Publications by authors named "Ruslan I Sadreyev"

84 Publications

A unique subset of glycolytic tumour-propagating cells drives squamous cell carcinoma.

Nat Metab 2021 Feb 22;3(2):182-195. Epub 2021 Feb 22.

The Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA.

Head and neck squamous cell carcinoma (SCC) remains among the most aggressive human cancers. Tumour progression and aggressiveness in SCC are largely driven by tumour-propagating cells (TPCs). Aerobic glycolysis, also known as the Warburg effect, is a characteristic of many cancers; however, whether this adaptation is functionally important in SCC, and at which stage, remains poorly understood. Here, we show that the NAD-dependent histone deacetylase sirtuin 6 is a robust tumour suppressor in SCC, acting as a modulator of glycolysis in these tumours. Remarkably, rather than a late adaptation, we find enhanced glycolysis specifically in TPCs. More importantly, using single-cell RNA sequencing of TPCs, we identify a subset of TPCs with higher glycolysis and enhanced pentose phosphate pathway and glutathione metabolism, characteristics that are strongly associated with a better antioxidant response. Together, our studies uncover enhanced glycolysis as a main driver in SCC, and, more importantly, identify a subset of TPCs as the cell of origin for the Warburg effect, defining metabolism as a key feature of intra-tumour heterogeneity.
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http://dx.doi.org/10.1038/s42255-021-00350-6DOI Listing
February 2021

Ibrutinib-Mediated Atrial Fibrillation Attributable to Inhibition of C-Terminal Src Kinase.

Circulation 2020 Dec 23;142(25):2443-2455. Epub 2020 Oct 23.

Cardiovascular Research Center (L.X., S.C., A.H., A.B., J.T., M.N., P.T.E., D.J.M.), Massachusetts General Hospital and Harvard Medical School, Boston, MA.

Background: Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood.

Methods: We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF.

Results: We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7; <0.0001).

Conclusions: These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.049210DOI Listing
December 2020

A MicroRNA Linking Human Positive Selection and Metabolic Disorders.

Cell 2020 Oct 14;183(3):684-701.e14. Epub 2020 Oct 14.

Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, USA; Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Positive selection in Europeans at the 2q21.3 locus harboring the lactase gene has been attributed to selection for the ability of adults to digest milk to survive famine in ancient times. However, the 2q21.3 locus is also associated with obesity and type 2 diabetes in humans, raising the possibility that additional genetic elements in the locus may have contributed to evolutionary adaptation to famine by promoting energy storage, but which now confer susceptibility to metabolic diseases. We show here that the miR-128-1 microRNA, located at the center of the positively selected locus, represents a crucial metabolic regulator in mammals. Antisense targeting and genetic ablation of miR-128-1 in mouse metabolic disease models result in increased energy expenditure and amelioration of high-fat-diet-induced obesity and markedly improved glucose tolerance. A thrifty phenotype connected to miR-128-1-dependent energy storage may link ancient adaptation to famine and modern metabolic maladaptation associated with nutritional overabundance.
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http://dx.doi.org/10.1016/j.cell.2020.09.017DOI Listing
October 2020

The lysine demethylase KDM4A controls the cell-cycle expression of replicative canonical histone genes.

Biochim Biophys Acta Gene Regul Mech 2020 10 13;1863(10):194624. Epub 2020 Aug 13.

Massachusetts General Hospital, Cancer Center and Harvard Medical School, Department of Medicine, 13th street bldg. 149, Charlestown, MA 02129, United States of America; Fox Chase Cancer Center, 333 Cottman Avenue West 260, Philadelphia, PA 19111, United States of America. Electronic address:

Chromatin modulation provides a key checkpoint for controlling cell cycle regulated gene networks. The replicative canonical histone genes are one such gene family under tight regulation during cell division. These genes are most highly expressed during S phase when histones are needed to chromatinize the new DNA template. While this fact has been known for a while, limited knowledge exists about the specific chromatin regulators controlling their temporal expression during cell cycle. Since histones and their associated mutations are emerging as major players in diseases such as cancer, identifying the chromatin factors modulating their expression is critical. The histone lysine tri-demethylase KDM4A is regulated over cell cycle and plays a direct role in DNA replication timing, site-specific rereplication, and DNA amplifications during S phase. Here, we establish an unappreciated role for the catalytically active KDM4A in directly regulating canonical replicative histone gene networks during cell cycle. Of interest, we further demonstrate that KDM4A interacts with proteins controlling histone expression and RNA processing (i.e., hnRNPUL1 and FUS/TLS). Together, this study provides a new function for KDM4A in modulating canonical histone gene expression.
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http://dx.doi.org/10.1016/j.bbagrm.2020.194624DOI Listing
October 2020

Liver X receptors are required for thymic resilience and T cell output.

