- Email: [email protected]
Do Ldb1 complexes mediate long range DNA interactions?
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
or at any of the few CpG-rich regions present in the human h-globin locus contained in the h-globinYAC model. This suggests an indirect silencing mechanism mediated by another gene(s). These results support an evolutionarily conserved role for MBD2 in silencing of the embryonic/fetal globin genes during vertebrate development. As such, MBD2 is potentially a molecular target for therapeutic reversal of globin gene switching. doi:10.1016/j.bcmd.2006.10.054
44 Transcriptional networks controlling blood stem cell development Bertie Gottgens CIMR, Cambridge, UK The importance of transcriptional networks in both normal and leukaemic stem cell biology is underlined by the large number of transcription factor genes that are disrupted as part of the pathogenesis of haematological malignancies. However, blood stem cell transcriptional networks are still poorly understood. Transcriptional control regions determine the connectivity of transcription factor networks. Therefore, to understand these networks, it is essential to identify transcriptional control regions, define their in vivo activity and characterise their molecular mechanisms. Work in my group employs both top-down and bottom-up approaches to identify and characterise regulatory elements of blood stem cell regulatory networks. To this end, we have developed a new suite of bioinformatics tools for the top-down computational identification of gene regulatory elements from the human genome sequence with predicted in vivo activity. In parallel, we are performing transgenic and molecular studies to dissect the transcriptional regulation of LMO2, Lyl1, SCL and endoglin, all key regulators of early blood development. Through these combined approaches we are continually expanding our regulatory network which currently consists of 12 genes with 33 network connections, all validated in vivo in transgenic mice. Future work will address how transcriptional networks are perturbed in specific subtypes of leukaemia and may thus open up new avenues for the development of targeted therapies. doi:10.1016/j.bcmd.2006.10.055
45 Do Ldb1 complexes mediate long range DNA interactions? Frank Grosveld Department of Cell Biology, Erasmus Medical Center, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands The Ldb1 protein is vital for the development of the hematopoietic lineage in vertebrates (Mukhopadhyay et al., 2003). It has previously been implicated in Drosophila to be
143
important in the long-range interaction between regulatory elements (Morcillo et al., 1997). Ldb1 was previously identified as interacting with a number of erythroid transcription factors including Gata1, Tal1 and Lmo2 (Wadman et al., 1997). We will discuss that Ldb1 is a central factor in two different transcription factor complexes that interact with each other in erythroid development and which change during development. Importantly these complexes contain a number of DNA binding proteins in particular bHLH factors, which suggest that Ldb1 may indeed be part of a complex (or complexes) that bridges the interaction between regulatory elements. We will also present our recent developments on the imaging of the b-globin locus using in situ fluorescence microscopy. doi:10.1016/j.bcmd.2006.10.056
46 Nuclear positioning of tissue-specific genes during hematopoietic differentiation of primary progenitors from human cord blood Claire Guillemin, Adeline Guais, Claire Francastel Institut Cochin, Hematology department, INSERM U567/CNRS UMR8104/Univeriste´ Paris V, Paris, France The multilineage potential of HSC is gradually lost as the stem cell differentiates to non-self renewing then lineagecommitted progenitors, a process accompanied by heritable changes in gene expression. These gene expression programs are determined not only by the availability of combinations of transcription factors, but also by modulation of higher order chromatin structure and, as observed more recently, by positioning in the nucleus relative to a ‘‘non-permissive’’ heterochromatin compartment. Our aim is to define the chromatin structure and global nuclear organization that underlies the self-renewal and multilineage potential of primitive hematopoietic cells. The question being do HSC maintain an open chromatin structure, allowing access to multilineage affiliated programs (which would gradually be lost as the cell differentiates), or are lineageaffiliated genes selectively opened during commitment and differentiation. In order to address this question, we compare the chromatin structure and nuclear organisation of key lymphoid- and myeloid-specific genes in multipotent human HSC and lineage committed progenitors. Multipotent (CD34+CD38 ) and lineage committed hematopoietic progenitors are isolated from umbilical cord blood and adult bone marrow based on the expression of surface markers. To study dynamic changes in chromatin structure and nuclear localisation, we also use in vitro culture systems to differentiate CD34+CD38 cells towards the B-lymphoid, granulocyte or erythroid lineages. Localization of tissue specific markers following differentiation of HSC in various hematopoietic lineages are followed by FISH. Comparison of ex vivo HSC and lineage-committed precursors will allow us to
