roles of KLF1 and KLF3 have been explored, the contribution of KLF8 to this regulatory network has been unknown. To investigate this, we have generated a mouse model with disrupted KLF8 expression. While these mice are viable, albeit with a reduced life span, mice lacking both KLF3 and KLF8 die at around embryonic day 14.5 (E14.5), indicative of a genetic interaction between these two factors. In the fetal liver, Klf3 Klf8 double mutant embryos exhibit greater dysregulation of gene expression than either of the two single mutants. In particular, we observe derepression of embryonic, but not adult, globin expression. Taken together, these results suggest that KLF3 and KLF8 have overlapping roles in vivo and participate in the silencing of embryonic globin expression during development.

A CpG mutational hotspot in a ONECUT binding site accounts for the prevalent variant of hemophilia B Leyden.

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, designated +1) account for the majority of Leyden cases and have been postulated to disrupt the binding of a transcriptional activator, the identity of which has remained elusive for more than 20 years. Here, we show that ONECUT transcription factors (ONECUT1 and ONECUT2) bind to the -5/-6 site. The various hemophilia B Leyden mutations that have been reported in this site inhibit ONECUT binding to varying degrees, which correlate well with their associated clinical severities. In addition, expression of F9 is crucially dependent on ONECUT factors in vivo, and as such, mice deficient in ONECUT1, ONECUT2, or both exhibit depleted levels of F9. Taken together, our findings establish ONECUT transcription factors as the missing hemophilia B Leyden regulators that operate through the -5/-6 site.

The CACCC-binding protein KLF3/BKLF represses a subset of KLF1/EKLF target genes and is required for proper erythroid maturation in vivo.

The CACCC-box binding protein erythroid Krüppel-like factor (EKLF/KLF1) is a master regulator that directs the expression of many important erythroid genes. We have previously shown that EKLF drives transcription of the gene for a second KLF, basic Krüppel-like factor, or KLF3. We have now tested the in vivo role of KLF3 in erythroid cells by examining Klf3 knockout mice. KLF3-deficient adults exhibit a mild compensated anemia, including enlarged spleens, increased red pulp, and a higher percentage of erythroid progenitors, together with elevated reticulocytes and abnormal erythrocytes in the peripheral blood. Impaired erythroid maturation is also observed in the fetal liver. We have found that KLF3 levels rise as erythroid cells mature to become TER119(+). Consistent with this, microarray analysis of both TER119(-) and TER119(+) erythroid populations revealed that KLF3 is most critical at the later stages of erythroid maturation and is indeed primarily a transcriptional repressor. Notably, many of the genes repressed by KLF3 are also known to be activated by EKLF. However, the majority of these are not currently recognized as erythroid-cell-specific genes. These results reveal the molecular and physiological function of KLF3, defining it as a feedback repressor that counters the activity of EKLF at selected target genes to achieve normal erythropoiesis.

Holding Tight: Cell Junctions and Cancer Spread.

Cell junctions are sites of intercellular adhesion that maintain the integrity of epithelial tissue and regulate signalling between cells. These adhesive junctions are comprised of protein complexes that serve to establish an intercellular cytoskeletal network for anchoring cells, in addition to regulating cell polarity, molecular transport and communication. The expression of cell adhesion molecules is tightly controlled and their downregulation is essential for epithelial-mesenchymal transition (EMT), a process that facilitates the generation of morphologically and functionally diverse cell types during embryogenesis. The characteristics of EMT are a loss of cell adhesion and increased cellular mobility. Hence, in addition to its normal role in development, dysregulated EMT has been linked to cancer progression and metastasis, the process whereby primary tumors migrate to invasive secondary sites in the body. This paper will review the current understanding of cell junctions and their role in cancer, with reference to the abnormal regulation of junction protein genes. The potential use of cell junction molecules as diagnostic and prognostic markers will also be discussed, as well as possible therapies for adhesive dysregulation.

Impaired B cell development in the absence of Krüppel-like factor 3.

