Biology subjects

Marques, A. C.

Publications and source records attributed to Marques, A. C..

9 recordsLinked to original sources

bioRxiv2022

A deep learning approach for improved detection of homologous recombination deficiency from shallow genomic profiles

Homologous Recombination Deficiency (HRD) is a predictive biomarker of poly-ADP ribose polymerase 1 inhibitors (PARPi) response. Most HRD detection methods are based on genome wide enumeration of scarring events and require deep genome sequence profiles (> 30x). The cost and workflow-specific biases introduced by these genome profiling methods currently limits clinical adoption of HRD testing. We introduce the Genomic Integrity Index (GII), a Convolutional Neuronal Network, that leverages features from low pass (1x) Whole Genome Sequencing data to distinguish HRD positive and negative samples. In a cohort of 230 ovarian and breast cancer, we found GII supports accurate stratification of samples yielding results that are highly concordant with state-of-the-art HRD detection methods (0.865<AUC<0.996) which require 50x deeper coverage. We conclude that the deep learning framework supporting GII allows accurate detection of HRD from shallow genome profiles, reducing biases and data generation costs making it uniquely suited for clinical applications.

cancer biology↗

bioRxiv2020

Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

BackgroundSARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution. ResultsWe used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020. ConclusionsWe present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.

genomics↗

bioRxiv2020

Noncanonical targeting contributes significantly to miRNA-mediated regulation.

Determining which genes are targeted by miRNAs is crucial to elucidate their contributions to diverse biological processes in health and disease. Most miRNA target prediction tools rely on the identification of complementary regions between transcripts and miRNAs. Whereas important for target recognition, the presence of complementary sites is not sufficient to identify transcripts targeted by miRNAs. Here, we describe an unbiased statistical genomics approach that explores genetically driven changes in gene expression between human individuals. Using this approach, we identified transcripts that respond to physiological changes in miRNA levels. We found that a much smaller fraction of mRNAs expressed in lymphoblastoid cell lines (LCLs) than what is predicted by other tools is targeted by miRNAs. We estimate that each miRNA has a relatively small number of targets. The transcripts we predict to be miRNA targets are enriched in AGO-binding and previously validated miRNAs target interactions, supporting the reliability of our predictions. Consistent with previous analysis, these targets are also enriched among dosage sensitive and highly controlled genes. Almost a third of genes we predict to be miRNA targets lack sequence complementarity to the miRNA seed region (noncanonical targets). These noncanonical targets have higher complementary with the miRNA 3' end. The impact of miRNAs on the levels of their canonical or noncanonical targets is identical supporting the relevance of this poorly explored mechanism of targeting.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genomics↗

bioRxiv2020

The activity of human enhancers is modulated by the splicing of their associated lncRNAs.

Pervasive enhancer transcription is at the origin of more than half of all long noncoding RNAs in humans. Transcription of enhancer-associated long noncoding RNAs (elncRNA) contribute to their cognate enhancer activity and gene expression regulation in cis. Recently, splicing of elncRNAs was shown to be associated with elevated enhancer activity. However, whether splicing of elncRNA transcripts is a mere consequence of accessibility at highly active enhancers or if elncRNA splicing directly impacts enhancer function, remains unanswered. We analysed genetically driven changes in elncRNA expression, in humans, to address this outstanding question. We showed that splicing related motifs within multi-exonic elncRNAs evolved under selective constraints during human evolution, suggesting the processing of these transcripts is unlikely to have resulted from transcription across spurious splice sites. Using a genome-wide and unbiased approach, we used nucleotide variants as independent genetic factors to directly assess the causal relationship that underpin elncRNA splicing and their cognate enhancer activity. We found that the splicing of most elncRNAs is associated with changes in chromatin signatures at cognate enhancers and target mRNA expression. We conclude that efficient and conserved processing of enhancer-associated elncRNAs contributes to enhancer activity.

genomics↗

bioRxiv2020

Translation is required for miRNA-dependent decay of endogenous transcripts.

Posttranscriptional repression by microRNA (miRNA) occurs through transcript destabilization or translation inhibition. Whereas RNA degradation explains most miRNA-dependent repression, transcript decay occurs co-translationally, raising questions regarding the requirement of target translation to miRNA-dependent transcript destabilization. To assess the contribution of translation to miRNA-mediated RNA destabilization, we decoupled these two molecular processes by dissecting the impact of miRNA loss of function on cytosolic long noncoding RNAs (lncRNAs). We show, that despite interacting with miRNA loaded RNA-induced silencing complex (miRISC), the steady state abundance and degradation rates of these endogenously expressed non-translated transcripts are minimally impacted by miRNA loss. To validate the requirement of translation for miRNA-dependent decay, we fused a miRISC bound lncRNA, whose levels are unaffected by miRNAs, to the 3end of a protein-coding gene reporter and show that this results in its miRNA-dependent transcript destabilization. Furthermore, analysis of the few lncRNAs whose levels are regulated by miRNAs revealed these tend to associate with translating ribosomes and are likely misannotated micropeptides, further substantiating the necessity of target translation for miRNA-dependent transcript decay. Our analyses reveal the strict requirement of translation for miRNA-dependent transcript destabilization and demonstrate that the levels of coding and noncoding transcripts are differently affected by miRNAs.

