Keith Durkin

Serial translocation by means of circular intermediates underlies colour sidedness in cattle

Colour sidedness is a dominantly inherited phenotype of cattle characterized by the polarization of pigmented sectors on the flanks, snout and ear tips. It is also referred to as ‘lineback’ or ‘witrik’ (which means white back), as colour-sided animals typically display a white band along their spine. Colour sidedness is documented at least since the Middle Ages and is presently segregating in several cattle breeds around the globe, including in Belgian blue and brown Swiss. Here we report that colour sidedness is determined by a first allele on chromosome 29 (Cs29), which results from the translocation of a 492-kilobase chromosome 6 segment encompassing KIT to chromosome 29, and a second allele on chromosome 6 (Cs6), derived from the first by repatriation of fused 575-kilobase chromosome 6 and 29 sequences to the KIT locus. We provide evidence that both translocation events involved circular intermediates. This is the first example, to our knowledge, of a phenotype determined by homologous yet non-syntenic alleles that result from a novel copy-number-variant-generating mechanism.

Deep sequencing reveals abundant noncanonical retroviral microRNAs in B-cell leukemia/lymphoma

Viral tumor models have significantly contributed to our understanding of oncogenic mechanisms. How transforming delta-retroviruses induce malignancy, however, remains poorly understood, especially as viral mRNA/protein are tightly silenced in tumors. Here, using deep sequencing of broad windows of small RNA sizes in the bovine leukemia virus ovine model of leukemia/lymphoma, we provide in vivo evidence of the production of noncanonical RNA polymerase III (Pol III)-transcribed viral microRNAs in leukemic B cells in the complete absence of Pol II 5′-LTR–driven transcriptional activity. Processed from a cluster of five independent self-sufficient transcriptional units located in a proviral region dispensable for in vivo infectivity, bovine leukemia virus microRNAs represent ∼40% of all microRNAs in both experimental and natural malignancy. They are subject to strong purifying selection and associate with Argonautes, consistent with a critical function in silencing of important cellular and/or viral targets. Bovine leukemia virus microRNAs are strongly expressed in preleukemic and malignant cells in which structural and regulatory gene expression is repressed, suggesting a key role in tumor onset and progression. Understanding how Pol III-dependent microRNAs subvert cellular and viral pathways will contribute to deciphering the intricate perturbations that underlie malignant transformation.

The bovine genomic DNA sequence data reveal three IGHV subgroups, only one of which is functionally expressed

A comprehensive analysis of cattle shotgun sequencing data reveals 36 immunoglobulin heavy chain variable genes. The previously described bovine subgroup IGHV1 contains 10 functional genes with a conserved promoter including the consensus octamer and several other transcription factor binding sites, intact exons and matching cDNA sequences. Subgroups IGHV2 and IGHV3 consist entirely of pseudogenes. Thus, the bovine germline IGHV repertoire is very limited. The IGHV genes are distributed in mammalian clans I and II, while no clan III genes were detected. Clan-specific PCR of genomic DNA from cattle, sheep, Eurasian elk, white-tailed deer, pig and dolphin indicates highly dynamic evolution of IGHV gene usage within Cetartiodactyla. The bovine germline IGHV repertoire was probably generated by recent duplications of an IGHV1-IGHV2 homology unit. Immunoglobulin heavy chain genes are largely incorrectly assembled in the current cattle genome versions Btau_4.2 and UMD_3.1. FISH experiments confirm an IGHV locus close to terminus of BTA21.

Cis-perturbation of cancer drivers by the HTLV-1/BLV proviruses is an early determinant of leukemogenesis

Human T-cell leukaemia virus type-1 (HTLV-1) and bovine leukaemia virus (BLV) infect T- and B-lymphocytes, respectively, provoking a polyclonal expansion that will evolve into an aggressive monoclonal leukaemia in ∼5% of individuals following a protracted latency period. It is generally assumed that early oncogenic changes are largely dependent on virus-encoded products, especially TAX and HBZ, while progression to acute leukaemia/lymphoma involves somatic mutations, yet that both are independent of proviral integration site that has been found to be very variable between tumours. Here, we show that HTLV-1/BLV proviruses are integrated near cancer drivers which they affect either by provirus-dependent transcription termination or as a result of viral antisense RNA-dependent cis-perturbation. The same pattern is observed at polyclonal non-malignant stages, indicating that provirus-dependent host gene perturbation contributes to the initial selection of the multiple clones characterizing the asymptomatic stage, requiring additional alterations in the clone that will evolve into full-blown leukaemia/lymphoma.

