Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci

Abstract

We have studied developmentally regulated patterns of histone acetylation at high resolution across approximately 54 kb of DNA containing three independently regulated but neighboring genetic loci. These include a folate receptor gene, a 16 kb condensed chromatin region, the chicken beta-globin domain and an adjacent olfactory receptor gene. Within these regions the relative levels of acetylation appear to fall into three classes. The condensed chromatin region maintains the lowest acetylation at every developmental stage. Genes that are inactive show similarly low levels, but activation results in a dramatic increase in acetylation. The highest levels of acetylation are seen at regulatory sites upstream of the genes. These patterns imply the action of more than one class of acetylation. Notably, there is a very strong constitutive focus of hyperacetylation at the 5' insulator element separating the globin locus from the folate receptor region, which suggests that this insulator element may harbor a high concentration of histone acetylases.

Fig. 1. Expression and chromatin structural changes in a 54 kb region containing the FR gene, β-globin gene and chicken olfactory receptor gene in cell lines derived from different erythropoietic stages and a non-erythroid cell. In the cells or cell lines listed, + or – indicates whether the gene in the map below is expressed or silent, respectively. P or A indicates the presence or absence of DNase I-hypersensitive sites. A scale map of the FR gene, condensed chromatin region, β-globin domain and chicken olfactory receptor gene is shown at the bottom. The locations of hypersensitive sites (HSA, HS4 and 3′HS) are indicated by large arrows. ‘Taqman probes’ denotes the name and location of primer pairs and Taqman probes used for analysis.

Fig. 2. Real-time PCR using Taqman probes. (A) A schematic of PCR in the presence of a Taqman probe. (1) Probes and primers anneal to target sequence. Taqman probes have two covalently linked fluorescent dyes: a reporter (R) and a quencher (Q). On the probe, the reporter dye emission is quenched. (2) During each extension cycle, the 5′→3′ exonuclease activity of Taq DNA polymerase cleaves the reporter dye from the probe. (3) Once separated from the quencher, the reporter dye emits its characteristic fluorescence, which is measured in every cycle by the ABI Prism 7700 sequence detector. (B) A representative panel of data generated by the ABI Prism 7700 sequence detector software for Taqman probe 5.613 on samples immunoprecipitated from 6C2 nuclei. IP indicates the signal for the fluorescent reporter detected in each cycle for the immunopreciptated target sequence. Ref indicates a similar analysis of the target sequence abundance in total input DNA. C_t indicates the cycle threshold number. Below the data panel is a sample calculation to determine the fold difference between the IP sample and the reference sample.

Fig. 3. Acetylation across three independently regulated domains of two erythroid lines and 10 day embryonic erythroid cells at different stages of development. The graphs show the relative chromatin immunoprecipitation of acetylated histone H3, acetylated histone H4 and specific acetylated histone H4 lysines of mono and di-nucleosomes from HD24 cells (A), 6C2 cells (B) and 10 day chicken embryo red blood cells (C). The y-axis indicates the fold difference between the input fraction and the bound fraction and the x-axis indicates position in kb relative to the end of the FR gene. Different line colors (top) correspond to acetylation of individual sites (H4 lysine 5, 8 or 12) or multi-acetylated H3 or H4. The lower section shows the name and location of primer pairs and Taqman probes used for analysis. Several DNase I-hypersensitive sites are indicated: HSA, HS4, HS3, HS2, HS1 and 3′HS.

Fig. 4. Acetylation in a non-erythroid cell line. The graph shows the relative chromatin immunoprecipitation of acetylated histone H3, acetylated histone H4 and specific acetylated histone H4 lysines of mono- and dinucleosomes from DT40, a chicken lymphocyte cell line. Designations and map are the same as in Figure 3.

Fig. 5. Two methods of preparation for chromatin used in immunoprecipitation of acetylated histones show similar patterns. (A) Comparison of the relative acetylation of histone H4 measured following two methods of preparation in 10 day chicken embryo erythrocytes. (B) The relative acetylation of Lys5 on histone H4 in 6C2 cells, compared for two different methods of chromatin preparation. In both (A) and (B), the black line shows data for chromatin that was fixed with 1% formaldehyde and sonicated to generate small fragments. The gray dashed line (data from Figure 3) shows results for chromatin prepared from nuclei digested with MNase and purified on a sucrose gradient to isolate mono- and dinucleosomes. Designations and map are the same as in Figure 3.

Fig. 6. Percentage of acetylated nucleosomes in total chromatin immunoprecipitated with specific antibodies. (A) Determination of the maximum percentage of immunoprecipitated nucleosomes acetylated at specific histones or histone sites. Each tube contains a fixed amount of antibody, always in excess over chromatin. Increasing amounts of chromatin are added and the fraction of IP material, M⁺, is measured (see Materials and methods). T is the amount of input chromatin at each point in the titration. T₀ is the amount of chromatin in the tube with the highest concentration. The immunoprecipitate (M⁺) as a fraction of T₀ (M⁺/T₀) is plotted against the input chromatin (T) as a fraction of T₀ (T/T₀). The line represents an average of data from separate titrations of the same antibody. The percentage of acetylated nucleosomes in total chromatin for a specific histone or histone sites is determined from the value of the slope (see Materials and methods). The linearity and non-zero slope of the plots are consistent with the presence in each tube of an antibody excess. This does not preclude the possibility that a sub-population of acetylated nucleosomes is not precipitable by the antibody. The fact that the line does not go through the origin shows that there is a fixed amount of material that precipitates in each tube. (B) A table of the percentage of acetylated nucleosomes in total chromatin. Specific histones and histone H4 acetylation sites are indicated above. The maximum percentage of nucleosomes immunoprecipitated by a specific anti-acetyl-histone antibody is indicated immediately below. (C) The absolute percentage of acetylation of histone H3 in 6C2 cells across the 54 kb region. The y-axis indicates the percentage of acetylation. All other designations are the same as in Figure 3.