Most of the known regulatory mechanisms that curb inflammatory gene expression target pre–transcription-initiation steps, and evidence for post-initiation regulation of inflammatory gene expression remains scarce. We found that the transcriptional repressor Hes1 suppressed production of CXCL1, a chemokine that is crucial for recruiting neutrophils. Hes1 negatively regulated neutrophil recruitment in vivo in a manner that was dependent on macrophage-produced CXCL1, and it attenuated the severity of inflammatory arthritis. Mechanistically, inhibition of Cxcl1 expression by Hes1 did not involve modification of transcription initiation. Instead, Hes1 inhibited signal-induced recruitment of the positive transcription-elongation complex P-TEFb and thereby prevented phosphorylation of RNA polymerase II at Ser2 and productive elongation. Thus, our results identify Hes1 as a homeostatic suppressor of inflammatory responses that exerts its suppressive function by regulating transcription elongation.
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We thank R. Kageyama (Koyto University) for Hes1fl/fl mice; K.F. Roby (University of Kansas Medical Center) for Cxcl1 reporter plasmids; L.B. Ivashkiv for discussion; S. Chakravarty, K. Au, S. Smith and J. Zhu for technical assistance; and C. Tang, J. Lu, Z. Zhang, X. Yang and J. Zhang (Peking University) for advice on bioinformatics analysis. Supported by the Ministry of Science and Technology of China (National Key Research Project 2015CB943201 to X.H.), the National Natural Science Foundation of China (Young Investigator Award 81422019 to X.H.), the Tsinghua-Peking Center for Life Sciences (X.H.), the Institute for Immunology at Tsinghua University (X.H.), The David Rosensweig HSS Genomics Center (M.C., I.R.),the Rheumatology Research Foundation (I.R.), the US Department of Defense (I.R.) and the US National Institutes of Health (I.R. and B.Z.).
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Integrated supplementary information
(a) Quantitative RT-PCR (qPCR) analysis of mRNA expression of Hes1 and Hey1 in BMDMs from Hey1+/+Hes1fl/fl (WT) and Hey1−/−Hes1fl/flMx1-Cre (DKO) littermates. (b,c) Heat maps of gene expression profiles of LPS-induced genes (b) and LPS-suppressed gene (c) in WT and DKO BMDMs treated ± LPS (2 ng/ml) for 3 h (cut off=2 fold). Microarray analysis was performed as described in Methods. Hes1-regulated genes in both positive and negative directions are shown in enlarged panels on the right. An individual gene may be represented more than once due to presence of multiple probe sets in microarrays. 3 determinants of the Cxcl1 gene are marked with arrows (b, enlarged). The regulated gene number corresponds to unique genes but may include ESTs with uncharacterized functions shown without identifiers. Genes were ranked according to the order of numeric identifiers of microarray probes. (d) qPCR analysis of Cxcl1 mRNA expression in WT and DKO BMDMs stimulated with 10 ng/ml of Pam3Cys (TLR2 agonist) or 1 μg/ml of R848 (TLR7/8 agonist) for 3 h. Cumulative data from four independent experiments (a) or representative data from two independent experiments (d) are shown as mean and s.d.. Error bars are technical triplicate determinants in d. ***P<0.001 (two-tailed Student’s paired t-test). N.D., not detected.
(a,e) qPCR analysis of mRNA expression of Cxcl1 (a) or Il12b (e) in BMDMs from WT (Hey1+/+) and Hey1 KO (Hey1−/−) littermates, Hes1+/+Mx1-Cre (WT) and Hes1fl/flMx1-Cre (Hes1 KO) mice, or WT and DKO mice. BMDMs were untreated or stimulated with 100 ng/ml of LPS (a) or 10 ng/ml of LPS (e) for 3 h. (b) qPCR analysis of Hes1 mRNA expression in BMDMs from Hes1+/+Mx1-Cre (WT) and Hes1fl/flMx1-Cre (Hes1 KO) littermates. (c) Immunoblotting analysis of Hes1 protein expression in nuclear extraction of BMDMs from WT and Hes1 KO mice. TATA binding protein (TBP) served as a loading control. (d) qPCR analysis of Cxcl1 mRNA expression in WT and Hes1 KO BMDMs stimulated with Pam3Cys (10 ng/ml) or R848 (1 μg/ml) for 3 h. Representative data is shown as means ± s.d. of technical triplicate determinants from two independent experiments (a,d,e). Cumulative data from 9 independent experiments are shown in b (means and s.d.). ****P<0.0001 (two-tailed Student’s paired t-test).
