CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus.

The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2). On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2-4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19 (refs 5, 6). This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.

Cell type-specific regulation of choline acetyltransferase gene expression. Role of the neuron-restrictive silencer element and cholinergic-specific enhancer sequences.

This study demonstrates the presence of positive and negative regulatory elements within a 2336-base pair-long region of the rat choline acetyltransferase (ChAT) gene promoter that cooperate to direct cell type-specific expression in cholinergic cells. A 21-base pair-long neuron-restrictive silencer element (NRSE) was identified in the proximal part of this region. This element was recognized by the neuron-restrictive silencer factor (NRSF), previously shown to regulate expression of other neuron-specific genes. The ChAT NRSE was inactive in both cholinergic and non-cholinergic neuronal cells, but repressed expression from a heterologous promoter in non-neuronal cells. Specific deletion of this element allowed ChAT gene promoter activity in non-neuronal cells, and overexpression of NRSF repressed ChAT gene promoter activity in cholinergic cells. The distal part of the ChAT gene promoter showed cholinergic-specific enhancing activity, which stimulated promoter activity in cholinergic cells, but was inactive in non-cholinergic neuronal and non-neuronal cells. This enhancer region suppressed the activity of the ChAT NRSE in cholinergic cells, even after NRSF overexpression. Thus, at least two kinds of regulatory elements cooperate to direct ChAT gene expression to cholinergic neurons, namely a neuron-restrictive silencer element and a cholinergic-specific enhancer.

Identification of potential target genes for the neuron-restrictive silencer factor.

The neuron-restrictive silencer factor (NRSF) represses transcription of several neuronal genes in nonneuronal cells by binding to a 21-bp element called the neuron-restrictive silencer element (NRSE). We have performed data base searches with a composite NRSE to identify additional candidate NRSF target genes. Twenty-two more genes, 17 of which are expressed mainly in neurons, were found to contain NRSE-like sequences. Many of these putative NRSEs bound NRSF in vitro and repressed transcription in vivo. Most of the neuronal genes identified contribute to the basic structural or functional properties of neurons. However, two neuronal transcription factor genes contain NRSEs, suggesting that NRSF may repress neuronal differentiation both directly and indirectly. Functional NRSEs were also found in several nonneuronal genes, implying that NRSF may play a broader role than originally anticipated.

Silencing is golden: negative regulation in the control of neuronal gene transcription.

Recent work has identified negative-acting DNA regulatory elements that function to prevent the expression of neuronal genes in non-neuronal cell types or in inappropriate neuronal subtypes. In some cases, the protein factors that interact with these silencer elements have been isolated and characterized. For example, the recently cloned silencer-binding factor NRSF/REST is a novel zinc-finger protein that interacts with silencer elements in a number of neuron-specific genes. These data suggest that negative regulation plays a major role in determining the diverse patterns of gene expression within the nervous system.

The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes.

The neuron-restrictive silencer factor (NRSF) binds a DNA sequence element, called the neuron-restrictive silencer element (NRSE), that represses neuronal gene transcription in nonneuronal cells. Consensus NRSEs have been identified in 18 neuron-specific genes. Complementary DNA clones encoding a functional fragment of NRSF were isolated and found to encode a novel protein containing eight noncanonical zinc fingers. Expression of NRSF mRNA was detected in most nonneuronal tissues at several developmental stages. In the nervous system, NRSF mRNA was detected in undifferentiated neuronal progenitors, but not in differentiated neurons. NRSF represents the first example of a vertebrate silencer protein that potentially regulates a large battery of cell type-specific genes, and therefore may function as a master negative regulator of neurogenesis.

A common silencer element in the SCG10 and type II Na+ channel genes binds a factor present in nonneuronal cells but not in neuronal cells.

