Project description:Rel and RelA proteins were stably expressed in the chicken DT40 pre-B cell line and analyzed by transcription profiling to identify transformation-impacting genes regulated by NF-kB’s oncogenic v-Rel and c-Rel proteins. This analysis uncovered both common and differential gene expression profiles in cells expressing Rel vs. RelA proteins, and revealed that Rel protein expression can lead to gene-specific transcriptional repression, as seen for key B-cell receptor (BCR) components and signaling molecules like B-cell linker (BLNK), the B-cell adaptor for PI3K (BCAP) and Igλ. These were also downregulated in cells expressing a transformation-competent chimeric RelA/v-Rel protein, suggesting a correlation with the capacity of Rel proteins to transform lymphocytes. DNA binding, ChIP and transformation assays indicate that downregulation of BLNK and BCAP is an important contributing factor to the malignant transformation of lymphocytes by Rel and suggest that gene repression may be as important as transcriptional activation for the transforming activity of Rel proteins. Keywords: Comparative transcriptional profiling
Project description:The NF-kB family of transcription factors orchestrates signal-induced gene expression in a diversity of cell types. Cellular responses to NF-kB activation are regulated at the level of cell- and signal-specificity, as well as differential use of family members (subunit specificity). Here we used time-dependent multi-omics to investigate selective functions of Rel and RelA, two closely related NF-kB proteins, in primary B lymphocytes activated via the B cell receptor. Despite large numbers of shared binding sites genome wide, Rel and RelA directed kinetically distinct cascades of gene expression in activated B cells. Single-cell RNA-Seq revealed marked heterogeneity of Rel- and RelA-specific responses and sequential binding of these factors was not a major mechanism of protracted transcription. Moreover, nuclear co-expression of Rel and RelA led to functional antagonism between the two factors. By rigorously identifying target genes of each NF-kB subunit, these studies provide insights into exclusive functions of Rel and RelA in immunity and cancer.
Project description:The NF-kB family of transcription factors orchestrates signal-induced gene expression in a diversity of cell types. Cellular responses to NF-kB activation are regulated at the level of cell- and signal-specificity, as well as differential use of family members (subunit specificity). Here we used time-dependent multi-omics to investigate selective functions of Rel and RelA, two closely related NF-kB proteins, in primary B lymphocytes activated via the B cell receptor. Despite large numbers of shared binding sites genome wide, Rel and RelA directed kinetically distinct cascades of gene expression in activated B cells. Single-cell RNA-Seq revealed marked heterogeneity of Rel- and RelA-specific responses and sequential binding of these factors was not a major mechanism of protracted transcription. Moreover, nuclear co-expression of Rel and RelA led to functional antagonism between the two factors. By rigorously identifying target genes of each NF-kB subunit, these studies provide insights into exclusive functions of Rel and RelA in immunity and cancer.
Project description:The NF-kB family of transcription factors orchestrates signal-induced gene expression in a diversity of cell types. Cellular responses to NF-kB activation are regulated at the level of cell- and signal-specificity, as well as differential use of family members (subunit specificity). Here we used time-dependent multi-omics to investigate selective functions of Rel and RelA, two closely related NF-kB proteins, in primary B lymphocytes activated via the B cell receptor. Despite large numbers of shared binding sites genome wide, Rel and RelA directed kinetically distinct cascades of gene expression in activated B cells. Single cell RNA-Seq revealed marked heterogeneity of Rel- and RelA-specific responses and sequential binding of these factors was not a major mechanism of protracted transcription. Moreover, nuclear co-expression of Rel and RelA led to functional antagonism between the two factors. By rigorously identifying target genes of each NF-kB subunit, these studies provide insights into exclusive functions of Rel and RelA in immunity and cancer.
Project description:The NF-kB family of transcription factors orchestrates signal-induced gene expression in a diversity of cell types. Cellular responses to NF-kB activation are regulated at the level of cell- and signal-specificity, as well as differential use of family members (subunit specificity). Here we used time-dependent multi-omics to investigate selective functions of Rel and RelA, two closely related NF-kB proteins, in primary B lymphocytes activated via the B cell receptor. Despite large numbers of shared binding sites genome wide, Rel and RelA directed kinetically distinct cascades of gene expression in activated B cells. Single-cell RNA-Seq revealed marked heterogeneity of Rel- and RelA-specific responses and sequential binding of these factors was not a major mechanism of protracted transcription. Moreover, nuclear co-expression of Rel and RelA led to functional antagonism between the two factors. By rigorously identifying target genes of each NF-kB subunit, these studies provide insights into exclusive functions of Rel and RelA in immunity and cancer.