Supplementary Figure 3 Hes1-mediated suppression of Cxcl1 expression is independent of Cre type and deletion method.
(a,d) qPCR analysis of mRNA expression of Hes1 in BMDMs from Hes1+/+Cre-ERT2 (WT) and Hes1fl/flCre-ERT2 (Hes1 KO) mice (a) or Hes1+/+Lyz2Cre (WT) and Hes1fl/flLyz2Cre (Hes1 KO) mice (d). (b) Immunoblotting analysis of Hes1 protein expression in nuclear extracts of BMDMs from Hes1+/+Cre-ERT2 (WT) and Hes1fl/flCre-ERT2 (Hes1 KO) mice. TATA binding protein (TBP) served as a loading control. (c,e) qPCR analysis of mRNA expression of Cxcl1 in Hes1+/+Cre-ERT2 (WT) and Hes1fl/flCre-ERT2 (Hes1 KO) mice (c) or Hes1+/+Lyz2Cre (WT) and Hes1fl/flLyz2Cre (Hes1 KO) mice (e) BMDMs stimulated with LPS (10 ng/ml) for 3 h (c) or indicated periods (e). Cumulative data from three independent experiments are shown in a (means and s.d). Representative data from at least two independent experiments are shown as mean and s.d. of technical triplicate determinants (c-e).*P<0.05 (two-tailed Student’s paired t-test).
(a) Schematic drawings of murine Hes1 domains and mutants. 3 amino acids (E43, K44 and R47) in the basic region were mutated to alanine to obtain a dominant-negative form of Hes1 (dnHes1) that cannot bind to DNA but can still dimerize with endogenous Hes1 to form a non-DNA-binding heterodimeric complex. Hes1 (∆HLH) and Hes1 (∆WPRW) mutants were generated by deletion of HLH domain (amino acids 48-92) and the last C-terminal 6 amino acids of Hes1 respectively. (b) Immunoblotting with an anti-FLAG antibody to confirm expression of wild type Hes1 (wtHes1) and Hes1 mutants in HEK293T cells. Cells were transiently transfected with recombinant pMx vectors expressing GFP, wtHes1 or Hes1 mutants. 24 hours post transfection, cells were harvested for immunoblotting analysis. p38 served as a loading control. (c) qPCR analysis of Cxcl1 mRNA expression in BMDMs transduced with retroviral particles expressing GFP, wtHes1 or Hes1 mutants and subsequently stimulated with LPS (10 ng/ml) for 1 h. Data are shown as mean ± s.d. of technical triplicate determinants and are representative of two independent experiments (c).
Supplementary Figure 5 Hes1 deficiency does not alter other peritoneal cell populations or neutrophil motility.
(a-c) Total numbers of macrophages (a), B cells (b), and monocytes (c) in peritoneal exudates of Hes1+/+Mx1-Cre (WT) and Hes1fl/flMx1-Cre (Hes1 KO) littermates treated with LPS (100 ng/mouse) for 4 h. Cell population of macrophages (F4/80+CD11b+), B cells (B220+CD11b−), and monocytes (Ly6C+CD11b+) were identified with cell surface markers by flow cytometry analysis (n=3). (d) Flow cytometry analysis of purity of isolated neutrophils (Ly6G and 7/4 double positive) from bone marrows of Hes1+/+Cre-ERT2 (WT) and Hes1fl/flCre-ERT2 (Hes1 KO) mice. (e) Number of neutrophils that migrated into outside compartment of transwells. Chemotaxis was induced by adding 100 ng/ml of murine Cxcl1 in the outside compartment for 1 h. (f) Chemotaxis index (CI) of neutrophils treated as in e. CI for each sample was calculated as the ratio of cell number in the outside compartment in cultures with Cxcl1 and cell number in medium only cultures (random spontaneous cell migration). (g) Numbers of Ly6G+7/4+ neutrophils (left panel) and F4/80+CD11b+ macrophages (right panel) in peritoneal exudates of C57/BL6/J mice 4 h after adoptive transfer of activated BMDMs from Hes1+/+Cre-ERT2 (WT) or Hes1fl/flCre-ERT2 (Hes1 KO) mice (n=3 biological replicates per group). Cumulative data are from three independent experiments (a-c) or are pooled from two independent experiments (d). Representative data from two independent are shown in e&f. Data shown are means and s.e.m (a-c, g) of biological repeats or mean and s.d. of technical triplicates (e,f). N.S., not significant; **P<0.01, (two-tailed Student’s paired t-test (g)).
