Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Griffith, J.W., Sokol, C.L. & Luster, A.D. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu. Rev. Immunol. 32, 659–702 (2014).
Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell 140, 805–820 (2010).
Kondo, T., Kawai, T. & Akira, S. Dissecting negative regulation of Toll-like receptor signaling. Trends Immunol. 33, 449–458 (2012).
Smale, S.T. Selective transcription in response to an inflammatory stimulus. Cell 140, 833–844 (2010).
Ivashkiv, L.B. Epigenetic regulation of macrophage polarization and function. Trends Immunol. 34, 216–223 (2013).
Rogatsky, I. & Adelman, K. Preparing the first responders: building the inflammatory transcriptome from the ground up. Mol. Cell 54, 245–254 (2014).
Kwak, H. & Lis, J.T. Control of transcriptional elongation. Annu. Rev. Genet. 47, 483–508 (2013).
Adelman, K. & Lis, J.T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat. Rev. Genet. 13, 720–731 (2012).
Zhou, Q., Li, T. & Price, D.H. RNA polymerase II elongation control. Annu. Rev. Biochem. 81, 119–143 (2012).
Adelman, K. et al. Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling. Proc. Natl. Acad. Sci. USA 106, 18207–18212 (2009).
Hargreaves, D.C., Horng, T. & Medzhitov, R. Control of inducible gene expression by signal-dependent transcriptional elongation. Cell 138, 129–145 (2009).
Gupte, R., Muse, G.W., Chinenov, Y., Adelman, K. & Rogatsky, I. Glucocorticoid receptor represses proinflammatory genes at distinct steps of the transcription cycle. Proc. Natl. Acad. Sci. USA 110, 14616–14621 (2013).
Kobayashi, T. & Kageyama, R. Expression dynamics and functions of Hes factors in development and diseases. Curr. Top. Dev. Biol. 110, 263–283 (2014).
Ishibashi, M. et al. Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects. Genes Dev. 9, 3136–3148 (1995).
Hu, X. et al. Integrated regulation of Toll-like receptor responses by Notch and interferon-gamma pathways. Immunity 29, 691–703 (2008).
Su, X. et al. Interferon-γ regulates cellular metabolism and mRNA translation to potentiate macrophage activation. Nat. Immunol. 16, 838–849 (2015).
Alvarez, Y. et al. Notch- and transducin-like enhancer of split (TLE)-dependent histone deacetylation explain interleukin 12 (IL-12) p70 inhibition by zymosan. J. Biol. Chem. 286, 16583–16595 (2011).
Shang, Y., Smith, S. & Hu, X. Role of Notch signaling in regulating innate immunity and inflammation in health and disease. Protein Cell 7, 159–174 (2016).
Zhang, W., Xu, W. & Xiong, S. Blockade of Notch1 signaling alleviates murine lupus via blunting macrophage activation and M2b polarization. J. Immunol. 184, 6465–6478 (2010).
Sodsai, P., Hirankarn, N., Avihingsanon, Y. & Palaga, T. Defects in Notch1 upregulation upon activation of T Cells from patients with systemic lupus erythematosus are related to lupus disease activity. Lupus 17, 645–653 (2008).
Antoniv, T.T. & Ivashkiv, L.B. Dysregulation of interleukin-10-dependent gene expression in rheumatoid arthritis synovial macrophages. Arthritis Rheum. 54, 2711–2721 (2006).
Teachey, D.T. et al. Targeting Notch signaling in autoimmune and lymphoproliferative disease. Blood 111, 705–714 (2008).
Fischer, A., Schumacher, N., Maier, M., Sendtner, M. & Gessler, M. The Notch target genes Hey1 and Hey2 are required for embryonic vascular development. Genes Dev. 18, 901–911 (2004).
Ström, A., Castella, P., Rockwood, J., Wagner, J. & Caudy, M. Mediation of NGF signaling by post-translational inhibition of HES-1, a basic helix-loop-helix repressor of neuronal differentiation. Genes Dev. 11, 3168–3181 (1997).
Kolaczkowska, E. & Kubes, P. Neutrophil recruitment and function in health and inflammation. Nat. Rev. Immunol. 13, 159–175 (2013).
Wipke, B.T. & Allen, P.M. Essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis. J. Immunol. 167, 1601–1608 (2001).
Richmond, A. NF-κB, chemokine gene transcription and tumour growth. Nat. Rev. Immunol. 2, 664–674 (2002).
Stender, J.D. & Glass, C.K. Epigenomic control of the innate immune response. Curr. Opin. Pharmacol. 13, 582–587 (2013).
Barboric, M., Nissen, R.M., Kanazawa, S., Jabrane-Ferrat, N. & Peterlin, B.M. NF-κB binds P-TEFb to stimulate transcriptional elongation by RNA polymerase II. Mol. Cell 8, 327–337 (2001).
Nicodeme, E. et al. Suppression of inflammation by a synthetic histone mimic. Nature 468, 1119–1123 (2010).
Min, I.M. et al. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. Genes Dev. 25, 742–754 (2011).
Jonkers, I., Kwak, H. & Lis, J.T. Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons. eLife 3, e02407 (2014).
