The extent to which the three-dimensional organization of the genome contributes to chromosomal translocations is an important question in cancer genomics. We generated a high-resolution Hi-C spatial ...organization map of the G1-arrested mouse pro-B cell genome and used high-throughput genome-wide translocation sequencing to map translocations from target DNA double-strand breaks (DSBs) within it. RAG endonuclease-cleaved antigen-receptor loci are dominant translocation partners for target DSBs regardless of genomic position, reflecting high-frequency DSBs at these loci and their colocalization in a fraction of cells. To directly assess spatial proximity contributions, we normalized genomic DSBs via ionizing radiation. Under these conditions, translocations were highly enriched in cis along single chromosomes containing target DSBs and within other chromosomes and subchromosomal domains in a manner directly related to pre-existing spatial proximity. By combining two high-throughput genomic methods in a genetically tractable system, we provide a new lens for viewing cancer genomes.
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► A high-resolution Hi-C spatial organization map of the G1-arrested mouse cell genome ► Genomic spatial heterogeneity contributes to recurrent chromosomal translocations ► Spatial organization of chromosomes promotes dominant translocations in cis ► Translocations are guided genome-wide by pre-existing spatial genome organization
The first high-resolution Hi-C map of the murine G1 nucleus combined with high-throughput genome-wide translocation sequencing reveals how chromatin spatial organization contributes to DSB-driven translocations.
The new category of MiT family translocation renal cell carcinoma has been included into the World Health Organization (WHO) classification in 2016. The MiT family translocation renal cell carcinoma ...comprises Xp11 translocation renal cell carcinoma harboring
gene fusions and t(6;11) renal cell carcinoma harboring
gene fusion. At the beginning, they were recognized in childhood; nevertheless, it has been demonstrated that these neoplasms can occur in adults as well. In the nineties, among Xp11 renal cell carcinoma,
,
, and
(
) were the first genes recognized as partners in
rearrangement. Recently, many other genes have been identified, and a wide spectrum of morphologies has been described. For this reason, the diagnosis may be challenging based on the histology, and the differential diagnosis includes the most common renal cell neoplasms and pure epithelioid PEComa/epithelioid angiomyolipoma of the kidney. During the last decades, many efforts have been made to identify immunohistochemical markers to reach the right diagnosis. To date, staining for PAX8, cathepsin K, and melanogenesis markers are the most useful identifiers. However, the diagnosis requires the demonstration of the chromosomal rearrangement, and fluorescent in situ hybridization (FISH) is considered the gold standard. The outcome of Xp11 translocation renal cell carcinoma is highly variable, with some patients surviving decades with indolent disease and others dying rapidly of progressive disease. Despite most instances of t(6;11) renal cell carcinoma having an indolent clinical course, a few published cases demonstrate aggressive behavior. Recently, renal cell carcinomas with
amplification have been described in connection with t(6;11) renal cell carcinoma. Those tumors appear to be associated with a more aggressive clinical course. For the aggressive cases of MiT family translocation carcinoma, the optimal therapy remains to be determined; however, new target therapies seem to be promising, and the search for predictive markers is mandatory.
Bacteria with two cell membranes (diderms) have evolved complex systems for protein secretion. These systems were extensively studied in some model bacteria, but the characterisation of their ...diversity has lagged behind due to lack of standard annotation tools. We built online and standalone computational tools to accurately predict protein secretion systems and related appendages in bacteria with LPS-containing outer membranes. They consist of models describing the systems' components and genetic organization to be used with MacSyFinder to search for T1SS-T6SS, T9SS, flagella, Type IV pili and Tad pili. We identified ~10,000 candidate systems in bacterial genomes, where T1SS and T5SS were by far the most abundant and widespread. All these data are made available in a public database. The recently described T6SS(iii) and T9SS were restricted to Bacteroidetes, and T6SS(ii) to Francisella. The T2SS, T3SS, and T4SS were frequently encoded in single-copy in one locus, whereas most T1SS were encoded in two loci. The secretion systems of diderm Firmicutes were similar to those found in other diderms. Novel systems may remain to be discovered, since some clades of environmental bacteria lacked all known protein secretion systems. Our models can be fully customized, which should facilitate the identification of novel systems.
SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. ...SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single‐molecule FRET to derive a model that couples ATP hydrolysis‐dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two‐helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the channel. The finger retracts during ATP hydrolysis, while the clamp domain of SecA tightens around the polypeptide, preserving progress of translocation. The clamp opens after phosphate release and allows passive sliding of the polypeptide chain through the SecA‐SecY complex until the next ATP binding event. This power‐stroke mechanism may be used by other ATPases that move polypeptides.
Synopsis
How AAA ATPases harness energy from ATP to promote directional translocation of polypeptides across membranes is poorly understood. Single‐molecule FRET reveals that bacterial SecA uses ATP hydrolysis to push a polypeptide segment into the SecY channel, and phosphate release to slide it through the SecA‐SecY complex.
SecA two‐helix finger and clamp domains move upon ATP binding, hydrolysis and phosphate release.
ATP binding moves the two‐helix finger towards the SecY channel, causing insertion of the first polypeptide segment into the channel.
Upon ATP hydrolysis, finger retraction and clamp domain closure around the translocating polypeptide ensure the translocation progress.
Clamp opening induced by phosphate release permits passive sliding of the polypeptide in either direction.
Single‐molecule FRET reveals that the bacterial SecA translocase uses ATP hydrolysis to push a polypeptide segment into the SecY channel, and phosphate release to slide it through the SecA‐SecY complex.