Monitoring molecular response in adult T-cell leukemia by high-throughput sequencing analysis of HTLV-1 clonality

Monitoring molecular response in adult T-cell leukemia by high-throughput sequencing analysis of HTLV-1 clonality

Elucidating the role of Bovine Leukemia Virus encoded micro-RNAs

Bovine Leukemia Virus (BLV) and its close relative Human T-cell leukemia virus-1 (HTLV-1) display similar patterns of pathogenesis and genome organisation. The natural host of BLV is cattle, however it is possible to experimentally infect sheep with the virus. Infected sheep develop tumors following a significantly reduced latency period compared to cattle (20 months on average), making for an attractive cancer model. Like HTLV-1, BLV mRNAs/proteins transcribed from the 5’ LTR are silenced in tumors. However, our group and others have recently reported the presence of five highly expressed micro-RNAs transcribed from the BLV genome via a noncanonical RNA polymerase III pathway in BLV induced tumors. It has been noted that one of these micro-RNAs (BLV-miR-B4-3p) shares a seed sequence with miR-29, a regulator of the tumor suppressor HBP1. This observation points to one potential role for a single micro-RNA, however the role of the remaining micro-RNAs remains to be uncovered. In order to further explore potential targets of the BLV micro-RNAs we have carried out high throughput RNA sequencing of a number of experientially induced ovine and natural bovine BLV tumors, in addition to ovine derived BLV tumor cell lines. As a result we have identified a target of the viral-microRNAs and have begun exploring its role in the life cycle of the virus and its potential contribution to tumorigenesis. Data describing the results obtained to date will be discussed.

Identification and characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts reveals their constitutive expression in leukemic and pre-leukemic clones

Bovine Leukemia Virus (BLV) is a deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells and produces a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about ∽5% develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The 5’LTRs of both the BLV and HTLV-1 proviruses are transcriptionally silent in tumors, however they are not entirely quiescent, with the HLTV-1 antisense transcript HBZ and the BLV microRNAs constitutively expressed in tumors. Here, using RNA-seq, we demonstrate that in addition to microRNAs, the BLV provirus also constitutively expresses two antisense transcripts in all BLV infected samples examined. The first transcript (AS1) has alternate potential polyadenylation sites generating a short transcript of ∽600bp (aS1-S) and a less abundant longer transcript of ∽2200bp (AS1-L). Alternative splicing also creates a second transcript of ∽400bp (AS2) utilizing the first exon of AS1. Production of AS transcripts from the 3’LTR was supported by reporter assays demonstrating that the BLV LTR has substantial and Tax-independent antisense promoter activity. BLV AS transcripts predominantly localize in the nucleus. Examination of protein coding potential showed AS2 to be non-coding, while the AS1-S/L transcripts coding potential is ambiguous, with a small potential open reading frame (ORF) of 264bp present. The AS1-L transcript overlaps the BLV microRNAs transcribed in the sense direction. Using high throughput sequencing of RNA-ligase-mediated (RLM) 5’ RACE products, we show that the perfect complementary between the transcripts leads to RNA-induced silencing complex (RISC) mediated cleavage of AS1-L. Furthermore, experiments using BLV proviruses where the microRNAs were removed or inverted point to additional transcriptional interactions between the two viral RNA species. Knock down of AS1-S/L using locked nucleic acids (LNAs) showed no obvious effect on the cells phenotype. While a detailed elucidation of the BLV antisense transcripts function remains in the future, the constitutive expression in all samples examined, points to a vital role for the transcripts in the life cycle and oncogenic potential of BLV.

Improving the methodology for the detection of proviral integration sites in the host genome via high throughput sequencing

Bovine Leukemia Virus (BLV) and the closely related Human T-cell leukemia virus-1 (HTLV-1) are deltaretrovirus that induce leukemia/lymphoma in about ~5% of infected individuals. The mechanisms responsible for cellular transformation have remained largely enigmatic as both viruses are largely transcriptionally silent in tumors and show multiple integration sites in the host genome. The recent application of high throughput sequencing to track proviral insertion sites in the host genome has provided a number of insights into the evolution of deltaretrovirus infections and the progression of tumor clones in deltaretrovirus induced leukemia/lymphoma. However the protocols currently utilised have a number of limitations, including relatively high sequencing costs, the use of custom sequencing primers, no examination of the region upstream of the provirus and limited dynamic range for determining clone abundance. We have developed an alternative high throughput sequencing protocol for identifying proviral integration sites in BLV and HTLV-1 infected individuals that uses off-the-shelf Illumina primers for the addition of adapters and indexes. This greatly simplifies the process of multiplexing libraries and does away with the need for custom sequencing primers. Additionally our approach assays the region upstream of the provirus in addition to the downstream region, giving additional information on the frequency of 5’ deletions in proviruses and increasing the dynamic range of the assay. We have tested the approach on over 1 BLV and HTLV-1 samples, representing both tumors and preleukemic stages. Our approach allowed for a more accurate determination of clone abundance in tumors and by assaying the 5’ end of the provirus identified clones overlooked with previously published methods. Finally, by facilitating greater multiplexing of libraries we have reduced the cost to a level where the technique may be attractive in a clinical setting.