Elsevier

Journal of Autoimmunity

Volume 96, January 2019, Pages 74-85
Journal of Autoimmunity

CRL4DCAF2 is required for mature T-cell expansion via Aurora B-regulated proteasome activity

https://doi.org/10.1016/j.jaut.2018.08.006Get rights and content

Highlights

  • DCAF2 expression was highly associated with multiple autoimmune diseases.

  • CRL4DCAF2 is essential for controlling G2/M phase transition and proteasome activity of mature T cells at M phase.

  • CRL4DCAF2 controlled 26S proteasome activity via an Aurora B-mediated novel mechanism.

Abstract

The proliferation of T cells in peripheral lymphoid tissues requires T cell receptor (TCR)-mediated cell cycle entry. However, the underlying mechanism regulating cell cycle progression in mature T cells is incompletely understood. Here, we have identified an E3 ubiquitin ligase, CRL4DCAF2, as a critical mediator controlling M phase exit in activated T cells. DCAF2 expression is induced upon TCR stimulation and its deficiency attenuates T cell expansion. Additionally, DCAF2 T cell-specific knockout mice display impaired peripheral T cell maintenance and reduced severity of various autoimmune diseases. Continuous H4K20me1 modification caused by DCAF2 deficiency inhibits the induction of Aurkb expression, which regulates 26S proteasome activity during G2/M phase. CRL4DCAF2 deficiency causes M phase arrest through proteasome-dependent mechanisms in peripheral T cells. Our findings establish DCAF2 as a novel target for T cell-mediated autoimmunity or inflammatory diseases.

Introduction

T cells, a type of white blood cells, play a critical role in adaptive immunity against infectious microorganisms and cancer. Hyperactive T cells are also responsible for autoimmune and inflammatory disorders [1]. Upon stimulation by self-antigens, deregulated T cells are activated to enter the cell cycle and proliferate. These deregulated T cells subsequently secrete various effecter cytokines to drive autoimmunity [2]. Consistent with the expansion of other eukaryotic cells, T cell expansion is also tightly controlled by multiple regulatory checkpoints throughout the cell cycle [3]. Entry into the cell cycle is a complex process controlled by the ordered expression and activation of various molecules, including cyclins and cyclin-dependent kinases (CDKs), and the phosphorylation of downstream substrates [4]. Once the signals that trigger the cell cycle are deregulated, immunological tolerance becomes disrupted, and this results in autoimmune responses [5,6]. Nonetheless, the coordinated mechanism of T cell receptor (TCR)-mediated cell cycle regulation remains incompletely investigated.

Current understanding reveal that the first checkpoint controls the transition of eukaryotic cells from the G1 phase into S phase and initiates DNA synthesis [7]. An active complex including Aurora, Survivin, mTOR, p70S6k and 4E-BP1 controls cell cycle progression at the G1/S phase transition [8]. The process of cell cycle in T cells is also tightly controlled by the action of several negative regulators, such as p21, p27, and p53 [[9], [10], [11], [12], [13]]. However, whether these molecules are also involved in the cycling of T cells and their negatively regulatory mechanisms remain controversial.

Ubiquitination is a critical post-translational modification that regulates T cell activation and cell cycle entry [14]. Various E3 ubiquitin ligases and deubiquitinases (DUBs), including Pellino 1, Itch, c-Cbl, Cbl-b, GRAIL, and OTUD7B, have been identified to negatively regulate TCR-CD28 signal transduction and prevent T cell-mediated autoimmune disease progression [[15], [16], [17], [18], [19]]. Ubiquitination also regulates cell cycle in T cells by controlling CDKs and their inhibitors (CDKIs). Mule-deficient mice develop severe experimental autoimmune encephalomyelitis (EAE) and show impaired antiviral immune responses by regulating Krüppel-like factor 4 (KLF4) stability, which is required for the entry of T cells into S phase [20]. Previous studies have shown that cullin ring-finger ubiquitin ligase-4 (CRL4) have multiple functions in maintaining cell cycle progression. CRL4 plays its physiological role by employing more than 90 DDB1-cullin 4-associated factors (DCAFs). Current evidence reveals that CRL4DCAF1 is involved in critical steps towards aberrant T cell expansion. CRL4DCAF1 promotes the destabilization of p53, which suppresses metabolism and cell cycle entry [21]. However, there is limited evidence regarding the physiological functions of other DCAFs in primary T cells.