J Exp Med 2020 10;217(10)

Center for Systems Biology and Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, MA.

The thymus is a primary lymphoid organ necessary for optimal T cell development. Here, we show that liver X receptors (LXRs)-a class of nuclear receptors and transcription factors with diverse functions in metabolism and immunity-critically contribute to thymic integrity and function. LXRαβ-deficient mice develop a fatty, rapidly involuting thymus and acquire a shrunken and prematurely immunoinhibitory peripheral T cell repertoire. LXRαβ's functions are cell specific, and the resulting phenotypes are mutually independent. Although thymic macrophages require LXRαβ for cholesterol efflux, thymic epithelial cells (TECs) use LXRαβ for self-renewal and thymocytes for negative selection. Consequently, TEC-derived LXRαβ protects against homeostatic premature involution and orchestrates thymic regeneration following stress, while thymocyte-derived LXRαβ limits cell disposal during negative selection and confers heightened sensitivity to experimental autoimmune encephalomyelitis. These results identify three distinct but complementary mechanisms by which LXRαβ governs T lymphocyte education and illuminate LXRαβ's indispensable roles in adaptive immunity.
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http://dx.doi.org/10.1084/jem.20200318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537384PMC
October 2020

A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction.

Nat Commun 2020 07 1;11(1):3282. Epub 2020 Jul 1.

Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.
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http://dx.doi.org/10.1038/s41467-020-17099-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329900PMC
July 2020

Distinct functions of tissue-resident and circulating memory Th2 cells in allergic airway disease.

J Exp Med 2020 09;217(9)

Airway Immunity Research Program, Massachusetts General Hospital, Harvard Medical School, Boston, MA.

Memory CD4+ T helper type 2 (Th2) cells drive allergic asthma, yet the mechanisms whereby tissue-resident memory Th2 (Th2 Trm) cells and circulating memory Th2 cells collaborate in vivo remain unclear. Using a house dust mite (HDM) model of allergic asthma and parabiosis, we demonstrate that Th2 Trm cells and circulating memory Th2 cells perform nonredundant functions. Upon HDM rechallenge, circulating memory Th2 cells trafficked into the lung parenchyma and ignited perivascular inflammation to promote eosinophil and CD4+ T cell recruitment. In contrast, Th2 Trm cells proliferated near airways and induced mucus metaplasia, airway hyperresponsiveness, and airway eosinophil activation. Transcriptional analysis revealed that Th2 Trm cells and circulating memory Th2 cells share a core Th2 gene signature but also exhibit distinct transcriptional profiles. Th2 Trm cells express a tissue-adaptation signature, including genes involved in regulating and interacting with extracellular matrix. Our findings demonstrate that Th2 Trm cells and circulating memory Th2 cells are functionally and transcriptionally distinct subsets with unique roles in promoting allergic airway disease.
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http://dx.doi.org/10.1084/jem.20190865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478729PMC
September 2020

Linking indirect effects of cytomegalovirus in transplantation to modulation of monocyte innate immune function.

Sci Adv 2020 Apr 22;6(17):eaax9856. Epub 2020 Apr 22.

Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

Cytomegalovirus (CMV) is an important cause of morbidity and mortality in the immunocompromised host. In transplant recipients, a variety of clinically important "indirect effects" are attributed to immune modulation by CMV, including increased mortality from fungal disease, allograft dysfunction and rejection in solid organ transplantation, and graft-versus-host-disease in stem cell transplantation. Monocytes, key cellular targets of CMV, are permissive to primary, latent and reactivated CMV infection. Here, pairing unbiased bulk and single cell transcriptomics with functional analyses we demonstrate that human monocytes infected with CMV do not effectively phagocytose fungal pathogens, a functional deficit which occurs with decreased expression of fungal recognition receptors. Simultaneously, CMV-infected monocytes upregulate antiviral, pro-inflammatory chemokine, and inflammasome responses associated with allograft rejection and graft-versus-host disease. Our study demonstrates that CMV modulates both immunosuppressive and immunostimulatory monocyte phenotypes, explaining in part, its paradoxical "indirect effects" in transplantation. These data could provide innate immune targets for the stratification and treatment of CMV disease.
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http://dx.doi.org/10.1126/sciadv.aax9856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176434PMC
April 2020

S-phase Enriched Non-coding RNAs Regulate Gene Expression and Cell Cycle Progression.

Cell Rep 2020 05;31(6):107629

Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Many proteins that are needed for progression through S-phase are produced from transcripts that peak in the S-phase, linking temporal expression of those proteins to the time that they are required in cell cycle. Here, we explore the potential roles of long non-coding RNAs in cell cycle progression. We use a sensitive click-chemistry approach to isolate nascent RNAs in a human cell line, and we identify more than 900 long non-coding RNAs (lncRNAs) whose synthesis peaks during the S-phase. More than 200 of these are long intergenic non-coding RNAs (lincRNAs) with S-phase-specific expression. We characterize three of these lincRNAs by knockdown and find that all three lincRNAs are required for appropriate S-phase progression. We infer that non-coding RNAs are key regulatory effectors during the cell cycle, acting on distinct regulatory networks, and herein, we provide a large catalog of candidate cell-cycle regulatory RNAs.
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http://dx.doi.org/10.1016/j.celrep.2020.107629DOI Listing
May 2020

Genome-wide analysis identifies -acting elements regulating mRNA polyadenylation and translation during vertebrate oocyte maturation.

RNA 2020 03 2;26(3):324-344. Epub 2020 Jan 2.

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.

Most cells change patterns of gene expression through transcriptional regulation. In contrast, oocytes are transcriptionally silent and regulate mRNA poly(A) tail length to control protein production. However, the genome-wide relationship of poly(A) tail changes to mRNA translation during vertebrate oocyte maturation is not known. We used Tail-seq and polyribosome analysis to measure poly(A) tail and translational changes during oocyte maturation in We identified large-scale poly(A) and translational changes during oocyte maturation, with poly(A) tail length changes preceding translational changes. Proteins important for completion of the meiotic divisions and early development exhibited increased polyadenylation and translation during oocyte maturation. A family of U-rich sequence elements was enriched near the polyadenylation signal of polyadenylated and translationally activated mRNAs. We propose that changes in mRNA polyadenylation are a conserved mechanism regulating protein expression during vertebrate oocyte maturation and that these changes are controlled by a spatial code of acting sequence elements.
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http://dx.doi.org/10.1261/rna.073247.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025505PMC
March 2020

Histone Lysine Methylation Dynamics Control DNA Copy-Number Amplification.

Cancer Discov 2020 02 27;10(2):306-325. Epub 2019 Nov 27.

Department of Medicine, Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, Massachusetts.

Acquired chromosomal DNA copy gains are a feature of many tumors; however, the mechanisms that underpin oncogene amplification are poorly understood. Recent studies have begun to uncover the importance of epigenetic states and histone lysine methyltransferases (KMT) and demethylases (KDM) in regulating transient site-specific DNA copy-number gains (TSSG). In this study, we reveal a critical interplay between a myriad of lysine methyltransferases and demethylases in modulating H3K4/9/27 methylation balance to control extrachromosomal amplification of the oncogene. This study further establishes that cellular signals (hypoxia and EGF) are able to directly promote amplification through modulation of the enzymes controlling copy gains. Moreover, we demonstrate that chemical inhibitors targeting specific KMTs and KDMs are able to promote or block extrachromosomal amplification, which identifies potential therapeutic strategies for controlling copy-number heterogeneity in cancer, and, in turn, drug response. SIGNIFICANCE: This study identifies a network of epigenetic factors and cellular signals that directly control DNA amplification. We demonstrate that chemical inhibitors targeting enzymes controlling this amplification can be used to rheostat copy number, which uncovers therapeutic opportunities for controlling DNA amplification heterogeneity and the associated drug response..
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http://dx.doi.org/10.1158/2159-8290.CD-19-0463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271978PMC
February 2020

Exercise reduces inflammatory cell production and cardiovascular inflammation via instruction of hematopoietic progenitor cells.