or at any of the few CpG-rich regions present in the human h-globin locus contained in the h-globinYAC model. This suggests an indirect silencing mechanism mediated by another gene(s). These results support an evolutionarily conserved role for MBD2 in silencing of the embryonic/fetal globin genes during vertebrate development. As such, MBD2 is potentially a molecular target for therapeutic reversal of globin gene switching. doi:10.1016/j.bcmd.2006.10.054
44 Transcriptional networks controlling blood stem cell development Bertie Gottgens CIMR, Cambridge, UK The importance of transcriptional networks in both normal and leukaemic stem cell biology is underlined by the large number of transcription factor genes that are disrupted as part of the pathogenesis of haematological malignancies. However, blood stem cell transcriptional networks are still poorly understood. Transcriptional control regions determine the connectivity of transcription factor networks. Therefore, to understand these networks, it is essential to identify transcriptional control regions, define their in vivo activity and characterise their molecular mechanisms. Work in my group employs both top-down and bottom-up approaches to identify and characterise regulatory elements of blood stem cell regulatory networks. To this end, we have developed a new suite of bioinformatics tools for the top-down computational identification of gene regulatory elements from the human genome sequence with predicted in vivo activity. In parallel, we are performing transgenic and molecular studies to dissect the transcriptional regulation of LMO2, Lyl1, SCL and endoglin, all key regulators of early blood development. Through these combined approaches we are continually expanding our regulatory network which currently consists of 12 genes with 33 network connections, all validated in vivo in transgenic mice. Future work will address how transcriptional networks are perturbed in specific subtypes of leukaemia and may thus open up new avenues for the development of targeted therapies. doi:10.1016/j.bcmd.2006.10.055
45 Do Ldb1 complexes mediate long range DNA interactions? Frank Grosveld Department of Cell Biology, Erasmus Medical Center, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands The Ldb1 protein is vital for the development of the hematopoietic lineage in vertebrates (Mukhopadhyay et al., 2003). It has previously been implicated in Drosophila to be
143
important in the long-range interaction between regulatory elements (Morcillo et al., 1997). Ldb1 was previously identified as interacting with a number of erythroid transcription factors including Gata1, Tal1 and Lmo2 (Wadman et al., 1997). We will discuss that Ldb1 is a central factor in two different transcription factor complexes that interact with each other in erythroid development and which change during development. Importantly these complexes contain a number of DNA binding proteins in particular bHLH factors, which suggest that Ldb1 may indeed be part of a complex (or complexes) that bridges the interaction between regulatory elements. We will also present our recent developments on the imaging of the b-globin locus using in situ fluorescence microscopy. doi:10.1016/j.bcmd.2006.10.056
46 Nuclear positioning of tissue-specific genes during hematopoietic differentiation of primary progenitors from human cord blood Claire Guillemin, Adeline Guais, Claire Francastel Institut Cochin, Hematology department, INSERM U567/CNRS UMR8104/Univeriste´ Paris V, Paris, France The multilineage potential of HSC is gradually lost as the stem cell differentiates to non-self renewing then lineagecommitted progenitors, a process accompanied by heritable changes in gene expression. These gene expression programs are determined not only by the availability of combinations of transcription factors, but also by modulation of higher order chromatin structure and, as observed more recently, by positioning in the nucleus relative to a ‘‘non-permissive’’ heterochromatin compartment. Our aim is to define the chromatin structure and global nuclear organization that underlies the self-renewal and multilineage potential of primitive hematopoietic cells. The question being do HSC maintain an open chromatin structure, allowing access to multilineage affiliated programs (which would gradually be lost as the cell differentiates), or are lineageaffiliated genes selectively opened during commitment and differentiation. In order to address this question, we compare the chromatin structure and nuclear organisation of key lymphoid- and myeloid-specific genes in multipotent human HSC and lineage committed progenitors. Multipotent (CD34+CD38 ) and lineage committed hematopoietic progenitors are isolated from umbilical cord blood and adult bone marrow based on the expression of surface markers. To study dynamic changes in chromatin structure and nuclear localisation, we also use in vitro culture systems to differentiate CD34+CD38 cells towards the B-lymphoid, granulocyte or erythroid lineages. Localization of tissue specific markers following differentiation of HSC in various hematopoietic lineages are followed by FISH. Comparison of ex vivo HSC and lineage-committed precursors will allow us to