Busslinger Group Research
Stem cell commitment in hematopoiesis
Acquired immunity to pathogens depends on the differentiation of B and T lymphocytes from hematopoietic stem cells, which is controlled by a multitude of transcription factors. We are interested in understanding the process by which transcription factors regulate the commitment of early hematopoietic progenitors to lymphoid lineages and control their subsequent differentiation to B and T cells. We investigate the underlying transcriptional control mechanisms by using mouse transgenic, cell biological, and genome-wide molecular approaches.
B cell commitment
- Figure 1 (Click to view legend)
A fundamental question in hematopoiesis is how stem cells and early progenitors become committed to a single developmental pathway and then differentiate into mature cell types of the selected lineage. The entry of lymphoid progenitors into the B cell lineage depends on several transcription factors, including STAT5, E2A, EBF1 and Pax5. STAT5, a downstream mediator of IL-7 signaling fulfills a permissive role by controlling cell survival in the early development of B cells. E2A and EBF1 function as B cell specification factors by activating B-lymphoid genes. Pax5, in turn, controls B cell commitment by restricting the developmental potential of hematopoietic progenitor cells to the B cell pathway. The function of these transcription factors is required throughout B lymphopoiesis, as E2A, EBF1 and Pax5 also control the generation of mature B cell types, including germinal center B cells (Figure 1). Notably, conditional Pax5 loss enables mature B cells from peripheral lymphoid organs to dedifferentiate to early uncommitted progenitors in the bone marrow, which subsequently develop into functional T cells. These experiments identified Pax5 as the critical B cell identity factor that maintain B-lineage commitment throughout B cell development.
T cell development
Signaling through the Notch1 receptor is essential for initiating the development of T cells in the thymus. Early T cell specification also depends on other transcription factors, such as GATA3 and E2A. However, little is known about target genes that mediate the effects of these transcriptional regulators in early T cell development. We therefore want to elucidate the molecular functions of these transcription factors in pro-T cells by conditional mutagenesis, gene expression profiling, and ChIP sequencing.
Transcriptional networks
- Figure 2 (Click to view legend)
Global genomic approaches are ideal for elucidating the transcriptional network controlling early development of B cells and T cells. To achieve this aim, we define the regulatory landscape of pro-B and pro-T cells by genome-wide mapping of DNase I hypersensitive sites, transcription start sites and chromatin modifications, in order to delineate active enhancers and promoters (Figure 2). By ChIP sequencing, we identify the binding sites of the different transcription factors at these regulatory elements (Figure 2). Conditional mutagenesis combined with mRNA sequencing is used to study the dependence of target gene expression on the different transcription factors. These genome-wide approaches have already provided important insights into the transcriptional network controlling early B cell development.
Spatial control of V(D)J recombination
- Figure 3 (Click to view legend)
The development of B cells and αβ T cells depends on functional rearrangement of the Igh and Igk or Tcrb and Tcra loci, respectively. All four loci are large in size (0.7 to 3 megabases), organized in a complex manner (Figure 3), and undergo reversible contraction by looping in rearranging lymphocytes. Locus contraction is thus a general mechanism that juxtaposes distantly located V genes of the large V gene cluster next to D or J segments, which facilitates synapse formation and V-(D)J recombination. Our previous work demonstrated that contraction of the Igh locus primarily depends on Pax5. Recently, we were able to show that Pax5 activates conserved intergenic repeats (PAIRs) in the distal VH gene cluster, which most likely functions as a novel regulatory element in controlling Igh locus contraction (Figure 3). We are currently elucidating the molecular mechanism of Igh locus contraction by investigating the function of such cis-regulatory elements and by identifying novel trans-acting factors involved in this process.