Onco-exaptation of an endogenous retroviral LTR drives IRF5 expression in Hodgkin lymphoma.

The transcription factor interferon regulatory factor 5 (IRF5) is upregulated in Hodgkin lymphoma (HL) and is a key regulator of the aberrant transcriptome characteristic of this disease. Here we show that IRF5 upregulation in HL is driven by transcriptional activation of a normally dormant endogenous retroviral LOR1a long terminal repeat (LTR) upstream of IRF5. Specifically, through screening of RNA-sequencing libraries, we detected LTR-IRF5 chimeric transcripts in multiple HL cell lines but not in normal B-cell controls. In HL, the LTR was in an open and hypomethylated epigenetic state, and we further show the LTR is the site of transcriptional initiation. Among HL cell lines, usage of the LTR promoter strongly correlates with overall levels of IRF5 mRNA and protein, indicating that LTR transcriptional awakening is a major contributor to IRF5 upregulation in HL. Taken together, oncogenic IRF5 overexpression in HL is the result of a specific LTR transcriptional activation. We propose that such LTR derepression is a distinct mechanism of oncogene activation ('onco-exaptation'), and that such a mechanism warrants further investigation in molecular and cancer research.

Potential mechanisms of endogenous retroviral-mediated genomic instability in human cancer.

Malignancy results from a complex combination of genetic and epigenetic changes, the full effects of which are still largely unknown. Here we summarize current knowledge of the origin, retrotranspositional activity, epigenetic state, and transcription of human endogenous retroviruses (HERVs), and then discuss the potential effects of their deregulation in cancer. Evidence suggests that cancer-associated epigenetic changes most likely underlie potential HERV-mediated effects on genome and transcriptome instability and may play a role in malignancy. Despite our currently limited understanding of the importance of HERVs or other transposable elements in cancer development, we believe that the emerging era of high-throughput sequencing of cancer genomes, epigenomes, and transcriptomes will provide unprecedented opportunities to investigate these roles in the future.

Keeping active endogenous retroviral-like elements in check: the epigenetic perspective.

Endogenous retrovirus-like elements, or ERVs, are an abundant component of all eukaryotic genomes. Their transcriptional and retrotranspositional activities have great potential for deleterious effects on gene expression. Consequences of such activity may include germline mutagenesis and cancerous transformation. As a result, mammalian genomes have evolved means of counteracting ERV transcription and mobilization. In this review, we discuss epigenetic mechanisms of ERV and LTR retrotransposon control during mouse development, focusing on involvement of DNA methylation, histone modifications, small RNAs and their interaction with one another. We also address relevance of research performed in the mouse system to human and challenges associated with studying repetitive families. (Part of a multi-author review).

Bacteria and cancer: cause, coincidence or cure? A review.

Research has found that certain bacteria are associated with human cancers. Their role, however, is still unclear. Convincing evidence links some species to carcinogenesis while others appear promising in the diagnosis, prevention or treatment of cancers. The complex relationship between bacteria and humans is demonstrated by Helicobacter pylori and Salmonella typhi infections. Research has shown that H. pylori can cause gastric cancer or MALT lymphoma in some individuals. In contrast, exposure to H. pylori appears to reduce the risk of esophageal cancer in others. Salmonella typhi infection has been associated with the development of gallbladder cancer; however S. typhi is a promising carrier of therapeutic agents for melanoma, colon and bladder cancers. Thus bacterial species and their roles in particular cancers appear to differ among different individuals. Many species, however, share an important characteristic: highly site-specific colonization. This critical factor may lead to the development of non-invasive diagnostic tests, innovative treatments and cancer vaccines.

Impact of transposable elements on the evolution of mammalian gene regulation.

Transposable elements (TEs) are present in all organisms and nearly half of the human and mouse genome is derived from ancient transpositions. This fact alone suggests that TEs have played a major role in genome organization and evolution. Studies undertaken over the last two decades or so clearly show that TEs of various kinds have played an important role in organism evolution. Here we review the impact TEs have on the evolution of gene regulation and gene function with an emphasis on humans. Understanding the mechanisms resulting in genomic change is central to our understanding of gene regulation, genetic disease and genome evolution. Full comprehension of these biological processes is not possible without an in depth knowledge of how TEs impact upon the genome.

