Publications by authors named "Harihar Basnet"

13 Publications

  • Page 1 of 1

Labeling and Isolation of Fluorouracil Tagged RNA by Cytosine Deaminase Expression.

Bio Protoc 2019 Nov 20;9(22):e3433. Epub 2019 Nov 20.

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

Tissues are comprised of different cell types whose interactions elicit distinct gene expression patterns that regulate tissue formation, regeneration, homeostasis and repair. Analysis of these gene expression patterns require methods that can capture as closely as possible the transcriptomes of cells of interest in their tissue microenvironment. Current technologies designed to study transcriptomics are limited by their low sensitivity that require cell types to represent more than 1% of the total tissue, making it challenging to transcriptionally profile rare cell populations rapidly isolated from their native microenvironment. To address this problem, we developed fluorouracil-tagged RNA sequencing (Flura-seq) that utilizes cytosine deaminase (CD) to convert the non-natural pyrimidine fluorocytosine to fluorouracil. Expression of CD and exposure to fluorocytosine generates fluorouracil and metabolically labels newly synthesized RNAs specifically in cells of interest. Fluorouracil-tagged RNAs can then be immunopurified and used for downstream analysis. Here, we describe the detailed protocol to perform Flura-seq both and . The robustness, simplicity and lack of toxicity of Flura-seq make this tool broadly applicable to many studies in developmental, regenerative, and cancer biology.
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http://dx.doi.org/10.21769/BioProtoc.3433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854007PMC
November 2019

L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer.

Nat Cancer 2020 Jan 13;1(1):28-45. Epub 2020 Jan 13.

Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Metastasis-initiating cells with stem-like properties drive cancer lethality, yet their origins and relationship to primary-tumor-initiating stem cells are not known. We show that L1CAM cells in human colorectal cancer (CRC) have metastasis-initiating capacity, and we define their relationship to tissue regeneration. L1CAM is not expressed in the homeostatic intestinal epithelium, but is induced and required for epithelial regeneration following colitis and in CRC organoid growth. By using human tissues and mouse models, we show that L1CAM is dispensable for adenoma initiation but required for orthotopic carcinoma propagation, liver metastatic colonization and chemoresistance. L1CAM cells partially overlap with LGR5 stem-like cells in human CRC organoids. Disruption of intercellular epithelial contacts causes E-cadherin-REST transcriptional derepression of L1CAM, switching chemoresistant CRC progenitors from an L1CAM to an L1CAM state. Thus, L1CAM dependency emerges in regenerative intestinal cells when epithelial integrity is lost, a phenotype of wound healing deployed in metastasis-initiating cells.
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http://dx.doi.org/10.1038/s43018-019-0006-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351134PMC
January 2020

Publisher Correction: TGF-β orchestrates fibrogenic and developmental EMTs via the RAS effector RREB1.

Nature 2020 02;578(7793):E11

Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41586-020-1956-yDOI Listing
February 2020

TGF-β orchestrates fibrogenic and developmental EMTs via the RAS effector RREB1.

Nature 2020 01 8;577(7791):566-571. Epub 2020 Jan 8.

Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Epithelial-to-mesenchymal transitions (EMTs) are phenotypic plasticity processes that confer migratory and invasive properties to epithelial cells during development, wound-healing, fibrosis and cancer. EMTs are driven by SNAIL, ZEB and TWIST transcription factors together with microRNAs that balance this regulatory network. Transforming growth factor β (TGF-β) is a potent inducer of developmental and fibrogenic EMTs. Aberrant TGF-β signalling and EMT are implicated in the pathogenesis of renal fibrosis, alcoholic liver disease, non-alcoholic steatohepatitis, pulmonary fibrosis and cancer. TGF-β depends on RAS and mitogen-activated protein kinase (MAPK) pathway inputs for the induction of EMTs. Here we show how these signals coordinately trigger EMTs and integrate them with broader pathophysiological processes. We identify RAS-responsive element binding protein 1 (RREB1), a RAS transcriptional effector, as a key partner of TGF-β-activated SMAD transcription factors in EMT. MAPK-activated RREB1 recruits TGF-β-activated SMAD factors to SNAIL. Context-dependent chromatin accessibility dictates the ability of RREB1 and SMAD to activate additional genes that determine the nature of the resulting EMT. In carcinoma cells, TGF-β-SMAD and RREB1 directly drive expression of SNAIL and fibrogenic factors stimulating myofibroblasts, promoting intratumoral fibrosis and supporting tumour growth. In mouse epiblast progenitors, Nodal-SMAD and RREB1 combine to induce expression of SNAIL and mesendoderm-differentiation genes that drive gastrulation. Thus, RREB1 provides a molecular link between RAS and TGF-β pathways for coordinated induction of developmental and fibrogenic EMTs. These insights increase our understanding of the regulation of epithelial plasticity and its pathophysiological consequences in development, fibrosis and cancer.
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http://dx.doi.org/10.1038/s41586-019-1897-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450666PMC
January 2020

