Biology subjects

Sivakumaran, H.

Publications and source records attributed to Sivakumaran, H..

6 recordsLinked to original sources

bioRxiv2023

CRISPR-Cas13d screens identify KILR a breast cancer risk-associated lncRNA that regulates DNA replication and repair.

Long noncoding RNAs (lncRNAs) have surpassed the number of protein-coding genes, yet the majority have no known function. We previously discovered >800 lncRNAs at regions identified by breast cancer genome-wide association studies (GWAS). Here, we performed a pooled CRISPR-Cas13d RNA knockdown screen to identify which of these lncRNAs altered cell proliferation. We found that KILR, a lncRNA that functions as a tumor suppressor, safeguards breast cells against uncontrolled proliferation. The half-life of KILR is significantly reduced by the risk haplotype, revealing an alternative mechanism by which variants alter cancer risk. We showed that KILR sequesters RPA1, a subunit of the RPA complex, required for DNA replication and repair. Reduced KILR expression promotes cell proliferation by increasing the available pool of RPA1 and the speed of DNA replication. Our findings confirm lncRNAs as mediators of breast cancer risk, emphasize the need to annotate noncoding transcripts in relevant cell types when investigating GWAS variants and provide a scalable platform for mapping phenotypes associated with lncRNAs.

cancer biology↗

bioRxiv2023

Regulation of Non-Canonical Proteins Encoded by Small Open Reading Frames via the Nonsense-Mediated Decay Pathway.

Immunotherapy interventions relies heavily on neoantigen availability. The human genome encodes non-canonical/mutant proteins that potentially contain neoantigenic peptides. Nevertheless, their typically low expression, potentially moderated by the Nonsense-Mediated Decay (NMD) pathway, restricts their therapeutic utility. In this study, we explored the NMD pathway influence on non-canonical/mutant protein expression, specifically focusing on UPF1 knockdown. We implemented proteogenomic approaches to ascertain if the encoding transcripts and their respective proteins were upregulated post-knockdown. Complementary to this, we conducted a comprehensive pan-cancer survey of UPF1 expression and an in vivo evaluation of UPF1 expression in Triple-Negative Breast Cancer (TNBC) tissue. Our empirical results delineated that UPF1 knockdown precipitates an increase in the transcription of non-canonical/mutant proteins, especially those originating from retained-introns, pseudogenes, long non-coding RNAs, and unannotated biotypes. Furthermore, the analysis revealed that UPF1 expression was conspicuously high across a range of neoplastic tissues, with protein levels notably amplified in patient derived TNBC tumours in comparison to adjacent tissues. Our study elucidates UPF1 functional role in attenuating transcriptional noise through the degradation of transcripts encoding non-canonical/mutant proteins. Interestingly, we observed an upregulation of the NMD pathway in cancer, potentially functioning as a "neoantigen masking" mechanism that subdues non-canonical/mutant protein expression. Suppressing this mechanism may unveil a new cadre of neoantigens accessible to the antigen presentation pathway. Our novel findings proffer a solid base for devising therapeutic strategies targeting UPF1 or the NMD pathway, given the pronounced presence of UPF1 in malignant cells, thus potentially augmenting immunotherapeutic responses in cancer.

cell biology↗

bioRxiv2022

CRISPR interference for Sequence-Specific Regulation of Fibroblast Growth Factor Receptor A in Schistosoma mansoni

Employing the flatworm parasite Schistosoma mansoni as a model, we report the first application of CRISPR interference (CRISPRi) in parasitic helminths for loss-of-function targeting the SmfgfrA gene which encodes the stem cell marker, fibroblast growth factor receptor A. SmFGFRA is essential for maintaining schistosome stem cells and critical in the schistosome-host interplay. The SmfgfrA gene was targeted in S. mansoni adult worms, eggs and schistosomula using a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor KRAB. We showed that SmfgfrA repression resulted in considerable phenotypic differences in the modulated parasites compared with controls, including reduced levels of SmfgfrA transcription and decreased protein expression of SmFGFRA, a decline in EdU (thymidine analog 5-ethynyl-2-deoxyuridine, which specifically stains schistosome stem cells) signal, and an increase in cell apoptosis. Notably, reduced SmfgfrA transcription was evident in miracidia hatched from SmfgfrA-repressed eggs, and resulted in a significant change in miracidial behavior, indicative of a durable repression effect caused by CRISPRi. Intravenous injection of mice with SmfgfrA-repressed eggs resulted in granulomas that were markedly reduced in size and a decline in the level of serum IgE, emphasizing the importance of SmFGFRA in regulating the host immune response induced during schistosome infection. Our findings show the feasibility of applying CRISPRi for effective, targeted transcriptional repression in schistosomes, and provide the basis for employing CRISPRi to selectively perturb gene expression in parasitic helminths on a genome-wide scale.

molecular biology↗

bioRxiv2021

CRISPR screens identify gene targets and drug repositioning opportunities at breast cancer risk loci

Genome-wide association studies (GWAS) have identified >200 loci associated with breast cancer (BC) risk. The majority of candidate causal variants (CCVs) are in non-coding regions and are likely to modulate cancer risk by regulating gene expression. We recently developed a scoring system, INQUISIT, to predict candidate risk genes at BC-risk loci. Here, we used pooled CRISPR activation and suppression screens to validate INQUISIT predictions, and to define the cancer phenotypes they mediate. We measured proliferation in 2D, 3D, and in immune-deficient mice, as well as the effect on the DNA damage response. We performed 60 CRISPR screens and identified 21 high-confidence INQUISIT predictions that mediate a cancer phenotype. We validated the direct regulation of a subset of genes by BC-risk variants using HiCHIP and CRISPRqtl. Furthermore, we show the utility of expression profiling for drug repurposing against these targets. We provide a platform for identifying gene targets of risk variants, and lay a blueprint of interventions for BC risk reduction and treatment.

cancer biology↗

bioRxiv2019

Chromatin interactome mapping at 139 independent breast cancer risk signals

Genome-wide association studies have identified 196 high confidence independent signals associated with breast cancer susceptibility. Variants within these signals frequently fall in distal regulatory DNA elements that control gene expression. We designed a Capture Hi-C array to enrich for chromatin interactions between the credible causal variants and target genes in six human mammary epithelial and breast cancer cell lines. We show that interacting regions are enriched for open chromatin, histone marks for active enhancers and transcription factors relevant to breast biology. We exploit this comprehensive resource to identify candidate target genes at 139 independent breast cancer risk signals, and explore the functional mechanism underlying altered risk at the 12q24 risk region. Our results demonstrate the power of combining genetics, computational genomics and molecular studies to rationalize the identification of key variants and candidate target genes at breast cancer GWAS signals.

molecular biology↗

bioRxiv2019

High-throughput allelic expression imbalance analyses identify candidate breast cancer risk genes

Fine-mapping of breast cancer GWAS regions has identified 195 high confidence signals containing more than 5,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements and thus may confer the risk by altering gene expression in cis. We analyzed allelic expression imbalance (AEI) of genes surrounding known breast cancer signals, using normal breast and breast tumor transcriptome data and imputed genotypes. Fourteen genes, including NTN4, were identified whose expression was associated with CCV genotype. We showed that CCVs at this signal were located within an enhancer that physically interacts with the NTN4 promoter. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. Here, we present the most comprehensive AEI analysis of breast cancer CCVs resulting in identification of new candidate risk genes.

genetics↗