Biomedical subjects

Stephen J Brandt

Publications and source records attributed to Stephen J Brandt.

9 recordsLinked to original sources

PubMed2006

Recruitment of the SWI/SNF protein Brg1 by a multiprotein complex effects transcriptional repression in murine erythroid progenitors.

SWI/SNF complexes are involved in both activation and repression of transcription. While one of two homologous ATPases, Brg1 [Brm (Brahma)-related gene 1] or Brm, is required for their chromatin remodelling function, less is known about how these complexes are recruited to DNA. We recently established that a DNA-binding complex containing TAL1/SCL, E47, GATA-1, LMO2 and Ldb1 stimulates P4.2 (protein 4.2) transcription in erythroid progenitors via two E box-GATA elements in the gene's proximal promoter. We show here that the SWI/SNF protein Brg1 is also associated with this complex and that both the E box and GATA DNA-binding sites in these elements are required for Brg1 recruitment. Further, Brg1 occupancy of the P4.2 promoter decreased with terminal erythroid differentiation in association with increased P4.2 transcription, while enforced expression of Brg1 in murine erythroleukaemia cells reduced P4.2 gene expression. Overexpression of Brg1 was associated with increased occupancy of the P4.2 promoter by the nuclear co-repressor mSin3A and HDAC2 (histone deacetylase 2) and with reduced histone H3 and H4 acetylation. Finally, a specific HDAC inhibitor attenuated Brg1-directed repression of P4.2 promoter activity in transfected cells. These results provide insight into the mechanism by which SWI/SNF proteins are recruited to promoters and suggest that transcription of P4.2, and most likely other genes, is actively repressed until the terminal differentiation of erythroid progenitors.

Acetylation↗

PubMed2006

Spliced isoforms of LIM-domain-binding protein (CLIM/NLI/Ldb) lacking the LIM-interaction domain.

LIM-domain-binding proteins (CLIM/NLI/Ldb) are nuclear cofactors for LIM homeodomain transcription factors (LIM-HDs) and LIM-only proteins (LMOs). The LIM-interaction domain (LID) of Ldb is located in the carboxy-terminal region and encoded by the last exon (exon 10) of Ldb genes. It is known that the mammalian CLIM1/Ldb2 gene has a splice isoform, named CLIM1b, lacking the LID. However, little is known about the nature of CLIM1b or the evolutionary conservation of this type of alternative splicing in amphibians and teleost fish. Here, we demonstrate that splice isoforms lacking the LID are also present in the Ldb1 genes of mammals, chick, and Xenopus, as well as in fish paralog Ldb4. All these splicing variations occur in intron 9 and exon 10. We observed that Ldb4b (splice isoform lacking LID) is localized in the nucleus when expressed in mammalian culture cells, and binds to Ldb4a (splice isoform containing LID) but not directly to LIM proteins. However, Ldb4b binds to LMO4 via Ldb4a when coexpressed in culture cells. We also found that mouse Ldb1b lacks the ability to activate protein 4.2 promoter, which is stimulated by LMO2 and Ldb1. These findings suggest that splice isoforms of Ldb lacking LID are potential regulators of Ldb function.

Alternative Splicing↗

PubMed2006

Transcriptional link between blood and bone: the stem cell leukemia gene and its +19 stem cell enhancer are active in bone cells.

Blood and vascular cells are generated during early embryogenesis from a common precursor, the hemangioblast. The stem cell leukemia gene (SCL/tal 1) encodes a basic helix-loop-helix transcription factor that is essential for the normal development of blood progenitors and blood vessels. We have previously characterized a panel of SCL enhancers including the +19 element, which directs expression to hematopoietic stem cells and endothelium. Here we demonstrate that SCL is expressed in bone primordia during embryonic development and in adult osteoblasts. Despite consistent expression in cells of the osteogenic lineage, SCL protein is not required for bone specification of embryonic stem cells. In transgenic mice, the SCL +19 core enhancer directed reporter gene expression to vascular smooth muscle and bone in addition to blood and endothelium. A 644-bp fragment containing the SCL +19 core enhancer was active in both blood and bone cell lines and was bound in vivo by a common array of Ets and GATA transcription factors. Taken together with the recent observation that a common progenitor can give rise to blood and bone cells, our results suggest that the SCL +19 enhancer targets a mesodermal progenitor capable of generating hematopoietic, vascular, and osteoblastic progeny.

