Long-read sequencing of an advanced cancer cohort resolves rearrangements, unravels haplotypes, and reveals methylation landscapes.

The Long-Read Personalized OncoGenomics (POG) dataset comprises a cohort of 189 patient tumors and 41 matched normal samples sequenced using the Oxford Nanopore Technologies PromethION platform. This dataset from the POG program and the Marathon of Hope Cancer Centres Network includes DNA and RNA short-read sequence data, analytics, and clinical information. We show the potential of long-read sequencing for resolving complex cancer-related structural variants, viral integrations, and extrachromosomal circular DNA. Long-range phasing facilitates the discovery of allelically differentially methylated regions (aDMRs) and allele-specific expression, including recurrent aDMRs in the cancer genes RET and CDKN2A. Germline promoter methylation in MLH1 can be directly observed in Lynch syndrome. Promoter methylation in BRCA1 and RAD51C is a likely driver behind homologous recombination deficiency where no coding driver mutation was found. This dataset demonstrates applications for long-read sequencing in precision medicine and is available as a resource for developing analytical approaches using this technology.

Genomic structures and regulation patterns at HPV integration sites in cervical cancer.

Human papillomavirus (HPV) integration has been implicated in transforming HPV infection into cancer, but its genomic consequences have been difficult to study using short-read technologies. To resolve the dysregulation associated with HPV integration, we performed long-read sequencing on 63 cervical cancer genomes. We identified six categories of integration events based on HPV-human genomic structures. Of all HPV integrants, defined as two HPV-human breakpoints bridged by an HPV sequence, 24% contained variable copies of HPV between the breakpoints, a phenomenon we termed heterologous integration. Analysis of DNA methylation within and in proximity to the HPV genome at individual integration events revealed relationships between methylation status of the integrant and its orientation and structure. Dysregulation of the human epigenome and neighboring gene expression in cis with the HPV-integrated allele was observed over megabase-ranges of the genome. By elucidating the structural, epigenetic, and allele-specific impacts of HPV integration, we provide insight into the role of integrated HPV in cervical cancer.

The genome sequence of the Loggerhead sea turtle, Caretta caretta Linnaeus 1758.

We present a genome assembly of Caretta caretta (the Loggerhead sea turtle; Chordata, Testudines, Cheloniidae), generated from genomic data from two unrelated females. The genome sequence is 2.13 gigabases in size. The assembly has a busco completion score of 96.1% and N50 of 130.95 Mb. The majority of the assembly is scaffolded into 28 chromosomal representations with a remaining 2% of the assembly being excluded from these.

Redesign of a fixture mount to be used as an impression coping and a provisional abutment as well.

PURPOSE: An integrated fixture mount/impression coping/ temporary abutment can provide many advantages for immediate loading of dental implants, such as simpler procedure, less chair time, cost reduction, and comfort for the patients. MATERIALS AND METHODS: A newly designed dental implant fixture mount (DIFMA) can be used as an impression coping for taking an immediate impression. An immediate load provisional prosthesis can then be fabricated shortly after implant placement to immediately load the implants. This fixture mount can also serve as a temporary abutment for immediate chair-side fabrication of provisional prosthesis. Two clinical cases are presented. RESULTS: A clinical case utilizing the fixture mount abutment (DIFMA)/implant assembly is presented. The precision of fitting between the impression copings and implants is secured with this system. The chair time for taking an immediate impression is greatly reduced. Less cost for the restoration is provided and patient comfort is delivered. CONCLUSIONS: More patient satisfaction can be conferred by employing the fixture mount in the process of immediate impression taking and as an immediate provisional abutment.

Proteomic analysis of proteins regulated by TRPS1 transcription factor in DU145 prostate cancer cells.

The aim of the present study was to identify proteins differentially regulated by TRPS1 in human prostate cancer cells in order to better understand the role of TRPS1 in prostate cancer development. The proteomes of androgen-independent DU145 prostate cancer cells, that do not express TRPS1 and of genetically engineered DU145 cells that stable and inducible express recombinant TRPS1 protein, were compared. Using two-dimensional electrophoresis followed by mass spectrometric analysis, 13 proteins that were differentially expressed between these two cell lines were identified. These proteins represent a dominant reduction of expression of antioxidant proteins, including superoxide dismutase, protein disulfide isomerase A3 precursor, endoplasmin precursor and annexin A2. Furthermore, regulation was observed for mitochondrion-associated proteins, glycolytic enzymes, a cytoskeleton-associated protein, a nuclear protein and proteins involved in apoptosis. Our data indicate that overexpression of TRPS1 protein is correlated with reduced protein expression of certain antioxidants. This suggests a possible involvement of TRPS1 in oxidative stress, and possibly in apoptosis in androgen-independent DU145 prostate cancer cells.

The TRPS1 transcription factor: androgenic regulation in prostate cancer and high expression in breast cancer.

