Characterization of the RNA content of chromatin

Abstract

Noncoding RNA (ncRNA) constitutes a significant portion of the mammalian transcriptome. Emerging evidence suggests that it regulates gene expression in cis or trans by modulating the chromatin structure. To uncover the functional role of ncRNA in chromatin organization, we deep sequenced chromatin-associated RNAs (CARs) from human fibroblast (HF) cells. This resulted in the identification of 141 intronic regions and 74 intergenic regions harboring CARs. The intronic and intergenic CARs show significant conservation across 44 species of placental mammals. Functional characterization of one of the intergenic CARs, Intergenic10, revealed that it regulates gene expression of neighboring genes through modulating the chromatin structure in cis. Our data suggest that ncRNA is an integral component of chromatin and that it may regulate various biological functions through fine-tuning of the chromatin architecture.

Figure 1.

Isolation of chromatin-associated RNA by sucrose gradient. (A) Flowchart showing a brief summary of the method used for isolation of chromatin RNA. (B) Chromatin isolated from MNase-treated human fibroblast nuclei was run on a 1% agarose gel after sucrose gradient fractionation. The lanes (from left to right) in the agarose gel represent DNA extracted from the chromatin fragments collected from the top to bottom fractions of the sucrose gradient. (C) Chromatin-bound RNA, isolated from the different gradient fractions, was used for semiquantitative RT-PCR with beta-actin primers, and the RT product was loaded according to the gradient fractionation in B; the extreme right lane is a positive control for RT-PCR.

Figure 2.

Basic characterization of chromatin-associated RNA. (A) Pie chart showing distribution of the sequencing reads over intronic, intergenic, and exonic portions of the human genome. (B) The table includes information about the aligned chromatin RNA sequencing reads and the detailed analysis of the CARs. (C) Distribution of sequencing reads in the exonic and intronic regions along chromosome 3.

Figure 3.

Intergenic CARs overlap with lincRNAs and associate with PRC2 and RCOR1 complexes. (A) The table shows the position of the CARs, overlapping the lincRNAs present on different chromosomes as indicated, and their association with PRC2 or RCOR1 complexes. (B) The bar graph shows the enrichment of intergenic CARs in the EZH2 pull-down fraction over IgG. The analysis was performed using quantitative RT-PCR with primers corresponding to four intergenic CARs. Details of the primers are given in Supplemental Table S5.

Figure 4.

CARs are conserved across species. Distribution of the proportion of CAR regions that overlap with conserved phastCons elements (44wayPlacental, UCSC Genome Browser) across intronic (A) and intergenic (C) CARs. Distributions of overlap with phastCons elements for the randomized intronic (B) and intergenic (D) control sets from matching genomic locations. (E) Distribution of overlap to randomly selected 1-kb exonic regions. Both intronic and intergenic CAR regions are significantly more conserved than control regions (P = 2.1 × 10⁻¹², P = 7.1 × 10⁻⁴, respectively).

Figure 5.

Validation of the intronic and intergenic CARs by ChRIP. Bar graphs show fold enrichment of the intronic (A) and intergenic (B) CARs over IgG in the histone H3 immunopurified chromatin. (Black bars) control cells; (gray bars) actinomycin D–treated cells. Beta-actin (ACTB) was used as a control, which did not show any significant chromatin enrichment in ChRIP assay.

Figure 6.

Functional validation of an intergenic CAR. (A) The chromosomal location of the Intergenic10 transcript relative to two closely located neighboring genes FANK1 and ADAM12 and distally located UROS and DOCK1 genes. The arrows indicate the direction of the transcription. The relative levels of Intergenic10 (B), ADAM12 (C), and FANK1 (D) in human fibroblast (HF), placenta (P), and brain (B), as measured by quantitative RT-PCR. (E) siRNA knockdown of the Intergenic10 RNA and its effect on the expression of the FANK1 and ADAM12 genes. Percent expression of FANK1 and ADAM12 genes in Integenic10 (gray bars) and control (black bars) siRNA-treated cells. The percentage expression in intergenic siRNA-treated cells is presented relative to control siRNA-treated cells. The distal genes UROS and DOCK1 did not show any significant down-regulation in siRNA-treated cells. RNA amounts were measured by quantitative RT-PCR from three independent experiments, and beta-actin was used for normalization. (F) The siRNA knockdown of Intergenic10 RNA in HF cells affects the active chromatin marks H3K4me2 over ADAM12 and FANK1 promoter. Levels of H3K4me2 were measured by chromatin immunoprecipitation (ChIP) in two independent experiments using antibody against HeK4me2. The enrichment of H3K4me2 was measured by qPCR using the promoter-specific primers. (Black bars) The H3K4me3 enrichment over IgG at the indicated promoters in control siRNA-treated cells; (gray bars) enrichment over IgG in Intergenic10 siRNA-treated cells. DOCK1 and UROS promoters were used as controls, showing no change in H3K4me2 marks.