CCCTC-binding factor and the transcription factor T-bet orchestrate T helper 1 cell-specific structure and function at the interferon-gamma locus

Abstract

How cell type-specific differences in chromatin conformation are achieved and their contribution to gene expression are incompletely understood. Here we identify a cryptic upstream orchestrator of interferon-gamma (IFNG) transcription, which is embedded within the human IL26 gene, compromised of a single CCCTC-binding factor (CTCF) binding site and retained in all mammals, even surviving near-complete evolutionary deletion of the equivalent gene encoding IL-26 in rodents. CTCF and cohesins occupy this element in vivo in a cell type-nonspecific manner. This element is juxtaposed to two other sites located within the first intron and downstream of Ifng, where CTCF, cohesins, and the transcription factor T-bet bind in a T helper 1 (Th1) cell-specific manner. These interactions, close proximity of other elements within the locus to each other and to the gene encoding interferon-gamma, and robust murine Ifng expression are dependent on CTCF and T-bet. The results demonstrate that cooperation between architectural (CTCF) and transcriptional enhancing (T-bet) factors and the elements to which they bind is required for proper Th1 cell-specific expression of Ifng.

Figure 1. Cell-specific chromatin accessibility and CTCF occupancy in the IFNG locus

(A) Schematic depicting the location of the human IL26 and IFNG genes and DNaseI hypersensitive sites found in human Th1 cells (downward arrows, shown with distance from the IFNG 5′ end) in relation to the orthologous gene and regulatory elements in the mouse Ifng locus reported previously (Schoenborn et al., 2007). Nearly all of the mouse IL26 gene has been deleted as a result of complex structural rearrangements, which include a set of tandem repeats shown just upstream of the mouse −70kb region where the CTCF-binding site orthologous to the human −63kb CTCF-binding site located in an intron of IL26 is present. Shown below is PhastCons conservation score and evolutionary conservation relative to mouse, rat, dog, and cow. (B) Chromatin accessibility shown as the density of mapped DNaseI cleavage sites (in a 150bp sliding window) from human naïve, Th1 and Th2 CD4+ T cells. DHSs correspond with peaks in the density profiles. (C) CTCF ChIP analysis in human Th1 cells. Results are the mean ± SD of 3 experiments. (D) Zoomed view of conserved CTCF binding motifs within DHSs located in human at −63 kb and mouse at −70kb relative to IFNG.

Figure 2. CTCF, cohesin, T-bet binding and DNA methylation state of CTCF-binding sites in the mouse Ifng locus

(A) CTCF ChIP analysis in mouse naive, Th1 and Th2 CD4+ T cells. Results are the mean ± SD of 3 experiments; results are shown relative to the binding of CTCF at −70kb in naive T cells, which represented 0.9±0.3 % of input and was assigned a value of 1. P values (calculated by Student's t test) ≤0.05 are shown. (B) Rad21 (cohesin) ChIP analysis in mouse naive, Th1 and Th2 CD4+ T cells. Results are the mean ± SD of 2 experiments for naive T cells and 3 experiments for Th1 and Th2 cells; results are shown relative to the binding of cohesin at −70kb in naive T cells, which represented 2.6±0.3 % of input and was assigned a value of 1. (C) CD4⁺ T cell subset-specific CpG methylation at the CTCF-binding elements as determined by bisulfite sequencing. Arrows mark the positions of predicted CTCF-binding sites. Hepatocytes were used as a control. Rows, sequences of cloned alleles; filled circles, methylated CpG; open circles, unmethylated CpG. Below plots, the fraction and percentage of unmethylated CpGs. (D) T-bet ChIP analysis in mouse naive, Th1 and Th2 CD4+ T cells. Results are the mean ± SD of 2 experiments for naive T cells and 4 experiments for Th1 and Th2 cells; results are shown relative to the binding of T-bet at the Ifng promoter (DHSI) in naive T cells, which represented 4.9±0.4 % of input and was assigned a value of 1. ***, p<0.005; **p<0.01; *p<0.05

Figure 3. Three-dimensional conformation of the mouse Ifng locus

Relative cross-linking frequencies between a fixed anchor fragment bearing the Ifng gene (A-C) or the +66 CTCF site (D) and other BglII fragments using primary mouse naïve, Th1 and Th2 CD4+ T cells (A,B,D), primary mouse Th1 cells with or without restimulation with PMA + ionomycin for 6 hr (B), or Th1 (AE7), Th2 (D10G4.1), T cell progenitor (EL-4 thymocyte) or erythroid progenitor (G1E) cell lines (C). Black shading represents the position of the anchor fragment, and the locations and widths of gray shading indicate the positions and sizes of the BglII fragments whose cross-linking frequency to the anchor fragments was assessed. The location of the mouse Ifng and Il22 genes and their orientation are shown in the cartoon above. Iltifb, a degenerate pseudogene derived from an inverted duplication of Il22 and located between Il22 and Ifng, and the annotated non-coding transcript Tmevpg1 located downstream of Ifng, are also shown (Schoenborn et al., 2007). Data (error bars, s.d.) are representative of three independent experiments. Each signal was normalized to control templates and to interactions within the Gapdh locus.

