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Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system

Abstract

In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of RUNX transcription factor paralogs with apparent functional redundancy. Here we asked what cell-type-specific biologies might be supported by the selective expression of RUNX paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional nonequivalence between RUNX paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain and evolutionary reconstruction suggested convergence of RUNT domain residues toward submaximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.

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Fig. 1: RUNX paralog evolution and RUNT domain conservation.
Fig. 2: Selective deployment of RUNX paralogs enables signal-responsive Langerhans cell differentiation.
Fig. 3: Selective deployment of RUNX paralogs enables signal-responsive induction of the Treg cell signature transcription factor FOXP3.
Fig. 4: Functional differences in Foxp3 regulation encoded by the RUNT domain.
Fig. 5: The RUNX1 and RUNX3 RUNT domains have different DNA binding affinities.
Fig. 6: Identification of residues that functionally distinguish paralogous RUNT domains.
Fig. 7: RUNT domain evolution towards submaximal strength.

Data availability

Source data are available online for Figs. 1–7 and Supplementary Figs. 2,5,6 and 8.

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Acknowledgements

We thank A. Warren (University of Cambridge), F. Kondrashov (Institute of Science and Technology Austria), D. Odom (CRUK Cambridge), T. Warnecke, P. Sarkies, S. Santos and B. Lenhard for discussion and advice; N. Speck (University of Pennsylvania) for conditional Runx1 mutants; J. Elliott, B. Patel and T. Adejumo for cell sorting; P. Leung for assistance; D. Djeghloul for help with immunofluorescence; and L. Game and her team for sequencing. This work was funded by the Medical Research Council UK (M.M. and A.G.F.), by Wellcome (grant 099276/Z/12/Z to M.M.) and the NIH (R01A116829 to T.S.).

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L.B. performed experiments, made figures and contributed to writing; V.R. performed experiments and made figures; R.A.S. analyzed data, made figures and contributed to writing; S.S. did experiments; D.B. analyzed data; G.D. analyzed data and made figures; T.C. analyzed data and made figures; M.G. performed experiments and made figures; M.C. provided materials and performed experiments; S.L.N. provided materials, conceptualized and supervised work; S.E. conceptualized and supervised work and performed experiments; D.S.R. provided materials and supervised work; T.S. conceptualized and supervised work, analyzed data and made figures; A.G.F. conceptualized work and contributed to writing; P.B. conceptualized and supervised work; and M.M. conceptualized and supervised work, analyzed data, made figures and contributed to writing.

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Correspondence to Matthias Merkenschlager.

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Supplementary Figure 1 RUNX1 promotes LH cell differentiation.

a) Immature DC preferentially express RUNX3. 293 cells transfected with RUNX3 or RUNX1 are used as a control for antibody specificity. Lamin is the loading control (left). Comparison of endogenous end retrovirally encoded Runx3 in immature DC (right). b) For Langerhans (LH) cell cultures, bone marrow cells were transduced with the indicated vector and 500×103 transduced cells were plated per well with GM-CSF ± TGF-β. The number of cells recovered on the day of staining for Langerhans cell markers is shown (3 biological replicates per condition ± s.d.). c) MHC class II expression by Spi1-deficient CD11c+ bone marrow cells transduced with RUNX-IRES-GFP or IRES-GFP control vector and cultured with GM-CSF and TGF-β. Left: one representative experiment of 3. Right: mean ± s.d. of 3 independent experiments. * P<0.05, ** P<0.01 by two-tailed T test Runx1 versus control vector (blue) and Runx3 versus control vector (red). Below: the distribution of GFP expression by MHC class II+ CD11c+ cells. Representative of 3 independent experiments. d) Expression of FLAG-RUNX by GFP-low, intermediate and high immature DC following retroviral transduction with RUNX3, RUNX1 or control vector. Lamin is the loading control. Representative of 2 independent experiments. e) CD11c+ MHC class II+ BM wild-type BM cells were transduced with RUNX-IRES-GFP or IRES-GFP control vector and cultured with GM-CSF and TGF-β and analyzed for EPCAM and DEC205 expression by flow cytometry. Numbers indicate the percentage of CD11c+ cells that expressed MHC class II, DEC205 and Epcam (LH cells). Representative of 3 independent experiments. f) As in e), mean ± s.d. of 3 independent experiments. * P<0.05, ** P<0.01, *** P<0.001 by two-tailed T test between Runx1 and -3 (black), Runx1 and control vector (blue), Runx3 and control vector (red). g) As in e) but in the absence of TGF-β. Representative of 3 independent experiments. h) As in f) but in the absence of TGF-β. Statistics as in f.

