Allele-specific programming of Npy and epigenetic effects of physical activity in a genetic model of depression.

Neuropeptide Y (NPY) has been implicated in depression, emotional processing and stress response. Part of this evidence originates from human single-nucleotide polymorphism (SNP) studies. In the present study, we report that a SNP in the rat Npy promoter (C/T; rs105431668) affects in vitro transcription and DNA-protein interactions. Genotyping studies showed that the C-allele of rs105431668 is present in a genetic rat model of depression (Flinders sensitive line; FSL), while the SNP's T-allele is present in its controls (Flinders resistant line; FRL). In vivo experiments revealed binding of a transcription factor (CREB2) and a histone acetyltransferase (Ep300) only at the SNP locus of the FRL. Accordingly, the FRL had increased hippocampal levels of Npy mRNA and H3K18 acetylation; a gene-activating histone modification maintained by Ep300. Next, based on previous studies showing antidepressant-like effects of physical activity in the FSL, we hypothesized that physical activity may affect Npy's epigenetic status. In line with this assumption, physical activity was associated with increased levels of Npy mRNA and H3K18 acetylation. Physical activity was also associated with reduced mRNA levels of a histone deacetylase (Hdac5). Conclusively, the rat rs105431668 appears to be a functional Npy SNP that may underlie depression-like characteristics. In addition, the achieved epigenetic reprogramming of Npy provides molecular support for the putative effectiveness of physical activity as a non-pharmacological antidepressant.

Dpp signaling thresholds in the dorsal ectoderm of the Drosophila embryo.

The dorsal ectoderm of the Drosophila embryo is subdivided into different cell types by an activity gradient of two TGF(&bgr;) signaling molecules, Decapentaplegic (Dpp) and Screw (Scw). Patterning responses to this gradient depend on a secreted inhibitor, Short gastrulation (Sog) and a newly identified transcriptional repressor, Brinker (Brk), which are expressed in neurogenic regions that abut the dorsal ectoderm. Here we examine the expression of a number of Dpp target genes in transgenic embryos that contain ectopic stripes of Dpp, Sog and Brk expression. These studies suggest that the Dpp/Scw activity gradient directly specifies at least three distinct thresholds of gene expression in the dorsal ectoderm of gastrulating embryos. Brk was found to repress two target genes, tailup and pannier, that exhibit different limits of expression within the dorsal ectoderm. These results suggest that the Sog inhibitor and Brk repressor work in concert to establish sharp dorsolateral limits of gene expression. We also present evidence that the activation of Dpp/Scw target genes depends on the Drosophila homolog of the CBP histone acetyltransferase.

The Rpd3 histone deacetylase is required for segmentation of the Drosophila embryo.

Previous studies have implicated histone deacetylation and chromatin condensation as critical mechanisms of transcription repression in yeast and mammals. A specific histone deacetylase, Rpd3, interacts with a variety of sequence-specific transcriptional repressors, including Mad-Max heterodimers and members of the nuclear receptor superfamily. Here, we present evidence that a strong hypomorphic mutation in the Drosophila Rpd3 gene causes embryonic lethality and a specific pair-rule segmentation phenotype. The analysis of a number of segmentation genes suggests that the repressor function of Even-skipped (Eve) may be diminished, causing an indirect loss of Ftz-mediated activation of engrailed. The relatively mild defects observed in Rpd3 mutants suggest that the recently identified Groucho and dCtBP corepressor proteins do not function solely through the recruitment of histone deacetylases. We discuss the possibility that Eve mediates multiple mechanisms of repression, so that Rpd3 mutants disrupt the regulation of just a subset of Eve target genes.

Target genes of homeodomain proteins.

