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
Affiliations
Philip J. Brown
Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, Oxford University, London, UK
Duncan M. Gascoyne
Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, Oxford University, London, UK
Linden Lyne
Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, Oxford University, London, UK
Hayley Spearman
Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, Oxford University, London, UK
Suet Ling Felce
Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, Oxford University, London, UK
Nora McFadden
Immunity and Cancer Laboratory, The Francis Crick Institute, Lincoln’s Inn Fields Laboratory, Lincoln’s Inn Fields, London, UK
Probir Chakravarty
Computational Biology Laboratory, The Francis Crick Institute, Lincoln’s Inn Fields Laboratory, Lincoln’s Inn Fields, London, UK
Sharon Barrans
Leeds Teaching Hospitals NHS Trust, HMDS, Leeds Cancer Centre, Kings College London, UK
Steven Lynham
Centre of Excellence for Mass Spectrometry, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, UK
Dinis P. Calado
Immunity and Cancer Laboratory, The Francis Crick Institute, Lincoln’s Inn Fields Laboratory, Lincoln’s Inn Fields, London, UK;Peter Gorer Department of Immunobiology, Kings College London, UK
Malcolm Ward
Centre of Excellence for Mass Spectrometry, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, UK
Alison H. Banham
Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, Oxford University, London, UK
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.
