Publications by authors named "Andrea Bredemeyer"

33 Publications

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis.

JACC Basic Transl Sci 2021 Feb 26. Epub 2021 Feb 26.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

There is ongoing debate as to whether cardiac complications of coronavirus disease 2019 (COVID-19) result from myocardial viral infection or are secondary to systemic inflammation and/or thrombosis. We provide evidence that cardiomyocytes are infected in patients with COVID-19 myocarditis and are susceptible to severe acute respiratory syndrome coronavirus 2. We establish an engineered heart tissue model of COVID-19 myocardial pathology, define mechanisms of viral pathogenesis, and demonstrate that cardiomyocyte severe acute respiratory syndrome coronavirus 2 infection results in contractile deficits, cytokine production, sarcomere disassembly, and cell death. These findings implicate direct infection of cardiomyocytes in the pathogenesis of COVID-19 myocardial pathology and provides a model system to study this emerging disease.
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http://dx.doi.org/10.1016/j.jacbts.2021.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909907PMC
February 2021

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis.

bioRxiv 2020 Nov 5. Epub 2020 Nov 5.

Epidemiological studies of the COVID-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. Nonetheless, little is understood regarding SARS-CoV-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. Here, we develop a human engineered heart tissue model and demonstrate that SARS-CoV-2 selectively infects cardiomyocytes. Viral infection is dependent on expression of angiotensin-I converting enzyme 2 (ACE2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. After infection with SARS-CoV-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. Consistent with these findings, autopsy tissue obtained from individuals with COVID-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. These findings establish human cardiomyocyte tropism for SARS-CoV-2 and provide an experimental platform for interrogating and mitigating cardiac complications of COVID-19.
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http://dx.doi.org/10.1101/2020.11.04.364315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654892PMC
November 2020

XLF and H2AX function in series to promote replication fork stability.

J Cell Biol 2019 07 23;218(7):2113-2123. Epub 2019 May 23.

Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY

XRCC4-like factor (XLF) is a non-homologous end joining (NHEJ) DNA double strand break repair protein. However, XLF deficiency leads to phenotypes in mice and humans that are not necessarily consistent with an isolated defect in NHEJ. Here we show that XLF functions during DNA replication. XLF undergoes cell division cycle 7-dependent phosphorylation; associates with the replication factor C complex, a critical component of the replisome; and is found at replication forks. XLF deficiency leads to defects in replication fork progression and an increase in fork reversal. The additional loss of H2AX, which protects DNA ends from resection, leads to a requirement for ATR to prevent an MRE11-dependent loss of newly synthesized DNA and activation of DNA damage response. Moreover, cells exhibit a marked dependence on the ATR kinase for survival. We propose that XLF and H2AX function in series to prevent replication stress induced by the MRE11-dependent resection of regressed arms at reversed replication forks.
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http://dx.doi.org/10.1083/jcb.201808134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605786PMC
July 2019

Ferroptotic cell death and TLR4/Trif signaling initiate neutrophil recruitment after heart transplantation.

J Clin Invest 2019 02 26;129(6):2293-2304. Epub 2019 Feb 26.

Department of Surgery and.

Non-apoptotic forms of cell death can trigger sterile inflammation through the release of danger-associated molecular patterns, which are recognized by innate immune receptors. However, despite years of investigation the mechanisms which initiate inflammatory responses after heart transplantation remain elusive. Here, we demonstrate that ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, decreases the level of pro-ferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamine, reduces cardiomyocyte cell death and blocks neutrophil recruitment following heart transplantation. Inhibition of necroptosis had no effect on neutrophil trafficking in cardiac grafts. We extend these observations to a model of coronary artery ligation-induced myocardial ischemia reperfusion injury where inhibition of ferroptosis resulted in reduced infarct size, improved left ventricular systolic function, and reduced left ventricular remodeling. Using intravital imaging of cardiac transplants, we uncover that ferroptosis orchestrates neutrophil recruitment to injured myocardium by promoting adhesion of neutrophils to coronary vascular endothelial cells through a TLR4/TRIF/type I IFN signaling pathway. Thus, we have discovered that inflammatory responses after cardiac transplantation are initiated through ferroptotic cell death and TLR4/Trif-dependent signaling in graft endothelial cells. These findings provide a platform for the development of therapeutic strategies for heart transplant recipients and patients, who are vulnerable to ischemia reperfusion injury following restoration of coronary blood flow.
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http://dx.doi.org/10.1172/JCI126428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546457PMC
February 2019

Tissue Resident CCR2- and CCR2+ Cardiac Macrophages Differentially Orchestrate Monocyte Recruitment and Fate Specification Following Myocardial Injury.

