- Email: [email protected]
NIH grant applications: BIG changes!
ABSTRACTS / Blood Cells, Molecules, and Diseases 38 (2007) 120 – 191
locus in vivo. Several lines of work will be presented including the following: While we have demonstrated that the LCR is necessary for high-level ser-5 phosphorylation of Pol II and activation of expression, how this correlates with the nuclear localization of the locus, and the mechanisms involved, remain to be defined. We have examined the relationship between nuclear localization and transcriptional activity of the endogenous murine B-globin locus during erythroid differentiation. We find that the B-globin locus progressively moves away from the nuclear periphery and to hyper-phosphorylated Pol II transcription factories with increasing maturation, and that this is facilitated by the LCR.Hispanic thalassemia, a naturally occurring deletion of the LCR plus 25 kb upstream results in the failure to activate the B-globin locus at the levels of chromatin structure, transcription and replication. Deletion of the LCR does not recapitulate this phenotype, raising the question of which non-LCR sequences are capable of establishing an open B-globin domain. Extending the LCR deletion to remove additional upstream sequences in the mouse, and in a human chromosome transfer system does not lead to a ‘‘closed’’ DNaseI domain. Thus the Hispanic phenotype may result from a combination of the deletion and the cellular milieu the chromosome has been exposed to. While several lines of evidence have suggested that the flanking HSs, HS-62.5 and 3VHS1, may play a role in the regulation of the locus, we wished to determine if these regions are essential for normal regulation of the locus in vivo. Deletion of these regions does not detectably affect B-globin expression during development, differentiation or stress. Thus, while these regions may play a role in regulating the structure or epigenetic modifications of the locus, this does not appear to have any functional significance in erythroid cells. doi:10.1016/j.bcmd.2006.10.014
4 Novel role for EKLF in megakaryocyte –erythroid differential lineage commitment James J. Bieker Mount Sinai School of Medicine, Brookdale Department of Molecular, Cell and Developmental Biology, New York, NY 10029, USA The megakaryocyte and erythrocyte lineages are proposed to derive from a common precursor, the megakaryocyte – erythroid progenitor (MEP). Strikingly, these two lineages share a number of commonalities with respect to transcription factors that are absolutely required (e.g., GATA1, FOG1, SCL, Gfi-1b). At the same time, the protein partners that form with these factors during differentiation can be significantly different between lineages. However, as these factors are all positively required for both lineages, we are still left with an incomplete picture of how lineages are differentially established during hematopoiesis. Erythroid Kru¨ppel-like Factor (EKLF; KLF1) is a zinc finger transcription factor that plays a critical role in erythroid
125
gene expression, with adult B-globin being a particularly wellstudied target for activation. EKLF is highly restricted in its expression pattern to hematopoietic organs such as the yolk sac, fetal liver, adult bone marrow, and the red pulp of the spleen. Recent studies have expanded its activation target repertoire to protein-stabilizing, heme biosynthetic pathway, and red cell membrane proteins. However, along with other cellular and molecular studies, they also suggest that there are genes that are repressed by EKLF. EKLF’s role is absolutely critical for the erythroid lineage, as supported by gene ablation studies, of which the most obvious effect is a profound B-thalassemia that leads to lethality in murine embryos at the time of the switch to adult B-globin expression. Enigmatically however, EKLF is also expressed in multipotential hematopoietic cell lines and in cultured primary hematopoietic cells. As a result, we sought to determine by both gain- and loss-of-function approaches whether EKLF might be playing a heretofore undiscovered role in hematopoietic lineage decisions. Most unexpectedly, we find that, unlike its cohorts within the erythroid lineage that are also required for megakaryocyte development, EKLF plays a negative role and point to its expression level playing a critical part in the bipotential lineage decisions that are made by the MEP. doi:10.1016/j.bcmd.2006.10.015
5 NIH grant applications: BIG changes! Terry Rogers Bishop NIDDK/NIH, USA In compliance with U.S. Public Law 106 – 107, the NIH is transitioning to all electronic communication, including submission of grant applications. This change will eventually eliminate paper-based data collection, improve data quality with electronic validations, reduce scanning, printing and dataentry costs and provide a more rapid means of communication between the NIH and the scientific community. In compliance with the President’s Management Agenda (2002), which requires ‘‘applicants for Federal Grants to apply for and manage grant funds online through a common web site’’, NIH (and all federal) grant applications must be submitted through Grants.gov on SF424 [R&R] forms. The purpose of this Break-Out Session is to tell you how these changes affect you, as an NIH grant applicant. A brief overview of the federal budget process as it relates to the NIH will be followed by a step-by-step description of the process to apply for an NIH grant and highlights of the SF424 form will be discussed. Examples of relevant Funding Opportunity Announcements (FOAs) will be discussed as examples. At the end of the Session, you will have a better understanding of your role as Principal Investigator of an NIH grant and of what happens to your grant at the NIH. doi:10.1016/j.bcmd.2006.10.016
