A defect in nucleosome remodeling prevents IL-12(p35) gene transcription in neonatal dendritic cells

Abstract

To gain insight into the inability of newborns to mount efficient Th1 responses, we analyzed the molecular basis of defective IL-12(p35) expression in human neonatal monocyte-derived dendritic cells (DCs). Determination of IL-12(p35) pre-mRNA levels by real-time RT-PCR revealed that transcriptional activation of the gene in lipopolysaccharide-stimulated neonatal DCs was strongly impaired compared with adult DCs. We next showed that p50/p65 and p65/p65 dimers interact with kB#1 site, a critical cis-acting element of the IL-12(p35) promoter. We found that LPS-induced p65 activation was similar in adult and newborn DCs. Likewise, in vitro binding activity to the Sp1#1 site, previously shown to be critical for IL-12(p35) gene activation, did not differ in adults and newborns. Since the accessibility to this Sp1#1 site was found to depend on nucleosome remodeling, we used a chromatin accessibility assay to compare remodeling of the relevant nucleosome (nuc-2) in adult and neonatal DCs. We observed that nuc-2 remodeling in neonatal DCs was profoundly impaired in response to lipopolysaccharide. Both nuc-2 remodeling and IL-12(p35) gene transcription were restored upon addition of recombinant interferon-gamma. We conclude that IL-12(p35) transcriptional repression in neonatal DCs takes place at the chromatin level.

Figure 1.

Deficient transcriptional activation of the IL-12(p35) gene in LPS-stimulated neonatal DCs. Adult (black bars) or neonatal DCs (white bars) were either incubated with medium alone or stimulated with LPS (1 μg/ml) and/or rIFN-γ (100 U/ml) for 5 h. IL-12(p35) pre-mRNA levels were normalized using β-actin mRNA levels and compared with unstimulated conditions. Data are shown as mean ± SEM of at least eight independent experiments on different donors. ***P < 0.001 compared with adult DCs.

Figure 2.

Physical and functional characterization of a κB site within the p35 proximal promoter region. (A) Competition assays. Nuclear extracts from LPS-stimulated adult DCs (10 μg) were incubated with radiolabeled κB#1wt probe in the absence or the presence of increasing concentrations of the indicated unlabeled competitor. Three protein–DNA complexes (A–C) were observed. (B) Supershift assays. DCs were incubated with medium alone or activated for 2 h with LPS. Nuclear extracts were incubated with the indicated specific antibody before adding the radiolabeled κB#1wt probe. NS, nonspecific. (C) κB#1 site is necessary for basal and inducible p35 promoter activity. RAW 264.7 cells were transiently transfected with 2 μg p35-lucWT or p35-mutκB#1 reporter plasmid and 20 ng pRL-TK as an internal control. Promoter activities were normalized using Renilla luciferase activities. Values represent the means ± SEM of three independent experiments performed in triplicates.

Figure 3.

Comparison of NF-κB and Sp1 binding activities in adult and neonatal DCs. (A) Assessment of NF-κB p65 activation in adult and neonatal DCs. DCs were left untreated or stimulated for 2 h with LPS. Results are presented as mean ± SEM from at least four different donors. (B) Constitutive binding activity to the Sp1#1 site in neonatal and adult DCs. DCs were left untreated or stimulated with LPS or LPS + IFN-γ for 2 h. Nuclear extracts (10 μg protein) were then isolated and incubated with radio-labeled Sp1#1wt probe from the p35 promoter (9). To ensure specificity of the binding, nuclear extracts from LPS + IFN-γ–stimulated adult DCs were incubated with radiolabeled Sp1#1wt probe in the presence of a 50-fold molar excess of unlabeled Sp1 consensus (CONS) or mutated consensus (CONS Mut) (9). The results are representative of three independent experiments. (C) Nuclear extracts (5 and 2.5 μg protein) from adult and neonatal LPS-stimulated DCs were incubated with radiolabeled Sp1#1wt probe from the p35 promoter. (D) Schematic representation of the nucleosome organization of the p35 promoter. Positioned nucleosomes (ovals) are indicated. nuc-2 (−310/−160, broken lined oval) is selectively remodeled on p35 transcriptional activation. Locations of the Sp1#1, Sp1#2, and the κB#1 sites are shown. The BstXI cleavage sites and the relative position of primer sets A and B, used for the CHART-PCR experiments, are also represented.

Figure 4.

Impaired nuc-2 remodeling in LPS-stimulated neonatal DCs. Accessibility of the IL-12(p35) promoter region protected by nuc-2 to BstXI was assessed by indirect end labeling and CHART-PCR. Adult and neonatal DCs were incubated with medium alone or stimulated with LPS and/or IFN-γ for 3 h. Intact nuclei were incubated with BstXI. (A) After genomic DNA purification and in vitro digestion with EcoRI, DNA samples were analyzed by Southern blotting and the indirect end labeling technique. The results are representative of five independent experiments performed with different blood donors. The different BstXI restriction sites located in regions of the p35 locus (nt –298, +117, +456) are indicated by arrows. (B) For CHART-PCR assays, genomic DNA samples were subjected to real-time analysis using primer sets A and B as described in Materials and Methods. Results are expressed as a percentage of the accessibility observed in the unstimulated digested samples. Data shown are the mean ± SEM obtained from 9 neonatal and 15 adult samples. ***P < 0.001 compared with adult DCs. NS, not significant. (C) nuc-2 remodeling is insensitive to α-amanitin or mithramycin. Adult DCs were pretreated or not with α-amanitin (5 μg/ml) or mithramycin (20 nM) for 1 h. Cells were then incubated with medium alone or stimulated with LPS and IFN-γ for 5 h (real-time RT-PCR experiments, left) or 3 h (CHART-PCR experiments, right).