Astrocyte- and hepatocyte-specific expression of genes from the distal serpin subcluster at 14q32.1 associates with tissue-specific chromatin structures

Abstract

The distal serpin subcluster contains genes encoding alpha1-antichymotrypsin (ACT), protein C inhibitor (PCI), kallistatin (KAL) and the KAL-like protein, which are expressed in hepatocytes, but only the act gene is expressed in astrocytes. We show here that the tissue-specific expression of these genes associates with astrocyte- and hepatocyte-specific chromatin structures. In hepatocytes, we identified 12 Dnase I-hypersensitive sites (DHSs) that were distributed throughout the entire subcluster, with the promoters of expressed genes accessible to restriction enzyme digestion. In astrocytes, only six DHSs were located exclusively in the 5' flanking region of the act gene, with its promoter also accessible to restriction enzyme digestion. The acetylation of histone H3 and H4 was found throughout the subcluster in both cell types but this acetylation did not correlate with the expression pattern of these serpin genes. Analysis of histone modifications at the promoters of the act and pci genes revealed that methylation of histone H3 on lysine 4 correlated with their expression pattern in both cell types. In addition, inhibition of methyltransferase activity resulted in suppression of ACT and PCI mRNA expression. We propose that lysine 4 methylation of histone H3 correlates with the tissue-specific expression pattern of these serpin genes.

Fig. 1. Tissue-specific expression of genes from the distal serpin subcluster in astrocytes and hepatic cells

Human primary astrocytes, hepatoma HepG2 and astrocytoma U373-MG cells were treated with IL-1 (10 ng/ml) and OSM (25 ng/ml), IL-6 (50 ng/ml), TNF (5 ng/ml) or PMA (10/ng/ml). RNA was isolated after 18 hours and subjected to Northern blot analysis using ACT cDNA (A), PCI exon II (B), and KAL exon II as probes. Bottom panel shows 28S RNA stained with ethidium bromide on the membrane. (D) Human astrocytes were transfected with plasmids pPCI(−421)CAT, pKAL(−693)CAT, pACT(−352)CAT and β-galactosidase expression vector as an internal control for transfection efficiency. One day post transfection the cells were stimulated with indicated cytokines, cultured for another 24 hours, and harvested. CAT activities were normalized to β-galactosidase activities, and are mean values ± S.E.M (3 independent experiments).

Fig. 2. DNase I-hypersensitive site map of the distal serpin subcluster in astrocytes, hepatoma, astrocytoma and carcinoma cells

(A) The map is drawn to scale, with position +1 defined as the transcription start site of the act gene. Exons are indicated as black boxes, a gray box represents IL-1 enhancer, the kal, pci, and act genes are also shown. Arrows indicate DHSs. Nuclei from astrocytes, hepatoma HepG2, astrocytoma U373-MG, and carcinoma HeLa cells were digested with increasing concentrations of DNase I, DNA was purified and digested with HindIII. DNA samples were analyzed by Southern blotting using the PRA7 “IL-1 enhancer” probe (B) or the SG2 “pci promoter” probe (C). The description and location of probes is listed in table I.

Fig. 3. Enzyme-accessibility at the IL-1 enhancer and promoter of the ACT gene in hepatoma, astrocytoma, and carcinoma cells

Nuclei from hepatoma HepG2, astrocytoma U373-MG, and carcinoma HeLa cells were digested with SacI (A) or PstI (B) as described in materials and methods section, DNA was purified and subsequently digested with NcoI. DNA samples were then analyzed by Southern blotting using the short enhancer (A) and promoter (B) probes indicated on the diagram.

Fig. 4. Enzyme accessibility within the transcribed regions of the act and pci genes

Nuclei from hepatoma HepG2 and astrocytoma U373-MG cells were digested with SacI as described in materials and methods section, DNA was purified and subsequently digested with BglII. DNA samples were analyzed by Southern blotting using the PCI exon II probe (A) or the ACT intron II probe (B) as indicated on the diagram. Asterisks (*) indicate double digest control samples (purified DNA was digested with SacI and either BglII or NcoI).

Fig. 5. The histone code within the distal serpin subcluster

HepG2, U373-MG, and HeLa cells were stimulated with IL-1 or OSM for 1 hour, chromatin was prepared, and equal amounts of chromatin were immunoprecipitated with specific antibodies as described in materials and methods section. Subsequently, DNA was purified and the act, pci and hla-dra gene promoter regions were amplified by PCR in the presence of ³²P dCTP. PCR products were separated in 12% native polyacrylamide gels, and exposed to phosphorimager screens. Input represents 2% of chromatin used for immunoprecipitation. Ab indicates nonspecific polyclonal antibodies. ProACT, proPCI and proDRA mark the PCR products derived from the act, pci and hla-dra promoters, respectively. (A) Total acetylation of histone H3 (Ac-H3) and H4 (Ac-H4). (B) Specific modifications of histone H3: dimethyl-(K4) (mK4H3), dimethyl-(K9) (mK9H3), acetyl-(K14) (AcK14H3), phospho-(S28) (pS28H3); and histone H4: acetyl-(K5) (AcK5H4), acetyl-(K8) (AcK8H4), and acetyl-(K12) (AcK12H4).

Fig. 6. Inhibition of methyltransferase activity suppresses serpin expression

Human hepatoma HepG2 cells were pretreated with 40 mM AdOx for 24 hours, and subsequently treated with IL-1 (10 ng/ml) or OSM (25 ng/ml). (A) RNA was isolated after 18 hours and subjected to Northern blot analysis using ACT cDNA, PCI exon II, and GAPDH cDNA as probes. Numbers represent results of densitometric analysis. (B) Chromatin was prepared as described in materials and methods and immunoprecipitated with anti-dimethyl-histone H3 (K4) or nonspecific antibodies (Ab). PCR products were separated in 12% native polyacrylamide gels, and exposed to phosphorimager screens. Input represents 2% of chromatin used for immunoprecipitation.

Fig. 7. Model of the tissue-specific expression of genes from the distal serpin subcluster

The kal, pci, and act genes are represented by black boxes. Acetylation of histones H3 and H4 across the entire serpin subcluster prevents silencing that otherwise takes place and leads to “closed” chromatin formation and the lack of expression observed in HeLa cells. Subsequently, HMT is recruited to the regions destined for transcription and this modification leads to tissue-specific gene expression.