Hemophilia B Leyden and once mysterious cis-regulatory mutations

Hemophilia B is a classic, monogenic blood clotting disease caused by mutations in the coagulation factor IX (F9) locus. Although interpreting mutations within the gene itself has been relatively straightforward, ascribing molecular mechanisms to the complete suite of mutations within the promoter region has proven somewhat difficult and has only recently been achieved. These mutations, which are clustered at discrete transcription factor binding sites, dynamically alter the developmental expression of F9 in different ways. They illustrate how single-nucleotide mutations in cis-regulatory regions can have drastic ramifications for the control of gene expression and in some instances be causative of disease. Here we present the human F9 promoter as a model example for which saturation mutation mapping has revealed the mechanisms of its regulation. Moreover, we suggest that the growing number of genome-wide studies of transcription factor activity will accelerate both the discovery and understanding of regulatory polymorphisms and mutations. © 2013 Elsevier Ltd

Hit and Run Transcriptional Repressors Are Difficult to Catch in the Act

© 2019 WILEY Periodicals, Inc. Transcriptional silencing may not necessarily depend on the continuous residence of a sequence-specific repressor at a control element and may act via a “hit and run” mechanism. Due to limitations in assays that detect transcription factor (TF) binding, such as chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), this phenomenon may be challenging to detect and therefore its prevalence may be underappreciated. To explore this possibility, erythroid gene promoters that are regulated directly by GATA1 in an inducible system are analyzed. It is found that many regulated genes are bound immediately after induction of GATA1 but the residency of GATA1 decreases over time, particularly at repressed genes. Furthermore, it is shown that the repressive mark H3K27me3 is seldom associated with bound repressors, whereas, in contrast, the active (H3K4me3) histone mark is overwhelmingly associated with TF binding. It is hypothesized that during cellular differentiation and development, certain genes are silenced by repressive TFs that subsequently vacate the region. Catching such repressor TFs in the act of silencing via assays such as ChIP-seq is thus a temporally challenging prospect. The use of inducible systems, epitope tags, and alternative techniques may provide opportunities for detecting elusive “hit and run” transcriptional silencing. Also see the video abstract here https://youtu.be/vgrsoP_HF3g

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. © 2014 Mak et al.; licensee BioMed Central Ltd

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 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

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

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. © The Author(s) 2013. Published by Oxford University Press

Krüppel-like factor 3 (KLF3/BKLF) is required for widespread repression of the inflammatory modulator galectin-3 (Lgals3)

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 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

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

Genes encoding the human b-like hemoglobin proteins undergo a developmental switch from fetal g-globin to adult b-globin expression around the time of birth. b-hemoglobinopathies, such as sickle-cell disease and b-thalassemia, result from mutations affecting the adult b-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 g-globin genes in adult erythroid cells is a promising therapy. The g-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

ENU-induced Mutation in the DNA-binding Domain of KLF3 Reveals Important Roles for KLF3 in Cardiovascular Development and Function in Mice

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

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