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β-hemoglobinopathies such as sickle cell disease (SCD) and β-thalassemia result from mutations in the adult HBB (β-globin) gene. Reactivating the developmentally silenced fetal HBG1 and HBG2 (γ-globin) genes is a therapeutic goal for treating SCD and β-thalassemia1. 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. The fetal globin gene repressors BCL11A and ZBTB7A directly bind γ-globin gene promoter regions. Repressor binding is disrupted by naturally occurring point mutations located upstream of the transcriptional start site that are associated with hereditary persistence of fetal hemoglobin.

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.

Highlights • GATA1 and FLI1 switch on the endogenous platelet factor 4 (Pf4) gene in fibroblasts. • Pf4 expression is maintained and increases over time. • PF4 protein is secreted and readily collected. • A new technology for the g0065neration of important biomolecules in heterologous cells., 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.

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 binding in vitro, it appears that in vivo FDs 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 the VEGF-A promoter 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.

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.

Genes encoding the human β-like hemoglobin proteins undergo a developmental switch from fetal γ-globin to adult β-globin expression around the time of birth. β-hemoglobinopathies, such as sickle-cell disease and β-thalassemia, result from mutations affecting the adult β-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 Kruppel-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.

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, Kruppel-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 Kruppel-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.

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.

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.

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.

The Lgals3 gene encodes a multifunctional β-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 Kruppel-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.

The development of mature adipocytes from preadipocyte precursor cells requires coordinated changes in gene expression. Management of these expression changes relies on the actions of both DNA-binding transcription factors and their coregulators. Recent studies have identified the corepressor C-terminal binding protein (CtBP) as a key transcriptional coregulator in adipose tissue. CtBP proteins work with several different partner proteins to regulate the development of both white and brown adipocytes. CtBP is of particular interest as it binds to NAD(+)/NADH and may respond to the metabolic state of the cell, thereby linking changes in nutrient levels to transcriptional outcomes.

Splenic marginal zone (MZ) B cells are a lineage distinct from follicular and peritoneal B1 B cells. They are located next to the marginal sinus where blood is released. Here they pick up antigens and shuttle the load onto follicular dendritic cells inside the follicle. On activation, MZ B cells rapidly differentiate into plasmablasts secreting antibodies, thereby mediating humoral immune responses against blood-borne type 2 T-independent antigens. As Krüppel-like factors are implicated in cell differentiation/function in various tissues, we studied the function of basic Krüppel-like factor (BKLF/KLF3) in B cells. Whereas B-cell development in the bone marrow of KLF3-transgenic mice was unaffected, MZ B-cell numbers in spleen were increased considerably. As revealed in chimeric mice, this occurred cell autonomously, increasing both MZ and peritoneal B1 B-cell subsets. Comparing KLF3-transgenic and nontransgenic follicular B cells by RNA-microarray revealed that KLF3 regulates a subset of genes that was similarly up-regulated/down-regulated on normal MZ B-cell differentiation. Indeed, KLF3 expression overcame the lack of MZ B cells caused by different genetic alterations, such as CD19-deficiency or blockade of B-cell activating factor-receptor signaling, indicating that KLF3 may complement alternative nuclear factor-κB signaling. Thus, KLF3 is a driving force toward MZ B-cell maturation.

This study identifies KLF3 as a transcriptional regulator of muscle genes and reveals a novel synergistic interaction between KLF3 and serum response factor (SRF). Using quantitative proteomics, KLF3 was identified as one of several candidate factors that recognize the MPEX control element in the Muscle creatine kinase (MCK) promoter. Chromatin immunoprecipitation analysis indicated that KLF3 is enriched at many muscle gene promoters (MCK, Myosin heavy chain IIa, Six4, Calcium channel receptor alpha-1, and Skeletal alpha-actin), and two KLF3 isoforms are upregulated during muscle differentiation. KLF3 and SRF physically associate and synergize in transactivating the MCK promoter independently of SRF binding to CArG motifs. The zinc finger and repression domains of KLF3 plus the MADS box and transcription activation domain of SRF are implicated in this synergy. Our results provide the first evidence of a role for KLF3 in muscle gene regulation and reveal an alternate mechanism for transcriptional regulation by SRF via its recruitment to KLF binding sites. Since both factors are expressed in all muscle lineages, SRF may regulate many striated- and smooth-muscle genes that lack known SRF control elements, thus further expanding the breadth of the emerging CArGome.