Figure 4. shRNA-mediated knockdown of CTCF impairs CTCF binding at the Ifng locus and the 3-dimensional conformation of the locus

Naive CD4⁺ T cells were cultured under Th1-polarizing conditions and transduced on day 1 with a bicistronic retroviral vector expressing GFP and CTCF#1, CTCF#2 or control (scrambled CTCF#1 sequence) shRNAs. GFP+ cells were purified by flow cytometric cell sorting on day 6 and CTCF mRNA and protein abundance were determined (A) by real-time RT-PCR normalized to β-actin and expressed as percentage of the control and (B) by Western blotting with tubulin serving as a loading control. (C) Relative cross-linking frequencies between the −70kb and +66 kb CTCF elements and (D) between Ifng as the fixed anchor fragment and other BglII fragments for Th1 cells transduced with CTCF#1, CTCF#2, or control retroviruses are shown by the red, blue, and black lines, respectively. Data (error bars, s.d.) are representative of 4 independent experiments. CTCF (E) and T-bet (F) occupancy at the Ifng locus in Th1 cells transduced with control or CTCF#1 shRNAs; only p values (calculated by Student's t test) ≤0.05 are shown. Data (error bars, s.d.) are representative of 4 independent experiments. ***p<0.005; *p<0.05.

Figure 5. shRNA-mediated knockdown of CTCF impairs Ifng expression

(A) Intracellular staining for the indicated cytokines in Th1 cells transduced with the control shRNA or CTCF shRNAs #1 or #2. The percentage of IFN-γ+ and IL-2+ cells among GFP+ Th1 cells and the mean fluorescence intensity (MFI) for cytokine containing cells are shown 6 hr after restimulation with PMA + ionomycin. (B) Concentrations (mean ± SD) of the indicated cytokines in culture supernatants measured by ELISA. (C) Expression of mRNA (mean ± SD) relative to cells transduced with the control shRNA for the indicated genes. (D) The percentage of Th1 cells containing T-bet protein and the amount as indicated by MFI are shown along with cells stained with an isotype control antibody.

Figure 6. Impaired Ifng expression and three-dimensional organization of the Ifng locus in T-bet-deficient Th1 cells

(A) Concentrations of IFN-γ (mean ± SD) in culture supernatants of wildtype (WT) and Tbx21-/- CD4+ T cells cultured in Th1 or Th2 conditions for 6 days. (B) Relative cross-linking frequencies between Ifng as the fixed anchor fragment and other BglII fragments and (C) between the −70kb CTCF or +66 CTCF element and the indicated BglII fragments for WT Th1 CD4⁺ T cells (dashed dark blue lines) and Tbx21-/- naive, Th1, and Th2 CD4⁺ T cells (black, red, and light blue lines, respectively). Results are presented as in Figure 3. (D) CTCF ChIP in naïve, Th1 and Th2 CD4+ T cells from WT or Tbx21-/- mice. Results are the mean ± SD of 2 experiments; results are shown relative to wildtype naïve T cells at −70kb, which represented 1.05±0.17% of input; only p values (calculated by Student's t test) ≤0.05 are shown. *p<0.05.

Figure 7. In T-bet-deficient Th1 cells, the histone deacetylase inhibitor trichostatiin A (TSA) restores IFN-γ production and 3-dimensional locus conformation but not CTCF occupancy at the Ifnglocus

(A) Concentrations of IFN-γ in culture supernatants, (B) Ifng mRNA expression, (C) intracellular IFN-γ and (D) CTCF ChIP for wildtype and T-bet-/- CD4+ T cells differentiated in Th1 conditions for 5 days with or without TSA. Data (error bars, s.d.) are representative of 4 independent experiments. *, p<0.05. (E) Proposed model for the three-dimensional conformation of the Ifng locus. In naïve CD4⁺ T cells, CTCF is bound primarily at −70kb and T-bet is not bound. Upon Th1 differentiation, T-bet binds to the Ifng promoter and to the CNS-34, CNS+18/20 and CNS+29 enhancers, and CTCF binds strongly at +1kb, in intron 1 of Ifng, and at +66kb in addition to −70kb. This binding contributes to and is required for the juxtaposition of each of these distal regulatory elements to Ifng and its promoter. In this active locus conformation, the CNS-34 enhancer is in close proximity to Ifng but more distant from the +1 and +66 CTCF-binding elements. Filled and open lollipops represent methylated and unmethylated CpGs, respectively.