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Biomedical subjects

Mikael Sigvardsson

Publications and source records attributed to Mikael Sigvardsson.

9 recordsLinked to original sources
PubMed2003

Probabilistic estimation of microarray data reliability and underlying gene expression.

BACKGROUND: The availability of high throughput methods for measurement of mRNA concentrations makes the reliability of conclusions drawn from the data and global quality control of samples and hybridization important issues. We address these issues by an information theoretic approach, applied to discretized expression values in replicated gene expression data. RESULTS: Our approach yields a quantitative measure of two important parameter classes: First, the probability P(sigma|S) that a gene is in the biological state sigma in a certain variety, given its observed expression S in the samples of that variety. Second, sample specific error probabilities which serve as consistency indicators of the measured samples of each variety. The method and its limitations are tested on gene expression data for developing murine B-cells and a t-test is used as reference. On a set of known genes it performs better than the t-test despite the crude discretization into only two expression levels. The consistency indicators, i.e. the error probabilities, correlate well with variations in the biological material and thus prove efficient. CONCLUSIONS: The proposed method is effective in determining differential gene expression and sample reliability in replicated microarray data. Already at two discrete expression levels in each sample, it gives a good explanation of the data and is comparable to standard techniques.

Algorithms↗
PubMed2003

Inhibition of p300/CBP by early B-cell factor.

Early B-cell factor (EBF) is a DNA binding protein required for early B-cell development. It activates transcription of several B-cell-specific genes, including the lambda5 gene, which encodes a protein necessary for signaling by the pre-B-cell receptor. In an effort to understand the mechanism by which EBF activates transcription, we examined its interaction with the coactivator protein p300/CBP. We found that two domains of EBF each bind the histone acetyltransferase (HAT)/CH3 domain of p300/CBP both in vitro and in vivo. Surprisingly, transcriptional activation by EBF was not sensitive to E1A, a potent p300/CBP inhibitor. In fact, overexpressed EBF mimicked E1A by severely repressing the activity of several other transcription factors, including E47, a protein that acts cooperatively with EBF to promote transcription of the lambda5 gene. This broad inhibitory profile correlated with EBF's ability to repress the HAT activity of p300/CBP in vivo and in vitro. However, such a repressed complex is not likely to form at the lambda5 promoter in vivo since (i) EBF could not bind p300/CBP and DNA simultaneously and (ii) the cooperativity imparted by E47 was sensitive to E1A. Our data reveal an intriguing inhibitory property of EBF-a property shared only by E1A, Twist, Pu.1, and the Hox family of homeodomain proteins-and suggest that E47 and EBF play distinct roles during lambda5 promoter activation.

3T3 Cells↗
PubMed2003

RNA analysis of B cell lines arrested at defined stages of differentiation allows for an approximation of gene expression patterns during B cell development.

The development of a mature B lymphocyte from a bone marrow stem cell is a highly ordered process involving stages with defined features and gene expression patterns. To obtain a deeper understanding of the molecular genetics of this process, we have performed RNA expression analysis of a set of mouse B lineage cell lines representing defined stages of B cell development using Affymetrix microarrays. The cells were grouped based on their previously defined phenotypic features, and a gene expression pattern for each group of cell lines was established. The data indicated that the cell lines representing a defined stage generally presented a high similarity in overall expression profiles. Numerous genes could be identified as expressed with a restricted pattern using dCHIP-based, quantitative comparisons or presence/absence-based, probabilistic state analysis. These experiments provide a model for gene expression during B cell development, and the correctly identified expression patterns of a number of control genes suggest that a series of cell lines can be useful tools in the elucidation of the molecular genetics of a complex differentiation process.

Animals↗
PubMed2002

Cloning and characterization of a promoter flanking the early B cell factor (EBF) gene indicates roles for E-proteins and autoregulation in the control of EBF expression.

The early B cell factor (EBF) is a transcription factor shown crucial for the development of B lymphocytes. The protein is expressed from the earliest stages of B cell development until the mature B cell stage, but the control elements responsible for the regulation of the gene are unknown. In this study, we report of the identification of a promoter region flanking the EBF gene. Several transcription start sites were identified by primer extension analysis in a region approximately 3.1 kb from the predicted ATG. Transient transfections revealed that this region was able to stimulate transcription of a reporter gene in B lymphoid and to a lesser extent, myeloid cells, but not in a pre-T cell line. The promoter was also able to functionally interact with E47, suggesting that the EBF gene may be a direct target for activation by E-proteins. In addition, functional binding of EBF to its own promoter was confirmed by EMSA and transfection assays indicating that the EBF protein may be involved in an autoregulatory loop. Finally, a tissue-restricted factor was able to bind an upstream regulatory region in B-lineage cells, further supporting the idea that the cloned promoter participates in the regulation of stage and lineage specific expression of the EBF gene.