Krüppel-like factor 3 (Klf3) is a member of the Klf family of transcription factors. Klfs are widely expressed and have diverse roles in development and differentiation. In this study, we examine the function of Klf3 in B cell development by studying B lymphopoiesis in a Klf3 knockout mouse model. We show that B cell differentiation is significantly impaired in the bone marrow, spleen, and peritoneal cavity of Klf3 null mice and confirm that the defects are cell autonomous. In the bone marrow, there is a reduction in immature B cells, whereas recirculating mature cells are noticeably increased. Immunohistology of the spleen reveals a poorly structured marginal zone (MZ) that may in part be caused by deregulation of adhesion molecules on MZ B cells. In the peritoneal cavity, there are significant defects in B1 B cell development. We also report that the loss of Klf3 in MZ B cells is associated with reduced BCR signaling strength and an impaired ability to respond to LPS stimulation. Finally, we show increased expression of a number of Klf genes in Klf3 null B cells, suggesting that a Klf regulatory network may exist in B cells.

The multi-zinc finger protein ZNF217 contacts DNA through a two-finger domain.

Classical C2H2 zinc finger proteins are among the most abundant transcription factors found in eukaryotes, and the mechanisms through which they recognize their target genes have been extensively investigated. In general, a tandem array of three fingers separated by characteristic TGERP links is required for sequence-specific DNA recognition. Nevertheless, a significant number of zinc finger proteins do not contain a hallmark three-finger array of this type, raising the question of whether and how they contact DNA. We have examined the multi-finger protein ZNF217, which contains eight classical zinc fingers. ZNF217 is implicated as an oncogene and in repressing the E-cadherin gene. We show that two of its zinc fingers, 6 and 7, can mediate contacts with DNA. We examine its putative recognition site in the E-cadherin promoter and demonstrate that this is a suboptimal site. NMR analysis and mutagenesis is used to define the DNA binding surface of ZNF217, and we examine the specificity of the DNA binding activity using fluorescence anisotropy titrations. Finally, sequence analysis reveals that a variety of multi-finger proteins also contain two-finger units, and our data support the idea that these may constitute a distinct subclass of DNA recognition motif.

PU.1 and Haematopoietic Cell Fate: Dosage Matters.

The ETS family transcription factor PU.1 is a key regulator of haematopoietic differentiation. Its expression is dynamically controlled throughout haematopoiesis in order to direct appropriate lineage specification. Elucidating the biological role of PU.1 has proved challenging. This paper will discuss how a range of experiments in cell lines and mutant and transgenic mouse models have enhanced our knowledge of the mechanisms by which PU.1 drives lineage-specific differentiation during haematopoiesis.

Cellular Reprogramming toward the Erythroid Lineage.

Haemoglobinopathies such as thalassaemia and sickle cell disease present a major health burden. Currently, the main forms of treatment for these diseases are packed red blood cell transfusions and the administration of drugs which act to nonspecifically reactivate the production of foetal haemoglobin. These treatments are ongoing throughout the life of the patient and are associated with a number of risks, such as limitations in available blood for transfusion, infections, iron overload, immune rejection, and side effects associated with the drug treatments. The field of cellular reprogramming has advanced significantly in the last few years and has recently culminated in the successful production of erythrocytes in culture. This paper will discuss cellular reprogramming and its potential relevance to the treatment of haemoglobinopathies.

The mammalian zinc finger transcription factor Krüppel-like factor 3 (KLF3/BKLF).

KLF3 is a member of the Krüppel-like factor (KLF) family of transcription factors. These proteins are classified by the presence of three C-terminal C2H2 zinc fingers that allow sequence-specific binding to CACCC boxes and GC-rich motifs found in the promoters, enhancers, and other control regions of target genes. KLFs have diverse biological roles, regulating proliferation, differentiation, and apoptosis in many tissues throughout development. KLF3 is a transcriptional repressor that binds the cofactor C-terminal binding protein, which in turn recruits a large repressor complex to mediate transcriptional silencing. In addition to an understanding of the molecular mechanisms that allow KLF3 to regulate the expression of its target genes, the biological roles of this transcription factor are now being defined. In agreement with the widespread expression pattern of this transcription factor, it is becoming clear that KLF3 is an important regulator of several biological processes, including adipogenesis, erythropoiesis, and B cell development.