Two distal downstream enhancers direct expression of the human apolipoprotein E gene to astrocytes in the brain

Abstract

Two distal downstream enhancers controlling astrocyte expression of the human apolipoprotein E (apoE) gene in the brain were identified by analysis of transgenic mice generated with various constructs of the apoE/C-I/C-IV/C-II gene cluster. In wild-type mice, the highest overall levels of apoE mRNA were found in astrocytes in the glomerular layer of olfactory bulbs and in Bergmann glia in the cerebellum. This pattern of expression was reproduced in transgenic mice expressing the entire human apoE gene cluster and also in transgenic mice expressing specific enhancer segments within the cluster. Expression of the human apoE transgene at these sites was specified by two enhancer domains: one enhancer is located 3.3 kb downstream of the apoE gene, and a duplication of this sequence is located 15 kb downstream of the apoE gene. Astrocyte enhancer activity was contained within 620 and 619 bp segments of these domains that show subtle differences in regional expression. In the absence of these distal enhancers, the apoE gene was not expressed in astrocytes. The relatively high levels of apoE expression at specific sites in the olfactory bulb and cerebellum suggest the presence of unique regulatory signals at these locations that may reflect common cellular properties and apoE gene functions. The localization of the two astrocytic enhancers reveals an unexpected complexity in the control of apoE production that is essential to understanding apoE function in the brain.

Fig. 1.

Transgenic constructs. The bold horizontal lines represent human genomic DNA. The apolipoprotein genes are illustrated as solid vertical lines for exons and open boxes for introns, and the scale in kilobases is shown. Transcription is in the 5′ to 3′ orientation for all genes. The 12 kb/11 kb duplication that yielded the apoC-I and apoC-I′ genes is shown as inverted brackets. The locations of hepatocyte-expressing hepatic control regions (HCR.1 and HCR.2) (Allan et al., 1995b, 1997) and astrocyte-expressing multienhancers (ME.1 and ME.2) are indicated by ovals. Theinverted lines illustrate segments downstream of the apoE gene that were deleted in preparing constructs as indicated in Materials and Methods. The HEG.EC1 construct deleted 1.0 kb and the HEG.EC4 construct deleted 1.6 kb from the intergenic region. The LE8 (0.06 kb), LE6 (0.77 kb), and LE2 (1.7 kb) fragments were derived from the LE1 (3.8 kb) fragment and ligated to HEG1. The 3′ end of HEG1 is located 1.7 kb downstream of the apoE gene. The 5′ end of LE1 is located 14.3 kb downstream of the apoE gene. The HEG.EC2 construct consists of an SphI–SphI subfragment of p198.KK.

Fig. 2.

Distribution of expression of endogenous apoE mRNA in nontransgenic control and p198.KK transgenic mouse brains. Horizontal and sagittal sections of nontransgenic control (A, B) and p198.KK transgenic (D, E) mouse brains were radioactively labeled by in situ hybridization and examined by autoradiography. A, B, Distribution pattern of endogenous mouse apoE mRNA (dark grains) as revealed by a mouse antisense probe. C, Hybridization to a sagittal brain section with a sense probe does not produce a signal. D, E,Distribution pattern of p198.KK human transgene apoE mRNA (dark grains) as revealed by a human antisense probe. F, Hybridization to a sagittal nontransgenic control brain section with a human antisense probe does not produce a signal.

Fig. 3.

Region-specific expression of endogenous and transgene apoE mRNA in nontransgenic control (A, D) and in p198.KK transgenic (B, E) mouse brains. Tissue sections were labeled by in situ hybridization and examined at 10× or 50× magnification by dark-field microscopy (A, B, D, E). Separate tissue sections were reacted with anti-GFAP, stained with hematoxylin/eosin, and visualized by bright-field microscopy at 50× or 100× magnification (C, F). A, B, D, E, The distribution of endogenous mouse and p198.KK transgenic mouse apoE mRNA is shown by white grains in the olfactory bulb and the cerebellum. C, GFAP-positive cells in the olfactory bulb are indicated by red staining. The granular (Gr), internal plexiform (IP), mitral (Mi), external plexiform (EP), glomerular (Glo), olfactory neuronal (ON) layers, and accessory olfactory bulb (AOB) are indicated. F, GFAP-positive processes radiating into the molecular layer from the interface of the molecular (Mol) and granular (Gr) layers.

Fig. 4.