Project description:The NF-kB family of transcription factors orchestrates signal-induced gene expression in a diversity of cell types. Cellular responses to NF-kB activation are regulated at the level of cell- and signal-specificity, as well as differential use of family members (subunit specificity). Here we used time-dependent multi-omics to investigate selective functions of Rel and RelA, two closely related NF-kB proteins, in primary B lymphocytes activated via the B cell receptor. Despite large numbers of shared binding sites genome wide, Rel and RelA directed kinetically distinct cascades of gene expression in activated B cells. Single-cell RNA-Seq revealed marked heterogeneity of Rel- and RelA-specific responses and sequential binding of these factors was not a major mechanism of protracted transcription. Moreover, nuclear co-expression of Rel and RelA led to functional antagonism between the two factors. By rigorously identifying target genes of each NF-kB subunit, these studies provide insights into exclusive functions of Rel and RelA in immunity and cancer.
Project description:Adaptive immunity and the five vertebrate NF-kB/Rel family members first appeared in cartilaginous fish, suggesting that NF-kB family expansion allowed the acquisition of new functions to regulate adaptive immune responses. Transcriptome profiling revealed that, even in macrophages, the NF-kB family member, c-Rel, most potently regulates a cytokine gene linked to adaptive immunity, Il12b, with limiting roles at key regulators of innate immunity. Neofunctionalization of c-Rel to regulate Il12b depends on its unique DNA-binding properties, which we examined using structural, biochemical, functional, and genomic approaches. Among our findings was functional c-Rel homodimer binding to motifs with little resemblance to canonical NF-kB motifs. To determine whether c-Rel’s unique binding properties drove c-Rel-RelA divergence, we compared binding properties in various vertebrate species. c-Rel-RelA binding properties diverged in mammals and amphibians but were comparable in earlier vertebrates, suggesting that divergent DNA binding emerged relatively late during vertebrate evolution to support increasing complexity of adaptive immune regulation.
Project description:Adaptive immunity and the five vertebrate NF-kB/Rel family members first appeared in cartilaginous fish, suggesting that NF-kB family expansion allowed the acquisition of new functions to regulate adaptive immune responses. Transcriptome profiling revealed that, even in macrophages, the NF-kB family member, c-Rel, most potently regulates a cytokine gene linked to adaptive immunity, Il12b, with limiting roles at key regulators of innate immunity. Neofunctionalization of c-Rel to regulate Il12b depends on its unique DNA-binding properties, which we examined using structural, biochemical, functional, and genomic approaches. Among our findings was functional c-Rel homodimer binding to motifs with little resemblance to canonical NF-kB motifs. To determine whether c-Rel’s unique binding properties drove c-Rel-RelA divergence, we compared binding properties in various vertebrate species. c-Rel-RelA binding properties diverged in mammals and amphibians but were comparable in earlier vertebrates, suggesting that divergent DNA binding emerged relatively late during vertebrate evolution to support increasing complexity of adaptive immune regulation.
Project description:c-Rel and RelA are members of the NF-kappaB family of transcription factors. They both have important roles in T cell activation. To discover where in the genome c-Rel and RelA bind and hence which genes they may directly target, we used ChIP-chip with EL4 cells stimulated with phorbol ester (PMA) and Ionomycin. We have identified regions in EL4 cells (background strain: C57BL/6N) activated for 2h or 8h by PMA and Ionomycin, that bind the transcription factors c-Rel and RelA.
Project description:c-Rel and RelA are members of the NF-kappaB family of transcription factors. They both have important roles in T cell activation. To discover where in the genome c-Rel and RelA bind and hence which genes they may directly target, we used ChIP-chip with EL4 cells stimulated with phorbol ester (PMA) and Ionomycin. We have identified regions in EL4 cells (background strain: C57BL/6N) activated for 2h or 8h by PMA and Ionomycin, that bind the transcription factors c-Rel and RelA. Immunoprecipitated samples from EL4 cells stimulated with PMA and ionomycin for 2 h and 8 h with antibodies against RelA or c-Rel respectively were used for ChIP-on-chip experiments. In addition, samples from total input and mock immunoprecipitation were used as controls. Biological triplicates were used.