genomics↗

bioRxiv2019

TIP5 safeguards genome architecture of ground-state pluripotent stem cells

Chromosomes have an intrinsic tendency to segregate into compartments, forming long-distance contacts between loci of similar chromatin states. However, how genome compartmentalization is regulated remains elusive. We analyzed two closely and developmentally related pluripotent cell types: ground-state ESCs that have an open and active chromatin and developmentally advanced ESCs that display a more closed and repressed state. We show that these two ESC types differ in their regulation of genome organization due to their differential dependency on TIP5, a component of the chromatin remodeling complex NoRC. We show that TIP5 interacts on ESC chromatin with SNF2H, DNA topoisomerase 2A (TOP2A) and cohesin. TIP5 associates with sub-domains within the active A compartment that strongly intersect through long-range contacts in ESCs. We found that only ground-state chromatin requires TIP5 to limit the invasion of active domains into repressive compartments. Depletion of TIP5 increased chromatin accessibility particularly at B compartments and decreased their repressive features. Furthermore, TIP5 acts as a barrier for the repressive H3K27me3 spreading, a process that also requires TOP2A activity. Finally, ground-state ESCs require TIP5 for growth, differentiation capacity, and correct expression of developmental genes. Our results revealed the propensity of open and active chromatin domains to invade repressive domains, an action counteracted by chromatin remodeling and the relief of chromatin torsional stress. This effort in controlling open/active chromatin domains is required to establish active and repressed genome partitioning and preserves cell function and identity.

molecular biology↗

bioRxiv2019

Three-dimensional chromatin interactions remain stable upon CAG/CTG repeat expansion

Expanded CAG/CTG repeats underlie thirteen neurological disorders, including myotonic dystrophy (DM1) and Huntingtons disease (HD). Upon expansion, CAG/CTG repeat loci acquire heterochromatic characteristics. This observation raises the hypothesis that repeat expansion provokes changes to higher order chromatin folding and thereby affects both gene expression in cis and the genetic instability of the repeat tract. Here we tested this hypothesis directly by performing 4C sequencing at the DMPK and HTT loci from DM1 and HD patient-derived cells. Surprisingly, chromatin contacts remain unchanged upon repeat expansion at both loci. This was true for loci with different DNA methylation levels and CTCF binding. Repeat sizes ranging from 15 to 1,700 displayed strikingly similar chromatin interaction profiles. Our findings argue that extensive changes in heterochromatic properties are not enough to alter chromatin folding at expanded CAG/CTG repeat loci. Moreover, the ectopic insertion of an expanded repeat tract did not change three-dimensional chromatin contacts. We conclude that expanded CAG/CTG repeats have little to no effect on chromatin conformation.

neuroscience↗

bioRxiv2019

LincRNA sequences are biased to counteract their translation

Long intergenic non-coding RNAs (lincRNAs) represent a large fraction of transcribed loci in eukaryotic genomes. Although classified as non-coding, most lincRNAs contain open reading frames (ORFs), and it remains unclear why cytoplasmic lincRNAs are not or very inefficiently translated. Here, we analysed signatures of repressed translation in lincRNA sequences from six eukaryotes. In species under stronger selection, i.e. fission yeast and worm, we detected significantly shorter ORFs than in intronic and non-transcribed control regions, a suboptimal sequence context around start codons for translation initiation, and trinucleotides ("codons") corresponding to less abundant tRNAs than codons in control regions, potentially impeding translation elongation. We verified that varying tRNA expression levels affect ribosome-binding to lincRNAs by analyzing data from five human cell lines. Notably, for three cell lines, codons in abundant cytoplasmic lincRNAs corresponded to lower expressed tRNAs than control codons, substantiating cell type-specific repression of lincRNA translation in human. Finally, comparing non-coding with peptide-encoding ORFs suggested that codon usage at the start of ORFs to be of particular relevance for ribosome-binding. The identified sequence signatures may assist distinguishing peptide- from real non-coding lincRNAs in a cell.

genomics↗

bioRxiv2019

The MITF-SOX10 regulated long non-coding RNA DIRC3 is a melanoma tumour suppressor

The MITF and SOX10 transcription factors regulate the expression of genes important for melanoma proliferation, invasion and metastasis. Despite growing evidence of the contribution of long noncoding RNAs (lncRNAs) in cancer, including melanoma, their functions within MITF-SOX10 transcriptional programmes remain poorly investigated. Here we identified 245 candidate melanoma associated lncRNAs whose loci are co-occupied by MITF-SOX10 and that are enriched at active enhancer-like regions. We characterise the function and molecular mechanism of action of one of these lncRNAs, Disrupted In Renal Carcinoma 3 (DIRC3), and show that it operates as a MITF-SOX10 regulated tumour suppressor. DIRC3 depletion in human melanoma cells leads to increased anchorage-independent growth, a hallmark of malignant transformation, whilst melanoma patients classified by low DIRC3 expression have decreased survival. DIRC3 is a nuclear lncRNA that functions locally to activate expression of its neighbouring IGFBP5 tumour suppressor through modulating chromatin structure and suppressing SOX10 binding to putative regulatory elements within the DIRC3 locus. In turn, DIRC3 dependent regulation of IGFBP5 impacts the expression of genes involved in multiple cancer associated processes. Our work indicates that lncRNA components of the MITF-SOX10 networks are an important new class of melanoma regulators and candidate therapeutic targets.

cancer biology↗