The tissue-specific methylation of human-specific endogenous retroviral long terminal repeats

A possible involvement of retroelements in the epigenetic regulation of human gene expression was considered by the example of methylation of long terminal repeats (LTRs) of the human endogenous retrovirus family K (HERV-K). The methylation status of six HERV-K LTRs was determined in various gene-enriched regions of the human genome. The methylation of four LTRs was shown to be tissue-specific. Our results correlated with published data on the tissue-specific changes in the expression level of human genes adjacent to the LTRs under study. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.     

Nucleotide sequences of the retroviral long terminal repeats and their adjacent regions.

The nucleotide sequences of the LTRs and their adjacent regions from 19 type C and one type B retrovirus were compared. Salient features are: (a) The R regions in the genomes of most of the type C retroviruses begin with GC and end with CA. (b) The mammalian type C retroviruses have a polyadenylation signal "AATAAA" in the R region, and most have a "CAT" box and a "TATA" box in the U3 region. (c) The avian type C retroviruses have an AATAAA sequence, and some also have "CAT-like" and "TATA-like" boxes, in the U3 region. (d) As with many transposable elements, the IR regions of the proviruses begin with TG and end with CA, and the DR sequences in the host genomes flanking the proviruses are different from one another. Although SNV is an avian retrovirus, the nucleotide sequences in the R, U5, TBS, and PU region are more similar to the mammalian type C than to the avian type C retroviruses.     

Factors affecting the performance of different long terminal repeats in the retroviral vector

The long terminal repeat (LTR) of retrovirus contains the nucleotide sequences that control gene expression. Although several different LTRs have been used in the context of retroviral vector, the activity of the various LTRs has not yet been systematically compared for their level of gene expression. We evaluated the effect of four different LTRs on gene expression using luciferase, stem cell factor, and enhanced green fluorescence protein as reporter genes. LTRs tested in this study were derived from Moloney murine leukemia virus, myeloproliferative sarcoma virus, murine stem cell virus, and spleen focus-forming virus. It was found that the level of gene expression is affected by not only LTRs but also the transgenes and the cell types in which gene expression occurs. Furthermore, the presence of other nucleotide sequences such as the internal ribosome entry site (IRES)-neo cassette could also significantly affect gene expression. Our results suggested that the LTR should be chosen carefully, more or less on an empirical basis.     

Factors that bind to adeno-associated virus terminal repeats.

We have identified and characterized a DNA-protein complex that forms with the adeno-associated virus (AAV) terminal repeats. The complex formed only if the terminal palindrome was in the covalently closed or hairpin configuration; little if any binding was detected with the open duplex form of the terminal repeat. This fact suggested that both secondary structure and primary sequence are essential elements of recognition. DNase I protection studies indicated that virtually all of the A-A' palindrome and significant portions of the B-B' and C-C' palindromes are protected. The postulated terminal resolution site of AAV also is protected. Restriction mapping of the sequences necessary for binding indicated that almost all of the terminal palindrome must be present for binding to occur. Hairpins which are similar in size and shape to the AAV termini did not exhibit competition for binding, and the complex formed only if AAV-infected extracts were used. Thus, the binding reaction is specific for AAV sequences. The viral-coded nonstructural proteins Rep78 and Rep68 comigrated with the DNA-protein complex on neutral acrylamide gels, suggesting that one or both of these proteins are components of the complex. The characteristics of the complex suggested that it has a role in AAV DNA replication.     

Surfactant protein D gene regulation. Interactions among the conserved CCAAT/enhancer-binding protein elements

Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following acute lung injury. Given the importance of CCAAT/enhancer-binding protein (C/EBP)-binding elements in the systemic acute-phase response and lung development and the expression of C/EBP isoforms by lung epithelial cells, we hypothesized that conserved C/EBP motifs in the near-distal and proximal promoters contribute to the regulation of SP-D expression by C/EBPs. Five SP-D motifs (-432, -340, -319, -140, and -90) homologous to the C/EBP consensus sequence specifically bound to C/EBPs in gel shift assays, and four of the five sites (-432, -340, -319, and -90) efficiently competed for the binding of C/EBPalpha, C/EBPbeta, or C/EBPdelta to consensus oligomers. Cotransfection of C/EBPalpha, C/EBPbeta, or C/EBPdelta cDNA in H441 lung adenocarcinoma cells significantly increased the luciferase activity of a wild-type SP-D promoter construct containing 698 bp of upstream sequence (SS698). Transfection of C/EBP also increased the level of endogenous SP-D mRNA in H441 cells. Transactivation of the reporter construct was abrogated by deletion of sequences upstream of -205. Independent site-directed mutagenesis of the sites at -432, -340, and -319 reduced C/EBP-mediated activation by approximately 50%, and mutagenesis of the site at -432 in combination with either of the tandem sites at -340 and -319 blocked activation. The conserved AP-1 element at -109 was required for maximal promoter activity, but not for the transactivation of SS698 by C/EBPs. Thus, interactions among C/EBP elements in the near-distal promoter can modulate the promoter activity of SP-D.     

Functional RNA polymerase II promoters in solitary retroviral long terminal repeats (LTR-IS elements).

LTR-IS elements are middle repetitive sequences in the mouse genome with structural features of solitary retroviral LTRs. In order to get some insight in the possible functional role of these sequences the promotor activity of two LTR-IS representatives differing by 105 bp in their U3 region was investigated. Gene fusions between LTR-IS sequences and the bacterial gene coding for chloramphenicol acetyl transferase (CAT) were transfected into mouse 3T6 cells and the expression of CAT was measured. It is shown that the LTR-IS sequences represent weak RNA polymerase II promoters which require enhancement by cis-or trans-activating factors.