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Co-Stimulation–Induced AP-1 Activity is Required for Chromatin Opening During T Cell Activation

Masashi Yukawa, Sajjeev Jagannathan, Andrey V. Kartashov, Xiaoting Chen, Matthew T. Weirauch, Artem Barski

Preprint posted on May 23, 2019 https://www.biorxiv.org/content/10.1101/647388v1

Article now published in Journal of Experimental Medicine at http://jem.rupress.org/content/early/2019/10/24/jem.20182009

Open sesame! Opening of chromatin following T cell stimulation

Selected by Jonny Coates

Context and background

The T cell receptor (TCR) is expressed on all T cells and is essential for the recognition of antigens presented by MHC molecules. Following antigen recognition, a complex signalling cascade is imitated (reviewed in (1) & (2), Fig. 1) leading to activation of the T cell.

Figure 1. TCR signalling pathway in T cells. Upon recognising antigen presented by MHC molecules, Zap-70 is activated which in turn phosphorylates downstream adapter proteins. Reproduced from Cell Signal with permission.

 

The outcome of TCR signalling is the activation and nuclear translocation of multiple transcription factors, such as NF-κB, NFAT and AP-1 (a complex of JUN and FOS). Although this signalling cascade is relatively well characterised, the outcomes of such signalling have mostly been investigated based on the interactions of these key transcription factors with specific genes. Thus, a more global view of the signalling pathway has been missing. In particular, how TCR signalling affects the accessibility of genes required for T cell functions remains an open question. In this preprint, Yukawa et al reveal that AP-1 is required for the opening of chromatin during T cell activation.

 

Key findings

  1. There are extensive epigenetic changes in response to T cell activation

The authors’ first aim was to investigate the extent of chromatin changes across the genome in response to T cell activation. Utilising ATAC-seq, they identified over 16,500 chromatin regions that opened within 60-hours of stimulation. There were a further 6000 regions that closed following stimulation. The authors focussed on the open regions to perform gene ontology analysis. This revealed that the open chromatin was initially near genes that were involved with T cell activation. However, at the later time points, chromatin that is associated with genes involved in T cell migration and metabolism became more open.

These open chromatin regions were thought to be acting in a regulatory capacity. The authors found that 334 of the open regions in activated T cells acted as super enhancers (combinations of enhancer elements with unusually high levels of H3K27 acetylation).

The authors next addressed the question of how this open chromatin is related to TCR signalling.

 

  1. NFAT and AP-1 bind to open chromatin regions

Utilising software algorithms, the authors found that DNA motifs of NFAT and AP-1 were enriched in the open chromatin of activated T cells. ChIP-seq enabled the confirmation of this observation. Over 70% of open chromatin regions in activated T cells were bound by AP-1 alone or in combination with NFAT. However, a key question remained; Is this remodelling due to direct actions of AP-1 or is AP-1 passively binding to the open regions?

The authors used the AP-1 dominant-negative protein A-FOS to reduce AP-1 levels (A-FOS sequesters JUN isoforms and thereby prevents the AP-1 complex formation). Inhibiting AP-1 reduced the amount of open chromatin regions following activation. Moreover, there was a downregulation of activation-inducible genes such as IFN-γ. Together, this suggests that AP-1 is required for the opening of chromatin.

Partial stimulation of T cells results in anergy, where the T cell becomes inactivated following incomplete stimulation. When the authors partially stimulated T cells, they found a reduction in the number of open chromatin regions and a decrease in H3K27Ac levels. This suggests that the chromatin remodelling is a result of TCR-stimulation and possibly mediated by AP-1.

 

Why I chose this paper

RNA sequencing approaches have revolutionised the life sciences, with single-cell sequencing now expanding that revolution further. However, there are many layers of regulation surrounding gene expression. Studies that investigate those layers of regulation are invaluable in progressing the field. Chromatin accessibility is hugely important for the transcription or repression of genes, representing one such layer of regulation. Yukawa et al demonstrate that following stimulation of the TCR, T cells rapidly induce opening of chromatin regions which rely on AP-1 signalling.

 

Open questions

  1. The authors nicely demonstrated that AP-1 is required for the open chromatin formation; however, it would be good to confirm this was a result of TCR activation by inhibiting ZAP70 or LCK (early proteins involved in conducting the TCR stimulation cascade). Have the authors considered the possibility of AP-1 originating from other sources?

 

  1. How is the chromatin accessibility affecting T cell effector functions?
    1. What is happening to the chromatin accessibility of key T cell effector genes?
    2. Could modulating this accessibility program T cell effector functions?

 

  1. What is the impact of inhibiting AP-1 on T cell differentiation or effector functions?
    1. Do these cells become exhausted or fail to differentiate?