Supplementary Figure 2 Naive CD4 T cells preferentially express RUNX1 and show increased Foxp3 induction in response to RUNX3, not RUNX1.

a) 293 cells were transfected with RUNX1, RUNX3 or control vector as controls for the specificity of antibodies to RUNX1 and -3. Lamin is a loading control (top). Levels of retrovirally encoded RUNX1 protein were comparable to endogenous RUNX1 protein in wild-type CD4 T cells (bottom). Representative of 2 independent experiments. b) The number of cells recovered on the day of staining for Foxp3 is shown (3 biological replicates per condition mean ± s.d. for full-length RUNX, mean of 2 biological replicates per condition for RUNT). c) Runx1-wild-type naive CD4 T cells were activated and transduced with RUNX1-IRES-GFP, RUNX3-IRES-GFP or control vector as in Fig. 3. Cells were cultured in the presence of TGF-β or with PI3K inhibitor LY294002 and mTOR inhibitor rapamycin. Foxp3 induction was evaluated by intracellular staining. The percentage of Foxp3+ cells is shown (mean ± s.d. of 3 independent experiments). Source data

Supplementary Figure 3 RUNT domain constructs used in this study.

a) FLAG tag (orange), linker and cloning site (green), RUNT domain (black) and STOP codon (*) are shown. The putative nuclear localization sequence immediately downstream of the RUNT domain42 was not included and no exogenous nuclear localization sequences were added. All constructs were verified by sequencing. £ corresponds to full ‘Ancestral’ at positions 59, 123, 157 and 168, but contains D53, V102 and S121 and has equivalent functional activity to full ‘Ancestral’ with E53, L102 and T121 (Supplementary Fig. 8a).@ corresponds to the ancestor of Runx2 and -3 in Fig. 7c and ‘alternative ancestral’ in Fig. 7e, contains D53, V102 and S121. $ corresponds to ‘early vertebrate RUNX3’ in Fig. 7c, e, contains D53, V102 and S121. b) To map differential impact of Runx1 and -3 on LH cell differentiation, BM cells were transduced with RUNX1-IRES-GFP, RUNX3-IRES-GFP or with vectors encoding the isolated RUNT domains of Runx1 (RUNT1-IRES-GFP) or Runx3 (RUNT3-IRES-GFP). Numbers indicate percentages of CD11c+ MHC class II+ DEC205+ Epcam+ LH cells. Note that while full-length Runx1 promotes LH cell differentiation more effectively than full-length Runx3, the isolated RUNT domains of Runx1 (RUNT1) and Runx3 (RUNT3) have indistinguishable dominant negative activity in this assay. One experiment.

Supplementary Figure 4 Estimation of RUNT domain DNA-binding affinities.

a) Replicate titration EMSAs for RUNT1 and RUNT3 complexed with CBFB1 (Fig. 5d), and titration EMSAs for the two chimeric proteins RUNT3 V123A and RUNT3 V168I complexed with CBFB1 binding to a probe derived from the Foxp3 promoter (inset). CBFB1 protein is 270 nM in binding reactions. b) EMSA probe sequence and dissociation constant (KD) estimates are shown. Titration EMSAs were performed in duplicate.