Circ Res 2019 01;124(2):263-278

From the Department of Medicine (G.B., A.B., A.L. Koenig, I.L., J.M., B.I., C.W., A. Kovacs, K.J.L.), Washington University School of Medicine, St. Louis, MO.

Rationale: Recent advancements have brought to light the origins, complexity, and functions of tissue-resident macrophages. However, in the context of tissue injury or disease, large numbers of monocytes infiltrate the heart and are thought to contribute to adverse remodeling and heart failure pathogenesis. Little is understood about the diversity of monocytes and monocyte-derived macrophages recruited to the heart after myocardial injury, including the mechanisms that regulate monocyte recruitment and fate specification.

Objective: We sought to test the hypothesis that distinct subsets of tissue-resident CCR2- (C-C chemokine receptor 2) and CCR2+ macrophages orchestrate monocyte recruitment and fate specification after myocardial injury.

Methods And Results: We reveal that in numerous mouse models of cardiomyocyte cell death (permanent myocardial infarction, reperfused myocardial infarction, and diphtheria toxin cardiomyocyte ablation), there is a shift in macrophage ontogeny whereby tissue-resident macrophages are predominately replaced by infiltrating monocytes and monocyte-derived macrophages. Using syngeneic cardiac transplantation to model ischemia-reperfusion injury and distinguish tissue-resident from recruited cell populations in combination with intravital 2-photon microscopy, we demonstrate that monocyte recruitment is differentially orchestrated by distinct subsets of tissue-resident cardiac macrophages. Tissue-resident CCR2+ macrophages promote monocyte recruitment through an MYD88 (myeloid differentiation primary response 88)-dependent mechanism that results in release of MCPs (monocyte chemoattractant proteins) and monocyte mobilization. In contrast, tissue-resident CCR2- macrophages inhibit monocyte recruitment. Using CD (cluster of differentiation) 169-DTR (diphtheria toxin receptor) and CCR2-DTR mice, we further show that selective depletion of either tissue-resident CCR2- or CCR2+ macrophages before myocardial infarction results in divergent effects on left ventricular function, myocardial remodeling, and monocyte recruitment. Finally, using single-cell RNA sequencing, we show that tissue-resident cardiac macrophages differentially instruct monocyte fate specification.

Conclusions: Collectively, these observations establish the mechanistic basis by which monocytes are initially recruited to the injured heart and provide new insights into the heterogeneity of monocyte-derived macrophages.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.314028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626616PMC
January 2019

MRI Is a DNA Damage Response Adaptor during Classical Non-homologous End Joining.

Mol Cell 2018 07 12;71(2):332-342.e8. Epub 2018 Jul 12.

Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY 10065, USA. Electronic address:

The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.
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http://dx.doi.org/10.1016/j.molcel.2018.06.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6083883PMC
July 2018

The human heart contains distinct macrophage subsets with divergent origins and functions.

Nat Med 2018 08 11;24(8):1234-1245. Epub 2018 Jun 11.

Center for Cardiovascular Research, Division of Cardiology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO, USA.

Paradigm-shifting studies in the mouse have identified tissue macrophage heterogeneity as a critical determinant of immune responses. In contrast, surprisingly little is known regarding macrophage heterogeneity in humans. Macrophages within the mouse heart are partitioned into CCR2- and CCR2+ subsets with divergent origins, repopulation mechanisms, and functions. Here, we demonstrate that the human myocardium also contains distinct subsets of CCR2- and CCR2+ macrophages. Analysis of sex-mismatched heart transplant recipients revealed that CCR2- macrophages are a tissue-resident population exclusively replenished through local proliferation, whereas CCR2+ macrophages are maintained through monocyte recruitment and proliferation. Moreover, CCR2- and CCR2+ macrophages have distinct functional properties, analogous to reparative CCR2- and inflammatory CCR2+ macrophages in the mouse heart. Clinically, CCR2+ macrophage abundance is associated with left ventricular remodeling and systolic function in heart failure patients. Collectively, these observations provide initial evidence for the functional importance of macrophage heterogeneity in the human heart.
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http://dx.doi.org/10.1038/s41591-018-0059-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082687PMC
August 2018

High-Throughput Screening Approach for Identifying Compounds That Inhibit Nonhomologous End Joining.

