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. 2014 Oct;15(10):957-64.
doi: 10.1038/ni.2985. Epub 2014 Sep 7.

Bcl-6 directly represses the gene program of the glycolysis pathway

Kenneth J Oestreich  1 , Kaitlin A Read  2 , Sarah E Gilbertson  3 , Kenneth P Hough  4 , Paul W McDonald  2 , Veena Krishnamoorthy  4 , Amy S Weinmann  5
Affiliations

Bcl-6 directly represses the gene program of the glycolysis pathway

Kenneth J Oestreich et al. Nat Immunol. 2014 Oct.

Abstract

Despite the increasing knowledge of the molecular events that induce the glycolysis pathway in effector T cells, very little is known about the transcriptional mechanisms that dampen the glycolysis program in quiescent cell populations such as memory T cells. Here we found that the transcription factor Bcl-6 directly repressed genes encoding molecules involved in the glycolysis pathway, including Slc2a1, Slc2a3, Pkm and Hk2, in type 1 helper T cells (TH1 cells) exposed to low concentrations of interleukin 2 (IL-2). Thus, Bcl-6 had a role opposing the IL-2-sensitive glycolytic transcriptional program that the transcription factors c-Myc and HIF-1α promote in effector T cells. Additionally, the TH1 lineage-specifying factor T-bet functionally antagonized the Bcl-6-dependent repression of genes encoding molecules in the glycolysis pathway, which links the molecular balance of these two factors to regulation of the metabolic gene program.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. IL-2 signaling regulates the expression of glycolysis pathway genes in CD8+ TC1 cells
Primary CD8+ T cells were cultured in TC1 polarizing conditions (IL-12 and anti-IL-4) and exposed to either high or low environmental IL-2 conditions (250 U/ml or 10 U/ml, respectively). RNA was isolated from the low (black bar) and high (white bar) IL-2 treated cells and transcript abundance for the indicated genes were determined by quantitative RT-PCR. Relative transcript expression was first normalized to the Rps18 (ribosomal protein S18) control and then the sample values were compared relative to the high IL-2 concentration (set to 1) for each independent experiment. At least three or four independent experiments were performed for all genes analyzed. All error bars represent standard error of the mean (SEM) *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test).
Figure 2
Figure 2. Glycolysis pathway genes are also regulated by environmental IL-2 conditions in CD4+ TH1 cells
Primary CD4+ T cells were cultured in TH1 polarizing conditions (IL-12 and anti-IL-4) with either low (black bars) or high (white bars) IL-2 concentrations as in Fig. 1. Transcript abundance for the indicated genes were determined by quantitative RT-PCR and represented as in Fig. 1. At least three or four independent experiments were performed for all genes analyzed. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test).
Figure 3
Figure 3. Bcl-6 directly represses genes in the glycolytic pathway
(a) EL4 T cells were transfected with the indicated promoter-reporter constructs in combination with either a Bcl-6 expression vector (black bars) or an empty vector control (white bars). Luciferase promoter-reporter values were normalized to a renilla control and expressed relative to the control sample (set to 1) for each experiment. (b) Primary CD4+ T cells cultured in TH1 conditions with high IL-2 concentrations were transfected with either a control (white bars) or Bcl-6 (black bars) expression vector. Relative transcript abundance was determined by quantitative RT-PCR analysis using the PrimePCR system customized with primers specific to the indicated genes. Samples were first normalized to the expression of Rps18 and then compared to the control sample (set to 1) in each independent experiment. (c) ChIP experiments were performed with TH1 polarized cells maintained in either low (black bars) or high (white bars) IL-2 conditions. Chromatin samples were immunoprecipitated with either an antibody specific to Bcl-6 or a nonspecific IgG antibody control. The indicated promoter regions were monitored by qPCR with promoter-specific primers. The Bcl-6-precipitated samples were first normalized to a standardized aliquot of the input chromatin for each condition, followed by subtraction of the IgG antibody control as the nonspecific background of the experiment to determine the Bcl-6 enrichment relative to the percent input. At least two (c) or three (a, b) independent experiments were performed. (a–c) Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test).
Figure 4
Figure 4. Genomic distribution of Bcl-6, HIF-1α, and c-Myc surrounding the loci for glycolysis pathway genes
