Polypyrimidine tract-binding proteins are essential for B cell development
- Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, United Kingdom
- Department of Biochemistry, University of Cambridge, United Kingdom
- Next Generation Sequencing Facility, The Babraham Institute, United Kingdom
- Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Germany
Figures
Figure 1 with 1 supplement
PTBP1 and PTBP3 are expressed throughout B cell development.
(A) Expression of PTBP1, PTBP2 and PTBP3 analysed by flow cytometry. Identification of different B cell developmental stages was carried out as shown in Figure 1—figure supplement 1B. (B) Geometric …
Figure 1—figure supplement 1
B cell developmental stages in the bone marrow.
(A) Representation of B cell development in the bone marrow using the Philadelphia nomenclature (Hardy and Hayakawa, 2003) including Hardy’s fractions (Fr) based on cell-surface markers (Hardy et …
Figure 2 with 1 supplement
Lack of B cells in the absence of PTBP1 and PTBP2.
(A) Numbers of B cells (B220+CD19+) in spleens of mice with the indicated genotypes. Data points are from individual mice. Arithmetic means are shown with lines. (B) PTBP1, PTBP2, PTBP3 and isotype …
Figure 2—figure supplement 1
Normal numbers of mature B cells in the absence of PTBP3.
(A) Numbers of B cells (B220+ CD19+) in spleens of mice with the indicated genotypes. Data points are from individual mice. Arithmetic means are shown with lines. Data shown are from one experiment. …
Figure 3 with 2 supplements
Absence of PTBP1 and PTBP2 blocks B cell development at the pro-B cell stage.
(A) Gating strategy based on cell-surface markers for developing B cells from bone marrow cells pre-gated on dump (Gr-1, CD11b, NK1.1, Siglec-F and F4/80)-negative live (eFluor780-) cells. (B) …
Figure 3—figure supplement 1
Absence of PTBP1 and PTBP2 blocks B cell development at the pro-B cell stage.
(A) Numbers of Pro-B cells gated on dump (Gr-1, CD11b, Siglec-F, F4/80, NK1.1)-negative, IgD-, IgM-, B220+, CD19+, cKIT+, CD25- bone marrow cells. Data shown are from two independent experiments …
Figure 3—figure supplement 2
Deletion of Ptbp1 and Ptbp3 results in normal B cell development in the bone marrow.
(A) Numbers of distinct developmental B cell stages in the bone marrow (two femurs) from mice with the indicated genotypes identified as: Pre-pro (IgD- IgM- B220+ CD43+ CD19-), Pro cKIT+ (IgD- IgM- …
Figure 4 with 2 supplements
PTBP1 and PTBP2 absence causes changes in mRNA abundance and AS.
(A) Differences in mRNA abundance in pairwise comparisons from pro-B cell transcriptomes. Shown are Log2Fold changes calculated with DESeq2 (Figure 4—source data 1). Red dots are values from genes …
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Figure 4—source data 1
Changes in mRNA abundance.
DESeq2 results shown in Figure 4A. Separate tabs show genes with significant differential (padj <0.05) mRNA abundance with a |log2 fold change| > 0.5 for the different pairwise comparisons carried out and also all the results obtained with DESeq2. Additional tabs show genes whose transcripts were bound by PTBP1 clusters at their 3’UTR.
- https://cdn.elifesciences.org/articles/53557/elife-53557-fig4-data1-v2.xls
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Figure 4—source data 2
Changes in AS.
Different tabs show inclusion level differences (IncLevelDifference) shown in Figure 4B for the three pairwise comparisons carried out. The first three tabs show significant (FDR < 0.05) alternative splicing events with an absolute inclusion level difference >0.1. ‘allresults’ tabs show all the results from rMATS. ‘PTBP1 bound’ tabs show those significantly differential splicing events that were bound in their vicinity by PTBP1 clusters.
- https://cdn.elifesciences.org/articles/53557/elife-53557-fig4-data2-v2.xls
Figure 4—figure supplement 1
Cell sorting strategy of pro-B cells.
Cell sorting strategy of FrB pro-B (B220+CD19+IgM-IgD-CD2-CD43highCD25-cKIT+CD24+CD249+) cells used to isolate RNA and carry out mRNAseq libraries. ‘Dump’ contains excluded cells stained with …
Figure 4—figure supplement 2
Transcriptome analysis of pro-B cells.
(A) Transcriptome correlation in mitogen-activated primary B cells and FrB pro-B cells. Dots show mean values of TPMs (Transcripts Per Million) from four or five biological replicates in …
Figure 5
PTBP1 regulates pathways associated with growth and proliferation.