SLAS Discov 2018 08 12;23(7):624-633. Epub 2017 Dec 12.

3 Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA.

DNA double-strand breaks (DSBs) are repaired primarily by homologous recombination (HR) or nonhomologous end joining (NHEJ). Compounds that modulate HR have shown promise as cancer therapeutics. The V(D)J recombination reaction, which assembles antigen receptor genes in lymphocytes, is initiated by the introduction of DNA DSBs at two recombining gene segments by the RAG endonuclease, followed by the NHEJ-mediated repair of these DSBs. Here, using HyperCyt automated flow cytometry, we develop a robust high-throughput screening (HTS) assay for NHEJ that utilizes engineered pre-B-cell lines where the V(D)J recombination reaction can be induced and monitored at a single-cell level. This approach, novel in processing four 384-well plates at a time in parallel, was used to screen the National Cancer Institute NeXT library to identify compounds that inhibit V(D)J recombination and NHEJ. Assessment of cell light scattering characteristics at the primary HTS stage (83,536 compounds) enabled elimination of 60% of apparent hits as false positives. Although all the active compounds that we identified had an inhibitory effect on RAG cleavage, we have established this as an approach that could identify compounds that inhibit RAG cleavage or NHEJ using new chemical libraries.
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http://dx.doi.org/10.1177/2472555217746324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997544PMC
August 2018

Pediatric and adult dilated cardiomyopathy represent distinct pathological entities.

JCI Insight 2017 Jul 20;2(14). Epub 2017 Jul 20.

Center for Cardiovascular Research, Division of Cardiology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

Pediatric dilated cardiomyopathy (DCM) is the most common indication for heart transplantation in children. Despite similar genetic etiologies, medications routinely used in adult heart failure patients do not improve outcomes in the pediatric population. The mechanistic basis for these observations is unknown. We hypothesized that pediatric and adult DCM comprise distinct pathological entities, in that children do not undergo adverse remodeling, the target of adult heart failure therapies. To test this hypothesis, we examined LV specimens obtained from pediatric and adult donor controls and DCM patients. Consistent with the established pathophysiology of adult heart failure, adults with DCM displayed marked cardiomyocyte hypertrophy and myocardial fibrosis compared with donor controls. In contrast, pediatric DCM specimens demonstrated minimal cardiomyocyte hypertrophy and myocardial fibrosis compared with both age-matched controls and adults with DCM. Strikingly, RNA sequencing uncovered divergent gene expression profiles in pediatric and adult patients, including enrichment of transcripts associated with adverse remodeling and innate immune activation in adult DCM specimens. Collectively, these findings reveal that pediatric and adult DCM represent distinct pathological entities, provide a mechanistic basis to explain why children fail to respond to adult heart failure therapies, and suggest the need to develop new approaches for pediatric DCM.
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http://dx.doi.org/10.1172/jci.insight.94382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518561PMC
July 2017

Deficiency of XLF and PAXX prevents DNA double-strand break repair by non-homologous end joining in lymphocytes.

Cell Cycle 2017 Feb 10;16(3):286-295. Epub 2016 Nov 10.

b Department of Pathology and Immunology , Washington University School of Medicine , St. Louis , MO , USA.