Nat Med 2019 11 7;25(11):1761-1771. Epub 2019 Nov 7.

Center for Systems Biology, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

A sedentary lifestyle, chronic inflammation and leukocytosis increase atherosclerosis; however, it remains unclear whether regular physical activity influences leukocyte production. Here we show that voluntary running decreases hematopoietic activity in mice. Exercise protects mice and humans with atherosclerosis from chronic leukocytosis but does not compromise emergency hematopoiesis in mice. Mechanistically, exercise diminishes leptin production in adipose tissue, augmenting quiescence-promoting hematopoietic niche factors in leptin-receptor-positive stromal bone marrow cells. Induced deletion of the leptin receptor in Prrx1-creER; Lepr mice reveals that leptin's effect on bone marrow niche cells regulates hematopoietic stem and progenitor cell (HSPC) proliferation and leukocyte production, as well as cardiovascular inflammation and outcomes. Whereas running wheel withdrawal quickly reverses leptin levels, the impact of exercise on leukocyte production and on the HSPC epigenome and transcriptome persists for several weeks. Together, these data show that physical activity alters HSPCs via modulation of their niche, reducing hematopoietic output of inflammatory leukocytes.
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http://dx.doi.org/10.1038/s41591-019-0633-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858591PMC
November 2019

Inducible histone K-to-M mutations are dynamic tools to probe the physiological role of site-specific histone methylation in vitro and in vivo.

Nat Cell Biol 2019 11 28;21(11):1449-1461. Epub 2019 Oct 28.

Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA.

Development and differentiation are associated with profound changes to histone modifications, yet their in vivo function remains incompletely understood. Here, we generated mouse models expressing inducible histone H3 lysine-to-methionine (K-to-M) mutants, which globally inhibit methylation at specific sites. Mice expressing H3K36M developed severe anaemia with arrested erythropoiesis, a marked haematopoietic stem cell defect, and rapid lethality. By contrast, mice expressing H3K9M survived up to a year and showed expansion of multipotent progenitors, aberrant lymphopoiesis and thrombocytosis. Additionally, some H3K9M mice succumbed to aggressive T cell leukaemia/lymphoma, while H3K36M mice exhibited differentiation defects in testis and intestine. Mechanistically, induction of either mutant reduced corresponding histone trimethylation patterns genome-wide and altered chromatin accessibility as well as gene expression landscapes. Strikingly, discontinuation of transgene expression largely restored differentiation programmes. Our work shows that individual chromatin modifications are required at several specific stages of differentiation and introduces powerful tools to interrogate their roles in vivo.
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http://dx.doi.org/10.1038/s41556-019-0403-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858577PMC
November 2019

Exploiting the Zonulin Mouse Model to Establish the Role of Primary Impaired Gut Barrier Function on Microbiota Composition and Immune Profiles.

Front Immunol 2019 19;10:2233. Epub 2019 Sep 19.

Department of Pediatric Gastroenterology and Nutrition, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital, Boston, MA, United States.

The balanced interplay between epithelial barrier, immune system, and microbiota maintains gut homeostasis, while disruption of this interplay may lead to inflammation. Paracellular permeability is governed by intercellular tight-junctions (TJs). Zonulin is, to date, the only known physiological regulator of intestinal TJs. We used a zonulin transgenic mouse (Ztm) model characterized by increased small intestinal permeability to elucidate the role of a primary impaired gut barrier on microbiome composition and/or immune profile. Ztm exhibit an altered gene expression profile of TJs in the gut compared to wild-type mice (WT): Claudin-15, Claudin-5, Jam-3, and Myosin-1C are decreased in the male duodenum whereas Claudin-15, Claudin-7, and ZO-2 are reduced in the female colon. These results are compatible with loss of gut barrier function and are paralleled by an altered microbiota composition with reduced abundance of the genus , known to have positive effects on gut barrier integrity and strengthening, and an increased abundance of the genus, associated to low-grade inflammatory conditions. Immune profile analysis shows a subtly skewed distribution of immune cell subsets toward a pro-inflammatory phenotype with more IL-17 producing adaptive and innate-like T cells in Ztm. Interestingly, microbiota "normalization" involving the transfer of WT microbiota into Ztm, did not rescue the altered immune profile. Our data suggest that a primary impaired gut barrier causing an uncontrolled trafficking of microbial products leads to a latent pro-inflammatory status, with a skewed microbiota composition and immune profile that, in the presence of an environmental trigger, as we have previously described (1), might promote the onset of overt inflammation and an increased risk of chronic disease.
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http://dx.doi.org/10.3389/fimmu.2019.02233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761304PMC
October 2020