locus in vivo. Several lines of work will be presented including the following: While we have demonstrated that the LCR is necessary for high-level ser-5 phosphorylation of Pol II and activation of expression, how this correlates with the nuclear localization of the locus, and the mechanisms involved, remain to be defined. We have examined the relationship between nuclear localization and transcriptional activity of the endogenous murine B-globin locus during erythroid differentiation. We find that the B-globin locus progressively moves away from the nuclear periphery and to hyper-phosphorylated Pol II transcription factories with increasing maturation, and that this is facilitated by the LCR.Hispanic thalassemia, a naturally occurring deletion of the LCR plus 25 kb upstream results in the failure to activate the B-globin locus at the levels of chromatin structure, transcription and replication. Deletion of the LCR does not recapitulate this phenotype, raising the question of which non-LCR sequences are capable of establishing an open B-globin domain. Extending the LCR deletion to remove additional upstream sequences in the mouse, and in a human chromosome transfer system does not lead to a ‘‘closed’’ DNaseI domain. Thus the Hispanic phenotype may result from a combination of the deletion and the cellular milieu the chromosome has been exposed to. While several lines of evidence have suggested that the flanking HSs, HS-62.5 and 3VHS1, may play a role in the regulation of the locus, we wished to determine if these regions are essential for normal regulation of the locus in vivo. Deletion of these regions does not detectably affect B-globin expression during development, differentiation or stress. Thus, while these regions may play a role in regulating the structure or epigenetic modifications of the locus, this does not appear to have any functional significance in erythroid cells. doi:10.1016/j.bcmd.2006.10.014
4 Novel role for EKLF in megakaryocyte –erythroid differential lineage commitment James J. Bieker Mount Sinai School of Medicine, Brookdale Department of Molecular, Cell and Developmental Biology, New York, NY 10029, USA The megakaryocyte and erythrocyte lineages are proposed to derive from a common precursor, the megakaryocyte – erythroid progenitor (MEP). Strikingly, these two lineages share a number of commonalities with respect to transcription factors that are absolutely required (e.g., GATA1, FOG1, SCL, Gfi-1b). At the same time, the protein partners that form with these factors during differentiation can be significantly different between lineages. However, as these factors are all positively required for both lineages, we are still left with an incomplete picture of how lineages are differentially established during hematopoiesis. Erythroid Kru¨ppel-like Factor (EKLF; KLF1) is a zinc finger transcription factor that plays a critical role in erythroid
125
gene expression, with adult B-globin being a particularly wellstudied target for activation. EKLF is highly restricted in its expression pattern to hematopoietic organs such as the yolk sac, fetal liver, adult bone marrow, and the red pulp of the spleen. Recent studies have expanded its activation target repertoire to protein-stabilizing, heme biosynthetic pathway, and red cell membrane proteins. However, along with other cellular and molecular studies, they also suggest that there are genes that are repressed by EKLF. EKLF’s role is absolutely critical for the erythroid lineage, as supported by gene ablation studies, of which the most obvious effect is a profound B-thalassemia that leads to lethality in murine embryos at the time of the switch to adult B-globin expression. Enigmatically however, EKLF is also expressed in multipotential hematopoietic cell lines and in cultured primary hematopoietic cells. As a result, we sought to determine by both gain- and loss-of-function approaches whether EKLF might be playing a heretofore undiscovered role in hematopoietic lineage decisions. Most unexpectedly, we find that, unlike its cohorts within the erythroid lineage that are also required for megakaryocyte development, EKLF plays a negative role and point to its expression level playing a critical part in the bipotential lineage decisions that are made by the MEP. doi:10.1016/j.bcmd.2006.10.015
5 NIH grant applications: BIG changes! Terry Rogers Bishop NIDDK/NIH, USA In compliance with U.S. Public Law 106 – 107, the NIH is transitioning to all electronic communication, including submission of grant applications. This change will eventually eliminate paper-based data collection, improve data quality with electronic validations, reduce scanning, printing and dataentry costs and provide a more rapid means of communication between the NIH and the scientific community. In compliance with the President’s Management Agenda (2002), which requires ‘‘applicants for Federal Grants to apply for and manage grant funds online through a common web site’’, NIH (and all federal) grant applications must be submitted through Grants.gov on SF424 [R&R] forms. The purpose of this Break-Out Session is to tell you how these changes affect you, as an NIH grant applicant. A brief overview of the federal budget process as it relates to the NIH will be followed by a step-by-step description of the process to apply for an NIH grant and highlights of the SF424 form will be discussed. Examples of relevant Funding Opportunity Announcements (FOAs) will be discussed as examples. At the end of the Session, you will have a better understanding of your role as Principal Investigator of an NIH grant and of what happens to your grant at the NIH. doi:10.1016/j.bcmd.2006.10.016