5' Untranslated Regions↗
PubMed2002

The human V-preB promoter is a target for coordinated activation by early B cell factor and E47.

The development of mature B lymphoid cells involves a highly orchestrated regulation of stage- and lineage-specific genes. In this study, we report an analysis of the human surrogate L chain VpreB promoter. The promoter has an overall homology of 56% to the mouse counterpart and displays a preB cell-restricted activity in transient transfections in cell lines. The promoter harbors three independent binding sites for early B cell factor (EBF) as defined by EMSA and supershift experiments. These sites were important for the full function of the promoter in a preB cell line, and chromatin immunoprecipitation experiments indicate that EBF interacts with the promoter in vivo. In addition to this, ectopic expression of EBF induces the activity of a reporter gene under control of the VpreB promoter in epithelioid HeLa cells, an effect augmented by coexpression of the basic-helix-loop helix transcription factor E47. The ability to interact directly with E47 was shared by the promoters controlling the human mb-1 and B29 genes. These data indicate that the human VpreB promoter is a direct target for activation by EBF and E47 and that functional collaboration between these proteins may be of great importance in human B cell development.

Animals↗
PubMed2002

The pT alpha promoter and enhancer are direct targets for transactivation by E box-binding proteins.

The pre-T alpha (pT alpha) gene encodes a protein that forms the pre-TCR together with the newly expressed TCR beta chain during a defined stage of early T cell development. The restricted expression of this gene has been suggested to depend on a promoter and an enhancer element containing E boxes, which are potential binding sites for basic helix-loop-helix (bHLH) proteins. We here report that increased expression of E47 and E12 mRNA in CD4(-)CD8(-)TCR(-) thymocytes correlates with the initiation of pT alpha transcription. Furthermore, electrophoretic mobility shift assays suggested that E47 binds directly to both the pT alpha promoter and enhancer in vitro. Ectopic expression of E47 in non-lymphoid HeLa cells resulted in a dose- and template-dependent activation of these control elements. In addition to this, ectopic expression of E47 in combination with the ets-protein Pu.1 and/or the Pu.1-interacting protein(Pip-1), resulted in a synergistic effect on the activity of the pT alpha control elements. Thus, we suggest that E47, Pu.1 and Pip-1 are directly involved in the regulation of pT alpha expression in early T cell development.

Animals↗
PubMed2002

Early B-cell factor (O/E-1) is a promoter of adipogenesis and involved in control of genes important for terminal adipocyte differentiation.

Olf-1/early B-cell factor (O/E-1) is a transcription factor important for B-lymphocyte and neuronal gene regulation. Here we report that all three known O/E genes (O/E-1, -2, and -3) are expressed in mouse adipose tissue and are upregulated during adipocyte differentiation. Forced expression of O/E-1 in either the preadipocyte cell line 3T3-L1 or mouse embryonic fibroblasts augmented adipogenesis, and constitutive expression of O/E-1 in uncommitted NIH 3T3 fibroblasts led to initiation of adipocyte differentiation. Furthermore, a dominant negative form of O/E-1 partially suppressed 3T3-L1 adipogenesis, indicating that expression from endogenous O/E target genes is required for 3T3-L1 terminal differentiation. Thus, our data point to the importance of O/E target genes for adipocyte differentiation and suggest a novel role for O/E-1 as an initiator and stimulator of adipogenesis.

Adipocytes↗
PubMed2002

Early B-cell factor, E2A, and Pax-5 cooperate to activate the early B cell-specific mb-1 promoter.

Previous studies have suggested that the early-B-cell-specific mb-1(Igalpha) promoter is regulated by EBF and Pax-5. Here, we used in vivo footprinting assays to detect occupation of binding sites in endogenous mb-1 promoters at various stages of B-cell differentiation. In addition to EBF and Pax-5 binding sites, we detected occupancy of a consensus binding site for E2A proteins (E box) in pre-B cells. EBF and E box sites are crucial for promoter function in transfected pre-B cells, and EBF and E2A proteins synergistically activated the promoter in transfected HeLa cells. Other data suggest that EBF and E box sites are less important for promoter function at later stages of differentiation, whereas binding sites for Pax-5 (and its Ets ternary complex partners) are required for promoter function in all mb-1-expressing cells. Using DNA microarrays, we found that expression of endogenous mb-1 transcripts correlates most closely with EBF expression and negatively with Id1, an inhibitor of E2A protein function, further linking regulation of the mb-1 gene with EBF and E2A. Together, our studies demonstrate the complexity of factors regulating tissue-specific transcription and support the concept that EBF, E2A, and Pax-5 cooperate to activate target genes in early B-cell development.

Animals↗