Jiří Hejnar

Similar integration but different stability of Alus and LINEs in the human genome

Alus and LINEs (LINE1) are widespread classes of repeats that are very unevenly distributed in the human genome. The majority of GC-poor LINEs reside in the GC-poor isochores whereas GC-rich Alus are mostly present in GC-rich isochores. The discovery that LINES and Alus share similar target site duplication and a common AT-rich insertion site specificity raised the question as to why these two families of repeats show such a different distribution in the genome. This problem was investigated here by studying the isochore distributions of subfamilies of LINES and Alus characterized by different degrees of divergence from the consensus sequences, and of Alus, LINEs and pseudogenes located on chromosomes 21 and 22. Young Alus are more frequent in the GC-poor part of the genome than old Alus. This suggests that the gradual accumulation of Alus in GC-rich isochores has occurred because of their higher stability in compositionally matching chromosomal regions. Densities of Alus and LINEs increase and decrease, respectively, with increasing GC levels, except for the telomeric regions of the analyzed chromosomes. In addition to LINEs, processed pseudogenes are also more frequent in GC-poor isochores. Finally, the present results on Alu and LINE stability/exclusion predict significant losses of Alu DNA from the GC-poor isochores during evolution, a phenomenon apparently due to negative selection against sequences that differ from the isochore composition.

The Receptor for the Subgroup C Avian Sarcoma and Leukosis Viruses, Tvc, Is Related to Mammalian Butyrophilins, Members of the Immunoglobulin Superfamily

ABSTRACT The five highly related envelope subgroups of the avian sarcoma and leukosis viruses (ASLVs), subgroup A [ASLV(A)] to ASLV(E), are thought to have evolved from an ancestral envelope glycoprotein yet utilize different cellular proteins as receptors. Alleles encoding the subgroup A ASLV receptors (Tva), members of the low-density lipoprotein receptor family, and the subgroup B, D, and E ASLV receptors (Tvb), members of the tumor necrosis factor receptor family, have been identified and cloned. However, alleles encoding the subgroup C ASLV receptors (Tvc) have not been cloned. Previously, we established a genetic linkage between tvc and several other nearby genetic markers on chicken chromosome 28, including tva. In this study, we used this information to clone the tvc gene and identify the Tvc receptor. A bacterial artificial chromosome containing a portion of chicken chromosome 28 that conferred susceptibility to ASLV(C) infection was identified. The tvc gene was identified on this genomic DNA fragment and encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. We subsequently cloned cDNAs encoding Tvc that confer susceptibility to infection by subgroup C viruses in chicken cells resistant to ASLV(C) infection and in mammalian cells that do not normally express functional ASLV receptors. In addition, normally susceptible chicken DT40 cells were resistant to ASLV(C) infection after both tvc alleles were disrupted by homologous recombination. Tvc binds the ASLV(C) envelope glycoproteins with low-nanomolar affinity, an affinity similar to that of binding of Tva and Tvb with their respective envelope glycoproteins. We have also identified a mutation in the tvc gene in line L15 chickens that explains why this line is resistant to ASLV(C) infection.

Retroviruses in foreign species and the problem of provirus silencing.

Retroviruses are known to integrate in the host cell genome as proviruses, and therefore they are prone to cell-mediated control at the transcriptional and posttranscriptional levels. This plays an important role especially after retrovirus heterotransmission to foreign species, but also to differentiated cells. In addition to host cell-mediated blocks in provirus expression, also so far undefined host specificities, deciding upon the pathogenic manifestation of retrovirus heterotransmission, are in play. In this respect, we discuss especially the occurrence of wasting disease and immunodeficiency syndrome, which we established also in avian species using avian leukosis virus subgroup C (ALV-C) inoculated in mid-embryogenesis in duck or chicken embryos. The problem of provirus downregulation in foreign species or in differentiated cells has been in the recent years approached experimentally. From a series of observations it became apparent that provirus downregulation is mediated by its methylation, especially in the region of proviral enhancer-promoter located in long terminal repeats (LTR). Several strategies have been devised in order to protect the provirus from methylation using LTR modification and/or introducing in the LTR sequence motifs acting as antimethylation tags. In such a way the expression of retroviruses and vectors in foreign species, as well as in differentiated cells, has been significantly improved. The complexity of the mechanisms involved in provirus downregulation and further possibilities to modulate it are discussed.

Epigenetic regulation of transcription and splicing of syncytins, fusogenic glycoproteins of retroviral origin

Syncytin-1 and -2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. We studied the epigenetic suppression of ERVWE1 and ERVFRDE1 5′-long terminal repeats by DNA methylation and chromatin modifications. Immunoprecipitation of the provirus-associated chromatin revealed the H3K9 trimethylation at transcriptionally inactivated syncytins in HeLa cells. qRT-PCR analysis of non-spliced ERVWE1 and ERVFRDE1 mRNAs and respective env mRNAs detected efficient splicing of endogenously expressed RNAs in trophoblastic but not in non-placental cells. Pointing to the pathogenic potential of aberrantly expressed syncytin-1, we have found deregulation of transcription and splicing of the ERVWE1 in biopsies of testicular seminomas. Finally, ectopic expression experiments suggest the importance of proper chromatin context for the ERVWE1 splicing. Our results thus demonstrate that cell-specific retroviral splicing represents an additional epigenetic level controling the expression of endogenous retroviruses.

