N-terminally truncated FOXP1 protein expression and alternate internal FOXP1 promoter usage in normal and malignant B cells Philip J Brown Duncan M Gascoyne Linden Lyne Hayley Spearman Suet Ling Felce Nora Mcfadden Probir Chakravarty Sharon Barrans Steven Lynham Dinis P Calado Malcolm Ward Alison H Banham 10779/crick.12608009.v1 https://crick.figshare.com/articles/journal_contribution/N-terminally_truncated_FOXP1_protein_expression_and_alternate_internal_FOXP1_promoter_usage_in_normal_and_malignant_B_cells/12608009 Strong FOXP1 protein expression is a poor risk factor in diffuse large B-cell lymphoma and has been linked to an activated B-cell-like subtype, which preferentially expresses short FOXP1 (FOXP1S) proteins. However, both short isoform generation and function are incompletely understood. Here we prove by mass spectrometry and N-terminal antibody staining that FOXP1S proteins in activated B-cell-like diffuse large B-cell lymphoma are N-terminally truncated. Furthermore, a rare strongly FOXP1-expressing population of normal germinal center B cells lacking the N-terminus of the regular long protein (FOXP1L) was identified. Exon-targeted silencing and transcript analyses identified three alternate 5' non-coding exons [FOXP1-Ex6b(s), FOXP1-Ex7b and FOXP1-Ex7c], downstream of at least two predicted promoters, giving rise to FOXP1S proteins. These were differentially controlled by B-cell activation and methylation, conserved in murine lymphoma cells, and significantly correlated with FOXP1S protein expression in primary diffuse large B-cell lymphoma samples. Alternatively spliced isoforms lacking exon 9 (e.g. isoform 3) did not encode FOXP1S, and an alternate long human FOXP1 protein (FOXP1AL) likely generated from a FOXP1-Ex6b(L) transcript was detected. The ratio of FOXP1L:FOXP1S isoforms correlated with differential expression of plasmacytic differentiation markers in U-2932 subpopulations, and altering this ratio was sufficient to modulate CD19 expression in diffuse large B-cell lymphoma cell lines. Thus, the activity of multiple alternate FOXP1 promoters to produce multiple protein isoforms is likely to regulate B-cell maturation. 2020-07-15 10:23:51 Alternative Splicing Animals Antigens, CD19 B-Lymphocytes Cell Line, Tumor Exons Forkhead Transcription Factors Gene Expression Regulation, Neoplastic Humans Lymphocyte Activation Lymphoma, Large B-Cell, Diffuse Mice Promoter Regions, Genetic Protein Interaction Domains and Motifs Protein Isoforms RNA, Messenger Repressor Proteins Calado FC001057 CB 1102 Cardiorespiratory Medicine and Haematology Immunology