SPLICE · Molecular mechanisms of transcriptional regulation of lymphocyte development by the E2A splice variants E12 and E47

Molecular mechanisms of transcriptional regulation of lymphocyte development by the E2A splice variants E12 and E47

The differentiation of highly specialized B and T cells from haematopoietic progenitor cells (HSC) is tightly regulated by the concerted action of several transcription factors. The E2A proteins E12 and E47, which arise through mutually exclusive splicing of two exons in the Tcfe2a gene, have been shown to be important for several developmental checkpoints during B and T cell development. Recently, we were able to show that the two E2A splice variants E12 and E47 have distinct functions during B cell development. While E47 is essential for developmental progression at the pre-pro-B cell stage, E12 is dispensable for early B cell development, commitment and maintenance. In contrast, both E12 and E47 are required at later stages in order to promote immunoglobulin lambda (Igλ) germline transcription as well as Igλ VJ gene rearrangement. Importantly, we found that at the Igλ locus the E2A proteins directly or indirectly promote the establishment of different histone modifications, which are crucial for the activation of Igλ germline transcription and rearrangement. We previously demonstrated that the two E2A splice variants fulfil distinct functions during B cell development, however the molecular mechanisms leading to E2A transcription, the regulation of variable splicing to either E12 or E47 mRNA and the mechanisms employed by the E12 and E47 proteins leading to target gene regulation have not been elucidated and are the subject of this project. In this project, we transferred cellular models, such as pro B cell cultures and E12/E47 deficient mice, as well as different methods, such as E2A chromatin immunoprecipitations (ChIP), which had been established before, to the host lab in Germany. Using these tools, we identified the E2A splice variant specific DNA binding properties by using ChIP-Sequencing in combination with E12- and E47-deficient pro B cells. Currently, we further elucidate the specific gene regulatory functions of E12 and E47 during B cell development, illuminating the need for two highly similar proteins, E12 and E47, for B cell development. In order to identify novel regulators of E12 or E47 splicing, we generated fluorescence-based splicing reporter constructs. These studies will for the first time investigate E2A splicing and might also identify splicing regulators, which are important for the splicing of other mRNAs. Finally, using mass spectroscopy of E47 containing protein complexes, we identified various transcriptional cofactors, such as CoREST1 or Dnmt1, which interact with E47. Taken together, these studies will lead to a better understanding of the regulation of E12/E47 splicing and of the molecular mechanisms used by the two highly similar proteins E12 and E47 in regulating B cell development.

Data: CORDIS, © European Union