Supplementary Figure 6 The Cxcl1 proximal promoter region is not sufficient for Hes1-mediated suppression.
(a) Sequence analysis of the core Cxcl1 gene promoter reveals 2 putative E-boxes and 4 NF-κB binding sites. (b) Relative Cxcl1 promoter-driven luciferase activities in RAW 264.7 cells co-transfected with a Cxcl1 promoter-derived (KC-701) reporter construct and a Hes1 expression plasmid (pCMV6-XL4-HES1) or an empty control vector (pCMV6-XL4). 24 hours post transfection, cells were untreated or stimulated with 100 ng/ml of LPS for 4 h, and cell lysates were analyzed for luciferase activities. Results were shown as relative firefly luciferase activities normalized to renilla luciferase activities. Data from one representative experiment out of three performed is shown. (c) Pol ChIP assays showing Pol II occupancy near the TSS regions of Il6 and Il12b genes in Hes1+/+Mx1-Cre (WT) and Hes1fl/fl, Mx1-Cre (Hes1 KO) BMDMs untreated (UT) or stimulated with 10 ng/ml of LPS for 1 h (LPS). Representative data as means and s.d. of technical triplicate determinants is shown in the left panels. Cumulative results from three independent experiments are shown in the right panels under LPS-stimulated conditions and relative occupancy in WT cells was set to 1. *P<0.05, two-tailed paired Student’s t-test.
Supplementary Figure 7 Global Pol II–distribution patterns in wild-type and Hes1-deficient macrophages.
(a,b) Heat map depiction of Pol II ChIP-seq reads in Hes1+/+Mx1-Cre (WT) and Hes1fl/flMx1-Cre (Hes1 KO) BMDMs untreated or stimulated with 10 ng/ml of LPS for 1 h. Genes longer than 750 bp are aligned around mouse RefSeq TSSs and ranked by decreasing Pol II ChIP-seq signals near TSSs (±150bp). Pol II distribution patterns of all genes and of LPS-induced genes are shown in (a) and (b) respectively. (c) Genes with enhanced Pol II occupancy on the gene body regions in Hes1-deficient BMDMs. The list was generated by calculating Pol II density throughout the gene body regions in Hes1 KO + LPS versus WT + LPS conditions. Data are obtained from two independent Pol II ChIP-seq experiments.
(a) qPCR analysis of Cxcl1 mRNA expression in Hes1+/+Cre-ERT2 (WT) and Hes1fl/flCre-ERT2 (Hes1 KO) BMDMs pretreated with DMSO or a CDK inhibitor flavopiridol (FP, 600 mM, dissolved in DMSO) for 30 min, followed by LPS (10 ng/ml) stimulation for the indicated periods. Representative data are shown as means ± s.d. of technical triplicate determinants from two independent experiments. (b) Immunoblotting analysis of CDK9 expression in Hes1+/+Mx1-Cre (WT) and Hes1fl/flMx1-Cre (Hes1 KO) BMDMs stimulated with LPS (10 ng/ml) for indicated periods. Total p38 levels served as loading control.Pol II productive elongation is controlled by the P-TEFb complex. (c) A model of Hes1-mediated suppression of Cxcl1 transcription elongation. In the presence of Hes1, relatively low level of P-TEFb is recruited to the Cxcl1 gene locus in response to LPS stimulation, concomitant with low level of Pol II S2 phosphorylation. Thereby, in activated macrophages, Hes1 functions as an endogenous brake restraining Cxcl1 transcription. In Hes1-deficient cells, removal of such brake leads to augmented recruitment of P-TEFb, which results in enhanced S2 phosphorylation of Pol II on the gene locus and, consequently, heightened productive elongation of the target gene.
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Shang, Y., Coppo, M., He, T. et al. The transcriptional repressor Hes1 attenuates inflammation by regulating transcription elongation. Nat Immunol 17, 930–937 (2016). https://doi.org/10.1038/ni.3486
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