Close

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 23 May 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: 2 August 2019 , updated on: 1 November 2019

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

Read preprint (No Ratings Yet)

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.

Have your say

Your email address will not be published.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage

Also in the genetics category:

Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University

This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.

 



List by Nándor Lipták

20th “Genetics Workshops in Hungary”, Szeged (25th, September)

In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf

 



List by Nándor Lipták

2nd Conference of the Visegrád Group Society for Developmental Biology

Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)

 



List by Nándor Lipták

EMBL Conference: From functional genomics to systems biology

Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020

 



List by Jesus Victorino

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.

 



List by Shikha Nayar
Close