Nuclear localization and histone acetylation: a pathway for chromatin opening and transcriptional activation of the human beta-globin locus

Abstract

We have investigated the mechanism, structural correlates, and cis-acting elements involved in chromatin opening and gene activation, using the human beta-globin locus as a model. Full transcriptional activity of the human beta-globin locus requires the locus control region (LCR), composed of a series of nuclease hypersensitive sites located upstream of this globin gene cluster. Our previous analysis of naturally occurring and targeted LCR deletions revealed that chromatin opening and transcriptional activity in the endogenous beta-globin locus are dissociable and dependent on distinct cis-acting elements. We now report that general histone H3/H4 acetylation and relocation of the locus away from centromeric heterochromatin in the interphase nucleus are correlated and do not require the LCR. In contrast, LCR-dependent promoter activation is associated with localized histone H3 hyperacetylation at the LCR and the transcribed beta-globin-promoter and gene. On the basis of these results, we suggest a multistep model for gene activation; localization away from centromeric heterochromatin is required to achieve general hyperacetylation and an open chromatin structure of the locus, whereas a mechanism involving LCR/promoter histone H3 hyperacetylation is required for high-level transcription of the beta-globin genes.

Figure 1

Different alleles of the human β-globin locus. Shown are the wild-type locus, present in the N-MEL and wt-MEL cell lines, the 5′HS2–5 deletion present in Δ2–5-MEL, and the Hispanic deletion allele present in T-MEL. Position of the five genes is represented by solid boxes, and strong hypersensitive sites by vertical arrows (for details, see Bulger et al. 1999). Transcription of the β gene in the wild-type allele is indicated by a horizontal arrow. Sequences analyzed in this study are indicated below each allele, and the corresponding primers for their amplification are listed in Materials and Methods.

Figure 2

Immunoprecipitation and duplex PCR assays. (A) Depletion of centromeric (heterochromatic) sequences and an inactive mouse gene in chromatin enriched for acetylated histone H4. Chromatin from MEL cells was immunoprecipitated with an antibody that detects all acetylated isoforms of histone H4 (αH4–Ac). Input and antibody-bound DNA (500 ng) were slot blotted and hybridized with an oligomer corresponding to a murine centromeric minor satellite repeat (R947, Kipling et al. 1994). This sequence is 2.8-fold less abundant in the antibody bound fraction. The same blot was rehybridized with a probe from the mouse amylase gene (generated by PCR with the primer pairs amy4 and amy6, see Materials and Methods). This pancreatic-specific gene, which is inactive in a red cell background, is slightly less abundant in chromatin enriched for acetylated H4. (B) Abundance of human and mouse globin sequences in chromatin enriched for acetylated histones was determined relative to the mouse amylase gene using a duplex PCR assay (see text and Fig. 3). One primer pair amplifies a sequence from the mouse amylase gene, the other pair amplifies either a human or mouse β-globin locus sequence. To determine conditions of linear amplification, serial dilutions of chromatin containing 0.5–4 ng of DNA were used as template. Shown are products and quantification for two representative primer pairs (βPr with amy4 + 6 and 3′β with amy4 + 5), revealing linear amplification of the total signal (bars) and a constant ratio (line) for the two products under these PCR conditions (see Materials and Methods). For the quantitative analysis of immunoprecipitated material shown in Figs. 3 and 4, 1–2 ng of DNA were used per reaction to ensure amplification in the linear range.

Figure 2

Immunoprecipitation and duplex PCR assays. (A) Depletion of centromeric (heterochromatic) sequences and an inactive mouse gene in chromatin enriched for acetylated histone H4. Chromatin from MEL cells was immunoprecipitated with an antibody that detects all acetylated isoforms of histone H4 (αH4–Ac). Input and antibody-bound DNA (500 ng) were slot blotted and hybridized with an oligomer corresponding to a murine centromeric minor satellite repeat (R947, Kipling et al. 1994). This sequence is 2.8-fold less abundant in the antibody bound fraction. The same blot was rehybridized with a probe from the mouse amylase gene (generated by PCR with the primer pairs amy4 and amy6, see Materials and Methods). This pancreatic-specific gene, which is inactive in a red cell background, is slightly less abundant in chromatin enriched for acetylated H4. (B) Abundance of human and mouse globin sequences in chromatin enriched for acetylated histones was determined relative to the mouse amylase gene using a duplex PCR assay (see text and Fig. 3). One primer pair amplifies a sequence from the mouse amylase gene, the other pair amplifies either a human or mouse β-globin locus sequence. To determine conditions of linear amplification, serial dilutions of chromatin containing 0.5–4 ng of DNA were used as template. Shown are products and quantification for two representative primer pairs (βPr with amy4 + 6 and 3′β with amy4 + 5), revealing linear amplification of the total signal (bars) and a constant ratio (line) for the two products under these PCR conditions (see Materials and Methods). For the quantitative analysis of immunoprecipitated material shown in Figs. 3 and 4, 1–2 ng of DNA were used per reaction to ensure amplification in the linear range.

