Epigenetic changes and suppression of the nuclear factor of activated T cell 1 (NFATC1) promoter in human lymphomas with defects in immunoreceptor signaling

Abstract

The nuclear factor of activated T cell 1 (Nfatc1) locus is a common insertion site for murine tumorigenic retroviruses, suggesting a role of transcription factor NFATc1 in lymphomagenesis. Although NFATc1 is expressed in most human primary lymphocytes and mature human T- and B-cell neoplasms, we show by histochemical stainings that NFATc1 expression is suppressed in anaplastic large cell lymphomas and classical Hodgkin's lymphomas (HLs). In HL cell lines, NFATc1 silencing correlated with a decrease in histone H3 acetylation, H3-K4 trimethylation, and Sp1 factor binding but with an increase in HP1 binding to the NFATC1 P1 promoter. Together with DNA hypermethylation of the NFATC1 P1 promoter, which we detected in all anaplastic large cell lymphoma and many HL lines, these observations reflect typical signs of transcriptional silencing. In several lymphoma lines, methylation of NFATC1 promoter DNA resulted in a "window of hypomethylation," which is flanked by Sp1-binding sites. Together with the under-representation of Sp1 at the NFATC1 P1 promoter in HL cells, this suggests that Sp1 factors can protect P1 DNA methylation in a directional manner. Blocking immunoreceptor signaling led to NFATC1 P1 promoter silencing and to a decrease in H3 acetylation and H3-K4 methylation but not DNA methylation. This shows that histone modifications precede the DNA methylation in NFATC1 promoter silencing.

Figure 1

Immunohistochemical stainings of human lymphomas showing the loss of NFATc1 expression in ALCL and cHL. A and B: Negative NFATc1 immunostaining of tumor cells in anaplastic lymphoma kinase-positive (A) and anaplastic lymphoma kinase-negative (B) systemic ALCL cases. C: Enteropathy-type T-cell lymphoma with strong cytosolic staining of NFATc1. D: Strong homogenous cytoplasmic NFATc1 positivity in a mantle B-cell lymphoma. E: Large Hodgkin’s and Reed-Sternberg cells in a cHL demonstrating the loss of NFATc1 expression. F: Nodular lymphocyte predominance Hodgkin’s lymphoma showing a strong membraneous NFATc1 positivity. Therefore, in contrast to cHL, primary nodular lymphocyte predominance Hodgkin’s lymphoma cells express NFATc1.

Figure 2

Hypermethylation of NFATC1 P1 promoter DNA in HL and ALCL cell lines. A: Scheme of the human chromosomal NFATC1 gene and its P1 promoter region. The inducible promoter P1 and the proximal polyA site that control NFATc1/αA expression are shown in red, the constitutive promoter P2 and the distal polyA2 site are shown in green. Horizontal arrows indicate transcription starts directed by the promoters P1 and P2, respectively. RSD, Rel similarity domain. The organization of NFATC1 gene in exons (Ex1, Ex2, etc.) is also indicated. Below, the organization of P1 promoter region is shown. CpG dinucleotides are marked by vertical dashes. The Sp1/Sp3 binding sites are shown as large black dots that flank the proximal and distal blocks of homology of P1 promoter region. Binding sites are indicated for inducible transcription factors within these blocks of homology. The binding of transcription factors to these sites has been shown in EMSAs (data not shown). The major start site of transcription is indicated by +1. B: DNA methylation at the CpG dinucleotides of distal block of homology of P1 promoter in human peripheral blood lymphocytes (PBL), Jurkat T leukemia cells, in the HL cells lines KM-H2, L1236, L428, and L540 and the ALCL lines Sup-M2, SR-786, and Karpas 299. Filled boxes indicate full DNA methylation, empty boxes no methylation, and half-filled boxes partial methylation. The binding sites for Sp1/Sp3 transcription factors are indicated. C: Typical Western immunoblots for the detection of NFATc1 expression in peripheral blood lymphocytes, Jurkat, KM-H2, L428, and L540 HL cells. The cells were either noninduced (−) or induced by T+I (+) for 4 hours. Equal protein loading was checked by Poinceau staining of membranes. A, B, and C mark the position of NFATc1 isoforms in blot of peripheral blood lymphocytes. T+I, TPA (10 ng/ml) and ionomycin (0.5 μM) induction for 4 hours.