Non-homologous end joining (NHEJ) is a major DNA double-strand break (DSB) repair pathway that functions in all phases of the cell cycle. NHEJ repairs genotoxic and physiological DSBs, such as those generated by ionizing radiation and during V(D)J recombination at antigen receptor loci, respectively. DNA end joining by NHEJ relies on the core factors Ku70, Ku80, XRCC4, and DNA Ligase IV. Additional proteins also play important roles in NHEJ. The XRCC4-like factor (XLF) participates in NHEJ through its interaction with XRCC4, and XLF deficiency in humans leads to immunodeficiency and increased sensitivity to ionizing radiation. However, XLF is dispensable for NHEJ-mediated DSB repair during V(D)J recombination in murine lymphocytes, where it may have redundant functions with other DSB repair factors. Paralog of XRCC4 and XLF (PAXX) is a recently identified NHEJ factor that has structural similarity to XRCC4 and XLF. Here we show that PAXX is also dispensable for NHEJ during V(D)J recombination and during the repair of genotoxic DSBs in lymphocytes. However, a combined deficiency of PAXX and XLF blocks NHEJ with a severity comparable to that observed in DNA Ligase IV-deficient cells. Similar to XLF, PAXX interacts with Ku through its C-terminal region, and mutations that disrupt Ku binding prevent PAXX from promoting NHEJ in XLF-deficient lymphocytes. Our findings suggest that the PAXX and XLF proteins may have redundant functions during NHEJ.
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http://dx.doi.org/10.1080/15384101.2016.1253640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323033PMC
February 2017

Modeling altered T-cell development with induced pluripotent stem cells from patients with RAG1-dependent immune deficiencies.

Blood 2016 08 14;128(6):783-93. Epub 2016 Jun 14.

Sunnybrook Research Institute, and Department of Immunology, University of Toronto, Toronto, ON, Canada;

Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3(--)CD4(-)CD8(-)CD7(+)CD5(+)CD38(-)CD31(-/lo)CD45RA(+) stage of T-cell development to reach the CD3(-/+)CD4(+)CD8(+)CD7(+)CD5(+)CD38(+)CD31(+)CD45RA(-) stage. Despite residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-β (TRB) and TCR-α (TRA) rearrangements of CD3(-)CD4(+)CD8(-) immature single-positive and CD3(+)CD4(+)CD8(+) double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/δ excision circles (TRECs), a marker of TRA/δ locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients.
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http://dx.doi.org/10.1182/blood-2015-10-676304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982452PMC
August 2016

DNA damage responses: beyond double-strand break repair.

Curr Biol 2015 Jan;25(1):R45-6

Division of Laboratory and Genomic Medicine, Department of Pathology and Immunology, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8118, St. Louis, MO 63110-1093, USA. Electronic address:

The RAG endonuclease generates DNA double strand breaks during antigen receptor gene assembly, an essential process for B- and T-lymphocyte development. However, a recent study reveals that RAG endonuclease activity affects natural killer cell function, demonstrating that such double strand breaks, and the responses they elicit, may have broad cellular effects.
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http://dx.doi.org/10.1016/j.cub.2014.11.024DOI Listing
January 2015

HCoDES reveals chromosomal DNA end structures with single-nucleotide resolution.

Mol Cell 2014 Dec 26;56(6):808-18. Epub 2014 Nov 26.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

The structure of broken DNA ends is a critical determinant of the pathway used for DNA double-strand break (DSB) repair. Here, we develop an approach involving the hairpin capture of DNA end structures (HCoDES), which elucidates chromosomal DNA end structures at single-nucleotide resolution. HCoDES defines structures of physiologic DSBs generated by the RAG endonuclease, as well as those generated by nucleases widely used for genome editing. Analysis of G1 phase cells deficient in H2AX or 53BP1 reveals DNA ends that are frequently resected to form long single-stranded overhangs that can be repaired by mutagenic pathways. In addition to 3' overhangs, many of these DNA ends unexpectedly form long 5' single-stranded overhangs. The divergence in DNA end structures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in end protection. Thus, the high-resolution end structures obtained by HCoDES identify features of DNA end processing during DSB repair.
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http://dx.doi.org/10.1016/j.molcel.2014.10.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272619PMC
December 2014

KAP-1 promotes resection of broken DNA ends not protected by γ-H2AX and 53BP1 in G₁-phase lymphocytes.