Figure 3

Comparative analysis of histone H3 and H4 acetylation at specific sequences in different alleles of the globin locus. Duplex PCR was performed on the input and bound fractions from the chromatin immunoprecipitation experiments. (see Fig. 2 and text). Three antibodies were used for immunoprecipitation. αH3–Ac recognizes histone H3 acetylated at lysines 9 and 14; αH4–Ac recognizes any acetylated isoforms of H4; and αH4–Ac8 is specific for H4 acetylated at lysine 8. To control for nonspecific binding, immunoprecipitation experiments were performed in parallel with rabbit preimmune serum (Pre), and a background signal of 10% or less compared with any antibody containing immunoprecipitation was observed. The figure shows PCR reactions from one representative experiment. Chromatin immunoprecipitation and PCR analysis were repeated at least twice with consistent results. Quantification reveals up to ninefold enrichment for a human globin sequence in N-MEL and wt-MEL (see Fig. 4) compared with the mouse amylase gene. HS2 is deleted in Δ2–5-MEL and T-MEL (see Fig. 1) and therefore could not be analyzed in these hybrids.

Figure 4

Quantification of duplex PCR results. The ratio of products obtained with β-globin and amylase primers was determined for each input and antibody-bound sample for each cell line. The globin/amylase ratio from each bound fraction was standardized by dividing by the globin/amylase ratio from the input material to determine enrichment or depletion of a globin sequence during the immunoprecipitation. Enrichment of the globin sequence over amylase is therefore reflected by a number >1 and a depletion <1. The X-axis is drawn at 1, which reflects no enrichment. (A) Results obtained with the αH4–Ac antibody, (B) the αH4–Ac8 antibody, and (C) the αH3–Ac antibody. The sequence for the HS2 primer set is deleted in T-MEL and Δ2–5. (nd) Not done.

Figure 5

Expression of β-globin mRNA. RT–PCR was performed with a primer pair which coamplifies the human and murine adult β-globin mRNA, followed by restriction enzyme digestion specific for the human product, allowing separation of the human and murine products by gel electrophoresis. Bands are quantified and signal intensities are corrected for the size difference between the digested human (hu) product and the undigested mouse (mo) product, but not for the number of alleles (mouse 2, human 1).

Figure 6

FISH analysis of the localization of the human β-globin locus in interphase MEL nuclei. (A) Examples of interphase nuclei from MEL cell hybrids carrying a human chromosome 11 containing a wild-type β-globin locus (N-MEL), Hispanic deletion (T-MEL), or deletion of 5′HS2–5 of the LCR (Δ2–5-MEL). DAPI is shown in blue, centromeres in red, and the β-globin locus in green. In MEL hybrids containing an open β-globin locus (N-MEL and Δ2–5-MEL), the green signal for the globin locus is found away from a red signal for centromeres, regardless of β-globin gene transcription. In contrast, the locus is in close proximity to centromeres in hybrids harboring a silent, closed locus (T-MEL). (B) Box plot representing the distance between the globin locus and the closest murine centromere cluster. Data were collected for each cell line from at least 35 interphase nuclei. Horizontal bars represent the 10th, 25th, 50th (median), 75th, and 90th percentiles, and p values for pairs of samples are indicated (see Materials and Methods). The distance between the globin locus and the closest murine centromere cluster is significantly higher when the locus is in an open configuration (N-MEL and Δ2–5-MEL), than when it is in a closed configuration (T-MEL).