The salivary microbiota as a diagnostic indicator of oral cancer: a descriptive, non-randomized study of cancer-free and oral squamous cell carcinoma subjects.

BACKGROUND: The purpose of the present investigation was to determine if the salivary counts of 40 common oral bacteria in subjects with an oral squamous cell carcinoma (OSCC) lesion would differ from those found in cancer-free (OSCC-free) controls. METHODS: Unstimulated saliva samples were collected from 229 OSCC-free and 45 OSCC subjects and evaluated for their content of 40 common oral bacteria using checkerboard DNA-DNA hybridization. DNA counts per ml saliva were determined for each species, averaged across subjects in the 2 subject groups, and significance of differences between groups determined using the Mann-Whitney test and adjusted for multiple comparisons. Diagnostic sensitivity and specificity in detection of OSCC by levels of salivary organisms were computed and comparisons made separately between a non-matched group of 45 OSCC subjects and 229 controls and a group of 45 OSCC subjects and 45 controls matched by age, gender and smoking history. RESULTS: Counts of 3 of the 40 species tested, Capnocytophaga gingivalis, Prevotella melaninogenica and Streptococcus mitis, were elevated in the saliva of individuals with OSCC (p < 0.001). When tested as diagnostic markers the 3 species were found to predict 80% of cancer cases (sensitivity) while excluding 83% of controls (specificity) in the non-matched group. Diagnostic sensitivity and specificity in the matched group were 80% and 82% respectively. CONCLUSION: High salivary counts of C. gingivalis, P. melaninogenica and S. mitis may be diagnostic indicators of OSCC.

Repetitive elements in the 5' untranslated region of a human zinc-finger gene modulate transcription and translation efficiency.

A substantial proportion of the human genome consists of repetitive sequences. Although most of these sequences are nonessential for the organism, retroelements, such as Alu sequences, L1s, and HERVs (human endogenous retroviruses), have recently been implicated in the regulation of various genes. Our laboratory previously identified a novel, alternatively spliced zinc-finger gene, ZNF177, which incorporates Alu L1, and HERV segments into the 5' untranslated region (UTR) of transcripts. In this study, we investigated the genomic structure and functional significance of the repetitive sequences in the 5' UTR of ZNF177 mRNAs. Using luciferase and GFP reporter constructs, we assessed the effect of the HERV, Alu, and L1 sequences on gene expression levels. Our results indicate that the presence of the retroelement sequences, particularly the Alu and L1 segments which form one 5' UTR exon, modifies the expression level of both reporter genes. We present evidence that the Alu and L1 sequences alter both the RNA and protein levels of reporter genes by increasing transcription efficiency while decreasing translation efficiency. Our findings indicate that the Alu and L1 repeats in the 5' UTR of ZNF177 exert a positive transcriptional enhancer effect, but repress translation of the zinc finger gene. In addition, our analysis of a 5' UTR database suggests that 4% of human 5' UTRs harbor Alu sequences, indicating that the expression of many genes might be influenced by Alu repeats. These results illustrate the complex regulatory effects that retroelements can have on human gene expression.

Ly49 and CD94/NKG2: developmentally regulated expression and evolution.

Murine natural killer (NK) cells express two families of MHC class I-specific receptors, namely the Ly49 family and CD94/NKG2 heterodimers. Stochastic co-expression of these receptors generates diverse receptor repertoires in adult NK-cell populations, whereas fetal NK cells have much more limited receptor diversity as they mostly express CD94/NKG2A but not Ly49. These receptors are also expressed on CD8-T cells and NK1.1+ T cells and regulate their functions, but their expression pattern on NK cells is significantly different from those on T cells. Thus, expression of Ly49 and CD94/NKG2 is developmentally regulated. NK cells acquire the Ly49 family of receptors in an orderly manner as they differentiate from bone marrow progenitors in vitro. Similarly, acquisition of CD94 and NKG2 by NK cells as they differentiate from embryonic stem cells is also orderly To gain insight into the mechanisms regulating Ly49 expression, potential regulatory regions of several Ly49 genes have been examined. Ly49 genes with different expression patterns have remarkably similar sequences in the putative regulatory regions. Finally, a functional Ly49 gene has been identified in baboon, and primate comparisons suggest that functional extinction of the Ly49 gene in the human lineage seems to have been a relatively recent event.

