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Pinello, Luca

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Pinello

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Luca

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Pinello, Luca
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Now showing 1 - 10 of 16
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    Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci
    (2017) Canver, Matthew C.; Lessard, Samuel; Pinello, Luca; Wu, Yuxuan; Ilboudo, Yann; Stern, Emily; Needleman, Austen; Galactéros, Frédéric; Brugnara, Carlo; Kutlar, Abdullah; McKenzie, Colin; Reid, Marvin; Chen, Diane D.; Das, Partha Pratim; Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo-Cheng; Lettre, Guillaume; Bauer, Daniel; Orkin, Stuart
    Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants from genome-wide association studies largely cluster in regulatory DNA. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating mutagenesis libraries with single or combinatorial nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, a locus associated with red blood cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false positive regions, which emphasizes the importance of off-target analysis in design of saturating mutagenesis experiments. Taken together, these data establish a widely applicable high-throughput and high-resolution methodology to reliably identify minimal functional sequences within large regions of disease- and trait-associated DNA.
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    STAT5 Outcompetes STAT3 To Regulate the Expression of the Oncogenic Transcriptional Modulator BCL6
    (American Society for Microbiology, 2013) Walker, Sarah; Nelson, Erik; Yeh, Jennifer; Pinello, Luca; Yuan, Guo-Cheng; Frank, David
    Inappropriate activation of the transcription factors STAT3 and STAT5 has been shown to drive cancer pathogenesis through dysregulation of genes involved in cell survival, growth, and differentiation. Although STAT3 and STAT5 are structurally related, they can have opposite effects on key genes, including BCL6. BCL6, a transcriptional repressor, has been shown to be oncogenic in diffuse large B cell lymphoma. BCL6 also plays an important role in breast cancer pathogenesis, a disease in which STAT3 and STAT5 can be activated individually or concomitantly. To determine the mechanism by which these oncogenic transcription factors regulate BCL6 transcription, we analyzed their effects at the levels of chromatin and gene expression. We found that STAT3 increases expression of BCL6 and enhances recruitment of RNA polymerase II phosphorylated at a site associated with transcriptional initiation. STAT5, in contrast, represses BCL6 expression below basal levels and decreases the association of RNA polymerase II at the gene. Furthermore, the repression mediated by STAT5 is dominant over STAT3-mediated induction. STAT5 exerts this effect by displacing STAT3 from one of the two regulatory regions to which it binds. These findings may underlie the divergent biology of breast cancers containing activated STAT3 alone or in conjunction with activated STAT5.
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    Predicting chromatin organization using histone marks
    (BioMed Central, 2015) Huang, Jialiang; Marco, Eugenio; Pinello, Luca; Yuan, Guo-Cheng
    Genome-wide mapping of three dimensional chromatin organization is an important yet technically challenging task. To aid experimental effort and to understand the determinants of long-range chromatin interactions, we have developed a computational model integrating Hi-C and histone mark ChIP-seq data to predict two important features of chromatin organization: chromatin interaction hubs and topologically associated domain (TAD) boundaries. Our model accurately and robustly predicts these features across datasets and cell types. Cell-type specific histone mark information is required for prediction of chromatin interaction hubs but not for TAD boundaries. Our predictions provide a useful guide for the exploration of chromatin organization. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0740-z) contains supplementary material, which is available to authorized users.
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    Functionally distinct patterns of nucleosome remodeling at enhancers in glucocorticoid-treated acute lymphoblastic leukemia
    (BioMed Central, 2015) Wu, Jennifer N.; Pinello, Luca; Yissachar, Elinor; Wischhusen, Jonathan W.; Yuan, Guo-Cheng; Roberts, Charles W. M.
    Background: Precise nucleosome positioning is an increasingly recognized feature of promoters and enhancers, reflecting complex contributions of DNA sequence, nucleosome positioning, histone modification and transcription factor binding to enhancer activity and regulation of gene expression. Changes in nucleosome position and occupancy, histone variants and modifications, and chromatin remodeling are also critical elements of dynamic transcriptional regulation, but poorly understood at enhancers. We investigated glucocorticoid receptor-associated (GR) nucleosome dynamics at enhancers in acute lymphoblastic leukemia. Results: For the first time, we demonstrate functionally distinct modes of nucleosome remodeling upon chromatin binding by GR, which we term central, non-central, phased, and minimal. Central and non-central remodeling reflect nucleosome eviction by GR and cofactors, respectively. Phased remodeling involves nucleosome repositioning and is associated with rapidly activated enhancers and induction of gene expression. Minimal remodeling sites initially have low levels of enhancer-associated histone modification, but the majority of these regions gain H3K4me2 or H3K27Ac to become de novo enhancers. Minimal remodeling regions are associated with gene ontologies specific to decreased B cell number and mTOR inhibition and may make unique contributions to glucocorticoid-induced leukemia cell death. Conclusions: Our findings form a novel framework for understanding the dynamic interplay between transcription factor binding, nucleosome remodeling, enhancer function, and gene expression in the leukemia response to glucocorticoids. Electronic supplementary material The online version of this article (doi:10.1186/s13072-015-0046-0) contains supplementary material, which is available to authorized users.
