Formation of a large, complex domain of histone hyperacetylation at human 14q32.1 requires the serpin locus control region

Abstract

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system to study cell-type-specific gene expression and chromatin structure. Activation of the serpin locus can be induced in vitro by transferring human chromosome 14 from non-expressing to expressing cells. Serpin gene activation in expressing cells is correlated with locus-wide alterations in chromatin structure, including the de novo formation of 17 expression-associated DNase I-hypersensitive sites (DHSs). In this study, we investigated histone acetylation throughout the proximal serpin subcluster. We report that gene activation is correlated with high levels of histone H3 and H4 acetylation at serpin gene promoters and other regulatory regions. However, the locus is not uniformly hyperacetylated, as there are regions of hypoacetylation between genes. Furthermore, genetic tests indicate that locus-wide controls regulate both gene expression and chromatin structure. For example, deletion of a previously identified serpin locus control region (LCR) upstream of the proximal subcluster reduces both gene expression and histone acetylation throughout the approximately 130 kb region. A similar down regulation phenotype is displayed by transactivator-deficient cell variants, but this phenotype can be rescued by transfecting the cells with expression cassettes encoding hepatocyte nuclear factor-1alpha (HNF-1alpha) or HNF-4. Taken together, these results suggest that histone acetylation depends on interactions between the HNF-1alpha/HNF-4 signaling cascade and the serpin LCR.

Figure 1

Genomic structure of the proximal serpin subcluster and surrounding regions. The proximal serpin subcluster (α1AT, ATR, CBG and ZPI) extends from ∼−45 to ∼+110 kb. This region contains 17 expression-associated DHSs, 9 constitutive DHSs, and 6 MARs. The positions of the PCR amplicons used in ChIP assays described in this report are indicated.

Figure 2

Histone acetylation in the proximal serpin subcluster in expressing, non-expressing and Δ8.0 mutant hybrid cells. Representative examples of duplex PCRs used to quantitate levels of histone H3 (αAcH3) and H4 (αAcH4) acetylation in expressing [F(14n)14], non-expressing [R(14n)6] and Δ8.0 mutant hybrid cells. Results for amplicons from the α1AT MAR, the α1AT hepatic promoter (α1AT_H), the CBG promoter (CBG_P), the CBG intron 1 MAR (CBG_IMAR), the ZPI promoter (ZPI_P), the KIAA1622 promoter (KIAA_P) and the ubiquitously expressed rP4H promoter (rP4H_P) are shown. All experimental determinations were normalized to signals from the non-expressed Goosecoid (GSC) control, which is ∼380 kb distal to α1AT on human chromosome 14. Input–input DNA. IgG-pre-immune serum control. The numbers below each lane are the levels of the test amplicon relative to the Goosecoid control amplicon in the same sample, normalized to the IgG control, as quantitated using a Typhoon PhosphorImager and Image Quant software.

Figure 3

Patterns of histone acetylation in the proximal serpin subcluster in expressing and non-expressing cells. The graph shows patterns of histone acetylation across the proximal serpin subcluster in expressing and non-expressing cells from distal (left) to proximal (right). The black and dark gray bars represent histone H3 and H4 acetylation, respectively, in expressing F(14n)14 cells, whereas the light gray and white bars represent histone H3 and H4 acetylation, respectively, in non-expressing R(14n)6 cells. Amplicons that were assayed in these experiments included promoter regions of the Centerin (CENT_P), APL-2 (APL-2_P), α1AT (A1AT_M and α1AT_H), CBG (CBG_P), ZPI (ZPI_P), KIAA 1622 (KIAA 1622_P), and rat prolyl-4-hydroxylase (rP4H_P) genes. Results from amplicons in the regions of expression-associated DNase I-hypersensitive sites at −45, −24 and −6 kb are also shown, as are those from the α1AT MAR, the ATR MAR and the CBG Intron 1 MAR, as well as an intergenic (INT) sequence between ATR and CBG. The positions of these amplicons on the genomic map of the region are shown in Figure 1. Each column represents the average of at least two independent determinations, with the standard deviations shown.

Figure 4

Cell-specific histone acetylation in the proximal subcluster requires the serpin locus control region. The graph shows patterns of histone acetylation across the proximal serpin subcluster in wild-type and Δ8.0 mutant cells from distal (left) to proximal (right). The black and dark gray bars represent histone H3 and H4 acetylation, respectively, in expressing F(14n)14 cells, whereas the light gray and white bars represent histone H3 and H4 acetylation, respectively, in mutant F(Δ8.0)1 cells. The amplicon designations are as described in the legend to Figure 3, and their genomic positions are shown in Figure 1. Each column represents the average of at least two independent determinations, with the standard deviations shown.

Figure 5

Histone acetylation in transactivator-deficient variant cells. (A and B) Representative examples of duplex PCRs used to quantitate levels of histone H3 (αAcH3) and H4 (αAcH4) acetylation in variant H11(14n)D cells (A) and H11(14n)E cells (B). Levels of histone H3 and H4 acetylation in H11(14n)E cells stably transfected with expression plasmids encoding HNF-4 [EH4, (A)] or HNF-1α [EH1, (B) are also shown. Results for amplicons from the α1AT macrophage promoter (α1AT_M), the α1AT hepatic promoter (α1AT_H), the CBG promoter (CBG_P), the ZPI promoter (ZPI_P), the KIAA1622 promoter (KIAA_P) and the ubiquitously expressed rP4H promoter (rP4H_P) are shown. All experimental determinations were normalized to signals from the non-expressed Goosecoid (GSC) control, which is ∼380 kb distal to α1AT on human chromosome 14. Input–input DNA. IgG- pre-immune serum control. The numbers below each lane are the levels of the test amplicon relative to the Goosecoid control amplicon in the same sample, normalized to the IgG control, as quantitated using a Typhoon PhosphorImager and Image Quant software. (C and D) Patterns of histone acetylation across the proximal serpin subcluster in wild-type, variant and rescued variant cells. The amplicon designations are as described in the legend to Figure 3, and their genomic positions are shown in Figure 1. The black and dark gray bars represent histone H3 and H4 acetylation, respectively, in wild-type F(14n)14 cells (C and D). The light gray and white bars represent histone H3 and H4 acetylation, respectively, in H11(14n)D (C) or H11(14n)E (D) variant cells. The small stripe bars and large stripe bars in (C) represent histone H3 and H4 acetylation, respectively, in the rescued variant EH4, an H11(14n)E derivative stably transfected with an expression plasmid encoding HNF-4. Note that ectopic expression of HNF-4 in these cells largely restored cell-specific histone acetylation at serpin gene promoters (50–70% of wild-type) but had little effect on histone acetylation at DHSs further upstream of α1AT (∼−24 kb and ∼−6 kb). The small stripe bars and large stripe bars in (D) represent histone H3 and H4 acetylation, respectively, in the rescued variant EH1, an H11(14n)E derivative stably transfected with an expression plasmid encoding HNF-1α. Ectopic expression of HNF-1α in these cells partially restored cell-specific histone acetylation at serpin promoters.