The RNA Helicase DDX6 Controls Cellular Plasticity by Modulating P-Body Homeostasis.

Cell Stem Cell 2019 11 3;25(5):622-638.e13. Epub 2019 Oct 3.

Department of Molecular Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA; Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA; Cancer Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA; Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA; Harvard Stem Cell Institute, 1350 Massachusetts Avenue, Cambridge, MA 02138, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, "hyper-pluripotent" state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency.
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http://dx.doi.org/10.1016/j.stem.2019.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247364PMC
November 2019

Salt-inducible kinases dictate parathyroid hormone 1 receptor action in bone development and remodeling.

J Clin Invest 2019 12;129(12):5187-5203

Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

The parathyroid hormone 1 receptor (PTH1R) mediates the biologic actions of parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP). Here, we showed that salt-inducible kinases (SIKs) are key kinases that control the skeletal actions downstream of PTH1R and that this GPCR, when activated, inhibited cellular SIK activity. Sik gene deletion led to phenotypic changes that were remarkably similar to models of increased PTH1R signaling. In growth plate chondrocytes, PTHrP inhibited SIK3, and ablation of this kinase in proliferating chondrocytes rescued perinatal lethality of PTHrP-null mice. Combined deletion of Sik2 and Sik3 in osteoblasts and osteocytes led to a dramatic increase in bone mass that closely resembled the skeletal and molecular phenotypes observed when these bone cells express a constitutively active PTH1R that causes Jansen's metaphyseal chondrodysplasia. Finally, genetic evidence demonstrated that class IIa histone deacetylases were key PTH1R-regulated SIK substrates in both chondrocytes and osteocytes. Taken together, our findings establish that SIK inhibition is central to PTH1R action in bone development and remodeling. Furthermore, this work highlights the key role of cAMP-regulated SIKs downstream of GPCR action.
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http://dx.doi.org/10.1172/JCI130126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877304PMC
December 2019

The Histone Deacetylase SIRT6 Restrains Transcription Elongation via Promoter-Proximal Pausing.

Mol Cell 2019 08 6;75(4):683-699.e7. Epub 2019 Aug 6.

The Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA; The MGH Center for Regenerative Medicine, Harvard Medical School, Boston, MA 02114, USA; The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address:

Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)-that phosphorylates NELF and the carboxyl terminal domain of Pol II-and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase.
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http://dx.doi.org/10.1016/j.molcel.2019.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6907403PMC
August 2019

TREM2 Acts Downstream of CD33 in Modulating Microglial Pathology in Alzheimer's Disease.

Neuron 2019 09 10;103(5):820-835.e7. Epub 2019 Jul 10.

Genetics and Aging Research Unit, McCance Center for Brain Health, Mass General Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA. Electronic address:

The microglial receptors CD33 and TREM2 have been associated with risk for Alzheimer's disease (AD). Here, we investigated crosstalk between CD33 and TREM2. We showed that knockout of CD33 attenuated amyloid beta (Aβ) pathology and improved cognition in 5xFAD mice, both of which were abrogated by additional TREM2 knockout. Knocking out TREM2 in 5xFAD mice exacerbated Aβ pathology and neurodegeneration but reduced Iba1 cell numbers, all of which could not be rescued by additional CD33 knockout. RNA-seq profiling of microglia revealed that genes related to phagocytosis and signaling (IL-6, IL-8, acute phase response) are upregulated in 5xFAD;CD33 and downregulated in 5xFAD;TREM2 mice. Differential gene expression in 5xFAD;CD33 microglia depended on the presence of TREM2, suggesting TREM2 acts downstream of CD33. Crosstalk between CD33 and TREM2 includes regulation of the IL-1β/IL-1RN axis and a gene set in the "receptor activity chemokine" cluster. Our results should facilitate AD therapeutics targeting these receptors.
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http://dx.doi.org/10.1016/j.neuron.2019.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728215PMC
September 2019

Single-Cell RNA-seq: Introduction to Bioinformatics Analysis.