Mol Cell Biol 2014 Aug 19;34(15):2811-21. Epub 2014 May 19.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA

The resection of broken DNA ends is required for DNA double-strand break (DSB) repair by homologous recombination (HR) but can inhibit normal repair by nonhomologous end joining (NHEJ), the main DSB repair pathway in G1-phase cells. Antigen receptor gene assembly proceeds through DNA DSB intermediates generated in G1-phase lymphocytes by the RAG endonuclease. These DSBs activate ATM, which phosphorylates H2AX, forming γ-H2AX in flanking chromatin. γ-H2AX prevents CtIP from initiating resection of RAG DSBs. Whether there are additional proteins required to promote resection of these DNA ends is not known. KRAB-associated protein 1 (KAP-1) (TRIM28) is a transcriptional repressor that modulates chromatin structure and has been implicated in the repair of DNA DSBs in heterochromatin. Here, we show that in murine G1-phase lymphocytes, KAP-1 promotes resection of DSBs that are not protected by H2AX and its downstream effector 53BP1. In these murine cells, KAP-1 activity in DNA end resection is attenuated by a single-amino-acid change that reflects a KAP-1 polymorphism between primates and other mammalian species. These findings establish KAP-1 as a component of the machinery that can resect DNA ends in G1-phase cells and suggest that there may be species-specific features to this activity.
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http://dx.doi.org/10.1128/MCB.00441-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135573PMC
August 2014

β-Catenin induces T-cell transformation by promoting genomic instability.

Proc Natl Acad Sci U S A 2014 Jan 26;111(1):391-6. Epub 2013 Dec 26.

Section of Rheumatology and Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL 60637.

Deregulated activation of β-catenin in cancer has been correlated with genomic instability. During thymocyte development, β-catenin activates transcription in partnership with T-cell-specific transcription factor 1 (Tcf-1). We previously reported that targeted activation of β-catenin in thymocytes (CAT mice) induces lymphomas that depend on recombination activating gene (RAG) and myelocytomatosis oncogene (Myc) activities. Here we show that these lymphomas have recurring Tcra/Myc translocations that resulted from illegitimate RAG recombination events and resembled oncogenic translocations previously described in human T-ALL. We therefore used the CAT animal model to obtain mechanistic insights into the transformation process. ChIP-seq analysis uncovered a link between Tcf-1 and RAG2 showing that the two proteins shared binding sites marked by trimethylated histone-3 lysine-4 (H3K4me3) throughout the genome, including near the translocation sites. Pretransformed CAT thymocytes had increased DNA damage at the translocating loci and showed altered repair of RAG-induced DNA double strand breaks. These cells were able to survive despite DNA damage because activated β-catenin promoted an antiapoptosis gene expression profile. Thus, activated β-catenin promotes genomic instability that leads to T-cell lymphomas as a consequence of altered double strand break repair and increased survival of thymocytes with damaged DNA.
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http://dx.doi.org/10.1073/pnas.1315752111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890837PMC
January 2014

Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination.

Mol Cell Biol 2013 Sep 8;33(18):3568-79. Epub 2013 Jul 8.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA-PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs(3A)) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpin-sealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease activity, and these functions can be regulated redundantly by the kinase activity of either ATM or DNA-PKcs.
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http://dx.doi.org/10.1128/MCB.00308-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753869PMC
September 2013

Recurrent hemizygous deletions in cancers may optimize proliferative potential.

Science 2012 Jul 24;337(6090):104-9. Epub 2012 May 24.

Department of Genetics, Harvard University Medical School, and Division of Genetics, Howard Hughes Medical Institute, Brigham and Women's Hospital, Boston, MA 02115, USA.

Tumors exhibit numerous recurrent hemizygous focal deletions that contain no known tumor suppressors and are poorly understood. To investigate whether these regions contribute to tumorigenesis, we searched genetically for genes with cancer-relevant properties within these hemizygous deletions. We identified STOP and GO genes, which negatively and positively regulate proliferation, respectively. STOP genes include many known tumor suppressors, whereas GO genes are enriched for essential genes. Analysis of their chromosomal distribution revealed that recurring deletions preferentially overrepresent STOP genes and underrepresent GO genes. We propose a hypothesis called the cancer gene island model, whereby gene islands encompassing high densities of STOP genes and low densities of GO genes are hemizygously deleted to maximize proliferative fitness through cumulative haploinsufficiencies. Because hundreds to thousands of genes are hemizygously deleted per tumor, this mechanism may help to drive tumorigenesis across many cancer types.
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http://dx.doi.org/10.1126/science.1219580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027969PMC
July 2012

Ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases have overlapping activities during chromosomal signal joint formation.

Proc Natl Acad Sci U S A 2011 Feb 18;108(5):2022-7. Epub 2011 Jan 18.