Homeodomain proteins are transcription factors that share a related DNA binding domain, the homeodomain. This class of proteins was first recognized in the fruitfly Drosophila melanogaster where they cause homeotic transformations such as a fly with four wings instead of two (Lewis EB. A gene complex controlling segmentation in Drosophila. Nature 1978;276:565-570 [Ref. 18]). They are now known to exist in all eukaryotes where they perform important functions during development. Given that homeodomain proteins are transcription factors, they control the expression of downstream genes to regulate development. Which genes are controlled by homeodomain proteins and how many of them are there? This review focuses on a recent paper by Liang and Biggin (Liang Z, Biggin MD. Eve and Ftz regulate a wide array of genes in blastoderm embryos: the selector homeoproteins directly or indirectly regulate most genes in Drosophila. Development 1998; 125:4471-4482 [Ref. 1]), which proposes that the Drosophila homeodomain proteins Even-skipped and Fushi-tarazu directly control the expression of the majority of genes in the Drosophila genome. An alternative view, that most genes are only indirectly affected by homeodomain proteins is also discussed.

Adenovirus E4 open reading frame 4-induced dephosphorylation inhibits E1A activation of the E2 promoter and E2F-1-mediated transactivation independently of the retinoblastoma tumor suppressor protein.

Previous studies have shown that the cell cycle-regulated E2F transcription factor is subjected to both positive and negative control by phosphorylation. Here we show that in transient transfection experiments, adenovirus E1A activation of the viral E2 promoter is abrogated by coexpression of the viral E4 open reading frame 4 (E4-ORF4) protein. This effect does not to require the retinoblastoma protein that previously has been shown to regulate E2F activity. The inhibitory activity of E4-ORF4 appears to be specific because E4-ORF4 had little effect on, for example, E4-ORF6/7 transactivation of the E2 promoter. We further show that the repressive effect of E4-ORF4 on E2 transcription works mainly through the E2F DNA-binding sites in the E2 promoter. In agreement with this, we find that E4-ORF4 inhibits E2F-1/DP-1-mediated transactivation. We also show that E4-ORF4 inhibits E2 mRNA expression during virus growth. E4-ORF4 has previously been shown to bind to and activate the cellular protein phosphatase 2A. The inhibitory effect of E4-ORF4 was relieved by okadaic acid, which inhibits protein phosphatase 2A activity, suggesting that E4-ORF4 represses E2 transcription by inducing transcription factor dephosphorylation. Interestingly, E4-ORF4 did not inhibit the transactivation capacity of a Gal4-E2F hybrid protein. Instead, E4-ORF4 expression appears to result in reduced stability of E2F/DNA complexes.

Transcriptional coregulators in development.

Small differences in the levels of an extracellular signaling molecule can specify cell fate during development. Threshold responses are often determined at the level of transcription. Cell-specific and spatially localized patterns of gene expression depend on combinations of sequence-specific activators and repressors that bind to extensive cis-regulatory regions. Different mechanisms for integrating this complex regulatory information are discussed, particularly the role of coregulatory proteins, which are recruited to the DNA template by sequence-specific transcription factors. Recent studies suggest that a growing set of coactivators and corepressors mediate communication between diverse upstream regulatory proteins and the core RNA polymerase II transcription complex.

The transcriptional co-activator proteins p300 and CBP stimulate adenovirus E1A conserved region 1 transactivation independent of a direct interaction.

p300 and CBP are two related transcriptional co-activator proteins required by many cellular transcription factors for activity. The adenovirus E1A protein binds p300 and CBP through its amino-terminus and conserved region (CR) 1. Fusing CR1 to a heterologous DNA-binding domain creates a potent transcriptional activator, suggesting that CR1 might activate transcription by recruiting p300/CBP to the promoter. We show that both p300 and CBP enhances CR1-dependent transactivation. However, this enhancement occurs independently of a direct interaction with E1A and does not correlate with the CR1 activator function.

In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive.

