Final Report Summary - PAX5TARGETS (Characterization of the b-cell-specific gene regulatory network by genome-wide identification of direct pax5 target genes)
All cells of the hematopoietic system develop from the hematopoietic stem cell through a series of alternative differentiation steps controlled by various transcription factors. These transcription factors lead to the activation of gene expression programs of the respective lineage while repressing alternative cell fates. B and T lymphocytes develop from a common progenitor cell called All lymphocytes progenitor (ALP) (1). Entry into the B cell lineage occurs through the differentiation of the ALP into a B lymphocyte progenitor (BLP) and subsequently a pro-B cell, the first B-lineage committed progenitor (2). Further critical developmental events take place during the differentiation of pro-B cells to mature B cells, which are ready to participate in an immune response.
It has been previously demonstrated that the ordered activity of the transcription factors E2A and Ebf1 is essential for instruction of progenitors to the B lineage, while Pax5 is essential for commitment and maintenance of the B cell identity (2). However, understanding how these functions are executed, and how the developmental process works at the system-level, requires elucidating the network of interactions between these and all other factors that control the process. The project described here represents a significant step toward this goal.
We have used several recently developed, unbiased, genome-wide techniques to perform our analyses.
First, we accurately quantified gene expression in B cell progenitors (ALP, BLP and pro-B cells) and in mature B cells by the RNA-sequencing technique (RNA-seq). Ongoing analyses of this data are allowing us to define all gene expression changes that take place during B cell development.
Second, the RNA-seq technique was used to quantify gene expression in uncommitted and committed progenitors where pax5 expression had been inactivated in vivo by conditional mutagenesis. This allowed us to determine all genes that require Pax5 for their correct expression during B cell development.
Third, we identified genome-wide binding sites of Pax5 by performing chromatin affinity precipitation of in-vivo biotinylated Pax5 with streptavidin beads coupled with Solexa DNA sequencing.
And fourth, we have integrated this information with genome-wide chromatin profiles (ChIP-seq), from which putative active cis-regulatory elements and promoters are identified. These analyses allowed comprehensive identification of Pax5 direct targets in pro-B and mature B cells. Our present analyses will tell us which of the gene expression changes that take place during B cell development are directly dependent on Pax5 function.
Analyses are still ongoing, but a few conclusions can already be drawn about the role of pax5 in the gene regulatory network that controls B cell development. Pax5 has a large number of direct targets both in pro-B and mature B cells, underscoring its role in maintenance of B cell identity. Transcription factors that bind in the proximity of Pax5 are currently being identified. Cooperation of pax5 with these transcription factors might explain why pax5 can function both as an activator or a repressor of its direct targets.
Pax5 also affects the expression of a large number of genes whose loci are not directly bound by this factor. These indirect effects can be explained by the fact that among the direct targets of pax5 we find several transcription factors that are expressed in the right cells stages. We hope to uncover, among these transcription factor, novel important regulators of B cell development. The door is now open to study the function of these novel transcription factors and to identify their direct targets in a similar fashion as described here for Pax5. Through these analyses we will be able to describe with increasing detail the molecular mechanism that controls B cell development.
References:
1. Inlay, M. A. et al., 2009, Genes and Development 23:2376-2381.
2. Busslinger, M. 2004. Annu. Rev. Immunol. 22:55-79.
It has been previously demonstrated that the ordered activity of the transcription factors E2A and Ebf1 is essential for instruction of progenitors to the B lineage, while Pax5 is essential for commitment and maintenance of the B cell identity (2). However, understanding how these functions are executed, and how the developmental process works at the system-level, requires elucidating the network of interactions between these and all other factors that control the process. The project described here represents a significant step toward this goal.
We have used several recently developed, unbiased, genome-wide techniques to perform our analyses.
First, we accurately quantified gene expression in B cell progenitors (ALP, BLP and pro-B cells) and in mature B cells by the RNA-sequencing technique (RNA-seq). Ongoing analyses of this data are allowing us to define all gene expression changes that take place during B cell development.
Second, the RNA-seq technique was used to quantify gene expression in uncommitted and committed progenitors where pax5 expression had been inactivated in vivo by conditional mutagenesis. This allowed us to determine all genes that require Pax5 for their correct expression during B cell development.
Third, we identified genome-wide binding sites of Pax5 by performing chromatin affinity precipitation of in-vivo biotinylated Pax5 with streptavidin beads coupled with Solexa DNA sequencing.
And fourth, we have integrated this information with genome-wide chromatin profiles (ChIP-seq), from which putative active cis-regulatory elements and promoters are identified. These analyses allowed comprehensive identification of Pax5 direct targets in pro-B and mature B cells. Our present analyses will tell us which of the gene expression changes that take place during B cell development are directly dependent on Pax5 function.
Analyses are still ongoing, but a few conclusions can already be drawn about the role of pax5 in the gene regulatory network that controls B cell development. Pax5 has a large number of direct targets both in pro-B and mature B cells, underscoring its role in maintenance of B cell identity. Transcription factors that bind in the proximity of Pax5 are currently being identified. Cooperation of pax5 with these transcription factors might explain why pax5 can function both as an activator or a repressor of its direct targets.
Pax5 also affects the expression of a large number of genes whose loci are not directly bound by this factor. These indirect effects can be explained by the fact that among the direct targets of pax5 we find several transcription factors that are expressed in the right cells stages. We hope to uncover, among these transcription factor, novel important regulators of B cell development. The door is now open to study the function of these novel transcription factors and to identify their direct targets in a similar fashion as described here for Pax5. Through these analyses we will be able to describe with increasing detail the molecular mechanism that controls B cell development.
References:
1. Inlay, M. A. et al., 2009, Genes and Development 23:2376-2381.
2. Busslinger, M. 2004. Annu. Rev. Immunol. 22:55-79.