Comparative analysis of the promoter regions and transcriptional start sites of mouse Ly49 genes.

Despite numerous studies on the function of Ly49 natural killer cell receptors in the mouse, relatively little is known about how these genes are regulated at the transcriptional level. In the present study, we sequenced and compared 800 bp of the promoter region of nine Ly49 genes from C57B1/6 mice. This comparison showed that there is a high degree of sequence identity between the genes, and also revealed a region which is conserved between the mouse genes and the human Ly49L gene, indicating a potential core promoter region. This analysis also found that Ly49B and H differ from the other genes in having long interspersed repetitive sequence in their promoter region which suggests a gene conversion or rearrangement involving these two genes. In addition, we performed 5' rapid amplification of cDNA ends on four Ly49 genes to localize transcriptional start sites. These experiments showed that the transcriptional initiation sites are heterogeneous for all of the genes examined, and that a large majority of Ly49G transcripts originate from the second exon as well as its first intron. Although potential TATA boxes have been previously identified for some of the genes, we did not find evidence that a majority of transcripts initiate at the expected distance downstream of these boxes. Our data suggest that differences in the location of transcriptional start sites contribute to the observed complexity in receptor repertoire patterns.

Evolution of natural killer cell receptors: coexistence of functional Ly49 and KIR genes in baboons.

Natural killer (NK) cells represent an important first line of defense against viruses and malignancy [1]. NK cells express a variety of inhibitory and activating receptors that interact with classical major histocompatibility complex (MHC) class I molecules on potential target cells and determine the NK cell response [2-4]. Mouse NK receptors are encoded by the C-type lectin multigene family Ly49. However, in humans, a completely different family of receptors, the immunoglobulin-like killer inhibitory receptors (KIRs), performs the same function [2-4]. One Ly49-like gene, Ly49L, exists in humans but is incorrectly spliced and assumed to be nonfunctional [5, 6]. Mouse KIR-like genes have not been found, and evidence suggests that the primate KIRs amplified after rodents and primates diverged [7, 8]. Thus, two structurally dissimilar families, Ly49 and KIR, have evolved to play similar roles in mouse and human NK cells. This apparent example of functional convergent evolution raises several questions. It is unknown, for example, when the Ly49L gene became nonfunctional and if this event affected the functional evolution of the KIRs. The distribution of these gene families in different mammals is unstudied, and it is not known if any species uses both types of receptors. Here, we demonstrate that the Ly49L gene shows evidence of conservation in other mammals and that the human gene likely became nonfunctional 6-10 million years ago. Furthermore, we show that baboon lymphocytes express both full-length Ly49L transcripts and multiple KIR genes.

Functional analysis of 5' and 3' regions of the closely related Ly49c and j genes.

The Ly49 multigene family consists of at least 14 closely related genes located in the natural killer (NK) gene complex on mouse Chromosome 6. Reverse transcriptase (RT)-PCR on single NK cells has shown that Ly49c is expressed on approximately 50% of NK cells, whereas the closely related Ly49j gene is expressed on 5-8% of NK cells. In this study, we examined three regions to determine whether they contain cis-acting elements involved in regulating the expression of these two closely related Ly49 genes within NK cells. Luciferase reporter assays in EL-4 cells suggested that the 5' regions of Ly49c and j contain promoter elements and repressor sequences. In addition, luciferase assays suggest that Ly49j also contains an active promoter in the first intron, although the transcripts produced from this promoter appear to be severely truncated. Finally, comparisons of the 3' noncoding regions of Ly49c and j revealed that the sequence of Ly49j diverges completely from Ly49c 130 bp downstream of the termination codon. The polyadenylation signal for Ly49j is located downstream of the Ly49c poly(A) site, which results in a much longer 3' untranslated region (UTR). When the Ly49j 3'UTR was used to provide the polyadenylation signal for the green fluorescent protein (GFP) reporter gene, GFP expression was reduced twofold. These results suggest that both internal promoters, repressors, and 3' regions play a role in regulating Ly49 gene expression.

Long terminal repeats are used as alternative promoters for the endothelin B receptor and apolipoprotein C-I genes in humans.