Transcription factors of the Sp/Klf (Kruppel-like factor) family regulate biological processes such as hematopoiesis, adipogenesis, and stem cell maintenance. Here we show that Bklf or Klf3 (Basic Kruppel-like factor) represses the Klf8 (Kruppel-like Factor 8) gene in vivo. Conversely, Eklf or Klf1 (Erythroid Kruppel-like factor) activates the Klf8 gene. Klf8 is driven by two promoters, both of which contain multiple CACCC sites. Klf3 can repress Klf1-mediated activation of both promoters. Chromatin immunoprecipitation experiments confirm that Klf3 occupies both Klf8 promoters in vivo. Interestingly, in Klf3 knock-out tissue Klf1 gains access, binds, and activates both Klf8 promoters. These results demonstrate direct competition between activating and repressing Klfs in vivo. Together with previous evidence that Klf1 directly activates the Klf3 gene, the results reveal an elaborate network of cross-talk within the Klf family. The recognition of cross-regulation and potential redundancy between Klf family members is critical to the interpretation of various Klf knock-out mice and the understanding of individual Klfs in particular contexts.

The Krüppel-like factor (Klfs) family of gene regulatory proteins are transcription factors implicated in many biological processes, including proliferation, apoptosis, differentiation and development. The characteristic feature of this family is the presence of three Krüppel-like zinc fingers, which bind to CACCC elements and GC-rich regions of DNA, to mediate activation and/or repression of transcription. In recent years several Klf knockout mice have been generated. The aim of this review is to outline the biological roles of those Klfs as deduced from the gene ablation studies.

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) -175TC 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.

Obesity is a major public health concern and a strong risk factor for insulin resistance, type 2 diabetes mellitus (T2DM), and cardiovascular disease. The last two decades have seen a reconsideration of the role of white adipose tissue (WAT) in whole body metabolism and insulin action. Adipose tissue-derived cytokines and hormones, or adipokines, are likely mediators of metabolic function and dysfunction. While several adipokines have been associated with obese and insulin-resistant phenotypes, a select group has been linked with insulin sensitivity, namely leptin, adiponectin, and more recently, adipolin. What is known about these insulin-sensitizing molecules and their effects in healthy and insulin resistant states is the subject of this review. There remains a significant amount of research to do to fully elucidate the mechanisms of action of these adipokines for development of therapeutics in metabolic disease.

Krüppel-like factor 3 (KLF3) is a transcriptional regulator that we have shown to be involved in the regulation of adipogenesis in vitro. Here, we report that KLF3-null mice are lean and protected from diet-induced obesity and glucose intolerance. On a chow diet, plasma levels of leptin are decreased, and adiponectin is increased. Despite significant reductions in body weight and adiposity, wild-type and knockout animals show equivalent energy intake, expenditure, and excretion. To investigate the molecular events underlying these observations, we used microarray analysis to compare gene expression in Klf3+/+ and Klf3−/− tissues. We found that mRNA expression of Fam132a, which encodes a newly identified insulin-sensitizing adipokine, adipolin, is significantly upregulated in the absence of KLF3. We confirmed that KLF3 binds the Fam132a promoter in vitro and in vivo and that this leads to repression of promoter activity. Further, plasma adipolin levels were significantly increased in Klf3−/− mice compared with wild-type littermates. Boosting levels of adipolin via targeting of KLF3 offers a novel potential therapeutic strategy for the treatment of insulin resistance.

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.

The CACCC-box binding protein erythroid Kruppel-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 Kruppel-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.

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.

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.

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.

KLF3 is a member of the Kruppel-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. © 2011 IUBMB IUBMB Life, 63(2): 86–93, 2011

Kruppel-like transcription factors (KLFs) comprise a family of gene regulatory proteins with diverse roles in cellular proliferation, survival, and differentiation. KLFs contain three characteristic, highly conserved C-terminal zinc fingers that coordinate sequence-specific DNA binding. Despite having highly homologous DNA binding domains, family members are able to regulate the expression of diverse target genes, resulting in both temporal and tissue-specific control of differentiation. To do this, KLFs have evolved distinct N-terminal regulatory domains that allow interaction with various co-regulators. This chapter describes the cofactors that interact with KLFs and outlines how these interactions potentiate or inhibit KLF transcriptional activity, how they help define target gene specificity, and how they dictate whether a gene is activated or repressed.

