Role of three-dimensional chromatin organization and spatial co-localization during B to plasma cell differentiation – PLASMADIFF-3D

Although the role of the complex network of transcription factors involved in PCD has been investigated, the mechanisms regulating key plasma cell differentiation transcription networks remain poorly known. Little is known about the dynamic and hierarchical nuclear architecture in B to plasma cell differentiation and how it could regulate fundamental processes during normal plasma cell differentiation. In this project, we seek to characterize and understand the epigenetic, remodeling, structural and architectural changes that affect chromatin during B to plasma cell differentiation using Hi-C, ERRBS, RNA-seq, ATAC-seq and ChIP-seq. We would like to reveal both the pathways involved in this remodeling and their downstream transcriptional impact.

ChIP sequencing to analyze changes of the histone marks are ongoing: H3K9ac, H3K36me3, H3K27me1 and H3K4me3 to follow active transcription, H3K4me1 and H3K27ac to follow enhancer activity, H3K9me3 to follow heterochromatin, H3K27me3 to follow Polycomb-silenced chromatin and H3K27me2 to follow inactive chromatin distinct from the previous two. We obtained high-resolution Hi-C maps of all stages including memory B cells, preplasmablasts, plasmablasts and plasma cells. Hi-C data have been analyzed and combined with bulk RNA-sequencing data of the B to PC differentiation model. These analyses revealed new regulatory contact networks dependent on the stage of normal B to PC differentiation.
To complete the characterization of epigenome modifications during B to PC differentiation, single-cell RNA seq of the B to PC differentiation model was also completed.

Based on our analyses of RNA seq, CHIP seq, Hi-C and ERBBS data, we started to investigate the biological role of BMI-1 during normal B to PC differentiation. Using a specific BMI1 inhibitor, we identified that BMI1 inhibition results in a significant blockade of B to PC differentiation. Biological characterizations are ongoing. Validation using shRNA and BMI1 overexpression have been planned after the lockdown.

Publications: Team 1 has submitted a manuscript related to the characterization of temporal transcriptional changes during normal B to PC differentiation to Blood.

Team 2 has focused on the Hi-C analysis and their relation ro bulk RNA-seq data of each differentiation stage, as well as the initial ChIP-seq data that were obtained with the H3K27me3 and H3K9me3 marks. Among others, striking Hi-C transitions were observed at key transcription factor coding genes that play pivotal roles in differentiation. A paper will be prepared once the Hi-C results can be jointly analyzed together with the whole set of epigenomic data, including ChIP-seq, ERBBS and RNA-seq.

We obtained the first high-resolution Hi-C maps of human B to plasma cell differentiation including memory B cells, preplasmablasts, plasmablasts and plasma cells. Hi-C data have been analyzed and combined with bulk RNA-sequencing data of the B to PC differentiation model. These analyses revealed new regulatory contact networks dependent on the stage of normal B to PC differentiation.A paper will be prepared once the Hi-C results can be jointly analyzed together with the whole set of epigenomic data, including ChIP-seq, ERBBS and RNA-seq.

We developed a comprehensive, temporal program of gene expression data encompassing human PCD from memory B cells, using RNA sequencing. Our results revealed 6,374 differentially expressed genes classified into four temporal gene expression patterns. A stringent pathway enrichment analysis of these gene clusters highlights known pathways but also pathways largely unknown in PCD, including the heme biosynthesis and the glutathione conjugation pathways. Additionally, our analysis revealed numerous novel transcriptional regulators and helicases with significant stage-specific overexpression and potential importance in PCD, including BATF2, BHLHA15/MIST1, EZH2, WHSC1/MMSET and BLM.

These data constitute a unique resource to progress in the understanding of the underlying molecular mechanisms that control PCD.

Patents:
1/ Iron-score and in vitro method for identifying high risk B-cell lymphoma subjects and therapeutic uses and methods. Nov 2019. EP19306436.
2/ Prognosis method of acute myeloid leukemia. April 2020. BR 107049.

The “PLASMADIFF-3D” project (36 months, 2 teams) will address scientific issues pertinent to the B-cell-lineage and their implications in immunopathology, with specific focus on antibody-secreting plasma cells. Antibody secreting cells are critical effector cells and long-lived sentinels for immune memory. B cell maturation should be tightly regulated to ensure efficient immune response without autoimmunity or immune deficiency. On the transcriptional level, the differentiation of B cells into plasma cells is associated with substantial and coordinated changes in the gene expression profile, which fall into two main categories: the loss of B cell-associated transcripts and the acquisition of plasma cell gene expression program. These changes are tightly guided by two sets of stage-specific transcription factors (TFs) that repress each other: i) B cell TFs (PAX5, BCL6 and BACH2) maintaining the B cell fate and ii) plasma cell TFs (IRF4, BLIMP1 and XBP1) that are required to extinguish the B cell genes and activate the antibody-secreting cell (ASC) program. Although the role of the complex network of transcription factors involved in PCD has been investigated, the mechanisms regulating key plasma cell differentiation transcription networks remain poorly known. Little is known about the dynamic and hierarchical nuclear architecture in B to plasma cell differentiation and how it could regulate fundamental processes during normal plasma cell differentiation. In this proposal, we seek to characterize and understand the epigenetic, remodeling, structural and architectural changes that affect chromatin during B to plasma cell differentiation. We would like to reveal both the pathways involved in this remodeling and their downstream transcriptional impact. The rationale of our project is in line with the identification of major nuclear architecture reorganization during plasma cell differentiation, which involves a significant modification of the size of the cell nucleus and the emergence of compact nuclear bodies.
We plan to identify upstream pathways mediating these changes and their downstream transcriptional impact. These studies will take advantage of a unique combination of powerful models and new technologies that are fully mastered by the two partners of the project.