Here we show that the TATA-binding protein (TBP) is localized in the nucleoplasm and in the nucleolus of mammalian cells, consistent with its known involvement in transcription by RNA polymerase I, II, and III. In the nucleolus of actively growing cells, TBP colocalizes with upstream binding factor (UBF) and RNA polymerase I at the sites of rRNA transcription. During mitosis, when rRNA synthesis is down-regulated, TBP colocalizes with TBP-associated factors for RNA polymerase I (TAF(I)s), UBF, and RNA polymerase I on the chromosomal regions containing the rRNA genes. Treatment of cells with a low concentration of actinomycin D inhibits rRNA synthesis and causes a redistribution of the rRNA genes that become concentrated in clusters at the periphery of the nucleolus. A similar redistribution was observed for the major components of the rRNA transcription machinery (i.e., TBP, TAF(I)s, UBF, and RNA polymerase I), which still colocalized with each other. Furthermore, anti-TBP antibodies are shown to coimmunoprecipitate TBP and TAF(I)63 in extracts prepared from untreated and actinomycin D-treated cells. Collectively, the data indicate that in vivo TBP/promoter selectivity factor, UBF, and RNA polymerase I remain associated with both active and inactive rRNA genes.

An adenovirus E1A transcriptional repressor domain functions as an activator when tethered to a promoter.

The adenovirus E1A protein contains three well conserved regions, designated conserved region (CR) 1, 2 and 3, which are important for the multiple activities ascribed to E1A. The CR3 domain constitutes a prototypic transcription activator, consisting of a promoter targeting region and a transactivating region. Here we demonstrate the existence of a second transactivating region located within amino acids 28 to 90 (essentially the CR1 domain) of the E1A protein. A fusion protein, containing the Gal4 DNA binding domain linked to CR1, was as efficient as the classical CR3 transactivator in activating transcription from a reporter plasmid containing Gal4 binding sites. However, competition experiments suggest that Gal/CR1 and Gal/CR3 work through different cellular targets. The E1A-243R protein has previously been extensively characterized as a repressor of transcription. Here we show that a Gal4 fusion protein expressing the CR1 domain is indeed sufficient for repression of SV40 enhancer activity. Collectively, our results suggest that CR1 functions as an activator if tethered to a promoter and as a repressor in the absence of promoter association.

Identification of B-cell antigenic sites on HIV-2 gp125.

Synthetic peptides were used to identify continuous antigenic sites on the external envelope glycoprotein gp125 of human immunodeficiency virus (HIV)-2. Initially, seven HIV-2-positive human serum samples were screened with 172 sequential nonapeptides containing a six-amino-acid overlap. This represents the entire gp125 molecule of HIV-2ISY. The antibody reactivity was found to be mainly restricted to 14 regions within gp125. Following these results, 33 longer peptides, 15-24 amino acids in length, were synthesized and tested against a larger number of samples. Eleven antigenic regions were thus identified. Two of these were detected within a region corresponding to the C1 region and four others within a region corresponding to the C2 region of HIV-1. The highest frequency of reactivity (90%) of 31 HIV-2 seropositive human serum samples was elicited by three peptides from a region corresponding to the V3 region of HIV-1. The C-terminal portion of this region was recognized by almost 80% of the samples. Reactive regions corresponding to the V4, V5, and N-terminal portion of V4 were also identified. A mouse monoclonal antibody reacting with gp125 was mapped to the N-terminal region of the molecule and was found to react with the sequence DVWNLFETS. The peptides were used to evaluate the antibody response of monkeys immunized with whole killed HIV-2 or simian immunodeficiency virus (SIV). The monkeys showed a pattern of reactivity similar to HIV-2-infected human serum samples. Postinfection samples from monkeys inoculated with HIV-2 or SIV reacted mainly to peptides from the V3 region. Two peptides were used to detect the seroconversion of two SIV-infected monkeys. Thus, we have demonstrated that human seroreactivity to HIV-2 gp125 occurs at a few distinct linear antigenic sites distributed at similar positions on the molecule as those in HIV-1 gp120.