To examine the potential regulatory involvement of retroelements in the human genome, we screened the transcribed sequences of GenBank and expressed sequence tag data bases with long terminal repeat (LTR) elements derived from different human endogenous retroviruses. These screenings detected human transcripts containing LTRs belonging to the human endogenous retrovirus-E family fused to the apolipoprotein CI (apoC-I) and the endothelin B receptor (EBR) genes. However, both genes are known to have non-LTR (native) promoters. Initial reverse transcription-polymerase chain reaction experiments confirmed and authenticated the presence of transcripts from both the native and LTR promoters. Using a 5'-rapid amplification of cDNA ends protocol, we showed that the alternative transcripts of apoC-I and EBR are initiated and promoted by the LTRs. The LTR-apoC-I fusion and native apoC-I transcripts are present in many of the tissues tested. As expected, we found apoC-I preferentially expressed in liver, where about 15% of the transcripts are derived from the LTR promoter. Transient transfections suggest that the expression is not dependent on the LTR itself, but the presence of the LTR increases activity of the apoC-I promoter from both humans and baboons. The native EBR-driven transcripts were also detected in many tissues, whereas the LTR-driven transcripts appear limited to placenta. In contrast to the LTR of apoC-I, the EBR LTR promotes a significant proportion of the total EBR transcripts, and transient transfection results indicate that the LTR acts as a strong promoter and enhancer in a placental cell line. This investigation reports two examples where LTR sequences contribute to increased transcription of human genes and illustrates the impact of mobile elements on gene and genome evolution.

The genomic organization of the mouse CD94 C-type lectin gene.

The mouse natural killer (NK) gene complex is located on chromosome 6 and contains a number of genes encoding C-type lectin receptors which have been found to regulate NK cell function. Among these are CD94 and four NKG2 genes. Like its human counterpart, the mouse CD94 protein associates with different NKG2 isoforms and recognizes the atypical MHC class I molecule Qa-1b. Here, the genomic organization of the mouse CD94 gene was determined by analysing a BAC clone containing the CD94 gene. The mouse CD94 gene contains six exons separated by five introns. Exons I and II encode the 5' untranslated region (UTR) and the transmembrane domain. Exon III encodes the stalk region and exons IV-VI encode the carbohydrate recognition domain (CRD). Furthermore, we cloned and sequenced the CD94 promoter region, and putative regulatory DNA elements were identified. Further studies on the CD94 promoter region may help to elucidate the restricted expression pattern of CD94 in NK cells and a subpopulation of T cells.

Novel mouse type D endogenous proviruses and ETn elements share long terminal repeat and internal sequences.

The repetitive ETn (early transposon) family of sequences represents an active "mobile mutagen" in the mouse genome. The presence of long terminal repeats (LTRs) and other diagnostic features indicate that ETns are retrotransposons but they contain no long open reading frames or documented similarity to the genes of known retroviruses or other retroelements. Thus, the mechanisms responsible for the mobility of this family have been unknown. In this study, we used computer searches to detect a small region of previously unrecognized type D retroviral pol homology within ETn elements. This small region was used to isolate two mouse endogenous proviral elements with gag, pro, and pol genes similar to simian type D viruses. This new family of mouse endogenous proviruses, termed MusD, is present in several hundred copies in the genome. Interestingly, the MusD LTRs, 3' internal region, and the 5' region expected to contain the packaging signal are very closely related to members of the ETn subfamily that have recently transposed. Analysis of different mouse strains indicates that MusD elements predate the existence of the mobile subfamily of ETns. These findings indicate that the ETn family was likely created via recombination events resulting in a near complete substitution of MusD coding sequences with unrelated DNA. Furthermore, these results suggest that ETn transcripts retrotranspose using proteins provided by MusD proviruses.

Endogenous retroviruses provide the primary polyadenylation signal for two new human genes (HHLA2 and HHLA3).

By screening the expressed sequence tag (EST) database, we identified transcripts of two new human genes that are polyadenylated within a long terminal repeat (LTR) of the HERV-H endogenous retrovirus family. The first gene, termed HHLA2, is represented by two EST clones and one cDNA clone, all of which have a polyadenylated LTR as their 3' end. The gene has an open reading frame (ORF) of 414 amino acids with three immunoglobulin-like domains and is expressed primarily in intestinal tissues, kidney, and lung. Seven small EST clones from several different tissues were found for the second gene, termed HHLA3. As with HHLA2, all HHLA3 ESTs utilized a HERV-H LTR as the polyadenylation signal. Three types of alternatively spliced HHLA3 transcripts that could encode proteins of 76, 121, or 153 amino acids were detected. Interestingly, the ORF for two of these transcripts continues into the LTR. For both HHLA2 and 3, no major human transcripts that utilized a non-LTR polyadenylation signal were detected. Analysis of RNA from baboon, which lacks the LTRs at these genomic loci, showed that the baboon HHLA2 and 3 genes use other polyadenylation signals. This study demonstrates that ancient retroviral insertions have assumed gene regulatory functions during the course of human evolution.