BACKGROUND
Fluorescence in situ hybridization (FISH) is the most widely used method for detecting unbalanced chromosome rearrangements in preimplantation embryos but it is known to have several ...technical limitations. We describe the clinical application of a molecular-based assay, array comparative genomic hybridization (array-CGH), to simultaneously screen for unbalanced translocation derivatives and aneuploidy of all 24 chromosomes.
METHODS
Cell biopsy was carried out on cleavage-stage embryos (Day 3). Single cells were first lysed and DNA amplified by whole-genome amplification (WGA). WGA products were then processed by array-CGH using 24sure + arrays, BlueGnome. Balanced/normal euploid embryos were then selected for transfer on Day 5 of the same cycle.
RESULTS
Twenty-eight consecutive cycles of preimplantation genetic diagnosis were carried out for 24 couples carrying 18 different balanced translocations. Overall, 187/200 (93.5%) embryos were successfully diagnosed. Embryos suitable for transfer were identified in 17 cycles (60.7%), with transfer of 22 embryos (mean 1.3 ± 0.5). Twelve couples achieved a clinical pregnancy (70.6% per embryo transfer), with a total of 14 embryos implanted (63.6% per transferred embryo). Three patients delivered three healthy babies, during writing, the other pregnancies (two twins and seven singletons) are ongoing beyond 20 weeks of gestation.
CONCLUSIONS
The data obtained demonstrate that array-CGH can detect chromosome imbalances in embryos, also providing the added benefit of simultaneous aneuploidy screening of all 24 chromosomes. Array-CGH has the potential to overcome several inherent limitations of FISH-based tests, providing improvements in terms of test performance, automation, sensitivity and reliability.
Protein synthesis, transport, and N-glycosylation are coupled at the mammalian endoplasmic reticulum by complex formation of a ribosome, the Sec61 protein-conducting channel, and ...oligosaccharyltransferase (OST). Here we used different cryo-electron microscopy approaches to determine structures of native and solubilized ribosome-Sec61-OST complexes. A molecular model for the catalytic OST subunit STT3A (staurosporine and temperature sensitive 3A) revealed how it is integrated into the OST and how STT3-paralog specificity for translocon-associated OST is achieved. The OST subunit DC2 was placed at the interface between Sec61 and STT3A, where it acts as a versatile module for recruitment of STT3A-containing OST to the ribosome-Sec61 complex. This detailed structural view on the molecular architecture of the cotranslational machinery for N-glycosylation provides the basis for a mechanistic understanding of glycoprotein biogenesis at the endoplasmic reticulum.
Background. Despite effective antiretroviral therapy (ART), patients with chronic human immunodeficiency virus (HIV) infection have increased microbial translocation and systemic inflammation. ...Alterations in the intestinal microbiota may play a role in microbial translocation and inflammation. Methods. We profiled the fecal microbiota by pyrosequencing the gene encoding 16S ribosomal RNA (rRNA) and measured markers of microbial translocation and systemic inflammation in 21 patients who had chronic HIV infection and were receiving suppressive ART (cases) and 16 HIV-uninfected controls. Results. The fecal microbial community composition was significantly different between cases and controls. The relative abundance of Proteobacteria, Gammaproteobacteria, Enterobacteriales, Enterobacteriaceae, Erysipelotrichi, Erysipelotrichales, Erysipelotrichaceae, and Barnesiella was significantly enriched in cases, whereas that of Rikenellaceae and Alistipes was depleted. The plasma soluble CD 14 level (sCD14) was significantly higher and the endotoxin core immunoglobulin M (IgM) level lower in cases, compared with controls. There were significant positive correlations between the relative abundances of Enterobacteriales and Enterobacteriaceae and the sCD14 level; the relative abundances of Gammaproteobacteria, Enterobacteriales, and Enterobacteriaceae and the interleukin 1β(IL-Iβ) level; the relative abundances of Enterobacteriales and Enterobacteriaceae and the interferon γ level; and the relative abundances of Erysipelotrichi and Barnesiella and the TNF-α level. There were negative correlations between endotoxin core IgM and IL-Iβ levels. Conclusions. Patients who have chronic HIV infection and are receiving suppressive ART display intestinal dysbiosis associated with increased microbial translocation and significant associations between specific taxa and markers of microbial translocation and systemic inflammation. This was an exploratory study, the findings of which need to be confirmed.
R-loops are DNA-RNA hybrids enriched at CpG islands (CGIs) that can regulate chromatin states
. How R-loops are recognized and interpreted by specific epigenetic readers is unknown. Here we show that ...GADD45A (growth arrest and DNA damage protein 45A) binds directly to R-loops and mediates local DNA demethylation by recruiting TET1 (ten-eleven translocation 1). Studying the tumor suppressor TCF21 (ref.
), we find that antisense long noncoding (lncRNA) TARID (TCF21 antisense RNA inducing promoter demethylation) forms an R-loop at the TCF21 promoter. Binding of GADD45A to the R-loop triggers local DNA demethylation and TCF21 expression. TARID transcription, R-loop formation, DNA demethylation, and TCF21 expression proceed sequentially during the cell cycle. Oxidized DNA demethylation intermediates are enriched at genomic R-loops and their levels increase upon RNase H1 depletion. Genomic profiling in embryonic stem cells identifies thousands of R-loop-dependent TET1 binding sites at CGIs. We propose that GADD45A is an epigenetic R-loop reader that recruits the demethylation machinery to promoter CGIs.
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