Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Clinical Trial
. 2014 Jan 24;289(4):2361-70.
doi: 10.1074/jbc.M113.523605. Epub 2013 Dec 2.

cAMP responsive element modulator (CREM) α mediates chromatin remodeling of CD8 during the generation of CD3+ CD4- CD8- T cells

Christian M Hedrich  1 , José C Crispín, Thomas Rauen, Christina Ioannidis, Tomohiro Koga, Noe Rodriguez Rodriguez, Sokratis A Apostolidis, Vasileios C Kyttaris, George C Tsokos
Affiliations
Clinical Trial

cAMP responsive element modulator (CREM) α mediates chromatin remodeling of CD8 during the generation of CD3+ CD4- CD8- T cells

Christian M Hedrich et al. J Biol Chem. .

Abstract

TCR-αβ(+)CD3(+)CD4(-)CD8(-) "double negative" T cells are expanded in the peripheral blood of patients with systemic lupus erythematosus (SLE) and lupus-prone mice. Double negative T cells have been claimed to derive from CD8(+) cells that down-regulate CD8 co-receptors and acquire a distinct effector phenotype that includes the expression of proinflammatory cytokines. This, along with the fact that double negative T cells have been documented in inflamed organs, suggests that they may contribute to disease expression and tissue damage. We recently linked the transcription factor cAMP responsive element modulator (CREM) α, which is expressed at increased levels in T cells from SLE patients and lupus prone MRL/lpr mice, with trans-repression of a region syntenic to the murine CD8b promoter. However, the exact molecular mechanisms that result in a stable silencing of both CD8A and CD8B genes remain elusive. Here, we demonstrate that CREMα orchestrates epigenetic remodeling of the CD8 cluster through the recruitment of DNA methyltransferase (DNMT) 3a and histone methyltransferase G9a. Thus, we propose that CREMα is essential for the expansion of double negative T cells in SLE. CREMα blockade may have therapeutic value in autoimmune disorders with DN T cell expansion.

