Signal transducer and activator of transcription 5 (STAT5) paralog dose governs T cell effector and regulatory functions
- National Institutes of Health, United States
Figures
Figure 1
Stat5b is required for immunological tolerance.
(A) Cartoon depicts the mutant mice used in this study. Genotypes are grouped according to total Stat5 alleles. (B) Bar graphs show averaged RBC, hematocrit and WBC counts. (C) Scatter plot shows …
Figure 2
Impact of STAT5 paralog deficiency on B and T cells.
(A) Bar graphs show averaged cell counts for spleens (top row) and lymph nodes (bottom row). Error bars indicate standard deviation. (B) Percentages of CD4+ T cells (CD3+ CD4+ CD8α-), CD8+ T cells …
Figure 3 with 1 supplement
Aberrant effector T cell responses in the absence of Stat5b.
(A) Contour plots show percentages of CD44low IL-7R+naive and CD44high effector/memory CD4+ T cells in the spleens of 8 week-old mice. (B) Scatter plots show percentages of naive CD4+ T cells in …
Figure 3—figure supplement 1
Impact of Stat5 allele depletion on effector T cell responses.
(A) Contour plots show percentages of CD44low IL-7R+ naive and CD44high effector/memory CD4+ T cells. (B) Contour plots show percentages of PD1+ CXCR5high T follicular helper cells. (A and B) Data …
Figure 4 with 1 supplement
T regulatory cell function is impaired in Stat5b-deficient mice.
(A) Contour plots show percentages of IL-2Rα+ cells within the FOXP3+ Treg compartment in spleens of 8 week-old mice. (B) Box plots show log2 fold changes in the ratio of IL-2Rα+/IL-2Rα- Treg (WT=0; …
Figure 4—figure supplement 1
Impact of Stat5 allele depletion on Treg cells.
Bar graphs show total numbers of Treg cells in spleens and LNs of 8 week old mice (4–6 mice per genotype). Number of Stat5a, Stat5b and total Stat5 alleles (i.e. genotype) is explained in the key …
Figure 5
Defective iTreg differentiation in the absence of Stat5b.
(A) Naive CD4+ T cells were cultured under iTreg-inducing conditions. Contour plots show percentages of FOXP3+ and IL-2+ cells. (B) Box plots show log2 fold changes for FOXP3+ and IL-2+ cells …
Figure 6 with 2 supplements
Redundancy and specificity of STAT5 paralogs for gene transcription.
(A) Cartoons depict the cell types and experimental conditions used for RNA-seq. (B) Histograms show STAT5 paralog preference for all STAT5-regulated transcripts. Those which were more influenced by …
Figure 6—figure supplement 1
Transcriptomic analysis of Stat5a- and Stat5b-deficient T cells.
(A) Cartoons depict the experimental conditions used for RNA-seq. CD4+ T cells were purified from WT and one-allele Stat5a- or Stat5b-deficient mice, then transcriptomes measured either directly ex …
Figure 6—figure supplement 2
Transcriptomic analysis of Stat5a- and Stat5b-deficient T cells.
Genome browser tracks show transcript abundance in WT (grey), Stat5a-deficient (blue) or Stat5b-deficient (orange) cells. Vertical RPKM scale varies from gene to gene (but not across experimental …
Figure 7 with 2 supplements
Influence of paralog dose on genomic distribution of STAT5.
(A) CD4+ T cells from WT, Stat5a/bhet and two-allele Stat5a- or Stat5b-deficient mice were cultured in the presence of IL-2, then processed for pan-STAT5 ChIP-seq. Bar graph shows the total number …
Figure 7—figure supplement 1
Transcription factor motifs associated with STAT5 binding peaks.
CD4+ T cells were isolated from WT, Stat5a/bhet and two-allele Stat5a- or Stat5b-deficient mice, then cultured in the presence of IL-2 and processed for ChIP-seq. Chart shows the top ten …
Figure 7—figure supplement 2
Correlation between STAT5 binding and transcription of Il2ra, Bcl2 and Bcl6.
(A) Effector CD4+ T cells from one-allele Stat5a- or Stat5b-deficient mice were cultured in the presence of IL-2 and processed for RNA-seq. (B) Effector CD4+ T cells from one-allele Stat5b-deficient …
Figure 8
Preservation of high affinity targets in the absence of STAT5B.
Circle plot relates STAT5 occupancy and STAT5-dependent transcription for genes bound in: (1) only WT cells (white circle), (2) WT and Stat5a/bhet, cells (grey circle), (3) WT, Stat5a/bhet, and Stat5…
Figure 9 with 3 supplements
Relative abundance of STAT5A and STAT5B in helper T cells.
(A) Total STAT5 protein was measured in naive, Treg, Tfh and effector/memory T cells. Donut charts indicate the percentage of total STAT5 protein accounted for by each paralog. Histograms show …
Figure 9—figure supplement 1
Relative abundance of STAT5A and STAT5B in helper T cells.
(A) Donut charts indicate the percentage total STAT5 mRNA (top) or protein (bottom) accounted for by each paralog in ex vivo naive or Treg cells. mRNA data are compiled from 2–3 RNA-seq replicates …
Figure 9—figure supplement 2
Stat5a and Stat5b are transcribed from opposite DNA strands.
Genome browser tracks show relative abundance and DNA strand origin of Stat5a and Stat5b mRNAs within mouse spleen, thymus and mammary gland. Transcripts originating from the minus (-) strand are …
Figure 9—figure supplement 3
Putative lymphocyte-restricted enhancers within the Stat5a/b locus.
Genome browser tracks display DNAse I hypersensitivity sites (DHS) within the mouse Stat5a/b locus. Shown are data for primary CD4+ T cells (naive, activated, Treg), as well as spleen, thymus and …
Figure 10 with 1 supplement
Paralog dose governs STAT5-driven gene transcription.
(A) CD4+ T cells from one-allele Stat5b-deficient mice were transduced with STAT5A retrovirus, then processed for RNA-seq. Contour plots (left) show correlation between the transduction marker …
Figure 10—figure supplement 1
STAT5 paralog dose tips the balance between effector and regulatory T cell programs.
CD4+ T cells from WT, Stat5a/bhet and one-allele Stat5b-deficient mice were were transduced with control or STAT5A retrovirus under (A) non-polarizing or (B) Th17-polarizing conditions. Contour …
Additional files
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Supplementary file 1
Read depth for sequencing experiments.
Spreadsheet reports QC-passing reads for all RNA-seq and ChIP-seq experiments.
- https://doi.org/10.7554/eLife.08384.023
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Supplementary file 2
RPKM values for transcriptome analysis of STAT5-deficient CD4+ T cells.
Spreadsheet includes RPKM values for all experimental groups and biological replicates.
- https://doi.org/10.7554/eLife.08384.024
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Supplementary file 3
Paralog preference calculations for STAT5-deficient CD4+ T cells.
Spreadsheet includes paralog preference calculations for all relevant cell types and experimental conditions.
- https://doi.org/10.7554/eLife.08384.025
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Supplementary file 4
STAT5 paralog-specific genes.
Spreadsheet lists STAT5 paralog-specific genes for all relevant cell types and experimental conditions.
- https://doi.org/10.7554/eLife.08384.026