Transcription factors of the Sp/Klf (Krüppel-like factor) family regulate biological processes such as hematopoiesis, adipogenesis, and stem cell maintenance. Here we show that Bklf or Klf3 (Basic Krüppel-like factor) represses the Klf8 (Krüppel-like Factor 8) gene in vivo. Conversely, Eklf or Klf1 (Erythroid Krüppel-like factor) activates the Klf8 gene. Klf8 is driven by two promoters, both of which contain multiple CACCC sites. Klf3 can repress Klf1-mediated activation of both promoters. Chromatin immunoprecipitation experiments confirm that Klf3 occupies both Klf8 promoters in vivo. Interestingly, in Klf3 knock-out tissue Klf1 gains access, binds, and activates both Klf8 promoters. These results demonstrate direct competition between activating and repressing Klfs in vivo. Together with previous evidence that Klf1 directly activates the Klf3 gene, the results reveal an elaborate network of cross-talk within the Klf family. The recognition of cross-regulation and potential redundancy between Klf family members is critical to the interpretation of various Klf knock-out mice and the understanding of individual Klfs in particular contexts.

Krüppel-like factors (KLFs) recognize CACCC and GC-rich sequences in gene regulatory elements. Here, we describe the disruption of the murine basic Krüppel-like factor gene (Bklf or Klf3). Klf3 knockout mice have less white adipose tissue, and their fat pads contain smaller and fewer cells. Adipocyte differentiation is altered in murine embryonic fibroblasts from Klf3 knockouts. Klf3 expression was studied in the 3T3-L1 cellular system. Adipocyte differentiation is accompanied by a decline in Klf3 expression, and forced overexpression of Klf3 blocks 3T3-L1 differentiation. Klf3 represses transcription by recruiting C-terminal binding protein (CtBP) corepressors. CtBPs bind NADH and may function as metabolic sensors. A Klf3 mutant that does not bind CtBP cannot block adipogenesis. Other KLFs, Klf2, Klf5, and Klf15, also regulate adipogenesis, and functional CACCC elements occur in key adipogenic genes, including in the C/ebpalpha promoter. We find that C/ebpalpha is derepressed in Klf3 and Ctbp knockout fibroblasts and adipocytes from Klf3 knockout mice. Chromatin immunoprecipitations confirm that Klf3 binds the C/ebpalpha promoter in vivo. These results implicate Klf3 and CtBP in controlling adipogenesis.

KLF3 is a Krüppel family zinc finger transcription factor with widespread tissue expression and no previously known role in heart development. In a screen for dominant mutations affecting cardiovascular function in N-ethyl-N-nitrosourea (ENU) mutagenized mice, we identified a missense mutation in the Klf3 gene that caused aortic valvular stenosis and partially penetrant perinatal lethality in heterozygotes. All homozygotes died as embryos. In the first of three zinc fingers, a point mutation changed a highly conserved histidine at amino acid 275 to arginine (Klf3H275R). This change impaired binding of the mutant protein to KLF3's canonical DNA binding sequence. Heterozygous Klf3H275R mutants that died as neonates had marked biventricular cardiac hypertrophy with diminished cardiac chambers. Adult survivors exhibited hypotension, cardiac hypertrophy with enlarged cardiac chambers, and aortic valvular stenosis. A dominant negative effect on protein function was inferred by the similarity in phenotype between heterozygous Klf3H275R mutants and homozygous Klf3 null mice. However, the existence of divergent traits suggested the involvement of additional interactions. We conclude that KLF3 plays diverse and important roles in cardiovascular development and function in mice, and that amino acid 275 is critical for normal KLF3 protein function. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans., Author Summary Cardiac defects are among the most common malformations in humans. Most causative genetic mutations remain unknown. To discover new causative genes important in cardiovascular development and function, we examined 1770 mice with randomly mutated genes and found a mutant with aortic valvular stenosis, and increased risk of fetal and neonatal death. Using linkage analysis and sequencing, we identified a protein-altering point mutation in the gene regulatory protein KLF3. Mice that survived into adulthood with one mutant copy of the Klf3 gene had low arterial blood pressure, enlarged hearts, and increased mortality due to heart failure. When both copies of the Klf3 gene was mutant, then embryos had heart defects, and all died before birth. KLF3 had no previously known role in heart development so to confirm these findings, we (1) knocked down klf3 expression in zebrafish embryos and (2) examined mice with a mutation that effectively eliminated the KLF3 protein. In both cases, cardiovascular dysfunction was observed. In conclusion, we have discovered that KLF3 plays diverse and important roles in cardiovascular development and function in mice. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans.

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.