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    BCL11A Enhancer Dissection by Cas9-Mediated in Situ Saturating Mutagenesis
    (Nature, 2015-09-16) Canver, Matthew C.; Smith, Elenoe; Pinello, Luca; Sanjana, Neville E.; Shalem, Ophir; Chen, Diane; Schupp, Patrick G.; Vinjamur, Divya S.; Garcia, Sara P.; Luc, Sidinh; Kurita, Ryo; Fujiwara, Yuko; Maeda, Takahiro; Yuan, Guo-Cheng; Zhang, Feng; Orkin, Stuart; Bauer, Daniel; Nakamura, Yukio
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    Multi-scale chromatin state annotation using a hierarchical hidden Markov model
    (Nature Publishing Group, 2017) Marco, Eugenio; Meuleman, Wouter; Huang, Jialiang; Glass, Kimberly; Pinello, Luca; Wang, Jianrong; Kellis, Manolis; Yuan, Guo-Cheng
    Chromatin-state analysis is widely applied in the studies of development and diseases. However, existing methods operate at a single length scale, and therefore cannot distinguish large domains from isolated elements of the same type. To overcome this limitation, we present a hierarchical hidden Markov model, diHMM, to systematically annotate chromatin states at multiple length scales. We apply diHMM to analyse a public ChIP-seq data set. diHMM not only accurately captures nucleosome-level information, but identifies domain-level states that vary in nucleosome-level state composition, spatial distribution and functionality. The domain-level states recapitulate known patterns such as super-enhancers, bivalent promoters and Polycomb repressed regions, and identify additional patterns whose biological functions are not yet characterized. By integrating chromatin-state information with gene expression and Hi-C data, we identify context-dependent functions of nucleosome-level states. Thus, diHMM provides a powerful tool for investigating the role of higher-order chromatin structure in gene regulation.
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    The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells
    (Springer Nature, 2016) Beyaz, Semir; Kim, Ji Hyung; Pinello, Luca; Xifaras, Michael E; Hu, Yu; Huang, Jialiang; Kerenyi, Marc A; Das, Partha Pratim; Barnitz, R Anthony; Herault, Aurelie; Dogum, Rizkullah; Haining, William; Yilmaz, Omer; Passegue, Emmanuelle; Yuan, Guo-Cheng; Orkin, Stuart; Winau, Florian
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    BRCA1 Recruitment to Transcriptional Pause Sites Is Required for R-Loop-Driven DNA Damage Repair
    (Cell Press, 2015) Hatchi, Elodie; Skourti-Stathaki, Konstantina; Ventz, Steffen; Pinello, Luca; Yen, Angela; Kamieniarz-Gdula, Kinga; Dimitrov, Stoil; Pathania, Shailja; McKinney, Kristine M.; Eaton, Matthew L.; Kellis, Manolis; Hill, Sarah J.; Parmigiani, Giovanni; Proudfoot, Nicholas J.; Livingston, David M.
    Summary The mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites.
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    GiniClust: detecting rare cell types from single-cell gene expression data with Gini index
    (BioMed Central, 2016) Jiang, Lan; Chen, Huidong; Pinello, Luca; Yuan, Guo-Cheng
    High-throughput single-cell technologies have great potential to discover new cell types; however, it remains challenging to detect rare cell types that are distinct from a large population. We present a novel computational method, called GiniClust, to overcome this challenge. Validation against a benchmark dataset indicates that GiniClust achieves high sensitivity and specificity. Application of GiniClust to public single-cell RNA-seq datasets uncovers previously unrecognized rare cell types, including Zscan4-expressing cells within mouse embryonic stem cells and hemoglobin-expressing cells in the mouse cortex and hippocampus. GiniClust also correctly detects a small number of normal cells that are mixed in a cancer cell population. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1010-4) contains supplementary material, which is available to authorized users.
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    The cohesin-associated protein Wapal is required for proper Polycomb-mediated gene silencing
    (BioMed Central, 2016) Stelloh, Cary; Reimer, Michael H.; Pulakanti, Kirthi; Blinka, Steven; Peterson, Jonathan; Pinello, Luca; Jia, Shuang; Roumiantsev, Sergei; Hessner, Martin J.; Milanovich, Samuel; Yuan, Guo-Cheng; Rao, Sridhar
    Background: The cohesin complex consists of multiple core subunits that play critical roles in mitosis and transcriptional regulation. The cohesin-associated protein Wapal plays a central role in off-loading cohesin to facilitate sister chromatid separation, but its role in regulating mammalian gene expression is not understood. We used embryonic stem cells as a model, given that the well-defined transcriptional regulatory circuits were established through master transcription factors and epigenetic pathways that regulate their ability to maintain a pluripotent state. Results: RNAi-mediated depletion of Wapal causes a loss of pluripotency, phenocopying loss of core cohesin subunits. Using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq), we determine that Wapal occupies genomic sites distal to genes in combination with CTCF and core cohesin subunits such as Rad21. Interestingly, genomic sites occupied by Wapal appear enriched for cohesin, implying that Wapal does not off-load cohesin at regions it occupies. Wapal depletion induces derepression of Polycomb group (PcG) target genes without altering total levels of Polycomb-mediated histone modifications, implying that PcG enzymatic activity is preserved. By integrating ChIP-seq and gene expression changes data, we identify that Wapal binding is enriched at the promoters of PcG-silenced genes and is required for proper Polycomb repressive complex 2 (PRC2) recruitment. Lastly, we demonstrate that Wapal is required for the interaction of a distal cis-regulatory element (CRE) with the c-Fos promoter. Conclusions: Collectively, this work indicates that Wapal plays a critical role in silencing of PcG target genes through the interaction of distal CREs with promoters. Electronic supplementary material The online version of this article (doi:10.1186/s13072-016-0063-7) contains supplementary material, which is available to authorized users.