Transcriptional regulation of GATA1

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138 Short interfering RNA-mediated knockdown of A-globin: Applications in B-thalassaemia Hsiao Phin Voon 1, Patrick Western 2, Jim Vadolas 1 1 Cell and Gene Therapy Research Group, Murdoch Childrens Research Institute, The University of Melbourne, Royal Children’s Hospital, Parkville 3052, Australia 2 ARC Centre of Excellence for Biotechnology and Development and Murdoch Children’s Research Institute, The University of Melbourne, Royal Children’s Hospital, Parkville 3052, Australia h-Thalassaemia is an inherited haemoglobinopathy arising from a mutated h-globin gene, resulting in reduced h-globin chain synthesis. Much of the pathology of this disease is due to excess h-globin chains forming toxic insoluble precipitates in erythroid progenitor cells resulting in premature cell death. In h-thalassaemia, decreased a-globin expression leads to milder symptoms, exemplified in individuals who co-inherit a-thalassaemia and hthalassaemia. One way of reducing a-globin synthesis is by using short interfering RNA (siRNA). Since numerous studies have shown promising results utilising siRNA in vitro and in vivo, we are developing an siRNA strategy for reducing a-globin as a therapy for h-thalassaemia. In this study, we set out to determine whether a-globin specific siRNA is able to mediate the destruction of murine a-globin mRNA in murine erythroleukemic cells (MEL). MEL cells were induced to haemoglobinise using 2% DMSO for 6 days then electroporated with synthetic a-globin specific siRNAs. Total RNA was harvested 24 and 48 h after transfection and analysed using real-time PCR. Primers specific for a-globin were used to determine expression of a-globin mRNA relative to h-actin expression. h-Globin mRNA expression was also monitored as a control for non-specific knockdown effects. Four a-globin-specific siRNA sequences (sia-1, sia-2, sia-3 and sia-4) were tested in this system. Three of these constructs generated significant reductions in a-globin expression at the mRNA level, reducing a-globin mRNA by between 40% and 80% as determined by real-time PCR. h-globin expression remained unaffected. In this study, we have identified several aglobin-specific siRNA sequences which are effective in generating knockdowns of murine a-globin mRNA. Our initial results indicate that siRNA can effectively direct the destruction of aglobin mRNA, a result which supports our approach of developing an siRNA-based therapy for h-thalassaemia. doi:10.1016/j.bcmd.2006.10.149

139 Transcriptional regulation of GATA1 R. Drissen, B. Guyot, W.G. Wood, C. Porcher, P. Vyas MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK Gain-of-function experiments show that the critical myeloid transcription factor GATA1 specifies erythroid cells, megakar-

yocytes at expense of granulocytes and macrophages (GM) from the common myeloid progenitor (CMP). In lineages specified by GATA1, sustained GATA1 expression is required for terminal maturation. Conversely, GATA1 expression has to be extinguished to allow the CMP to differentiate to the GM lineages. Therefore, one important component in dissecting the molecular control of myeloid lineage specification will be to define how GATA1 expression is regulated. The level of GATA1 is mainly controlled transcriptionally. Thus, dissecting the molecular basis of the transcriptional regulation of GATA1 is likely to provide an instructive entry point to understanding how myeloid lineages are specified. GATA1 expression is first detected in the CMP. Here, we show that murine(m) GATA1 mRNA rise 6 fold as murine CMP differentiate to MEP and another 4-fold as MEP differentiate to Ter-119+ erythroid cells. By contrast, GATA1 mRNA levels only rise 2-fold when MEP differentiate to primary megakaryocytes. To dissect the molecular basis of erythroid-specific GATA1 expression, we have been identifying the DNA sequences regulating GATA1 expression. Previously, we and others showed that an upstream enhancer (mHS 3.5) is cooperate with sequences near the mGata1 gene (the promoter (IE) and an intron element (mHS + 3.5)) to direct erythroid-specific GATA1 expression in transgenic mice. Though a mGata1 transgene regulated by mHS 3.5/IE/mHS + 3.5 is able to grossly rescue erythropoiesis in GATA1 knock out mice, germ line deletion of mHS 3.5 only ablates megakaryocyte GATA1 expression leaving red cell GATA1 expression unaffected. This suggests other cis-elements in the mGata1 locus can substitute for mHS 3.5 in red cells (but not megakaryocytes). Recently, we identified another erythroid-specific DNase I hypersensitive site (HS), mHS 25, that has enhancer activity in erythroid cell lines and where the chromatin associated with it is hyperacetylated at histone H3 and H4. We now demonstrate by fine DNase I HS mapping that mHS 25 is more complex, being composed of two closely adjacent DNase I HS 500 bp apart (mHS 25 and mHS 26). These HSs are present only in primary red cells but not in other primary hemopoietic and non-hemopoietic cells. H3 and H4 associated with both sites are hyperacetylated only in Ter-119+ cells. In addition, we tested mHS25/6 function in mice transgenic for a mGata1-LacZ reporter construct regulated by mHS 25/6/IE/mHS + 3.5. h-Galactosidase expression was quantitated in myeloid lineages by FACS analysis using lineage-specific antibodies and the hgalactosidase substrate FDG. 6/7 F0 transgenic embryos expressed h-galactosidase in 5– 23% of fetal liver Ter-119+ cells (< 0.2% of non-transgenic Ter-119+ stained with FDG). FDG staining was not detected in CD61+Mac1 megakaryocytes or in Mac-1+ macrophages. Erythroid-specific reporter gene expression was conformed in bone marrow samples in two independent lines of mice transgenic for this reporter construct. Chromatin immunoprecipitation (ChIP) analysis demonstrated that the transcription factors GATA1, SCL and its heterodimeric partner E2A, the SCL-interacting protein Ldb1 and LMO2 all bind in vivo to mHS 25/6 only

ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191

in Ter-119+ cell (and not primary megakaryocytes or neutrophils). We and others have previously shown that these transcription factors exist in a multi-protein complex that can activate gene expression. The combination of these findings provides firm evidence that mHS 25/6 is an erythroid-specific GATA1 enhancer in primary murine erythroid cells. Finally, to begin understand the relative roles of mHS 3.5 and mHS 25 during erythroid differentiation from multi-potential myeloid progenitors, we show by DNase I HSS mapping and ChIP analysis that in the multi-potential myeloid cell line FDCP-mix, only mHS 3.5 is present (but not mHS 25/6) and that it binds GATA1/SCL/E2A/Ldb1/ LMO2 in vivo. Only later in Ter-119+ cells are both mHS 3.5 and mHS 25/6 seen with GATA1/SCL/E2A/ Ldb1/LMO2 detected at these sites. This observation suggests a hierarchical utilisation of these two mGata1 cis-elements during erythroid differentiation. doi:10.1016/j.bcmd.2006.10.150

140 Screening for DNase I hypersensitive sites with Q-PCR in a 6q23 quantitative trait locus influencing fetal haemoglobin levels in adults Karin Wahlberg, Jie Jiang, Steve Best, Stephan Menzel, Swee Lay Thein Department of Molecular Haematology, Division of Gene and Cell based Therapy, King’s College London School of Medicine, Kings College Hospital, London, UK A major ameliorating factor in sickle cell disease and hthalassemia is the inherent ability to produce fetal haemoglobin (HbF, a2g2). We have previously mapped a quantitative trait locus (QTL) controlling HbF levels in an extended Asian –Indian kindred to a 1.5 Mb interval on chromosome 6q23 that contains five protein-coding genes – ALDH8A1, HBS1L, MYB, AHI1 and PDE7B [1]. Recently our group, through expression profiling of cultured erythroid progenitors, has shown that MYB participates in the regulation of fetal haemoglobin expression [2]. In addition, the insertion of a transgene in the intergenic region upstream of myb in mice leads to a reduced expression of myb specifically in erythrocyte and platelet progenitors [3]. These findings suggest a myb-regulating region, upstream and distal of myb, which could be of importance for fetal haemoglobin levels. DNase I hypersensitive sites are known to coincide with regulatory regions. We have used a quantitative real-time PCR method [4] to screen for DNase I hypersensitive sites in a 20 kb region located at the transgenic insertion site. Experimental data show a high degree of sensitivity to DNase I over the entire 20 kb region and so far 6 hypersensitive sites have been identified. The hypersensitive sites coincide with areas of high sequence conservation between humans and other mammals and include motifs for transcription factor binding sites. The regulatory potential of

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the region will be tested further in a luciferase reporter assay. References Close, J., Game, L., Clark, B., Bergounioux, J., Gerovassili, A., Thein, S. L. Genome annotation of a 1.5 Mb region of human chromosome 6q23 encompassing a quantitative trait locus for fetal hemoglobin expression in adults, BMC. Genomics 5(1) (2004) 33. Jiang, J., Best, S., Menzel, S., Silver, N., Lai, M. I., Surdulescu, G. L., Spector, T. D., Thein, S. L. cMYB is involved in the regulation of fetal hemoglobin production in adults. Blood 108(3) (2006) 1077 – 1083. Mukai, H.Y, Motohashi, H., Suzuki, N., Ohneda, O., Nagasawa, T., Yamamoto, M. Peturbation of hematopoiesis as a consequence of transgene insertion into proximity of cmyb gene, Abstracts of the 14th Conference on Hemoglobin Switching: Blood Cells, M, 2004. McArthur, M., Gerum, S., Stamatoyannopoulos, G. Quantification of DNaseI-sensitivity by real-time PCR: quantitative analysis of DNaseI-hypersensitivity of the mouse beta-globin LCR. J. Mol. Biol. 313(1) (2001) 27 – 34. doi:10.1016/j.bcmd.2006.10.151

141 Multiple functions for alpha hemoglobin stabilizing protein (AHSP) in hemoglobin synthesis and homeostasis Mitchell J. Weiss The Children’s Hospital of Philadelphia and The University of Pennsylvania, Philadelphia, PA, USA AHSP binds a hemoglobin (Hb) to maintain its structure and limit its prooxidant activities. In addition, AHSP binds and stabilizes apo –a-globin, which lacks heme. Previously, we demonstrated that Ahsp / mice exhibit hemolytic anemia with Hb precipitates. Through interbreeding of mutant strains, we also showed that loss of AHSP exacerbates h thalassemia. Together, these studies indicate that AHSP binds and neutralizes potentially toxic excess a-globin known to be synthesized in normal erythroid cells, and to a greater extent, in h thalassemic ones. However, additional functions may exist. In particular, AHSP – a-globin complexes may also promote HbA synthesis. To test this, we depleted the pool of excess a-globin in Ahsp / mice by interbreeding with a thalassemic ones. Compared to mice with either mutation alone, compound mutants missing both AHSP and one aglobin allele (Ahsp / // a-globin +/++) exhibited more severe erythroid phenotypes, including worsened anemia, hypochromia, Hb instability and ineffective erythropoiesis. Pulse labeling of double-mutant reticulocytes showed that a to h globin synthetic ratios were unaffected by loss of AHSP, but precipitation of both a and h nascent chains into cell membranes was strongly enhanced. These data indicate that AHSP is important for erythropoiesis and Hb production even