Keywords: CD8; CREMα; Chromatin; Chromatin Histone Modification; DNA Methylation; Double Negative T Cells; Epigenetics; Histone Methylation; Histone Modification; SLE.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
DNA methylation through DNMT3a regulates CD8 expression. CD8A and CD8B mRNA expression was assessed by quantitative RT-PCR after forced CpG-DNA methylation through DNMT3a (24 h).
FIGURE 2.
FIGURE 2.
CD8 expression is determined by DNA methylation. A, CD4+ and CD8+ T cells from healthy humans and MRL/lpr mice were sorted for the assessment of DNA methylation. Murine DN T cells were sorted; human DN T cells were induced by TCR stimulation. In human (left) and murine (right) CD4+ T cells, the CD8 cluster was methylated. In CD8+ T cells from both species, the CD8 cluster was largely unmethylated. Stimulated human CD8+ T cells and murine DN T cells exhibited increased DNA methylation of the CD8 cluster. B, recruitment of the DNMT3a to the CD8 cluster was assessed using ChIP. In human and murine DN T cells, DNMT3a is recruited to the CD8 cluster. DNA methylation of the human CD8 cluster is more homogenous when compared with mice. In both species DNMT3a recruitment is particularly strong at CNS2, -6, -7, and -8.
FIGURE 3.
FIGURE 3.
CREMα recruits DNMT3a to the CD8 cluster. A, DNMT3a knock-down results in increased CD8A and CD8B mRNA expression in human CD8+ T cells (24 h) when compared with controls (left: CD8A, right: CD8B, first to third lanes). CREMα reduces CD8A and CD8B mRNA expression (24h) (fourth and fifth lanes). DNMT3a knock-down reverses CREMα-mediated suppression of CD8A and CD8B mRNA (24 h) (sixth lane). B, CD8A and CD8B mRNA expression in response to CREMα with or without DNMT3a knock-down are reflected by DNA methylation.
FIGURE 4.
FIGURE 4.
CD8 expression is reflected by H3K27 and H3K9 methylation. CD4+ and CD8+ T cells from healthy humans and MRL/LPR mice were sorted for the assessment of histone methylation of the CD8 cluster, using ChIP. Murine DN T cells were sorted; human DN T cells were induced by TCR stimulation. In human (left) and murine (right) CD4+ T cells, histone H3 was tri-methylated at residues lysine 27 (H3K27me3) (A) and at lysine 9 (H3K9me3) (B) over the entire CD8 cluster. In CD8+ T cells from both species, almost no H3K9me3 orH3K27me3 was detected, whereas induced human and murine DN T cells exhibited an increase in H3K27me3 and H3K9me3. Histone H3K9 and H3K27 methylation is more consistent over the murine CD8 cluster, whereas histone H3 methylation peaks around CNS2, -6, -7, and -8 in human DN T cells.
FIGURE 5.
FIGURE 5.
CREMα interacts with G9a mediating chromatin remodeling of the CD8 cluster. A and B, based on histone methylation patterns, the recruitment of G9a to the CD8 cluster has been tested, using ChIP. In both healthy humans (A) and MRL/lpr mice (B), the recruitment of G9a to the CD8 cluster in DN T cells is largely increased when compared with CD8+ T cells. C, forced expression of the histone methyltransferase G9a mediates a significant reduction of CD8A and CD8B mRNA expression through a significant increase of H3K9me3 and H3K27me3 (D) (*, indicates p < 0.05).
FIGURE 6.
FIGURE 6.
CREMα recruits G9a to the CD8 cluster. A, CREMα recruitment to CNS2, -7, and -8 is enhanced in CD8+ T cells from SLE patients. B, in analogy to SLE T cells, CREMα expression is increased in T cells from MRL/lpr mice (upper panel). In DN but not in CD8+ T cells, CREMα is recruited to CNS2, -7, and -8 suggesting CREMα is involved in the down-regulation of CD8 (lower panel). C, HEK293T cells were transfected with empty pcDNA3.1 plasmids. Proteins were co-immunoprecipitated with anti-CREMα or DNMT3a antibodies as indicated. Lysates were subjected to Western blotting with G9a antibodies. Representative results from one of three independent experiments are displayed. D, an interaction between CREMα and G9a has been established applying PLA. Ex vivo isolated CD8+ T cells exhibit interactions between CREMα and G9a that are enhanced after TCR stimulation (120 h). The number of PLA signals per unstimulated (NS) or stimulated (ST) CD8+ T cell was quantified using ImageJ software. Displayed is the number of signals per CD8+ T cell in 50 visual fields from 5 independent experiments (lower panel). E, G9a knock-down results in an increase of CD8A and CD8B mRNA expression in primary human CD8+ T cells (24 h) when compared with controls siRNA (left, CD8A; right, CD8B, first to third lanes). CREMα reduces CD8A and CD8B mRNA expression (24h) (fourth and fifth lanes). G9a knock-down reverses the CREMα effects on CD8A and CD8B (sixth lane). F and G, CD8A and CD8B expression patterns in response to CREMα with or without G9a knock-down are reflected by histone H3K9 (F) and H3K27 (G) methylation of CNS2, -7, and -8.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hedrich C. M., Rauen T., Crispin J. C., Koga T., Ioannidis C., Zajdel M., Kyttaris V. C., Tsokos G. C. (2013) cAMP responsive element modulator (CREM)α trans-represses the transmembrane glycoprotein CD8 and contributes to the generation of CD3+CD4−CD8− T cells in health and disease. J. Biol. Chem. 288, 31880–31887 - PMC - PubMed
    1. Hostert A., Tolaini M., Festenstein R., McNeill L., Malissen B., Williams O., Zamoyska R., Kioussis D. (1997) A CD8 genomic fragment that directs subset-specific expression of CD8 in transgenic mice. J. Immunol. 158, 4270–4281 - PubMed
    1. Kieffer L. J., Yan L., Hanke J. H., Kavathas P. B. (1997) Appropriate developmental expression of human CD8β in transgenic mice. J. Immunol. 159, 4907–4912 - PubMed
    1. Bilic I., Koesters C., Unger B., Sekimata M., Hertweck A., Maschek R., Wilson C. B., Ellmeier W. (2006) Negative regulation of CD8 expression via Cd8 enhancer-mediated recruitment of the zinc finger protein MAZR. Nat. Immunol. 7, 392–400 - PMC - PubMed
    1. Kioussis D., Ellmeier W. (2002) Chromatin and CD4, CD8A and CD8B gene expression during thymic differentiation. Nat. Rev. Immunol. 2, 909–919 - PubMed

Publication types

MeSH terms