Animals↗

PubMed2005

Translating the histone code into leukemia.

The "histone code" is comprised of the covalent modifications of histone tails that function to regulate gene transcription. The post-translational modifications that occur in histones within the regulatory regions of genes include acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP-ribosylation. These modifications serve to alter chromatin structure and accessibility, and to act as docking sites for transcription factors or other histone modifying enzymes. Several of the factors that are disrupted by chromosomal translocations associated with hematological malignancies can alter the histone code in a gene-specific manner. Here, we discuss how the histone code may be disrupted by chromosomal translocations, either directly by altering the activity of histone modifying enzymes, or indirectly by recruitment of this type of enzyme by oncogenic transcription factors. These alterations in the histone code may alter gene expression pattern to set the stage for leukemogenesis.

Amino Acid Motifs↗

PubMed2005

Tal1/SCL binding to pericentromeric DNA represses transcription.

Tal1/SCL is a basic helix-loop-helix transcription factor critical for normal hematopoiesis. To understand the mechanisms underlying transcriptional regulation by Tal1/SCL, we combined an in vitro DNA binding strategy and an in vivo chromatin immunoprecipitation analysis to search for Tal1/SCL target regions in K562 erythroleukemia cells. A 0.4-kb genomic DNA clone containing two Tal1/SCL binding E-boxes and GATA- and SATB1-binding motifs (EEGS) was identified that localized to the pericentromeric region with high homology to satellite 2 DNA. Pericentric DNA is related to heterochromatin and gene inactivation. We found that Tal1/SCL could complex with the histone H3 lysine 9 (H3K9)-specific methyltransferase Suv39H1. Binding of Tal1/SCL to EEGS chromatin correlated with hypermethylation of H3K9 and the association of heterochromatin protein HP1 to this region. In Rep4 reporter gene assays, EEGS affected repression in a manner dependent on the expression level of Tal1/SCL that was accompanied by increased H3K9 methylation in chromatin associated with EEGS and a linked promoter. A specific histone deacetylase inhibitor, trichostatin A, relieved Tal1/SCL-mediated repression by EEGS. In addition, SATB1 bound EEGS chromatin and promoted Tal1/SCL EEGS-dependent repression. We expand the list of potential interacting partners for Tal1/SCL by demonstrating direct associations of Tal1/SCL with SATB1 and with Suv39H1. These results reveal a novel mechanism of action for Tal1/SCL and implicate heterochromatin-like silencing via a cis-acting binding motif for transcriptional repression.

Amino Acid Motifs↗

PubMed2005

Differential gene expression in anaplastic lymphoma kinase-positive and anaplastic lymphoma kinase-negative anaplastic large cell lymphomas.

Anaplastic large cell lymphoma (ALCL) is an aggressive large T- or null-cell lymphoma. Most ALCLs arising in children and young adults express a constitutively active receptor tyrosine kinase, anaplastic lymphoma kinase (ALK). Anaplastic large cell lymphomas lacking ALK are clinically heterogeneous and their pathogenesis is unknown. This study is the first complementary DNA (cDNA) microarray analysis using RNA extracted from tumor tissue (7 ALK+ ALCLs and 7 ALK- ALCLs) to identify genes differentially expressed or shared between the ALK+ and ALK- tumors. Unsupervised hierarchical clustering using the top 11 most statistically significant discriminator cDNAs correctly grouped all ALK+ and ALK- tumors. Hierarchical clustering analysis using the 44 cDNAs with the greatest differential expression between ALK+ and ALK- RNAs grouped 6 of 7 ALK+ ALCLs together and 1 ALK+ ALCL with the ALK- group. In general, ALK+ tumors overexpress genes encoding signal transduction molecules (SYK , LYN , CDC37) and underexpress transcription factor genes (including HOXC6 and HOX A3 ) compared with the ALK- group. Cyclin D3 was overexpressed in the ALK+ group and the cell cycle inhibitor p19INK4D was decreased in the ALK- group, suggesting different mechanisms of promoting G 1 /S transition. Both groups had similar proliferation rates. Genes highly expressed in both ALK- and ALK+ ALCLs included kinases (LCK, protein kinase C, vav2, and NKIAMRE) and antiapoptotic molecules, suggesting possible common pathogenetic mechanisms as well.