Preferential integration of human immunodeficiency virus type 1 into genes, cytogenetic R bands and GC-rich DNA regions: insight from the human genome sequence

After entering a cell during infection, the human immunode¢ciency virus type 1 (HIV-1) undergoes a series of steps including reverse transcription of its genome and culminating in integration of proviral DNA into the host chromosomes. The further fate of the individual provirus is to a great extent in£uenced by the e⁄ciency of provirus transcription, dependent upon the site of its integration [1]. How HIV-1 and other retroviruses choose their integration sites is still far from completely understood. It seems that the integration is not strictly speci¢c, because most or all genomic regions are potential targets, but neither is it a random event. Locally, there are up to several hundred-fold di¡erences in the usage of target sites due to the local DNA structure, bending, distortion and wrapping around nucleosomes (reviewed in [2]). The non-randomness on the scale of genomic regions has been much less addressed [3]. Methods employed to study in vivo retrovirus integration sites include restriction enzyme digestions and blotting, £uorescence in situ hybridization, PCRbased assays, and most importantly cloning and sequencing the virus^host integration junctions. Most studies analyze only a small number of integration sites, or focus on selected genomic regions. To date, the most representative study is provided by Carteau et al. [4]. It lists a set of 61 HIV-1 integration site sequences obtained after short experimental infection of the human T-cell line SupT1. Of these, 59 sequences are available in GenBank, together with 104 control genomic sequences for comparison. The authors analyzed the sequences using the nr, dbEST and MONTH databases as of November 1997. They concluded that there is no signi¢cant di¡erence between integration sites and controls, except that centromeric alphoid repeats are selectively absent at integration sites. The availability of the human genome sequence [5] creates a great opportunity for a new genome-wide analysis of these data. By mapping the exact positions of the integration sites we can analyze large DNA regions £anking the proviruses and describe the genomic features present. We used the BLAT program to map the genomic positions of integration sites in the most recent GoldenPath assembly of 6 August 2001 (http://genome.ucsc.edu). Of 59 sequences available in GenBank, we succeeded in mapping 48, where the level of homology was satisfactory (Table 1 in supplementary material on the web; http://www.elsevier.com/PII/ S0014579302026121). For each mapped integration we collected several genomic features available in the GoldenPath assembly. The ¢rst was the presence of transcribed sequences, either as the ‘known protein coding genes’ category (from the RefSeq project) or as the ‘human mRNAs from GenBank’ category. In addition, 800 cytogenetic band resolution is available, light or dark according to Giemsa staining. Next we calculated the GC level of 100 kb regions surrounding the integration sites symmetrically and the gene densities along these £anking regions. We compared these data with the whole genome summary statistics that we calculated for the GoldenPath assembly and looked for any di¡erences indicating possible integration preference. In our analysis, 54.2% (26 of 48) of the mapped integration sites fall in genes, which is signi¢cantly higher compared to the genome average calculated as 22.2% (P6 0.00001, M2 test). For the broader category of mRNAs this comparison is 68.7% to 30.7% (P6 0.00001, M2 test). This implies that potentially transcribed regions represent strongly preferred targets for HIV-1 integration. Out of the 33 integrations in transcription units, 18 and 15 are in sense and antisense orientation, respectively, 28 map to introns, two to exons, two to 5P untranslated regions (UTR) and one to a 3P UTR. Giemsa light (R) and dark (G) bands were targeted in 68.7% and 31.2%, compared to genome averages of 44.0% and 48.5%, respectively, estimated from the GoldenPath assembly (P6 0.003, M2 test). The average GC content of 100 kb regions £anking the integration sites was calculated to be 44.4%, higher than the whole genome average of 41.0%. The distribution of integrations is clearly biased, with more hits belonging to the GCricher genomic regions (Fig. 1).

The Core Element of a CpG Island Protects Avian Sarcoma and Leukosis Virus-Derived Vectors from Transcriptional Silencing

ABSTRACT Unmethylated CpG islands are known to keep adjacent promoters transcriptionally active. In the CpG island adjacent to the adenosine phosphoribosyltransferase gene, the protection against transcriptional silencing can be attributed to the short CpG-rich core element containing Sp1 binding sites. We report here the insertion of this CpG island core element, IE, into the long terminal repeat of a retroviral vector derived from Rous sarcoma virus, which normally suffers from progressive transcriptional silencing in mammalian cells. IE insertion into a specific position between enhancer and promoter sequences led to efficient protection of the integrated vector from silencing and gradual CpG methylation in rodent and human cells. Individual cell clones with IE-modified reporter vectors display high levels of reporter expression for a sustained period and without substantial variegation in the cell culture. The presence of Sp1 binding sites is important for the protective effect of IE, but at least some part of the entire antisilencing capacity is maintained in IE with mutated Sp1 sites. We suggest that this strategy of antisilencing protection by the CpG island core element may prove generally useful in retroviral vectors.