(A) Ebf1, Foxo1 and IL7r mRNA abundance in FrB pro-B cells from control, P1sKO and P1P2dKO mice. (B) PTBP1 binding (iCLIP data) to the Foxo1 3’UTR. (C) IL-7R (CD127) geometric mean fluorescent …
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Figure 5—source data 1
Gene ontology enrichment analysis.
- https://cdn.elifesciences.org/articles/53557/elife-53557-fig5-data1-v2.xls
Figure 6 with 2 supplements
Enhanced entry into S-phase and block at G2 in P1P2dKO pro-B cells.
(A) EdU and BrdU sequential labelling experimental set up to distinguish early, late and post S-phase cells. (B) Flow cytometry data of the different stages of S-phase in pro-B cells (B220+CD19+IgD-s…
Figure 6—figure supplement 1
Cell cycle analysis in FrB and FrC pro-B cells.
(A) Identification strategy of proliferating FrB and FrC pro-B cells amongst bone marrow cells from mice with the indicated genotypes. Events shown on the left were pre-gated on eFluor780- live IgM-,…
Figure 6—figure supplement 2
Cell cycle analysis in pro-B cells.
(A) Gating strategy to identify pre-pro-, pro- and early-pre-B cells in EdU and BrdU sequential labelling experiments in live bone marrow cells from the indicated genotypes. (B) Early-, late- and …
Figure 7 with 1 supplement
PTBP1 controls CDK activity in FrB pro-B cells.
(A) Intracellular flow cytometry with anti-p27 antibody or control isotype staining detected with an anti-rabbit AF647-conjugated secondary antibody. (B) Median fluorescence intensities (MFI) from …
Figure 7—figure supplement 1
PTBP1 controls CDK activity in FrC pro-B cells.
Intracellular staining for p27 (A), pT592-SAMHD1 and pS807/S811-RB (B) amongst FrC pro-B cells. FrC pro-B cells in distinct phases of the cell cycle were identified as shown in Figure 6—figure …
Figure 8 with 2 supplements
PTBP1 controls expression of genes important for S-phase entry.
(A) Log2-fold changes in mRNA abundance in the indicated pairwise comparisons for all tested genes (all genes), genes with increased abundance in S-phase (S) and G2/M-phases (G2/M). Grey dots show …
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Figure 8—source data 1
DESeq2 results for genes shown to have high mRNA expression levels in S or G2/M phases (Giotti et al., 2019) in the three pair-wise comparisons shown in Figure 8A.
- https://cdn.elifesciences.org/articles/53557/elife-53557-fig8-data1-v2.xls
Figure 8—figure supplement 1
PTBP1 binding to target transcripts.
PTBP1 binding (iCLIP data) to 3’UTRs.
Figure 8—figure supplement 2
c-MYC staining in FrC pro-B cells.
(A) Intracellular c-MYC or control isotype staining of FrC pro-B cells in different stages of the cell cycle identified as shown in Figure 6—figure supplement 1A. (B) Median fluorescence intensities …
Figure 9
PTBP1 controls expression of genes important for mitosis.
(A) mRNA abundance of the indicated genes. Genes whose names are in yellow and bold are predicted to have reduced mRNA abundance due to changes in AS triggering NMD upon Ptbp1 and Ptbp2 deletion. …
Tables
Table 1
mRNA expression levels of cell cycle regulators in mitogen activated mature B cells and FrB pro-B cells.
Values shown are arithmetic mean Transcripts Per Million (TPMs) from four and five mRNAseq libraries for activated mature B cells and for FrB pro-B cells, respectively.
| Activated mature B cells | FrB pro-B cells | |
|---|---|---|
| Myc | 155.8 | 315.3 |
| Ccnd2 | 128.5 | 52.7 |
| Btg2 | 51.5 | 109.3 |
| Rbl1 | 19.3 | 127.0 |
| Cdc25b | 66.8 | 190.1 |
| Ect2 | 17.8 | 198.9 |
| Kif2c | 30.0 | 151.6 |
| Kif22 | 35.6 | 344.3 |
Additional files
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Supplementary file 1
PTBP1 binding sites (xlinks).
- https://cdn.elifesciences.org/articles/53557/elife-53557-supp1-v2.csv.zip
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Supplementary file 2
PTBP1 binding sites (clusters).
- https://cdn.elifesciences.org/articles/53557/elife-53557-supp2-v2.csv
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Supplementary file 3
Key resources table.
- https://cdn.elifesciences.org/articles/53557/elife-53557-supp3-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/53557/elife-53557-transrepform-v2.docx