Adolescent↗

PubMed2003

Identification of a TAL1 target gene reveals a positive role for the LIM domain-binding protein Ldb1 in erythroid gene expression and differentiation.

The TAL1 (or SCL) gene, originally identified from its involvement by a recurrent chromosomal translocation, encodes a basic helix-loop-helix transcription factor essential for erythropoiesis. Although presumed to regulate transcription, its target genes are largely unknown. We show here that a nuclear complex containing TAL1, its DNA-binding partner E47, zinc finger transcription factor GATA-1, LIM domain protein LMO2, and LIM domain-binding protein Ldb1 transactivates the protein 4.2 (P4.2) gene through two E box GATA elements in its proximal promoter. Binding of this complex to DNA was dependent on the integrity of both E box and GATA sites and was demonstrated to occur on the P4.2 promoter in cells. Maximal transcription in transiently transfected cells required both E box GATA elements and expression of all five components of the complex. This complex was shown, in addition, to be capable of linking in solution double-stranded oligonucleotides corresponding to the two P4.2 E box GATA elements. This DNA-linking activity required Ldb1 and increased with dimethyl sulfoxide-induced differentiation of murine erythroleukemia (MEL) cells. In contrast, enforced expression in MEL cells of dimerization-defective mutant Ldb1, as well as wild-type Ldb1, significantly decreased E box GATA DNA-binding activities, P4.2 promoter activity, and accumulation of P4.2 and beta-globin mRNAs. These studies define a physiologic target for a TAL1- and GATA-1-containing ternary complex and reveal a positive role for Ldb1 in erythroid gene expression and differentiation.

Animals↗

PubMed2002

Phosphorylation by mitogen-activated protein kinase mediates the hypoxia-induced turnover of the TAL1/SCL transcription factor in endothelial cells.

The basic helix-loop-helix transcription factor TAL1 (or SCL), originally identified from its involvement by a chromosomal rearrangement in T-cell acute lymphoblastic leukemia, is required for hematopoietic development. TAL1 also has a critical role in embryonic vascular remodeling and is expressed in endothelial cells postnatally, although little is known about its function or regulation in this cell type. We report here that the important proangiogenic stimulus hypoxia stimulates phosphorylation, ubiquitination, and proteasomal breakdown of TAL1 in endothelial cells. Tryptic phosphopeptide mapping and chemical inhibitor studies showed that hypoxia induced the mitogen-activated protein kinase-mediated phosphorylation of a single serine residue, Ser(122), in the protein, and site-directed mutagenesis demonstrated that Ser(122) phosphorylation was necessary for hypoxic acceleration of TAL1 turnover in an immortalized murine endothelial cell line. Finally, whereas TAL1 expression was detected in endothelial cells from both large and small vessels, hypoxia-induced TAL1 turnover was observed only in microvascular endothelial cells. Besides their implications for TAL1 function in angiogenic processes, these results demonstrate that a protein kinase(s) important for mitogenic signaling is also utilized in hypoxic endothelial cells to target a transcription factor for destruction.

Animals↗

PubMed2002

New insights into erythropoiesis.

Commitment of hematopoietic cells to the erythroid lineage involves the actions of several transcription factors, including TAL1, LMO2, and GATA-2. The differentiation of committed erythroid progenitor cells involves other transcription factors, including NF-E2 and EKLF. Upon binding erythropoietin, the principal regulator of erythropoiesis, cell surface erythropoietin receptors dimerize and activate specific intracellular kinases, including Janus family tyrosine protein kinase 2, phosphoinositol-3 kinase, and mitogen-activated protein kinase. Important substrates of these kinases are tyrosines in the erythropoietin receptors themselves and the signal transducer and transcription activator proteins. Erythropoietin prevents erythroid cell apoptosis. Some of the apoptotic tendency of erythroid cells can be attributed to proapoptotic molecules produced by hematopoietic cells, macrophages, and stromal cells. Cell divisions accompanying terminal erythroid differentiation are finely controlled by cell cycle regulators, and disruption of these terminal divisions causes erythroid cell apoptosis. In reticulocyte maturation, regulated degradation of internal organelles involves a lipoxygenase, whereas survival requires the antiapoptotic protein Bcl-x.

Animals↗