Diversity of NK cell receptor repertoire in adult and neonatal mice.

Murine NK cytotoxicity is regulated by two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2. We developed a single-cell RT-PCR method to analyze expression of all known Ly49 and NKG2A genes in individual NK cells and determined the receptor repertoires of NK cells from adult and neonatal (1-wk-old) C57BL/6 mice. In adult mouse NK cells, up to six different receptors were coexpressed in random combinations. Of 62 NK cells examined, 42 different patterns of receptor expression were observed. Most of them expressed at least one Ly49, whereas NKG2A was detected in 32% of the cells. Over 75% of them expressed Ly49C, I, or NKG2A, which are thought to recognize self-class I MHC (H-2b). Coexpression of multiple Ly49 receptors and NKG2A was stochastic. In contrast, very few neonatal NK cells expressed any Ly49, but almost 60% of them expressed NKG2A. These results demonstrate that adult NK cells are quite heterogeneous and have diverse receptor repertoires. They also suggest that the expression of NKG2A precedes Ly49 expression in NK cell ontogeny, and NKG2A is a major inhibitory receptor in neonatal NK cells.

Isolation of a human endogenous retroviral HERV-H element with an open env reading frame.

About 100 elements of the human endogenous retroviral HERV-H family have full-length env genes potentially coding for Env proteins with sequences highly similar to the immunosuppressive peptide CKS-17 from the MLV transmembrane protein p15E. However, previously sequenced HERV-H env genes have contained stop codons or framehifts. To isolate elements with open env reading frames, we first tried to assess the diversity of HERV-H env genes by comparing PCR-generated env sequences from genomic DNA with published HERV-H sequences. A region at the beginning of env displayed a similarity of 84-98% among 15 different elements. We then used a probe from one of the PCR-generated clones, 98% similar to the consensus sequence in this region, to screen a human genomic lambda library. Three HERV-H elements displaying ca. 98% identity in the env gene were isolated and were shown to have integrated relatively recently, after the divergence of the orangutan and the african great ape lineages. One of these elements, HERV-H19, had a 1752-bp open env reading frame, producing a 77-kDa Env protein in in vitro translation reactions. This is the first demonstration of a coding competent member of the HERV-H family. These findings raise the possibility that HERV-H Env proteins may play a biological role in human cells.

Expression analysis of new Ly49 genes: most transcripts of Ly49j lack the transmembrane domain.

Five new Ly49 genes, named Ly49j-n, have recently been identified in C57BL/6 mice. This study examined the expression of three of these new genes, Ly49j, k, and n. To determine whether the Ly49j, k, and n genes were transcribed, gene-specific primers were used to amplify cDNA clones for each gene from C57BL/6 interleukin-2-activated natural killer (NK) cell cDNA. A full-length cDNA for Ly49j was detected which encodes a 267 amino acid protein and shares approximately 96% nucleotide identity with Ly49c and i. COS cells transfected with the Ly49j cDNA were shown to react with the monoclonal antibody 8H7, suggesting that the gene likely encodes a functional protein. Many different sized Ly49k and n transcripts were observed, although it is likely that they do not encode functional proteins due to missing exons or severe truncations in the open reading frames. Interestingly, the most abundant Ly49j transcript detected was shown to lack exon 3, which encodes the transmembrane domain. Similar studies performed on the same source of NK cell cDNA using Ly49c- and i-specific primers revealed the presence of transmembrane-less Ly49i transcripts, although at a much lower frequency than observed for Ly49j. We also detected Ly49g and h transcripts lacking the transmembrane domain. Despite the absence of the transmembrane region, the resulting Ly49 transcripts maintain their open reading frames, and therefore could potentially encode cytoplasmic proteins with a role in NK cell function.