Tumor induction by the LTR, v-src, LTR DNA in four B (MHC) congenic lines of chickens

We report that the cloned DNA harboring the long terminal repeat (LTR), v-src, LTR proviral structure is tumorigenic in chickens of the Prague congenic lines. The growth rate of these tumors is by far the highest in the recombinant CC.R1 line, the B haplotype of which is composed of the B-F/L4 and B-G12 subregions originating from different naturally occurring haplotypes. Some of the tumors induced by the LTR, v-src, LTR DNA are repeatedly transplantable in syngeneic chickens, maintain unaltered provirus, and express v-src mRNA. Differences in the response to challenge with Rous sarcoma virus (RSV) and LTR, v-src, LTR DNA on a given experimental model are compared and possible involvement of an interaction between B-F/L and B-G region gene is considered. Regression of the LTR, v-src, LTR DNA-induced tumors did not prevent the formation and growth of tumors induced subsequently by RSV.

src-specific immunity in inbred chickens bearing v-src DNA- and RSV-induced tumors

The growth pattern (progression/regression) of v-src DNA- and Rous sarcoma virus (RSV)-induced tumors was analogous on a panel of inbred chicken lines. The decisive role of the major histocompatibility complex [Mhc(B)] alleles in resistance to the progression of these tumors was formally proved in segregating backcross populations. The immune mechanism of tumor regression was demonstrated by both in vivo and in vitro assays. A protective effect of v-src-specific immunity against RSV challenge was shown in Rous sarcoma regressor line CB (B12/B12). Immune cells from regressors of v-src DNA-induced tumors can protect syngeneic hosts from the development of tumor after challenge with both v-src DNA and RSV. Suppression of RSV-induced tumor cell growth in vitro was also achieved by the use of cocultivation with spleen cells from chickens in which v-src DNA-induced tumors had regressed. This in vitro sarcoma-specific response was Mhc(B)-restricted. Chickens of the congenic Rous sarcoma progressor line CC (B4/B4) are sometimes able to regress v-src DNA-induced tumors, but immune cells can only slow the growth of v-src DNA-induced tumors in syngeneic hosts. This suggests that the primary reason for the susceptibility of CC chickens is a weak v-src-specific immune response. Furthermore, some of the v-src DNA-induced tumors were transplantable across the Mhc(B) barrier. The growth of tumor allografts was able to be facilitated when immunological tolerance to the B-F/L region antigens (class I and class II) had been established. This demonstrated that a high tumorigenicity of the transplantable tumor was not due to the lack of Mhc(B) antigens on tumor cells.

The transcription factor EGR1 regulates metastatic potential of v-src transformed sarcoma cells.

Metastatic spreading of cancer cells is a highly complex process directed primarily by the interplay between tumor microenvironment, cell surface receptors, and actin cytoskeleton dynamics. To advance our understanding of metastatic cancer dissemination, we have developed a model system that is based on two v-src transformed chicken sarcoma cell lines—the highly metastatic parental PR9692 and a non-metastasizing but fully tumorigenic clonal derivative PR9692-E9. Oligonucleotide microarray analysis of both cell lines revealed that the gene encoding the transcription factor EGR1 was downregulated in the non-metastatic PR9692-E9 cells. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from parental PR9692 cells. Microarray analysis of EGR1 reconstituted cells revealed the activation of genes that are crucial for actin cytoskeleton contractility (MYL9), filopodia formation (MYO10), the production of specific extracellular matrix components (HAS2, COL6A1-3) and other essential pro-metastatic abilities.

Transcriptional provirus silencing as a crosstalk of de novo DNA methylation and epigenomic features at the integration site

The autonomous transcription of integrated retroviruses strongly depends on genetic and epigenetic effects of the chromatin at the site of integration. These effects are mostly suppressive and proviral activity can be finally silenced by mechanisms, such as DNA methylation and histone modifications. To address the role of the integration site at the whole-genome-scale, we performed clonal analysis of provirus silencing with an avian leucosis/sarcoma virus-based reporter vector and correlated the transcriptional silencing with the epigenomic landscape of respective integrations. We demonstrate efficient provirus silencing in human HCT116 cell line, which is strongly but not absolutely dependent on the de novo DNA methyltransferase activity, particularly of Dnmt3b. Proviruses integrated close to the transcription start sites of active genes into the regions enriched in H3K4 trimethylation display long-term stability of expression and are resistant to the transcriptional silencing after over-expression of Dnmt3a or Dnmt3b. In contrast, proviruses in the intergenic regions tend to spontaneous transcriptional silencing even in Dnmt3a−/− Dnmt3b−/− cells. The silencing of proviruses within genes is accompanied with DNA methylation of long terminal repeats, whereas silencing in intergenic regions is DNA methylation-independent. These findings indicate that the epigenomic features of integration sites are crucial for their permissivity to the proviral expression.