In this study, we identified DCAF2 as an essential component of T cell proliferation and T cell-mediated autoimmune responses. CRL4DCAF2 has been considered to induce the degradation of p21, SETD8 and Checkpoint kinase 1 (CHK1) by a ubiquitin-dependent mechanism and to promote cell cycle progression [[22], [23], [24]]. Despite extensive in vitro studies, the in vivo biological functions of CRL4DCAF2 have remained largely unknown due to the embryonic lethality of the conventional DCAF2 knockout (KO) mice. Therefore, to clarify the function of CRL4DCAF2 in T cell-mediated inflammation, we generated mice specific knockout DCAF2 in T cells for this study. Our studies led to the discovery of a central role for CRL4DCAF2 in the control of T cell proliferation and M phase exit. DCAF2 T cell-specific KO mice displayed impaired expansion of peripheral T cells coupled with a reduction in autoimmune symptoms. High-throughput RNA sequencing indicated that the loss of DCAF2 disrupted Aurora B induction and cause M phase arrest. Interestingly, we found that Aurora B physically interacted with the 26S proteasome and regulated proteasome activity, which is important for G2/M phase transition. By chromatin immunoprecipitation sequencing (CHIP-seq) assays, we further demonstrated that DCAF2 depletion attenuated Aurka and Aurkb induction by enhancing the H4K20me1 modification at their promoters. Therefore, our findings establish CRL4DCAF2 as a critical regulator of cell cycle progression in mature T cells and suggest DCAF2 as a therapeutic target for T cell-mediated autoimmune diseases.

Section snippets

TCR triggered DCAF2 induction

To evaluate the potential role of CRL4DCAF2 in inflammatory disease, we first analyzed DCAF2 expression in peripheral blood mononuclear cells (PBMCs) from patients with inflammatory bowel disease (IBD). Compared with that in healthy controls, DCAF2 mRNA level was clearly increased in PBMCs from Crohn's disease (CD) or ulcerative colitis (UC) (Fig. 1a). The hallmark of IBD is an aberrant mucosal infiltration by innate immune cells and adaptive immune cells including effector T cells. Although

Discussion

The clonal expansion of antigen-specific T cells is essential for the induction of effective adaptive immune responses. A key process in T cell proliferation is cell cycle entry, which ensures proper cell division. The cycling of T cells is tightly controlled by the ordered expression of cyclin/CDK complexes [4,7]. These kinases undergo periodic proteolysis to maintain the integrity of T cells. Two crucial E3 enzymes are responsible for regulating cell cycle progression and mediating the

Mice

Dcaf2fl/fl mice were generated by the Model Animal Resource Information Platform, Model Animal Research Center of Nanjing University. Embryonic stem cells used to generate this mouse strain were purchased from the European Conditional Mouse Mutagenesis Program (ES cell clone EPD0842_C05). The Dcaf2-floxed mice were further crossed with Cd4-Cre mice (all from Jackson Laboratory, C57BL/6 background) to generate T cell conditional DCAF2 KO (Dcaf2f/fCd4-Cre, TKO). Rag1−/− mice were from Model

ELISA and qRT-PCR

Supernatants of in vitro cell cultures were analyzed by ELISA using a commercial assay system (eBioScience). For qRT-PCR, total RNA was isolated using TRI reagent (Molecular Research Center, Inc.) and subjected to cDNA synthesis using RNase H-reverse transcriptase (Invitrogen) and oligo (dT) primers. qRT-PCR was performed in triplicates, using iCycler Sequence Detection System (Bio-Rad) and iQTM SYBR Green Supermix (Bio-Rad). The expression of individual genes was calculated by a standard curve

Author contributions

K.F. and F.W. performed the research, prepared the Figures, and wrote the manuscript; Y.L., L.C., Z.G., Y.Z., J.D., T.H., J.Z., R.L., X. M., H.F. and X.G. contributed experiments; J.J. supervised the work, prepared the Figures and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Acknowledgements

We thank Dr. Heng-Yu Fan, Dr. Fangwei Wang and Dr. Hai Song for expression plasmid and Tp53flox/flox mice. This study was supported by the National Key Research and Development Program of China (2018YFD0500100), the National Natural Science Foundation of China (81572651/81771675), the Fundamental Research Funds for the Central Universities (2016QN81013), the Thousand Young Talents Plan of China, and the Zhejiang University Special Fund for Fundamental Research.

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