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA.

Lymphocyte antigen receptor gene assembly occurs through the process of V(D)J recombination, which is initiated when the RAG endonuclease introduces DNA DSBs at two recombining gene segments to form broken DNA coding end pairs and signal end pairs. These paired DNA ends are joined by proteins of the nonhomologous end-joining (NHEJ) pathway of DSB repair to form a coding joint and signal joint, respectively. RAG DSBs are generated in G1-phase developing lymphocytes, where they activate the ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases to orchestrate diverse cellular DNA damage responses including DSB repair. Paradoxically, although Atm and DNA-PKcs both function during coding joint formation, Atm appears to be dispensible for signal joint formation; and although some studies have revealed an activity for DNA-PKcs during signal joint formation, others have not. Here we show that Atm and DNA-PKcs have overlapping catalytic activities that are required for chromosomal signal joint formation and for preventing the aberrant resolution of signal ends as potentially oncogenic chromosomal translocations.
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http://dx.doi.org/10.1073/pnas.1013295108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033293PMC
February 2011

A genome-wide camptothecin sensitivity screen identifies a mammalian MMS22L-NFKBIL2 complex required for genomic stability.

Mol Cell 2010 Nov 4;40(4):645-57. Epub 2010 Nov 4.

Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

Replication stress involving collision of replisomes with camptothecin (CPT)-stabilized DNA-Topoisomerase I adducts activates an ATR-dependent pathway to promote repair by homologous recombination. To identify human genes that protect cells from such replication stress, we performed a genome-wide CPT sensitivity screen. Among numerous candidate genes are two previously unstudied proteins: the ankyrin repeat protein NFKBIL2 and C6ORF167 (MMS22L), distantly related to yeast replication stress regulator Mms22p. MMS22L and NFKBIL2 interact with each other and with FACT (facilitator of chromatin transcription) and MCM (minichromosome maintenance) complexes. Cells depleted of NFKBIL2 or MMS22L are sensitive to DNA-damaging agents, load phosphorylated RPA onto chromatin in a CTIP-dependent manner, activate the ATR/ATRIP-CHK1 and double-strand break repair signaling pathways, and are defective in HR. This study identifies MMS22L-NFKBIL2 as components of the replication stress control pathway and provides a resource for discovery of additional components of this pathway.
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http://dx.doi.org/10.1016/j.molcel.2010.10.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3006237PMC
November 2010

Regulation of hematopoietic stem cell differentiation by a single ubiquitin ligase-substrate complex.

Nat Immunol 2010 Mar 17;11(3):207-15. Epub 2010 Jan 17.

Howard Hughes Medical Institute and Department of Pathology, New York, New York, USA.

Hematopoietic stem cell (HSC) differentiation is regulated by cell-intrinsic and cell-extrinsic cues. In addition to transcriptional regulation, post-translational regulation may also control HSC differentiation. To test this hypothesis, we visualized the ubiquitin-regulated protein stability of a single transcription factor, c-Myc. The stability of c-Myc protein was indicative of HSC quiescence, and c-Myc protein abundance was controlled by the ubiquitin ligase Fbw7. Fine changes in the stability of c-Myc protein regulated the HSC gene-expression signature. Using whole-genome genomic approaches, we identified specific regulators of HSC function directly controlled by c-Myc binding; however, adult HSCs and embryonic stem cells sensed and interpreted c-Myc-regulated gene expression in distinct ways. Our studies show that a ubiquitin ligase-substrate pair can orchestrate the molecular program of HSC differentiation.
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http://dx.doi.org/10.1038/ni.1839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2825759PMC
March 2010

Histone H2AX stabilizes broken DNA strands to suppress chromosome breaks and translocations during V(D)J recombination.

J Exp Med 2009 Nov 2;206(12):2625-39. Epub 2009 Nov 2.

Cell and Molecular Biology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre-B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igkappa DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-alpha/delta locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs.
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http://dx.doi.org/10.1084/jem.20091320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2806628PMC
November 2009

Aberrantly resolved RAG-mediated DNA breaks in Atm-deficient lymphocytes target chromosomal breakpoints in cis.

