1993 — 1997 |
Brandt, Stephen J. |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Scl Gene and Hematopoietic Development
The proposed research will investigate the expression and functions of the protein products of the SCL gene during hematopoietic development in the mouse. This gene was first identified at the breakpoint of a chromosomal translocation in human T-cell leukemia and is a member of a family of transcription factors whose members are recognized to be regulators of differentiation. The results of these experiments should provide insight into basic mechanisms of hematopoiesis. Since activation of this gene by chromosomal rearrangement is the most common genetic lesion in acute T-cell leukemia, these studies should also contribute to our understanding of leukemogenesis. The first specific aim of this work is to characterize the translational products of the murine SCL gene in hematopoietic cells. The protein products of the murine SCL gene will be characterized in hematopoietic cell lines and mouse tissues by radioimmunoprecipitation and Western blot analysis. Subcellular localization of SCL proteins will be determined by transient expression in COS cells and in cell lines stably expressing individual proteins. The second specific aim is to characterize the transcriptional and translational products of the murine SCL gene during hematopoietic development. SCL mRNA expression will be studied with RNase protection, in situ hybridization, and the polymerase chain reaction (PCR) and SCL protein expression by immunocytochemistry in staged mouse embryos. The transcriptional and translational products of the gene will also be characterized in an in vitro model of hematopoietic development using embryonic stem (ES) cell-derived embryoid bodies. The third specific aim is to define the actions of SCL proteins in hematopoietic development. This will be investigated in gain of function and loss of function mutants of ES cells made to undergo differentiation. The fourth specific aim is to determine the ability of SCL proteins to function as transcriptional regulators during hematopoietic development. The ability of SCL proteins to transactivate artificial and, if identified, physiologic targets will be determined. The possibility that certain SCL proteins may have different transcriptional potencies and act as transcriptional antagonists will be investigated. Cellular targets will be identified using a functional strategy based on the isolation of SCL-inducible promoters, and the ability of different SCL isoforms to activate such promoters will be defined.
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1996 — 1997 |
Brandt, Stephen J. |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Molecular Basis of the Chediak-Higashi Syndrome
Oculocutaneous albinism represents a heterogeneous group of genetic disorders characterized by reduced to absent production of melanin pigment in association with specific changes in the developing eye. In addition to primary abnormalities in melanin synthesis, reduced pigmentation may be a component of a multisystemic process. The Chediak-Higashi syndrome (CHS) is an autosomal recessive disorder characterized by partial oculocutaneous albinism, immunodeficiency, and a marked predisposition to lymphoproliferative disease. The gene responsible for the well- characterized mouse model of CHS, beige (bg), was recently isolated by positional cloning, providing the first opportunity for understanding this disorder at a molecular level. The molecular basis of this cause of oculocutaneous albinism will be investigated here by a genetic complementation approach involving the retrovirus-mediated expression of antisense RNA to bg coding and 3 - untranslated sequences in normal mouse melanocytes and of a full-length sense cDNA in beige mouse melanocytes and CHS patient-derived fibroblast and lymphoblast cell lines. These studies will provide functional confirmation that the CHS gene has been identified, complement efforts at identifying mutations in the human ortholog of the bg gene and in other bg alleles, determine whether more than one complementation group exists in this disorder, and generate valuable reagents for investigating the function of this gene in melanosomal biogenesis.
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1998 — 2011 |
Brandt, Stephen J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation and Function of the Tal1/Scl Gene
DESCRIPTION (provided by applicant): Aberrant expression of the TAL1 (or SCL) gene is one of the most frequent gain-of-function mutations in T-cell acute lymphoblastic leukemia. This helix-loop-helix (HLH) transcription factor is also important in hematopoietic specification during embryogenesis and differentiation of the erythroid and megakaryocytic lineages postnatally. TAL1 contributes to a DNA-binding complex that contains an HLH DNA-binding partner, the GATA- 1 transcription factor, a LIM-only protein, and the LIM domain-binding protein Ldb1 and recognizes an E box- GATA DNA sequence motif. Work in the current funding period identified additional members of this complex, including the SWI/SNF protein Brg1, corepressors ETO2 and MTGR-1, and Single-Stranded DNA-Binding Protein-2 and -3. While considerable information is available about TAL1 and GATA-1, much less is known about the functions of the non-DNA-binding members of this complex. This renewal application will test the hypotheses that Ldb1 contributes importantly to the transcription of TAL1- and GATA-1 target genes and that its ability to homo-oligomerize is important for long-range control of erythroid gene expression. The first specific aim is to determine the importance of Ldb1 expression for E box-GATA DNA-binding activity, gene expression, and differentiation of murine erythroid progenitors. These studies will determine the effects of reducing Ldb1 expression on the abundance of the E box-GATA DNA-binding complex and its affinity for DNA, transcription of select target genes of the TAL1- and GATA-1-containing complex, and transcription factor occupancy, RNA polymerase II recruitment, and histone acetylation at the promoters of these genes in two in vitro models of erythroid cell differentiation. The second specific aim is to determine the contribution of Ldb1 homodimer formation to E box-GATA DNA-binding activity, short-range control of gene expression, and cellular differentiation. These studies will define the minimal domain required in Ldb1 homodimerization, determine the importance of Ldb1 homodimerization for E box-GATA DNA-binding activity, develop a specific polypeptide inhibitor of Ldb1 dimerization, and test the effect of this inhibitor on E box-GATA DNA-binding activity, erythroid gene expression, and terminal differentiation. The third specific aim is to determine the importance of Ldb1 homodimerization in long-range control of gene expression. These studies will address whether Ldb1 mediates long-range interaction of the upstream regulatory regions and promoter of the mouse beta-globin (maj) gene and identify additional loci in the mouse genome occupied by Ldb1 to elucidate its role in regulation of their transcription. The results of these studies will advance basic understanding of erythroid differentiation, have relevance to other cellular programs regulated by LIM domain and HLH proteins, and provide insights into fundamental mechanisms of transcriptional regulation and leukemogenesis. Project Narrative: The studies proposed in this application are highly relevant to public health. In addition to advancing understanding of how red blood cells are made, which is applicable to the disorder of red cell production known as anemia, this work could lead to new treatment approaches for T-cell acute lymphoblastic leukemia and cancers of the breast and oral cavity.