 

  1. How are the observed epigenetic changes occurring?
    1. Inhibition of AP-1 prevented the open chromatin formation but the authors do not expand on how AP-1 is controlling chromatin accessibility. What histone modifying enzymes is AP-1 activating/inhibiting?

 

  1. What about the closed regions? The regions that are closing are just as important but these do not appear to be discussed. It would be interesting to get the authors’ opinions on what genes are being repressed upon TCR activation and what they think this means in terms of T cell effector functions.

 

References

  1. Courtney AH, Lo W-L, Weiss A. TCR Signaling: Mechanisms of Initiation and Propagation. Trends Biochem Sci. 2018;43(2):108–23.
  2. Gaud G, Lesourne R, Love PE. Regulatory mechanisms in T cell receptor signalling. Nat Rev Immunol. 2018 Aug;18(8):485.

 

 

 

 

 

Tags: chromatin, t cell, tcr

Posted on: 2nd August 2019 , updated on: 1st November 2019

doi: https://doi.org/10.1242/prelights.12120

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  • Author's response

    Masashi Yukawa & Artem Barski shared

    Thank you for highlighting our work!

    Here are the responses to your questions:

    1. In addition to TCR itself, the key input for AP-1 nuclear translocation is the CD28 co-stimulatory signal (according to our western blots of nuclear proteins). The IL2-ERK signaling pathway also could be a source of AP-1 (Indeed, anergy induction can be blocked by IL-2). However, the autocrine effects are likely to be small at the early timepoints that we used. We love your idea of using inhibitors, which will allow us to obtain more information about respective inputs of various pathways into AP-1 induction.
    2. Effector genes such as IL2 and IFNG tend to have many sites of chromatin remodeling that also become H3K27 – acetylated during T cell activation. Our experiments with CD28 co-stimulation showed that the lack of chromatin opening and the disappearance of H3K27Ac cooccurred and led to repression of effector genes during T cell activation.
    3. We did not test for exhaustion and differentiation of T cells electroporated with A-FOS. However, our culture system skews towards the Th1 lineage. RNA-Seq analysis showed that TBX21 and IFNG are strongly suppressed in the A-FOS–inhibited cells. Earlier results from our and other laboratories suggest that co-stimulatory blockade leads to a strong decrease in T cell proliferation and to T cell anergy 1,2.
    4. We did not conduct these experiments, but the work from the Greenberg laboratory showed that AP-1 can recruit most of the SWI/SNF chromatin remodeling complex proteins 3.
    5. Indeed, proper chromatin closing is also essential for normal T cell activation. We demonstrate the loss of chromatin accessibility and decreased expression at genes such as KLF2 and CXCR4. We observed enrichment for KLF and EGR motifs at the sites of chromatin closing.

    Thank you again for your interest in our work, your questions, and the opportunity for additional discussion!

    -Masashi and Artem

     

    References

    1. Wells AD, Walsh MC, Bluestone JA, Turka LA. Signaling through CD28 and CTLA-4 controls two distinct forms of T cell anergy. J. Clin. Invest. 2001;108(6):895–904.
    2. Rochman Y, Yukawa M, Kartashov AV, Barski A. Functional Characterization of Human T Cell Hyporesponsiveness Induced by CTLA4-Ig. PLoS ONE. 2015;10(4):e0122198–18.
    3. Vierbuchen T, Ling E, Cowley CJ, et al. AP-1 Transcription Factors and the BAF Complex Mediate Signal-Dependent Enhancer Selection. Molecular Cell. 2017;68(6):1067–1082.e12.

     

    Update 01/11/19

    This preprint has now been published in the Journal of Experimental Medicine (http://jem.rupress.org/content/early/2019/10/24/jem.20182009). We would like to congratulate the authors and extend our thanks for their engagement with this preLight. We asked the authors a follow-up question and received the response below.

    Q: In your view, what were the most important improvements in your study as a result of peer review?

    Artem Barski:

    The reviewers asked us to provide two pieces of data in addition to more browser shots and textual clarifications:

    1. Evidence that protein electroporation into T cells does not affect activation (some of these figures did not become a part of this paper, but will be published separately in a more technical manuscript) .
    2. ChIP-Seq for AP-1 in T cells that were not activated or activated in the presence of A-FOS dominant-negative or in the absence of CD28 signaling. Because ChIP-seq is a relative method (Lov´en et al., 2012), in the situations where global change in protein levels occurs (e.g., AP-1in GFP-electroporated vs. A-FOS–electroporated T cells), an external normalization is required. To compare ChIP enrichment for AP-1 and NFAT1 between GFP- and A-FOS–electroporated cells (Fig. 5 G) or cells with or without CD28 co-stimulation (Fig. 6 D and S5 B), normalization was performed by spiking in Drosophila genomic DNA.

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