TRPS1 mRNA is more highly expressed in androgen-dependent lymph node carcinoma of prostate-fast growing colony (LNCaP-FGC) compared with androgen-independent lymph node carcinoma of prostate-lymph node original (LNCaP-LNO) prostate cancer cell lines. Furthermore, TRPS1 mRNA expression is down-regulated by androgens in LNCaP-FGC cells, a process mediated by the androgen receptor (AR). Here, we present TRPS1 protein expression in human prostate cancer material derived from a panel of six androgen-dependent and eight androgen-independent human prostate cancer xenografts. TRPS1 protein is expressed in all androgen-dependent xenografts, which also express AR and prostate-specific antigen (PSA). Androgen withdrawal by castration resulted in an increase in TRPS1 protein in two androgen-dependent xenografts, indicating relieved repression by action of AR. TRPS1 protein is expressed in four androgen-independent xenografts and is low or absent in the other four androgen-independent xenografts. Androgen withdrawal by castration demonstrates that TRPS1 protein levels remain the same in 1 androgen-independent xenograft, most likely due to the lack of AR expression. These data show that TRPS1 protein expression is regulated by androgens via the AR in human prostate cancer xenografts. Analysis of TRPS1 mRNA expression in normal and tumour tissue of the prostate and 18 other human tissues, showed that TRPS1 had the highest mRNA expression levels in normal and tumour tissues of breast. In addition, high TRPS1 mRNA and protein expression levels were observed in four out of five human breast cancer cell lines. In conclusion, TRPS1 protein expression is down-regulated by androgens in human prostate cancer, and analysis of TRPS1 mRNA expression levels in several human tissues showed that the highest levels were observed in normal and tumour breast tissue.

The atypical GATA protein TRPS1 represses androgen-induced prostate-specific antigen expression in LNCaP prostate cancer cells.

Prostate-specific antigen (PSA) is considered as an important marker for prostate cancer. Regulation of PSA gene expression is mediated by androgens bound to androgen receptors via androgen response elements (AREs) in its promoter and far upstream enhancer regions. In addition, GATA proteins contribute to PSA gene transcription by interacting with GATA motifs present in the PSA enhancer sequence. The TRPS1 gene contains a single GATA zinc finger domain and not only binds to forward consensus GATA motifs but also to an inverse GATA motif overlapping the ARE III in the far upstream enhancer of the PSA gene. Overexpression of TRPS1 in androgen-dependent human LNCaP prostate cancer cells inhibited the transcription of a transiently transfected PSA enhancer/promoter-driven luciferase reporter construct. Furthermore, overexpression of TRPS1 reduced the androgen-induced endogenous PSA levels secreted in culture medium of LNCaP cells. Our results suggest a role of TRPS1 in androgen regulation of PSA gene expression.

The RING finger protein RNF4, a co-regulator of transcription, interacts with the TRPS1 transcription factor.

The TRPS1 gene encodes a repressor of GATA-mediated transcription. Mutations in this gene cause the tricho-rhino-phalangeal syndromes, but the affected pathways are unknown. In a yeast two-hybrid screen with the C-terminal part of the murine Trps1 protein as bait, we obtained three yeast clones encoding two overlapping fragments of the 194 amino acids RING finger protein 4 (Rnf4). The overlap narrows down the Trps1-binding region within Rnf4 to amino acids 6-65. This region in Rnf4 is also known to interact with several proteins including steroid receptors. By using truncated Trps1 constructs, the Rnf4-binding region in Trps1 could be assigned to amino acids 985-1184 of 1281. This 200 amino acid region of Trps1 does not contain any predicted protein-protein interacting motif. Complex formation between the human proteins TRPS1 and RNF4 was verified by co-immunoprecipitation from transfected and native mammalian cells. Confocal laser-scanning microscopy revealed that the endogenous proteins are located in distinct structures of the nucleus. Using a luciferase reporter assay, we could demonstrate that the repressional function of TRPS1 is inhibited by RNF4. This finding suggests that RNF4 is a negative regulator of TRPS1 activity.

Nuclear interaction of the dynein light chain LC8a with the TRPS1 transcription factor suppresses the transcriptional repression activity of TRPS1.

The TRPS1 gene codes for a 1281 amino acids nuclear transcription factor with an unusual combination of different types of zinc finger motifs, including GATA-type DNA-binding and IKAROS-like zinc fingers. TRPS1 is a repressor of GATA-regulated genes and implicated in the human tricho-rhino-phalangeal syndromes. We found that two distinct regions of TRPS1 can physically interact with the dynein light chain 8 protein, LC8a, that are at least 458 amino acids apart from each other. Region A covers 89 amino acids (635-723), spanning three potential C(2)H(2) zinc finger structures, and region B covers the 100 most C-terminal amino acids (1182-1281) containing the IKAROS-like motif. LC8a is known to interact with more than 10 different molecules, both proteins and nucleic acids. In most cases, LC8a was identified as a transport molecule in the cytoplasm. Interestingly, we found that LC8a co-localizes with TRPS1 in dot-like structures in the cell nucleus. In an electrophoretic mobility shift assay we could show that the interaction of LC8a and TRPS1 lowers the binding of TRPS1 to the GATA consensus sequence. In addition, GATA-regulated reporter gene assay indicated that LC8a is able to suppress the transcriptional repression activity of TRPS1.