Saponaro, M. et al. RECQL5 controls transcript elongation and suppresses genome instability associated with transcription stress. Cell 157, 1037–1049 (2014).
Nilson, K.A. et al. THZ1 reveals roles for Cdk7 in co-transcriptional capping and pausing. Mol. Cell 59, 576–587 (2015).
Williams, L.H. et al. Pausing of RNA polymerase II regulates mammalian developmental potential through control of signaling networks. Mol. Cell 58, 311–322 (2015).
Imayoshi, I., Shimogori, T., Ohtsuka, T. & Kageyama, R. Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline. Development 135, 2531–2541 (2008).
Chinenov, Y. et al. Role of transcriptional coregulator GRIP1 in the anti-inflammatory actions of glucocorticoids. Proc. Natl. Acad. Sci. USA 109, 11776–11781 (2012).
Xu, H. et al. Notch-RBP-J signaling regulates the transcription factor IRF8 to promote inflammatory macrophage polarization. Nat. Immunol. 13, 642–650 (2012).
Swamydas, M. & Lionakis, M.S. Isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments. J. Vis. Exp. 77, e50586 (2013).
Lima, T.F. et al. Warifteine, an alkaloid purified from Cissampelos sympodialis, inhibits neutrophil migration in vitro and in vivo. J. Immunol. Res. 2014, 752923 (2014).
Zhang, X., Goncalves, R. & Mosser, D.M. The isolation and characterization of murine macrophages. Curr. Protoc. Immunol. 14, 14.11 (2008).
De Filippo, K., Henderson, R.B., Laschinger, M. & Hogg, N. Neutrophil chemokines KC and macrophage-inflammatory protein-2 are newly synthesized by tissue macrophages using distinct TLR signaling pathways. J. Immunol. 180, 4308–4315 (2008).
Wongchana, W., Lawlor, R.G., Osborne, B.A. & Palaga, T. Impact of Notch1 deletion in macrophages on proinflammatory cytokine production and the outcome of experimental autoimmune encephalomyelitis. J. Immunol. 195, 5337–5346 (2015).
Monach, P.A., Mathis, D. & Benoist, C. The K/BxN arthritis model. Curr. Protoc. Immunol. 15, 15.22 (2008).
Li, S. et al. RBP-J imposes a requirement for ITAM-mediated costimulation of osteoclastogenesis. J. Clin. Invest. 124, 5057–5073 (2014).
Sadik, C.D., Kim, N.D., Iwakura, Y. & Luster, A.D. Neutrophils orchestrate their own recruitment in murine arthritis through C5aR and FcγR signaling. Proc. Natl. Acad. Sci. USA 109, E3177–E3185 (2012).
Son, D.S. & Roby, K.F. Interleukin-1α-induced chemokines in mouse granulosa cells: impact on keratinocyte chemoattractant chemokine, a CXC subfamily. Mol. Endocrinol. 20, 2999–3013 (2006).
Giannopoulou, E.G. & Elemento, O. An integrated ChIP-seq analysis platform with customizable workflows. BMC Bioinformatics 12, 277 (2011).
Liao, Y., Smyth, G.K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Acknowledgements
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.).
Author information
Authors and Affiliations
Contributions
Y.S. designed the study, performed experiments, analyzed data and wrote the manuscript; M.C. performed Pol II ChIP-seq experiments; T.H. analyzed Pol II ChIP-seq data and wrote methods for bioinformatics analysis; F.N. constructed retroviral plasmids, performed retroviral related experiments and prepared mice for experiments; L.Y. performed CDK9 and Hes1 ChIP-seq experiments; L.K. performed flow cytometry and analysis; B. Zhang and Y.Q. contributed to bioinformatics analysis; C.J. prepared mice for experiments; B. Zhao provided K/BxN serum and advice on experiments; M.G. provided Hey1−/− mice; I.R. provided advice on experiments and wrote the manuscript; and X.H. conceptualized the project, designed the study, supervised experiments and wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Hes1 and Hey1 suppress inflammatory gene expression in a selective manner.
(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.
Supplementary Figure 2 Regulation of TLR-induced expression of Cxcl1 and Il12b by Hes1 in BMDMs.
(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).
Supplementary Figure 4 Hes1 mutants lose Cxcl1-suppressive capacity.
(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.
Supplementary Figure 8 Hes1 suppresses Cxcl1 transcription elongation via targeting P-TEFb.
(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.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–8 and Supplementary Table 1 (PDF 1638 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ni.3486
This article is cited by
-
The HDAC inhibitor domatinostat induces type I interferon α in Merkel cell carcinoma by HES1 repression
Journal of Cancer Research and Clinical Oncology (2023)
-
Macrophage Notch1 inhibits TAK1 function and RIPK3-mediated hepatocyte necroptosis through activation of β-catenin signaling in liver ischemia and reperfusion injury
Cell Communication and Signaling (2022)
-
Targeting the transcription factor HES1 by L-menthol restores protein phosphatase 6 in keratinocytes in models of psoriasis
Nature Communications (2022)
-
GeneWalk identifies relevant gene functions for a biological context using network representation learning
Genome Biology (2021)
-
Effect of dietary supplementation with yeast cell wall extracts on performance and gut response in broiler chickens
Journal of Animal Science and Biotechnology (2020)