ID1 Mediates Escape from TGFβ Tumor Suppression in Pancreatic Cancer.

Cancer Discov 2020 01 3;10(1):142-157. Epub 2019 Oct 3.

Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York.

TGFβ is an important tumor suppressor in pancreatic ductal adenocarcinoma (PDA), yet inactivation of TGFβ pathway components occurs in only half of PDA cases. TGFβ cooperates with oncogenic RAS signaling to trigger epithelial-to-mesenchymal transition (EMT) in premalignant pancreatic epithelial progenitors, which is coupled to apoptosis owing to an imbalance of SOX4 and KLF5 transcription factors. We report that PDAs that develop with the TGFβ pathway intact avert this apoptotic effect via ID1. family members are expressed in PDA progenitor cells and encode components of a set of core transcriptional regulators shared by PDAs. PDA progression selects against TGFβ-mediated repression of . The sustained expression of uncouples EMT from apoptosis in PDA progenitors. AKT signaling and mechanisms linked to low-frequency genetic events converge on to preserve its expression in PDA. Our results identify ID1 as a crucial node and potential therapeutic target in PDA. SIGNIFICANCE: Half of PDAs escape TGFβ-induced tumor suppression without inactivating the TGFβ pathway. We report that expression is selected for in PDAs and that ID1 uncouples TGFβ-induced EMT from apoptosis. ID1 thus emerges as a crucial regulatory node and a target of interest in PDA..
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http://dx.doi.org/10.1158/2159-8290.CD-19-0529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954299PMC
January 2020

Flura-seq identifies organ-specific metabolic adaptations during early metastatic colonization.

Elife 2019 03 26;8. Epub 2019 Mar 26.

Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States.

Metastasis-initiating cells dynamically adapt to the distinct microenvironments of different organs, but these early adaptations are poorly understood due to the limited sensitivity of in situ transcriptomics. We developed fluorouracil-labeled RNA sequencing (Flura-seq) for in situ analysis with high sensitivity. Flura-seq utilizes cytosine deaminase (CD) to convert fluorocytosine to fluorouracil, metabolically labeling nascent RNA in rare cell populations in situ for purification and sequencing. Flura-seq revealed hundreds of unique, dynamic organ-specific gene signatures depending on the microenvironment in mouse xenograft breast cancer micrometastases. Specifically, the mitochondrial electron transport Complex I, oxidative stress and counteracting antioxidant programs were induced in pulmonary micrometastases, compared to mammary tumors or brain micrometastases. We confirmed lung metastasis-specific increase in oxidative stress and upregulation of antioxidants in clinical samples, thus validating Flura-seq's utility in identifying clinically actionable microenvironmental adaptations in early metastasis. The sensitivity, robustness and economy of Flura-seq are broadly applicable beyond cancer research.
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http://dx.doi.org/10.7554/eLife.43627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440742PMC
March 2019

Metastatic Latency and Immune Evasion through Autocrine Inhibition of WNT.

Cell 2016 Mar;165(1):45-60

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Metastasis frequently develops years after the removal of a primary tumor, from a minority of disseminated cancer cells that survived as latent entities through unknown mechanisms. We isolated latency competent cancer (LCC) cells from early stage human lung and breast carcinoma cell lines and defined the mechanisms that suppress outgrowth, support long-term survival, and maintain tumor-initiating potential in these cells during the latent metastasis stage. LCC cells show stem-cell-like characteristics and express SOX2 and SOX9 transcription factors, which are essential for their survival in host organs under immune surveillance and for metastatic outgrowth under permissive conditions. Through expression of the WNT inhibitor DKK1, LCC cells self-impose a slow-cycling state with broad downregulation of ULBP ligands for NK cells and evasion of NK-cell-mediated clearance. By expressing a Sox-dependent stem-like state and actively silencing WNT signaling, LCC cells can enter quiescence and evade innate immunity to remain latent for extended periods.
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http://dx.doi.org/10.1016/j.cell.2016.02.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808520PMC
March 2016

LSD1n is an H4K20 demethylase regulating memory formation via transcriptional elongation control.