Proc Natl Acad Sci U S A 2009 Oct 9;106(43):18339-44. Epub 2009 Oct 9.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Canonical chromosomal translocations juxtaposing antigen receptor genes and oncogenes are a hallmark of many lymphoid malignancies. These translocations frequently form through the joining of DNA ends from double-strand breaks (DSBs) generated by the recombinase activating gene (RAG)-1 and -2 proteins at lymphocyte antigen receptor loci and breakpoint targets near oncogenes. Our understanding of chromosomal breakpoint target selection comes primarily from the analyses of these lesions, which are selected based on their transforming properties. RAG DSBs are rarely resolved aberrantly in wild-type developing lymphocytes. However, in ataxia telangiectasia mutated (ATM)-deficient lymphocytes, RAG breaks are frequently joined aberrantly, forming chromosomal lesions such as translocations that predispose (ATM)-deficient mice and humans to the development of lymphoid malignancies. Here, an approach that minimizes selection biases is used to isolate a large cohort of breakpoint targets of aberrantly resolved RAG DSBs in Atm-deficient lymphocytes. Analyses of this cohort revealed that frequently, the breakpoint targets for aberrantly resolved RAG breaks are other DSBs. Moreover, these nonselected lesions exhibit a bias for using breakpoints in cis, forming small chromosomal deletions, rather than breakpoints in trans, forming chromosomal translocations.
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http://dx.doi.org/10.1073/pnas.0902545106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775277PMC
October 2009

The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart.

Genes Dev 2009 Oct 30;23(20):2415-25. Epub 2009 Sep 30.

Howard Hughes Medical Institute and Department of Genetics, Harvard Medical School, Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

The integrity of genomic DNA is continuously challenged by the presence of DNA base lesions or DNA strand breaks. Here we report the identification of a new DNA damage response protein, SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), which is a member of the SNF2 family and is mutated in Schimke immunoosseous dysplasia (SIOD). We demonstrate that SMARCAL1 directly interacts with Replication protein A (RPA) and is recruited to sites of DNA damage in an RPA-dependent manner. SMARCAL1-depleted cells display sensitivity to DNA-damaging agents that induce replication fork collapse, and exhibit slower fork recovery and delayed entry into mitosis following S-phase arrest. Furthermore, SIOD patient fibroblasts reconstituted with SMARCAL1 exhibit faster cell cycle progression after S-phase arrest. Thus, the symptoms of SIOD may be caused, at least in part, by defects in the cellular response to DNA replication stress.
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http://dx.doi.org/10.1101/gad.1832309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2764500PMC
October 2009

Formation of dynamic gamma-H2AX domains along broken DNA strands is distinctly regulated by ATM and MDC1 and dependent upon H2AX densities in chromatin.

Mol Cell 2009 May;34(3):298-310

Cell and Molecular Biology Graduate Program, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate gamma-H2AX. Here, we demonstrate that gamma-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which gamma-H2AX forms does not spread as DSBs persist; rather, gamma-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate gamma-H2AX, only ATM promotes gamma-H2AX formation to maximal distance and maintains gamma-H2AX densities. MDC1 is essential for gamma-H2AX formation at high densities near DSBs, but not for generation of gamma-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of gamma-H2AX formed. Our data suggest that H2AX fuels a gamma-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX.
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http://dx.doi.org/10.1016/j.molcel.2009.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2744111PMC
May 2009

Intrathymic proliferation wave essential for Valpha14+ natural killer T cell development depends on c-Myc.

Proc Natl Acad Sci U S A 2009 May 7;106(21):8641-6. Epub 2009 May 7.

Committee on Immunology, Department of Medicine, Rheumatology, University of Chicago, Chicago, IL 60637, USA.