We have localized a cell type-specific silencer element in the SCG10 gene by deletion analysis. This neural-restrictive silencer element (NRSE) selectively represses SCG10 expression in nonneuronal cells and tissues. The NRSE contains a 21 bp region with striking homology to a sequence present in a silencer domain of the rat type II sodium channel (NaII), another neuron-specific gene. We have identified a sequence-specific protein(s) that binds the SCG10 NRSE, as well as the homologous element in the NaII gene. A point mutation in the NRSE that abolishes binding of this neural-restrictive silencer-binding factor (NRSBF) in vitro also eliminates silencing activity in vivo. NRSBF is present in nuclear extracts from nonneuronal cells but not in extracts from neuronal cells, suggesting that the neuron-specific expression of SCG10 reflects, at least in part, the absence or inactivity of this protein. These data identify the NRSE as a potentially general DNA element for the control of neuron-specific gene expression in vertebrates.

Interferon and antibody titrations using haemagglutinating Togaviridae and trypsinized human erythrocytes.

Several Togaviridae of the alphavirus and flavivirus genera agglutinate trypsinized human group O erythrocytes (THOE) (Shortridge and Hu, 1976). Haemagglutinin titers of Semliki Forest virus (SFV) and Japanese encephalitis virus (JEV) measured with THOE were equivalent to, if not higher than, those obtained with Embden gander erythrocytes, even with unextracted haemagglutinin. Results obtained with THOE in JEV haemagglutination-inhibition tests on sera taken from a previously infected individual over a 20-yr period were similar to those measured during the initial JEV infection. The inhibition of SFV haemagglutinin production as measured with THOE was a very sensitive bioassay for chicken interferon: interferon titers were 6- to 10-fold higher than those obtained with the vesicular stomatitis virus plaque-reduction method. The generally greater availability of human erythrocytes (including those stabilized with glutaraldehyde), the simplicity of the trypsin treatment, and the possibility of using unextracted haemagglutinin recommend this technique for use with haemagglutinating Togaviridae.

Protection against Japanese encephalitis virus in mice and hamsters by treatment with carboxymethylacridanone, a potent interferon inducer.

A low-molecular-weight chemical inducer of interferon, 10-carboxymethyl-9-acridanone (CMA), effectively prevented death caused by Japanese encephalitis virus (JEV) injected peripherally into weanling mice and baby hamsters. Marked reductions in mortality were seen in mice when a single dose of CMA was administered intraperitoneally, subcutaneously, or intramuscularly to animals challenged intraperitoneally with JEV. The degree of protection was dependent on dose and time of administration of CMA in relation to virus challenge: all hamsters given CMA on the same day as JEV survived, with lesser although still significant protection when CMA was given one or two days after JEV. Viremia, an important characteristic of the pathogenesis of natural JEV infection, was reduced nearly 10,000-fold in hamsters treated with CMA. Thus, in the experimental animal models developed for these studies, CMA provided marked therapeutic and prophylactic effect against JEV.

High-yield interferon induction by 10-carboxymethyl-9-acridanone in mice and hamsters.

10-Carboxymethyl-9-acridanone (CMA) was shown to be a very potent interferon inducer in young and old Swiss albino as well as congenitally asplenic mice. This compound (molecular weight, 275) induced titers of interferon in plasma comparable to those obtained with the best viral inducers, attaining > 400,000 U/ml in mice in 2 to 3 h. CMA concentrations were highest in mouse plasma 1 h after intraperitoneal or oral delivery. Induction was dose dependent over a wide range. Intraperitoneal, subcutaneous, or intramuscular injections of CMA gave comparable ranges of interferon titers. Oral delivery by gavage gave lower titers (65,000 U/ml), but higher than those reported with other low-molecular-weight interferon inducers in mice. CMA injected into week-old hamsters (which usually produce iterferon poorly) induced levels of interferon as high as 6,400 U per ml of plasma in a dose-dependent fashion, but kinetics of interferon induction was less rapid than in mice. The mouse and hamster interferons induced by CMA had physical characteristics similar to those of virus-induced interferons of the homologous species. The unusually high yields of interferon obtainable with this chemical inducer suggest its use for further experimental antiviral or antitumor therapy.

Bluetongue virus, an exceptionally potent interferon inducer in mice.

The attenuated American BT-8 strain of bluetongue virus is 5 to 10 times more potent an interferon inducer than any other viral or nonviral agent reported to date, including as much as 600,000 units/ml of plasma by 8 h after intravenous injection.