Nat Neurosci 2015 Sep 27;18(9):1256-64. Epub 2015 Jul 27.

Howard Hughes Medical Institute, Department of Medicine, University of California, San Diego, La Jolla, California, USA.

We found that a neuron-specific isoform of LSD1, LSD1n, which results from an alternative splicing event, acquires a new substrate specificity, targeting histone H4 Lys20 methylation, both in vitro and in vivo. Selective genetic ablation of LSD1n led to deficits in spatial learning and memory, revealing the functional importance of LSD1n in neuronal activity-regulated transcription that is necessary for long-term memory formation. LSD1n occupied neuronal gene enhancers, promoters and transcribed coding regions, and was required for transcription initiation and elongation steps in response to neuronal activity, indicating the crucial role of H4K20 methylation in coordinating gene transcription with neuronal function. Our results indicate that this alternative splicing of LSD1 in neurons, which was associated with altered substrate specificity, serves as a mechanism acquired by neurons to achieve more precise control of gene expression in the complex processes underlying learning and memory.
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http://dx.doi.org/10.1038/nn.4069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625987PMC
September 2015

Tyrosine phosphorylation of histone H2A by CK2 regulates transcriptional elongation.

Nature 2014 Dec 24;516(7530):267-71. Epub 2014 Sep 24.

Howard Hughes Medical Institute, Department of Medicine, University of California San Diego, La Jolla, California 92093, USA.

Post-translational histone modifications have a critical role in regulating transcription, the cell cycle, DNA replication and DNA damage repair. The identification of new histone modifications critical for transcriptional regulation at initiation, elongation or termination is of particular interest. Here we report a new layer of regulation in transcriptional elongation that is conserved from yeast to mammals. This regulation is based on the phosphorylation of a highly conserved tyrosine residue, Tyr 57, in histone H2A and is mediated by the unsuspected tyrosine kinase activity of casein kinase 2 (CK2). Mutation of Tyr 57 in H2A in yeast or inhibition of CK2 activity impairs transcriptional elongation in yeast as well as in mammalian cells. Genome-wide binding analysis reveals that CK2α, the catalytic subunit of CK2, binds across RNA-polymerase-II-transcribed coding genes and active enhancers. Mutation of Tyr 57 causes a loss of H2B mono-ubiquitination as well as H3K4me3 and H3K79me3, histone marks associated with active transcription. Mechanistically, both CK2 inhibition and the H2A(Y57F) mutation enhance H2B deubiquitination activity of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, suggesting a critical role of this phosphorylation in coordinating the activity of the SAGA complex during transcription. Together, these results identify a new component of regulation in transcriptional elongation based on CK2-dependent tyrosine phosphorylation of the globular domain of H2A.
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http://dx.doi.org/10.1038/nature13736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461219PMC
December 2014

YAP mediates crosstalk between the Hippo and PI(3)K–TOR pathways by suppressing PTEN via miR-29.

Nat Cell Biol 2012 Dec;14(12):1322-9

Department of Pharmacology and Moores Cancer Center, School of Medicine, University of California at San Diego, La Jolla, California 92093, USA.

Organ development is a complex process governed by the interplay of several signalling pathways that have critical functions in the regulation of cell growth and proliferation. Over the past years, the Hippo pathway has emerged as a key regulator of organ size. Perturbation of this pathway has been shown to play important roles in tumorigenesis. YAP, the main downstream target of the mammalian Hippo pathway, promotes organ growth, yet the underlying molecular mechanism of this regulation remains unclear. Here we provide evidence that YAP activates the mammalian target of rapamycin (mTOR), a major regulator of cell growth. We have identified the tumour suppressor PTEN, an upstream negative regulator of mTOR, as a critical mediator of YAP in mTOR regulation. We demonstrate that YAP downregulates PTEN by inducing miR-29 to inhibit PTEN translation. Last, we show that PI(3)K–mTOR is a pathway modulated by YAP to regulate cell size, tissue growth and hyperplasia. Our studies reveal a functional link between Hippo and PI(3)K–mTOR, providing a molecular basis for the coordination of these two pathways in organ size regulation.
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http://dx.doi.org/10.1038/ncb2615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019071PMC
December 2012

Dual recognition of phosphoserine and phosphotyrosine in histone variant H2A.X by DNA damage response protein MCPH1.

