Widespread, exceptionally high levels of histone H3 lysine 4 trimethylation largely mediate "privileged" gene expression

Abstract

We examined the molecular determinants for sustained high-level expression of "privileged" genes, defined as the 0.03% most highly expressed genes within any specific cell. We identified histone modifications by chromatin immunoprecipitation analyses on Keratin 8, the most highly expressed gene in the human breast cancer cell line, MCF-7, based on serial analysis of gene expression. Quantitative comparisons to the "normal" counterpart cell line, MCF-10A, expressing 350-fold lower levels of Keratin 8 and other breast cancer cell lines expressing higher levels were performed using real-time PCR. Extraordinarily high levels of trimethyl histone H3 lysine 4 (H3K4) were found primarily in the first intron of the Keratin 8 gene stretching from 400 to 2000 bp downstream from the promoter in all breast cancer cells lines but not in MCF-10A cells. The highest levels of histone H3K4 trimethylation in MCF-7 cells ranged from 70% to 80% over input within 1200 bp of this region. Knockdown of mixed-lineage leukemia (MLL), the specific methyltransferase for histone H3K4, with MLL-specific siRNA decreased histone H3K4 trimethylation on the Keratin 8 gene and decreased Keratin 8 mRNA levels. Histone H3K4 trimethylation mediates approximately 86% of the elevated, sustained expression of the Keratin 8 gene in MCF-7 cells.

Figure 1

Keratin 8 mRNA levels in MCF-7, SK-BR-3, T-47D, and MCF-10A cells. The expression levels of Keratin 8 mRNA were quantified by real-time RT-PCR and normalized to the housekeeping gene β-actin. Results are expressed as the ratio relative to Keratin 8 mRNA level in MCF-10A cells. All data are presented as the means ± SD of three independent experiments. *p < 0.01 versus MCF-10A by Student’s t-test.

Figure 2

Histone H3 K4 trimethylation within the Keratin 8 gene in MCF-7 and MCF-10A cells. ChIP assays were performed with anti-trimethyl K4 Histone H3. The amount of precipitated DNA was quantified relative to input as described in Materials and Methods. The data are presented as the means ± SD of two independent experiments. The initiation site of TATA box is designated at +1.

Figure 3

Histone modifications within the Keratin 8 gene locus in MCF-7 and MCF-10A cells. ChIP assays were performed with anti-acetyl histone H4 (A), anti-acetyl histone H2B (B), anti-acetyl histone H3 K9 (C), or anti-dimethyl histone H3 K9 (D). Normal Rabbit IgG served as the negative control (E). The amount of precipitated DNA was quantified relative to input as described in Materials and Methods. All data are presented as the means ± SD of two independent experiments. The initiation site of TATA box is designated at +1. (F) Summary and comparison of the histone modifications examined on the Keratin 8 gene in MCF-7 cells.

Figure 3

Histone modifications within the Keratin 8 gene locus in MCF-7 and MCF-10A cells. ChIP assays were performed with anti-acetyl histone H4 (A), anti-acetyl histone H2B (B), anti-acetyl histone H3 K9 (C), or anti-dimethyl histone H3 K9 (D). Normal Rabbit IgG served as the negative control (E). The amount of precipitated DNA was quantified relative to input as described in Materials and Methods. All data are presented as the means ± SD of two independent experiments. The initiation site of TATA box is designated at +1. (F) Summary and comparison of the histone modifications examined on the Keratin 8 gene in MCF-7 cells.

Figure 4

Histone H3 K4 trimethylation within the Keratin 8 gene in SK-BR-3 and T-47D cells. ChIP assays were performed with anti-trimethyl K4 histone H3 in SK-BR-3 cells (A) or in T-47D cells (B). The amount of precipitated DNA was quantified relative to input as described in Materials and Methods. The data are presented as the means ± SD of two independent experiments. The initiation site of TATA box is designated at +1.

Figure 5

Effect of MLL knockdown on Keratin 8 mRNA levels in MCF-7, SK-BR-3, T-47D, and MCF-10A cells. Cells were transfected with MLL siRNA (+) or with control siRNA (−) and harvested 72 h after transfection. (A) Treatment with MLL siRNA results in a decrease of MLL mRNA levels compared to controls. Results are expressed as the percent ratio relative to Keratin 8 mRNA level in control cells. All data are presented as the means ± SD of three independent experiments. (B) Treatment with MLL siRNA results in a decrease of MLLc protein levels compared to controls. Whole cell lysate was subjected to Western blotting using anti-MLLc antibody. β-Actin served as the loading control. (C) Analyses of the effect of MLL knockdown on Keratin 8 mRNA levels. Results are expressed as the ratio relative to the Keratin 8 mRNA level in control MCF-10A cells. All data are presented as the means ± SD of three independent experiments. *p < 0.0001 versus control by Student’s t-test.

Figure 6

Effect of MLL knockdown on Histone H3 K4 trimethylation within the Keratin 8 gene. MCF-7 (A), SK-BR-3 (B), T-47D (C), and MCF-10A (D) cells were transfected with MLL siRNA or unrelated control siRNA and used for ChIP assays 72 h after transfection. The amount of precipitated DNA was quantified relative to input as described in Materials and Methods. All data are presented as the means ± SD of two independent experiments. The initiation site of TATA box is designated at +1.