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

Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification

Non-invasive epigenome editing is a promising strategy for engineering gene expression programs, yet potency, specificity, and persistence remain challenging. Here we show that effective epigenome editing is gated at single-base precision via 'keyhole' sites in endogenous regulatory DNA. Synthetic repressors targeting promoter keyholes can ablate gene expression in up to 99% of primary cells with single-gene specificity and can seamlessly repress multiple genes in combination. Transient exposure of primary T cells to keyhole repressors confers mitotically heritable silencing that persists to the limit of primary cultures in vitro and for at least 4 weeks in vivo, enabling manufacturing of cell products with enhanced therapeutic efficacy. DNA recognition and effector domains can be encoded as separate proteins that reassemble at keyhole sites and function with the same efficiency as single chain effectors, enabling gated control and rapid screening for novel functional domains that modulate endogenous gene expression patterns. Our results provide a powerful and exponentially flexible system for programming gene expression and therapeutic cell products

Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification

Non-invasive epigenome editing is a promising strategy for engineering gene expression programs, yet potency, specificity, and persistence remain challenging. Here we show that effective epigenome editing is gated at single-base precision via 'keyhole' sites in endogenous regulatory DNA. Synthetic repressors targeting promoter keyholes can ablate gene expression in up to 99% of primary cells with single-gene specificity and can seamlessly repress multiple genes in combination. Transient exposure of primary T cells to keyhole repressors confers mitotically heritable silencing that persists to the limit of primary cultures in vitro and for at least 4 weeks in vivo, enabling manufacturing of cell products with enhanced therapeutic efficacy. DNA recognition and effector domains can be encoded as separate proteins that reassemble at keyhole sites and function with the same efficiency as single chain effectors, enabling gated control and rapid screening for novel functional domains that modulate endogenous gene expression patterns. Our results provide a powerful and exponentially flexible system for programming gene expression and therapeutic cell products

Differential regulation of the alpha-globin locus by Kruppel-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. </p

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

doi:10.1155/2011/501464 Review Article Cellular Reprogramming toward the Erythroid Lineage

Copyright © 2011 Laura J. Norton et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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