ORCID Profile
0000-0003-0202-8018
Current Organisation
Royal Holloway University of London
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Publisher: Wiley
Date: 17-01-2014
DOI: 10.1096/FJ.13-246637
Abstract: Expression of fetal γ-globin in adulthood ameliorates symptoms of β-hemoglobinopathies by compensating for the mutant β-globin. Reactivation of the silenced γ-globin gene is therefore of substantial clinical interest. To study the regulation of γ-globin expression, we created the GG mice, which carry an intact 183-kb human β-globin locus modified to express enhanced green fluorescent protein (eGFP) from the Gγ-globin promoter. GG embryos express eGFP first in the yolk sac blood islands and then in the aorta-gonad mesonephros and the fetal liver, the sites of normal embryonic hematopoiesis. eGFP expression in erythroid cells peaks at E9.5 and then is rapidly silenced (>95%) and maintained at low levels into adulthood, demonstrating appropriate developmental regulation of the human β-globin locus. In vitro knockdown of the epigenetic regulator DNA methyltransferase-1 in GG primary erythroid cells increases the proportion of eGFP(+) cells in culture from 41.9 to 74.1%. Furthermore, eGFP fluorescence is induced >3-fold after treatment of erythroid precursors with epigenetic drugs known to induce γ-globin expression, demonstrating the suitability of the Gγ-globin eGFP reporter for evaluation of γ-globin inducers. The GG mouse model is therefore a valuable model system for genetic and pharmacologic studies of the regulation of the β-globin locus and for discovery of novel therapies for the β-hemoglobinopathies.
Publisher: Elsevier BV
Date: 2014
DOI: 10.1016/J.BBAGRM.2013.11.007
Abstract: Runx1 is a transcription factor essential for definitive hematopoiesis. In all vertebrates, the Runx1 gene is transcribed from two promoters: a proximal promoter (P2), and a distal promoter (P1). We previously found that runx1 expression in a specific hematopoietic cell population in zebrafish embryos depends on cohesin. Here we show that zebrafish runx1 is directly bound by cohesin and CCCTC binding factor (CTCF) at the P1 and P2 promoters, and within the intron between P1 and P2. Cohesin initiates expression of runx1 in the posterior lateral mesoderm and influences promoter use, while CTCF represses its expression in the newly emerging cells of the tail bud. The intronic binding sites for cohesin and CTCF coincide with histone modifications that confer enhancer-like properties, and two of the cohesin/CTCF sites behaved as insulators in an in vivo assay. The identified cohesin and CTCF binding sites are likely to be cis-regulatory elements (CREs) for runx1 since they also recruit RNA polymerase II (RNAPII). CTCF depletion excluded RNAPII from two intronic CREs but not the promoters of runx1. We propose that cohesin and CTCF have distinct functions in the regulation of runx1 during zebrafish embryogenesis, and that these regulatory functions are likely to involve runx1 intronic CREs. Cohesin (but not CTCF) depletion enhanced RUNX1 expression in a human leukemia cell line, suggesting conservation of RUNX1 regulation through evolution.
Publisher: Wiley
Date: 26-12-2013
DOI: 10.1096/FJ.13-242669
Abstract: The clinical symptoms of hemoglobin disorders such as β-thalassemia and sickle cell anemia are significantly ameliorated by the persistent expression of γ-globin after birth. This knowledge has driven the discovery of important regulators that silence γ-globin postnatally. Improved understanding of the γ- to β-globin switching mechanism holds the key to devising targeted therapies for β-hemoglobinopathies. To further investigate this mechanism, we used the murine erythroleukemic (MEL) cell line containing an intact 183-kb human β-globin locus, in which the (G)γ- and β-globin genes are replaced by DsRed and eGFP fluorescent reporters, respectively. Following RNA interference (RNAi)-mediated knockdown of two key transcriptional regulators, Myb and BCL11A, we observed a derepression of γ-globin, measured by DsRed fluorescence and qRT-PCR (P<0.001). Interestingly, double knockdown of Myb and DNA methyltransferase 1 (DNMT1) resulted in a robust induction of ε-globin, (up to 20% of total β-like globin species) compared to single knockdowns (P<0.001). Conversely, double knockdowns of BCL11A and DNMT1 enhanced γ-globin expression (up to 90% of total β-like globin species) compared to single knockdowns (P<0.001). Moreover, following RNAi treatment, expression of human β-like globin genes mirrored the expression levels of their endogenous murine counterparts. These results demonstrate that Myb and BCL11A cooperate with DNMT1 to achieve developmental repression of embryonic and fetal β-like globin genes in the adult erythroid environment.
Publisher: Springer New York
Date: 03-09-2014
DOI: 10.1007/978-1-4939-1652-8_18
Abstract: Bacterial artificial chromosome (BAC) reporter cell lines are generated through stable transfection of a BAC reporter construct wherein the gene of interest is tagged with a reporter gene such as eGFP. The large capacity of BACs (up to 350 kb of genomic sequence) enables the inclusion of all regulatory elements that ensure appropriate regulation of the gene of interest. Furthermore, the reporter gene allows the expression of the gene of interest to be readily detected by flow cytometry. Cell lines can also be easily cultured for extended periods with minimal cost. These features of BAC reporter cell lines make them highly amenable for use in high-throughput screening of large drug libraries for compounds that induce the expression of the gene of interest. This chapter describes a method for generation of BAC reporter cell lines that are suitable as cellular assay systems in high-throughput screening. Briefly, this method involves (A) generation of cell clones stably transfected with a BAC reporter construct, (B) selection of "candidate" cell clones based on the responsiveness to known inducers, (C) confirmation of the integrity of the BAC reporter construct integrated within the candidate clones, and (D) assessment of the developmental regulation of the BAC reporter construct. As an ex le, we describe the generation of a BAC reporter cell line containing the human β-globin locus modified to express γ-globin as eGFP for use as a cellular reporter assay for screening of drugs that can reactivate expression of developmentally silenced γ-globin for the treatment of β-hemoglobin disorders.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Betty Kao.