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2002 — 2006 |
Brandt, Stephen J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation and Function of the Tal 1/Scl Gene
DESCRIPTION (provided by applicant): Misexpression of the TAL1/SCL gene is the most frequent gain-of-function mutation observed in T-cell acute lymphoblastic leukemia. Gene knockout and overexpression studies have demonstrated that this basic helix-loop-helix transcription factor is essential for specification of blood cell formation and vascular remodeling during embryogenesis, generation of all hematopoietic cell types in the adult, and terminal differentiation of the erythroid and megakaryocytic lineages. Studies from our laboratory have demonstrated that TAL1 can interact in a reciprocal manner with corepressor and coactivator complexes in differentiating murine erythroleukemia (MEL) cells, that posttranslational modification can modify TAL1 interaction with the nuclear corepressor mSin3A, and that the Protein 4.2 gene is a physiologic target subject to sequential repression and activation by TAL1-containing complexes in differentiating MEL cells. This renewal application will investigate the hypothesis that TAL1 has experimentally separable functions determined by its interaction with specific coregulator complexes and will test a model of TAL1 action in erythroid cells that has TAL1-mediated transcriptional repression inhibiting differentiation and/or stimulating proliferation and TAL1-directed transactivation promoting terminal differentiation. The first aim is to determine the structural requirements for select TAL1-coregulator interactions. Residues critical for TAL1 interaction with the LIM-only protein LMO2 and corepressor mSin3A will be determined through mutagenesis, and specific interaction-defective alleles of TAL1 will be identified. The second aim is to determine the importance of specific TAL1 -coregulator interactions in the expression of a TAL1 target gene. TAL1's interaction with components of corepressor and coactivator complexes will be investigated on the Protein 4.2 promoter in living cells, and the effects of trans-dominant TAL1 and coregulator mutants will be determined on Protein 4.2 gene expression. The third aim is to determine the importance of TAL1-coregulator interactions in erythroid differentiation. The effects of interaction-defective mutants of TAL1, a dominant negative mutant of a coregulator present in TAL1-containing complexes (L.dbl), and experimental chimeras of TAL1 and potent activation and repression domains will be tested on erythroid proliferation and differentiation in two experimental models and in primary murine hematopoietic cells. The results of these studies will advance basic understanding of hematopoietic differentiation and provide insights into mechanisms of leukemogenesis.
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2004 — 2006 |
Brandt, Stephen J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Analysis of Viral Cyclin
DESCRIPTION (provided by applicant): The proposed research investigates how Kaposi's sarcoma herpesvirus (KSHV)-encoded cyclin (K-cyclin) regulates transcription. K-cyclin associates predominantly in the transcriptionally active fraction of chromatin along with other proteins that have a role in gene activation suggesting that K-cyclin may also activate transcription. K-cyclin binds a DNA consensus found in promoters regulated by retinoblastoma protein/Sp/Kruppel-like factor (Rb/Sp/KLF) complexes. K-cyclin/cdkG complexes phosphorylate the chromatin-associated transcriptional coactivator, p300 and exogenous expression of p300 along with K-cyclin synergistically activates NF-kappa B-dependent transcription, a process known to require p300 for activation. These findings suggest that K-cyclin/cdkG complexes may be responsible for the constitutive activation of NF-kappa B in primary effusion lymphoma, a tumor caused by KSHV. Collectively, these preliminary studies suggest that K-cyclin may not only deregulate the cell cycle, but may also modulate transcription of cellular and possibly viral genes. The investigator proposes to investigate the role of K-cyclin as a transcriptional regulator by addressing two specific hypotheses. In specific aim 1, the hypothesis that K-cyclin through sequence-specific interactions with KRE-containing promoters activates actives genes normally repressed by the Rb/Sp/KLF protein complexes will be examined. In specific aim 2, the hypothesis that independent of DNA-binding, K-cyclin regulates transcription through protein-protein interactions, which may be cyclin-dependent kinase (cdk) dependent and independent will be examined. Understanding how K-cyclin modulates transcription will expand current knowledge regarding the role of this highly conserved gamma 2-herpesvirus protein in the pathogenesis of diseases linked to KSHV infection. This knowledge may lead to novel therapeutic approaches for the treatment of KSHV-related malignancies.
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