Supplementary Figure 5 Impact of the RUNT3 V168I substitution on Foxp3 induction.

Runx1-wild-type naive CD4 T cells were activated and transduced with IRES-GFP constructs encoding the isolated RUNT domains of RUNX1, RUNX3, RUNX3 V123A, RUNX3 V168I or control vector. Cells were cultured with TGF-β or PI3K inhibitor LY294002 and mTOR inhibitor rapamycin. Foxp3 induction was evaluated by intracellular staining (mean ± s.d. of 3 independent experiments). Source data

Supplementary Figure 6 Functional differences between paralogous RUNT domains are partially offset by compensatory amino acid substitutions.

The impact of RUNT domain residue substitutions was assessed by dominant negative activity on Foxp3 induction in naive CD4 T cells. The X axis shows dominant negative activity as in Fig. 7c (n = 4 to 20, mean ± s.d. of 3 to 10 independent experiments. Two-tailed Student’s T test p<0.005 for RUNT3 versus all other constructs). Source data

Supplementary Figure 7 Reconstruction of the ancestral RUNT domain sequence.

a) Phylogenetic tree of the Runx family, with topology extracted from the EnsemblCompara tree. b) The EnsemblCompara tree was built using the TreeBeST algorithm (see methods). After removal of spurious sequences, the dataset for RUNX contains 188 sequences from the three paralogs (RUNX1, -2 and -3) and from organisms basal to the vertebrates (ascidians, nematode and fruit fly). The tree was rooted using the protostomian species (nematodes and fruit fly). Branch supports were estimated by an approximate likelihood ratio test (see methods). The names at the tips correspond to the Ensembl Protein ID. c) Sequence and posterior probability (in brackets) of the ancestral RUNT domain of Runx1, -2 and -3. S77 p(S) = 0.56, p(T) = 0.39; V87 p(V) = 0.72, p(I) = 0.26; L102 p(L) = 0.86, p(V) = 0.13; T121 p(T) = 0.89, p(S) = 0.11; V123 p(V) = 0.99; I168 p(I) = 0.74, p(V) = 0.26. d) Posterior probabilities for other RUNT domains described in this paper.

Supplementary Figure 8 RUNT domain residues E/D53, L/V102 and T/S121 confer equivalent dominant negative activity.

a) Dominant negative activity of the reconstructed ancestral RUNT domain featuring P59, V123, P157 and I168 and E53, L102 and T121 compared with a RUNT domain that features the Runx3 aa D53, V102 and S121 alongside the putative ancestral P59, V123, P157 and I168. The X axis shows dominant negative activity as in Fig. 7c. See Supplementary Fig. 3a for sequences. Mean ± s.d. of 6 independent experiments, P = 0.175 by two-tailed Student’s T test. b) Functional evolution of RUNT domain residues starting from an ancestral sequence with Ile in position 168 (alternat. ancestr.). Blue letters and arrows indicate amino acid substitutions that reduce RUNT domain activity (n = 6 to 20, mean ± s.d. of 3 to 10 independent experiments. The X axis shows dominant negative activity as in panel c). Constructs to test the activity of the alternative ancestral RUNT domain and early vertebrate Runx3 contained P59, L102 and T121, which in the context of the ancestral RUNT domain were functionally equivalent to E53, L102, and T121 (Supplementary Fig. 8a). See Supplementary Fig. 3a for sequences. Two-tailed Student’s T test RUNT3 versus early vertebrate Runx3: P = 9.52 x 10-5, RUNT3 versus alternative ancestral RUNT: P = 4.85 x 10-6, RUNT3 versus RUNT1 P = 4.89 x 10-17. Source data

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Bruno, L., Ramlall, V., Studer, R.A. et al. Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system. Nat Immunol 20, 1372–1380 (2019). https://doi.org/10.1038/s41590-019-0471-5

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