Dynamic changes in histone H3 Lys 9 methylation occurring at tightly regulated inducible inflammatory genes

Abstract

Methylation of histone H3 at Lys 9 is causally linked to formation of heterochromatin and to long-term transcriptional repression. We report an unexpected pattern of H3 Lys 9 methylation occurring at a subset of inducible inflammatory genes. This pattern is characterized by relatively low constitutive levels of H3 Lys 9 methylation that are erased upon activation and restored concurrently with post-induction transcriptional repression. Changes in H3 Lys 9 methylation strongly correlate with RNA polymerase II recruitment and release. In particular, remethylation correlates with RNApolII release more strongly than does histone deacetylation. We propose that, by generating a window of time in which transcription is permitted, dynamic modulation of H3 Lys 9 methylation adds an additional regulatory level to transcriptional activation of tightly controlled inducible genes.

Figure 1

Kinetics of LPS-induced gene expression in human monocyte-derived DCs. (A) Steady-state mRNA levels of LPS-induced genes were analyzed by RT-PCR. Dendritic cells were stimulated with 100 ng/ml LPS as indicated. (B) RNApolII recruitment to the same genes was analyzed by Chromatin Immunoprecipitation (ChIP) assay using an antibody directed against the RNApolII large subunit Rpb1. Immunoprecipitated DNA was analyzed by PCR using promoter-specific primers. The peak of RNApolII recruitment to the IkBα, IL-8 and MIP-1α genes occurs at earlier time points (0.5–2 h) than those shown in the figure. (C) Promoter-specific histone H3 and H4 acetylation was investigated by ChIP assays carried out with antibodies against acetylated H3 (Ac-Lys 9/Lys 14) and H4. The antibody against hyperacetylated H4 has a preference towards tetra- and tri-acetylated H4.

Figure 2

Dynamic changes in histone H3 Lys 9 methylation levels at the promoters of tightly regulated inducible genes. (A) DCs were stimulated with LPS as indicated and bulk levels of H3 methylated at Lys 9 were analyzed by Western blot. (B) Lys 9 methylation of H3 at the promoters of the genes under investigation was analyzed by ChIP assay. The left panel (Input) shows the relative amounts of the probed regions contained in the starting chromatin extracts. The right panel shows ChIP analysis of specific association of H3 methylated at Lys 9 with the promoters investigated. The induction of the genes we analyzed is asynchronous in LPS-stimulated DCs. It is therefore possible that the apparent gradation in H3 Lys 9 methylation changes (and in RNApolII recruitment) reflects different kinetics with which such changes occur in individual cells of the population analyzed. (C) LPS-stimulated monocytes express very low levels of ELC, MDC, and IL-12p40 mRNAs (transcripts can be detected at a higher number of PCR cycles). No detectable H3 Lys 9 demethylation was observed in these cells (right). (D) Density of H3 Lys 9 methylation at inducible euchromatic genes with respect to heterochromatic genes. Signals obtained in the Me/Lys 9 ChIP assay were compared to serial twofold dilutions of a fraction of the input. PCR was carried out at a lower number of cycles than in the experiment shown in B,C to maintain the signals obtained with differentially methylated promoters in a linear range. (E) ChIP analysis of promoter-specific H3 Lys 4 methylation at inducible genes. Promoter-specific levels of H3 Lys 4 methylation were analyzed by ChIP assay using an antibody recognizing H3 methylated at Lys 4. The negative controls for these experiments were parallel samples in which the immunoprecipitation step was carried out without antibody (data not shown). In these samples, only background levels of promoter sequences were detectable after 40 cycles of PCR.

Figure 3

Histone H3 methylation at Lys 9 across the ELC/SLC locus. Sequence data were retrieved from the National Center for Biotechnology Information (NCBI; accession no. 17451799). (Left) A schematic representation of the region containing the SLC and ELC genes. Cent., centromere; Tel., telomere. Positions of the sets of primers (A–F) used are shown. (Middle) ChIP assays carried out with an anti-methyl H3 Lys 9 antibody. In the right panels, anti-acetyl H3 ChIP assays show the levels of H3 acetylation over the same region. Input: PCR products obtained with each set of primers using as template a fraction of the ChIP inputs (untreated samples). In DCs from this donor a complete remethylation of the ELC gene and surrounding regions was observed at 48 h after LPS stimulation. The distances between the regions amplified are A > B 3.6 Kbp; B > C 6.5 Kbp; C > D 4.5 Kbp; D > E 4.2 Kbp; E > F 4.6 Kbp.

Figure 4

A schematic representation of histone methylation and acetylation changes occurring at a subset of inducible genes. RNApolII recruitment and release kinetics are shown in parallel to histone acetylation and methylation changes occurring at a subset of inducible inflammatory genes.