Shown are images derived from the UCSC genome browser displaying ChIP-seq tracks for BCOR (blue), SMRT (purple), two independent Bcl-6 antibodies (black), c-Myc ChIP-seq peaks from ENCODE (orange), and HIF-1α in hypoxic conditions (green). The boxes represent the BED file location of the significant ChIP-seq peaks for each experiment. Genes are displayed below the browser image, with the grey arrow indicating the direction of transcription. The cell types and antibodies for each ChIP-seq experiment are indicated to the left of the tracks. See Methods section for the GSE accession numbers for the individual ChIP-seq datasets,,.
Figure 5
Figure 5. The association of c-Myc and HIF-1α inversely correlates with Bcl-6 binding at the promoters for genes involved in glycolysis
(a, b) ChIP experiments were performed with TH1 polarized cells maintained in either low (black bars) or high (white bars) IL-2 conditions. Chromatin samples were immunoprecipitated with either an antibody specific to (a) c-Myc, (b) HIF-1α or (a, b) a nonspecific IgG antibody control. The indicated promoter regions were monitored by qPCR. The c-Myc or HIF-1α-precipitated samples were normalized and graphically represented as described in Fig. 3c. (a, b) Three independent experiments were performed and error bars represent the SEM. The P value for (a) Tpi1 was 0.0577 and for (b) Plod2 was 0.0981, which were not quite statistically significant (NS). (a, b) NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test).
Figure 6
Figure 6. The relative balance between T-bet and Bcl-6 regulates the expression of genes involved in the glycolysis pathway
(a) Primary wild-type (white bars) or Tbx21−/− (black bars) CD4+ T cells were cultured in TH1 polarizing conditions and transcript abundance for the indicated genes were determined by quantitative RT-PCR and normalized as described in Fig. 1. (b) Primary CD4+ T cells purified from either wild-type (white bars) or Tbx21−/− (black bars) mice were treated as described in (a). Chromatin samples were immunoprecipitated with an antibody specific to either H3K9Ac or a nonspecific IgG antibody control. The indicated promoter regions were monitored by qPCR and the samples were normalized and represented as in Fig. 3c. (c) Primary wild-type (white bars) or Tbx21−/− CD4+ T cells were cultured in TH1 polarizing conditions and either high or low IL-2 as indicated. Lactate production was monitored from equal cell numbers within each treatment condition. (a, b) Three or (c) four independent experiments were performed and error bars represent SEM. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test).
Figure 7
Figure 7. The C-terminal domain of T-bet inhibits the Bcl-6-dependent repression of a subset of glycolysis pathway genes
(a) V5-epitope tagged wild-type T-bet, T-bet(300–530), or an empty vector control were transfected into A20 lymphoma B cells. Lysates from each transfected sample were immunoprecipitated with a V5 antibody and the co-precipitation of endogenous Bcl-6 was detected in an immunoblot analysis using a Bcl-6-specific antibody. (b) A20 B cells were transfected with an empty vector control (black bars), T-bet(120–331) (grey bars) or T-bet(300–530) (white bars) in combination with the 3x-Bcl6-promoter reporter construct. The luciferase promoter-reporter values were normalized to a co-transfected renilla control and expressed relative to the control sample (set to 1). (c) A20 B cells were transfected with an empty vector control (black bars), T-bet(120–331) (grey bars), or T-bet(300–530) (white bars) in combination with the indicated promoter-reporter constructs. (d) EL4 T cells were transfected with an empty vector control, T-bet(300–530), or T-bet(300–530) and Bcl-6 in combination with the indicated promoter-reporter constructs. (c, d) The luciferase data were normalized and represented as in (b). (e) Ramos human B cells were transfected with either an empty vector control (black bars) or T-bet(300–530) (white bars) and stimulated with anti-CD40. Transcript amounts for the indicated endogenous genes were analyzed by quantitative RT-PCR and normalized to the Rps18 control and the expression was compared relative to the control sample (set to 1). (a–e) Three independent experiments were performed and (b–e) error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test).
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Comment in

  • Bcl-6 gets T cells off the sugar.
    Man K, Kallies A. Man K, et al. Nat Immunol. 2014 Oct;15(10):904-5. doi: 10.1038/ni.2993. Nat Immunol. 2014. PMID: 25232813 No abstract available.
  • Signalling: BCL-6 curbs glycolysis.
    Leavy O. Leavy O. Nat Rev Immunol. 2014 Oct;14(10):650-1. doi: 10.1038/nri3749. Epub 2014 Sep 19. Nat Rev Immunol. 2014. PMID: 25234149 No abstract available.

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