Functional collaboration between HLH transcription factors in B cell development.

The cells in B cell development can be divided into several subgroups or fractions, e.g. early-pro-B-, pro-B, large-pre-B-, small-pre-B-, immature and mature B cells, where the stages reflect the maturity degree of the cells. This characterization is based on the expression of intracellular markers such as the recombination activating genes 1 and 2(Rag 1 and Rag 2), the terminal deoxynucleotidyl transferase gene (TdT) and the expression of surface markers such as the components of the pre-B and B cell receptor (pre-BCR and BCR) as well as the rearrangement status of the heavy- and light chain genes. The expression of these genes is strongly regulated and controlled, otherwise it will lead to disruptions in the B cell development. One important way of maintaining this is by the interaction of proteins denoted transcription factors (TFs) and the genes regulatory sequences, i.e. promoter and enhancer sequences. This interaction, where the TFs help to stabilize and activate the transcriptional machinery, is called transcriptional regulation. Several TFs have been shown to play a crucial role in this regulation process during B cell development, since disruption of genes encoding these TFs causes developmental blocks and disruptions. Two of these factors are the Early B cell Factor (EBF) and E47. Using the mouse EBF cDNA as a probe, we managed to clone the human homologue in a human pre-B cell cDNA library. This made possible a study of the role of this protein during the human B cell development. In this thesis we suggest that EBF and E47 have conserved target sequences and function between man and mouse. This evolutionary conservation reflects their importance during B cell development. Furthermore, there is an interaction during transcription between the two TFs that in many cases result in a synergistic cooperation, reflected in the activation degree of the gene in question. Several target genes are components of the pre-BCR and positioned in different chromosomes, suggesting that EBF is a pleiotropic activator of genes encoding the pre-BCR. Also, we were able to clone a promoter region upstream the EBF gene. This sequence contain binding sites for both E47 and EBF, suggesting that E47 is a key regulator in the transcription of EBF and that EBF auto-regulates it self in a loop. This also suggests that E47 is placed upstream of EBF in the hierarchy of transcriptional factors that participates in the transcriptional regulation of B cell development

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

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

Pax6 promotes neurogenesis in human neural stem cells.

During brain embryogenesis, transcription factors drive stem cells towards neuronal fate. Here we show that the transcription factor Pax6 increased in vitro generation of neurons from striatal but not cortical neural stem cells (NSCs), derived from 6 to 9 weeks old human fetuses, without affecting survival and proliferation. Overexpression of mouse Pax6 produced increased numbers of GABA+ and DARPP-32+ (characteristic of striatum) but not glutamate+ neurons (characteristic of cortex). Pax6-overexpressing cells survived and migrated to the same extent as control cells at 1 month after intrastriatal transplantation into newborn rats and generated more neuroblasts. Overexpression of mouse Pax6 in human NSCs also leads to altered levels of lineage-appropriate genes as revealed by Q-PCR. Our data suggest that Pax6 function is conserved between species since its overexpression activates similar genes in mouse and human NSCs. Also, that Pax6 overexpression in striatal NSCs increases the number of neurons but their region-specificity is maintained

Pearson correlation analysis of micro-array data allows for the identification of genetic targets for early B-cell factor.

B lymphocyte development is a complex biological process critically dependent on the transcription factor early B cell factor (EBF). To deepen understanding of the roles for EBF in this process, we have used Pearson correlation analysis to evaluate microarray data from a set of mouse B lymphoid cell lines representing different stages of development. Comparing the expression pattern of EBF to that of the other genes in the data set revealed that VpreB1, mb-1, and lambda5, all known target genes, presented high correlation values to EBF. High correlations were also seen for the VpreB3 and CD19 genes and biochemical as well as functional data supported that they are target genes for EBF even though the expression of CD19 was critically dependent of Pax-5. We also obtained evidence for extensive collaborative actions of EBF and E47 even though microarray analysis of hematopoetic progenitor cells ectopically expressing these proteins suggested that they activated only a subset of pre-B cell restricted genes

Gene expression analysis suggests that EBF-1 and PPAR gamma 2 induce adipogenesis of NIH-3T3 cells with similar efficiency and kinetics

Differentiation of multipotent mesenchymal stem cells into lipid-accumulating adipocytes is a physiological process induced by transcription factors in combination with hormonal stimulation. We have used Affymetrix microarrays to compare the adipogenic differentiation pathways of NIH-3T3 fibroblasts induced to undergo in vitro differentiation by ectopic expression of early B cell factor (EBF)-1 or peroxisome proliferator-activated receptor (PPAR)gamma 2. These experiments revealed that commitment to the adipogenic pathway in the NIH-3T3 cells was not reflected in gene expression until 4 days after induction of differentiation. Furthermore, gene expression patterns at the earlier time points after stimulation indicated that EBF-1 and PPAR gamma 2 induced different sets of genes, while the similarities increased upon differentiation, and that several genes linked to adipocyte differentiation were also transiently induced in the vector-transduced cells. These data suggest that the initial activation of genes associated with adipocyte development is independent of commitment to the adipogenic pathway and that EBF-1 and PPAR gamma 2 induce adipocyte differentiation with comparable kinetics and efficiency

Gene expression analysis suggests that EBF-1 and PPARγ2 induce adipogenesis of NIH-3T3 cells with similar efficiency and kinetics

Differentiation of multipotent mesenchymal stem cells into lipid-accumulating adipocytes is a physiological process induced by transcription factors in combination with hormonal stimulation. We have used Affymetrix microarrays to compare the adipogenic differentiation pathways of NIH-3T3 fibroblasts induced to undergo in vitro differentiation by ectopic expression of early B cell factor (EBF)-1 or peroxisome proliferator-activated receptor (PPAR)gamma 2. These experiments revealed that commitment to the adipogenic pathway in the NIH-3T3 cells was not reflected in gene expression until 4 days after induction of differentiation. Furthermore, gene expression patterns at the earlier time points after stimulation indicated that EBF-1 and PPAR gamma 2 induced different sets of genes, while the similarities increased upon differentiation, and that several genes linked to adipocyte differentiation were also transiently induced in the vector-transduced cells. These data suggest that the initial activation of genes associated with adipocyte development is independent of commitment to the adipogenic pathway and that EBF-1 and PPAR gamma 2 induce adipocyte differentiation with comparable kinetics and efficiency