Curr Protoc Mol Biol 2019 06;127(1):e92

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts.

Quantitative analysis of single-cell RNA sequencing (RNA-seq) is crucial for discovering the heterogeneity of cell populations and understanding the molecular mechanisms in different cells. In this unit we present a bioinformatics workflow for analyzing single-cell RNA-seq data with a few current publicly available computational tools. This workflow is focused on the interpretation of the heterogeneity from single-cell transcriptomes as well as the identification of cell clusters and genes that are differentially expressed between clusters. © 2019 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmb.92DOI Listing
June 2019

Human gut derived-organoids provide model to study gluten response and effects of microbiota-derived molecules in celiac disease.

Sci Rep 2019 05 7;9(1):7029. Epub 2019 May 7.

Mucosal Immunology and Biology Research Center and Center for Celiac Research and Treatment, Department of Pediatrics, Massachusetts General Hospital, Boston, MA, USA.

Celiac disease (CD) is an immune-mediated disorder triggered by gluten exposure. The contribution of the adaptive immune response to CD pathogenesis has been extensively studied, but the absence of valid experimental models has hampered our understanding of the early steps leading to loss of gluten tolerance. Using intestinal organoids developed from duodenal biopsies from both non-celiac (NC) and celiac (CD) patients, we explored the contribution of gut epithelium to CD pathogenesis and the role of microbiota-derived molecules in modulating the epithelium's response to gluten. When compared to NC, RNA sequencing of CD organoids revealed significantly altered expression of genes associated with gut barrier, innate immune response, and stem cell functions. Monolayers derived from CD organoids exposed to gliadin showed increased intestinal permeability and enhanced secretion of pro-inflammatory cytokines compared to NC controls. Microbiota-derived bioproducts butyrate, lactate, and polysaccharide A improved barrier function and reduced gliadin-induced cytokine secretion. We concluded that: (1) patient-derived organoids faithfully express established and newly identified molecular signatures characteristic of CD. (2) microbiota-derived bioproducts can be used to modulate the epithelial response to gluten. Finally, we validated the use of patient-derived organoids monolayers as a novel tool for the study of CD.
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http://dx.doi.org/10.1038/s41598-019-43426-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505524PMC
May 2019

Exploration of CTCF post-translation modifications uncovers Serine-224 phosphorylation by PLK1 at pericentric regions during the G2/M transition.

Elife 2019 01 24;8. Epub 2019 Jan 24.

Department of Molecular Biology, Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, United States.

The zinc finger CCCTC-binding protein (CTCF) carries out many functions in the cell. Although previous studies sought to explain CTCF multivalency based on sequence composition of binding sites, few examined how CTCF post-translational modification (PTM) could contribute to function. Here, we performed CTCF mass spectrometry, identified a novel phosphorylation site at Serine 224 (Ser-P), and demonstrate that phosphorylation is carried out by Polo-like kinase 1 (PLK1). CTCF Ser-P is chromatin-associated, mapping to at least a subset of known CTCF sites. CTCF Ser-P accumulates during the G2/M transition of the cell cycle and is enriched at pericentric regions. The phospho-obviation mutant, S224A, appeared normal. However, the phospho-mimic mutant, S224E, is detrimental to mouse embryonic stem cell colonies. While ploidy and chromatin architecture appear unaffected, S224E mutants differentially express hundreds of genes, including p53 and p21. We have thus identified a new CTCF PTM and provided evidence of biological function.
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http://dx.doi.org/10.7554/eLife.42341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361588PMC
January 2019

Whole Genome Next-Generation Sequencing Mutation Identification in Pseudomonas aeruginosa.