Proc Natl Acad Sci U S A 2012 Sep 20;109(36):14381-6. Epub 2012 Aug 20.

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.

Tyr142, the C-terminal amino acid of histone variant H2A.X is phosphorylated by WSTF (Williams-Beuren syndrome transcription factor), a component of the WICH complex (WSTF-ISWI chromatin-remodeling complex), under basal conditions in the cell. In response to DNA double-strand breaks (DSBs), H2A.X is instantaneously phosphorylated at Ser139 by the kinases ATM and ATR and is progressively dephosphorylated at Tyr142 by the Eya1 and Eya3 tyrosine phosphatases, resulting in a temporal switch from a postulated diphosphorylated (pSer139, pTyr142) to monophosphorylated (pSer139) H2A.X state. How mediator proteins interpret these two signals remains a question of fundamental interest. We provide structural, biochemical, and cellular evidence that Microcephalin (MCPH1), an early DNA damage response protein, can read both modifications via its tandem BRCA1 C-terminal (BRCT) domains, thereby emerging as a versatile sensor of H2A.X phosphorylation marks. We show that MCPH1 recruitment to sites of DNA damage is linked to both states of H2A.X.
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http://dx.doi.org/10.1073/pnas.1212366109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437875PMC
September 2012

Monomeric structure of the cardioprotective chemokine SDF-1/CXCL12.

Protein Sci 2009 Jul;18(7):1359-69

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) directs leukocyte migration, stem cell homing, and cancer metastasis through activation of CXCR4, which is also a coreceptor for T-tropic HIV-1. Recently, SDF-1 was shown to play a protective role after myocardial infarction, and the protein is a candidate for development of new anti-ischemic compounds. SDF-1 is monomeric at nanomolar concentrations but binding partners promote self-association at higher concentrations to form a typical CXC chemokine homodimer. Two NMR structures have been reported for the SDF-1 monomer, but only one matches the conformation observed in a series of dimeric crystal structures. In the other model, the C-terminal helix is tilted at an angle incompatible with SDF-1 dimerization. Using a rat heart explant model for ischemia/reperfusion injury, we found that dimeric SDF-1 exerts no cardioprotective effect, suggesting that the active species is monomeric. To resolve the discrepancy between existing models, we solved the NMR structure of the SDF-1 monomer in different solution conditions. Irrespective of pH and buffer composition, the C-terminal helix remains tilted at an angle with no evidence for the perpendicular arrangement. Furthermore, we find that phospholipid bicelles promote dimerization that necessarily shifts the helix to the perpendicular orientation, yielding dipolar couplings that are incompatible with the NOE distance constraints. We conclude that interactions with the alignment medium biased the previous structure, masking flexibility in the helix position that may be essential for the distinct functional properties of the SDF-1 monomer.
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http://dx.doi.org/10.1002/pro.167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775206PMC
July 2009

Structural basis of CXCR4 sulfotyrosine recognition by the chemokine SDF-1/CXCL12.

Sci Signal 2008 Sep 16;1(37):ra4. Epub 2008 Sep 16.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

Stem cell homing and breast cancer metastasis are orchestrated by the chemokine stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4. Here, we report the nuclear magnetic resonance structure of a constitutively dimeric SDF-1 in complex with a CXCR4 fragment that contains three sulfotyrosine residues important for a high-affinity ligand-receptor interaction. CXCR4 bridged the SDF-1 dimer interface so that sulfotyrosines sTyr7 and sTyr12 of CXCR4 occupied positively charged clefts on opposing chemokine subunits. Dimeric SDF-1 induced intracellular Ca2+ mobilization but had no chemotactic activity; instead, it prevented native SDF-1-induced chemotaxis, suggesting that it acted as a potent partial agonist. Our work elucidates the structural basis for sulfotyrosine recognition in the chemokine-receptor interaction and suggests a strategy for CXCR4-targeted drug development.
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http://dx.doi.org/10.1126/scisignal.1160755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692298PMC
September 2008