Distribution of expression of human apoE mRNA in transgenic mouse brains. Horizontal (A, C, E, G, I, K) and sagittal (B, D, F, H, J, L) sections from the brains of transgenic mice generated with HEG1, HEG.LE1, HEG.LE6, HEG.LE8, HEG.EC1, and HEG.EC4 constructs were examined by in situ hybridization followed by autoradiography.

Fig. 5.

Region-specific expression of human apoE mRNA in transgenic mouse brains after in situ hybridization. Tissue sections from the brains of HEG.LE1 transgenic mice were analyzed by dark-field (A, B, C) or bright-field (D) microscopy. A, B, Olfactory bulb at 10× and 50× magnification. ON, Olfactory neuronal layer; Glo, glomerular layer; EP, external plexiform layer. C, D, Molecular and granular layers of the cerebellum at 50× and 100× magnification. BG, Bergmann glia nuclei; P, Purkinje cell nuclei.

Fig. 6.

Immunohistochemistry of transgenic mouse brains was detected by fluorescence microscopy. A, Antibody labeling for human apoE protein in HEG.LE1 transgenic mice is shown ingreen (A1, A4, A7), endogenous mouse GFAP is shown in red (A2, A5, A8), and double antibody labeling for both antibodies, which colocalize, is shown inyellow (A3, A6, A9). Sections are shown for double antibody labeling of human apoE and mouse GFAP in astrocytes surrounding glomeruli in the olfactory bulb (A1, A2, A3) and in Bergmann glia of the cerebellum (A4, A5, A6). As a control, sections are shown for dual labeling of human apoE and mouse GFAP in Bergmann glia of the cerebellum (A7, A8, A9) in HEG1 transgenic mice. B, Antibody labeling for human apoE in the thalamus of HEG.LE8 transgenic mice is shown in green(B1), mouse GFAP is shown in red (B2), and double antibody labeling of human apoE and mouse GFAP is shown inyellow (B3).

Fig. 7.

Distribution of expression of endogenous apoE mRNA in ME.2 (in HEG.LE8) (A) and ME.1 (in HEG.EC1 and HEG.EC4) (B) transgenic mouse brains. Horizontal and sagittal sections of transgenic mouse brains were radioactively labeled by in situ hybridization and examined at 50× magnification by dark-field microscopy. The distribution patterns of human transgene apoE mRNA (white grains) are shown as revealed by a human antisense probe. Arrows indicate representative cell bodies showing intense apoE mRNA signal. Signal location and tissue integrity were confirmed by phase-contrast imaging (A5–A8, B5–B8).A5, B5, Cerebellum. Wh, White matter;Gr, granular layer; Mol, molecular layer.A6, B6, Olfactory bulb. Gr, Granular layer;Mi, mitral layer; EP, external plexiform layer;Glo, glomerular layer; ON, olfactory neuronal layer. A7, B7, Hippocampus. Al, Alveus;Or, oriens; CA, cornu ammons; Py,pyramidal; Ra, radiens. A8, B8, Thalamus.

Fig. 8.

ME domains of the apoE gene cluster in humans and mice. Dark boxes show regions of identity, anddashes show deletions.

Fig. 9.

Reporter transgene expression vectors. The regulatory components of the vector were derived from the human apoE gene cluster, and details of vector sequences are described in Materials and Methods. The 3.8 kb LE1 and the 0.6 kb LE8 fragments were inserted into the internal flanking polylinker.

Fig. 10.

Astrocyte enhancer control of reporter gene expression in GFP.LE1 and GFP.LE8 mice. Transgenic expression of the constructs shown in Figure 9 was detected by fluorescence microscopy.A, Olfactory bulb. EP, External plexiform layer;Glo, glomerular layer; ON, olfactory neuronal layer. B, Cerebellum. BG, Bergmann glia;P, Purkinje cells.

Fig. 11.

Astrocyte enhancer control of reporter gene expression. Transgenic expression of the GFP.LE8 construct shown in Figure 9 was detected by fluorescence microscopy (A, C, E, G). Signal location and tissue integrity were confirmed by phase imaging (B, D, F, H). A, B, Cerebellum.Wh, White matter; Gr, granular layer. C, D, Olfactory bulb. Gr, Granular layer; Mi, mitral layer; EP, external plexiform layer; Glo, glomerular layer. E, F, Hippocampus. Al, Alveus;Or, oriens; CA, cornu ammons; Py, pyramidal; Ra, radiens. G, H, Thalamus.