Curr Protoc Mol Biol 2018 10 21;124(1):e69. Epub 2018 Sep 21.

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts.

Identification of spontaneous or chemically induced bacterial mutations is a powerful tool for investigation of molecular mechanisms, including the mechanism of action of novel antibiotics. However, a major bottleneck to this approach has been the identification of the causative mutation underlying a phenotype of interest. Until recently, this has required time-consuming genetic analysis. However, the advent of relatively inexpensive and rapid next-generation sequencing (NGS) technologies has revolutionized the correlation of bacterial phenotypes and genotypes. In this article we describe a simple bioinformatics pipeline to identify differences between sequenced bacterial genomes. We also describe the procedures involved in growing, extracting, and purifying DNA, and preparation of sequencing libraries for one bacterial species, Pseudomonas aeruginosa. Similar protocols will be applicable to other bacterial species. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmb.69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168388PMC
October 2018

RNA-seq: Basic Bioinformatics Analysis.

Curr Protoc Mol Biol 2018 10 17;124(1):e68. Epub 2018 Sep 17.

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts.

Quantitative analysis of gene expression is crucial for understanding the molecular mechanisms underlying genome regulation. RNA-seq is a powerful platform for comprehensive investigation of the transcriptome. In this unit, we present a general bioinformatics workflow for the quantitative analysis of RNA-seq data and describe a few current publicly available computational tools applicable at various steps of this workflow. These tools comprise a pipeline for quality assessment and quantitation of RNA-seq data that starts from raw sequencing files and is focused on the identification and analysis of genes that are differentially expressed between biological conditions. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmb.68DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168365PMC
October 2018

Robust interferon signature and suppressed tissue repair gene expression in synovial tissue from patients with postinfectious, Borrelia burgdorferi-induced Lyme arthritis.

Cell Microbiol 2019 02 17;21(2):e12954. Epub 2018 Oct 17.

Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.

In most patients with Lyme arthritis (LA), antibiotic therapy results in Borrelia burgdorferi pathogen elimination, tissue repair, and return to homeostasis. However, despite spirochetal killing, some patients develop proliferative synovitis, characterised by synovial hyperplasia, inflammation, vascular damage, and fibrosis that persists for months to several years after antibiotic treatment, called postinfectious LA. In this study, we characterised the transcriptomes of postinfectious LA patients' synovial tissue, the target tissue of the immune response. High-throughput RNA sequencing to a depth of ~30 million reads per sample was used to profile gene expression in synovial tissue from 14 patients with postinfectious LA, compared with eight patients with other types of chronic inflammatory arthritis and five with minimally inflammatory osteoarthritis (OA). Synovium from postinfectious LA and other inflammatory arthritides shared gene signatures associated with antigen presentation, innate immune responses, and cell-mediated immune activation, whereas these responses were diminished in OA synovium. Unique to postinfectious LA was a particularly robust interferon-gamma (IFNγ) signature. Moreover, this heightened IFNγ signature inversely correlated with expression of genes involved in repair of damaged tissue, including genes associated with stromal cell proliferation and differentiation, neovascularisation, and extracellular matrix synthesis, which were markedly suppressed in postinfectious LA. Transcriptional observations were confirmed by cytokine profiling, histologic analyses, and clinical correlations. We propose that in patients with postinfectious LA, overexpression of IFNγ in synovium prevents appropriate repair of tissue damaged by B. burgdorferi infection, blocking return to tissue homeostasis long after completion of antibiotic therapy and resolution of active infection.
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http://dx.doi.org/10.1111/cmi.12954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724218PMC
February 2019

Prospective Isolation of Poised iPSC Intermediates Reveals Principles of Cellular Reprogramming.

Cell Stem Cell 2018 Aug 12;23(2):289-305.e5. Epub 2018 Jul 12.