Figure 3

Re-expression of NFATc1, BOB.1/OBF1, PLCγ2, and Syk RNAs on inhibition of DNA methylation by 5-aza-dC. KM-H2, SUP-M2, L428, or L540 cells were treated for 4 days with 1, 2.5, or 5 μmol/L 5-aza-dC, and the RNA of cells was isolated for the detection of specific RNA concentrations in real-time PCR assays (see details in Materials and Methods). The “fold change” refers to the expression of corresponding RNAs in nontreated cells. All measurements were performed as triplicates, and one typical experiment from two assays is shown.

Figure 4

Binding sites of transcription factors Sp1 and Sp3 mark the borders of DNA methylation within the P1 NFATC1 promoter. A: Factor binding to the two Sp1/Sp3 sites around the positions −585 and −825 that flank a “window of hypomethylation” (see Figure 2B) within the distal block of homology of the P1 promoter. In EMSAs, the binding of Sp1 and Sp3 in nuclear protein extracts from Jurkat cells is demonstrated by competition with a 50-fold excess of an unlabeled consensus Sp1-binding site (lanes 2 and 6) and by supershifts with Abs specific for Sp1 (lanes 3 and 7, labeled by arrows) and Sp3 (see lanes 4 and 8). In lanes 9 to 16, EMSAs were performed with nuclear protein from uninduced cells (−) or from Jurkat, KM-H2, L428, and L540 cells induced by T+I for 4 hours (+). B: Sp1/Sp3 binding to the −15 and −210 sites of P1 promoter in the proximal block of homology. Nuclear proteins from induced Jurkat cells were incubated with an oligonucleotide probe of site −15 (lanes 1 to 10), of site −210 (lanes 21 to 29) or a Sp1 consensus site (lanes 11 to 20). In supershift assays, Sp1- and Sp3-specific Abs (αSp1 and αSp3) were added as indicated. For competition, a 5- or 50-fold excess of unlabeled oligonucleotides of a tandemly arranged Sp1/3 site from the P2 promoter (Sp1/2_tn), a consensus Sp1/Sp3 site (cSp1/3), a mutated Sp1/3 consensus site (mSp1/3), or a Sp1/3₋₁₅ site were added to the incubations as indicated. C: DNA methylation does not impair Sp1/Sp3 binding. A chemically synthesized −210 oligonucleotide probe methylated at all C residues (methyl-Sp1/3) was incubated with nuclear protein from Jurkat cells and assayed as in A and B. For competition, a 5- or 50-fold molar excess was added of cSp1/3, a consensus Sp1/Sp3 site, mSp1/3, a mutated Sp1/3 consensus site, and methyl-Sp1/3, a Sp1/3₋₂₁₀ site that was methylated at all Cs. D: Detection of Sp1 expression in Jurkat, KM-H2, L428, and L540 cells by Western blot immunoassays. Nuclear proteins were analyzed from uninduced cells (lanes 1, 3, 5, and 7) or from cell induced by T+I for 4 hours (lanes 2, 4, 6, and 8). Comparable protein loading of lanes of the gel was detected by staining of filters with Poinceau red. E: Detection of Sp1 binding to the P1 and DHFR promoters in vivo by ChIP assays. Nuclear proteins were analyzed from uninduced cells (lanes 1, 3, 5, and 7) or from cells induced as in D (lanes 2, 4, 6, and 8).

Figure 5

Reduction of histone H3 acetylation, H3-K4 trimethylation, and Sp1 binding in the HL cell line L540 in which NFATc1 expression is suppressed. ChIP assays. Jurkat cells and the HL cell lines L540 and KMH2, either noninduced (−) or induced by T+I for 4 hours (+) were cross-linked by formaldehyde, and their nuclei were isolated and sonicated. The resulting chromatin fragments were immunoprecipitated using Abs specific for acetylated histone H3, trimethylated H3-K4, di- and trimethylated H3-K9, HP1, or Sp1. After DNA isolation, promoter DNAs were amplified by PCR using primers for the distal block of homology of the P1, the β-actin, or the DHFR promoters followed by gel electrophoresis.

Figure 6

Inhibition of T-cell receptor signaling by CsA results in a block of NFATc1 expression and decrease in histone H3 acetylation and H3-K4 trimethylation at the murine Nfatc1, but not Nfatc2, promoter. A: Western immunoblot assays of whole protein extracts from EL-4 T lymphoma cells pretreated with 100 ng/ml CsA for 48 hours followed by induction with T+I for 6 hours in the absence or presence of CsA. Polyclonal Abs raised against the N-terminal halves of either NFATc1 or c2 were used for immunodetection. B: ChIP assays using chromatin preparations from EL-4 cells that were treated as in A. In PCRs, primers for the detection of Nfatc1 P1 or Nfatc2 promoter DNAs were used (see Materials and Methods).