Department of Molecular Biology, Cancer Center, and Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA; Harvard Stem Cell Institute, 1350 Massachusetts Avenue, Cambridge, MA 02138, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Cellular reprogramming converts differentiated cells into induced pluripotent stem cells (iPSCs). However, this process is typically very inefficient, complicating mechanistic studies. We identified and molecularly characterized rare, early intermediates poised to reprogram with up to 95% efficiency, without perturbing additional genes or pathways, during iPSC generation from mouse embryonic fibroblasts. Analysis of these cells uncovered transcription factors (e.g., Tfap2c and Bex2) that are important for reprogramming but dispensable for pluripotency maintenance. Additionally, we observed striking patterns of chromatin hyperaccessibility at pluripotency loci, which preceded gene expression in poised intermediates. Finally, inspection of these hyperaccessible regions revealed an early wave of DNA demethylation that is uncoupled from de novo methylation of somatic regions late in reprogramming. Our study underscores the importance of investigating rare intermediates poised to produce iPSCs, provides insights into reprogramming mechanisms, and offers a valuable resource for the dissection of transcriptional and epigenetic dynamics intrinsic to cell fate change.
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http://dx.doi.org/10.1016/j.stem.2018.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086589PMC
August 2018

Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism.

Nat Cell Biol 2018 07 25;20(7):811-822. Epub 2018 Jun 25.

Center for Cancer Research, Massachusetts General Hospital, Boston, MA, USA.

G protein α (GNAS) mediates receptor-stimulated cAMP signalling, which integrates diverse environmental cues with intracellular responses. GNAS is mutationally activated in multiple tumour types, although its oncogenic mechanisms remain elusive. We explored this question in pancreatic tumourigenesis where concurrent GNAS and KRAS mutations characterize pancreatic ductal adenocarcinomas (PDAs) arising from intraductal papillary mucinous neoplasms (IPMNs). By developing genetically engineered mouse models, we show that Gnas cooperates with Kras to promote initiation of IPMN, which progress to invasive PDA following Tp53 loss. Mutant Gnas remains critical for tumour maintenance in vivo. This is driven by protein-kinase-A-mediated suppression of salt-inducible kinases (Sik1-3), associated with induction of lipid remodelling and fatty acid oxidation. Comparison of Kras-mutant pancreatic cancer cells with and without Gnas mutations reveals striking differences in the functions of this network. Thus, we uncover Gnas-driven oncogenic mechanisms, identify Siks as potent tumour suppressors, and demonstrate unanticipated metabolic heterogeneity among Kras-mutant pancreatic neoplasms.
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http://dx.doi.org/10.1038/s41556-018-0122-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044476PMC
July 2018

Cell of origin dictates aggression and stem cell number in acute lymphoblastic leukemia.

Leukemia 2018 08 18;32(8):1860-1865. Epub 2018 Apr 18.

Department of Pathology, Massachusetts General Hospital Research Institute, Boston, MA, 02114, USA.

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http://dx.doi.org/10.1038/s41375-018-0130-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620784PMC
August 2018

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors.

Stem Cell Reports 2018 05;10(5):1505-1521

Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address:

Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7 cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.
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http://dx.doi.org/10.1016/j.stemcr.2018.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995754PMC
May 2018

The surveillance of pre-mRNA splicing is an early step in RNAi of endogenous genes.

Genes Dev 2018 05 8;32(9-10):670-681. Epub 2018 May 8.

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.

RNAi pathways detect and silence foreign nucleic acids such as viruses as well as endogenous genes in many species. The phylogenetic profile across eukaryotes of proteins that mediate key steps in RNAi is correlated with the profiles of multiple mRNA splicing proteins and with intron number, suggesting that RNAi may surveil mRNA splicing to detect the divergent or absent introns of viruses. Here we examine the role of mRNA splicing in RNAi We found that viable null mutations in U1 and U2 small nuclear ribonucleic protein (snRNP)-specific splicing factor genes cause defects in RNAi. The U1A ortholog is required for normal ERGO-1 Argonaute class 26G siRNA biogenesis, -splicing of the transcript, and targeting of poorly conserved gene transcripts by WAGO Argonaute class 22G siRNAs. We found that gene transcripts engaged by the siRNA-generating machinery are poorly conserved, possess few introns, and often have introns that are divergent from introns with strong consensus splicing sites found in highly conserved genes. We present biochemical evidence that RNAi targeted transcripts are tightly bound to spliceosomes. These findings suggest multiple layers of regulation by the spliceosome at early steps of small RNA-mediated gene silencing.
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http://dx.doi.org/10.1101/gad.311514.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004069PMC
May 2018