The molecular requirements for invariant Valpha14-bearing natural killer T cells (iNKT) in the thymus are poorly understood. A minute population of approximately 500 newly selected CD69(+)CD24(+) stage 0 (ST0) iNKT cells gives rise to approximately 100 times more CD44(neg/lo)CD24(-) stage 1 (ST1) cells, which then generate similar frequencies of CD44(hi)CD24(-) stage 2 (ST2) and mature iNKT cells. Although the increased number of ST1 compared with ST0 cells indicates the initiation of a proliferation wave in the very early stages of iNKT cell development, details about the controlling mechanism are currently lacking. Here, we show that the transcription factor c-Myc is required for iNKT cell development. Conditional ablation of c-Myc in double-positive thymocytes specifically impacted iNKT but not conventional T cell development. Within the iNKT population, a progressive reduction of iNKT cells was observed starting at ST1 (approximately 50-fold) and ST2 (approximately 350-fold), with a complete lack of mature cells in thymus, spleen, and liver. ST0/ST1 c-Myc-deficient iNKT cells showed reduced proliferation. In contrast, annexin V staining did not reveal increased apoptosis, and transgenic overexpression of BCL-2 did not rescue iNKT cell development in c-Myc-deficient mice. Moreover, expression of known iNKT differentiation factors such as Plzf and Gata3 was not dramatically altered. These, findings provide compelling evidence that c-Myc mediates an intrathymic proliferation wave immediately after agonist selection of iNKT cells and illustrate the importance of this expansion for the generation of mature iNKT cells in vivo.
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http://dx.doi.org/10.1073/pnas.0812255106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2689024PMC
May 2009

MRN complex function in the repair of chromosomal Rag-mediated DNA double-strand breaks.

J Exp Med 2009 Mar 16;206(3):669-79. Epub 2009 Feb 16.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

The Mre11-Rad50-Nbs1 (MRN) complex functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) at postreplicative stages of the cell cycle. During HR, the MRN complex functions directly in the repair of DNA DSBs and in the initiation of DSB responses through activation of the ataxia telangiectasia-mutated (ATM) serine-threonine kinase. Whether MRN functions in DNA damage responses before DNA replication in G0/G1 phase cells has been less clear. In developing G1-phase lymphocytes, DNA DSBs are generated by the Rag endonuclease and repaired during the assembly of antigen receptor genes by the process of V(D)J recombination. Mice and humans deficient in MRN function exhibit lymphoid phenotypes that are suggestive of defects in V(D)J recombination. We show that during V(D)J recombination, MRN deficiency leads to the aberrant joining of Rag DSBs and to the accumulation of unrepaired coding ends, thus establishing a functional role for MRN in the repair of Rag-mediated DNA DSBs. Moreover, these defects in V(D)J recombination are remarkably similar to those observed in ATM-deficient lymphocytes, suggesting that ATM and MRN function in the same DNA DSB response pathways during lymphocyte antigen receptor gene assembly.
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http://dx.doi.org/10.1084/jem.20081326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699138PMC
March 2009

DNA double-strand breaks activate a multi-functional genetic program in developing lymphocytes.

Nature 2008 Dec 12;456(7223):819-23. Epub 2008 Oct 12.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

DNA double-strand breaks are generated by genotoxic agents and by cellular endonucleases as intermediates of several important physiological processes. The cellular response to genotoxic DNA breaks includes the activation of transcriptional programs known primarily to regulate cell-cycle checkpoints and cell survival. DNA double-strand breaks are generated in all developing lymphocytes during the assembly of antigen receptor genes, a process that is essential for normal lymphocyte development. Here we show that in murine lymphocytes these physiological DNA breaks activate a broad transcriptional program. This program transcends the canonical DNA double-strand break response and includes many genes that regulate diverse cellular processes important for lymphocyte development. Moreover, the expression of several of these genes is regulated similarly in response to genotoxic DNA damage. Thus, physiological DNA double-strand breaks provide cues that can regulate cell-type-specific processes not directly involved in maintaining the integrity of the genome, and genotoxic DNA breaks could disrupt normal cellular functions by corrupting these processes.
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http://dx.doi.org/10.1038/nature07392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2605662PMC
December 2008

Aberrant V(D)J recombination in ataxia telangiectasia mutated-deficient lymphocytes is dependent on nonhomologous DNA end joining.

J Immunol 2008 Aug;181(4):2620-5

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

During lymphocyte Ag receptor gene assembly, DNA cleavage by the Rag proteins generates pairs of coding and signal ends that are normally joined into coding joints and signal joints, respectively, by the classical nonhomologous end-joining (NHEJ) pathway of DNA double strand break repair. Coding and signal ends can also be aberrantly joined to each other, generating hybrid joints, through NHEJ or through NHEJ-independent pathways, such as Rag-mediated transposition. Hybrid joints do not participate in the formation of functional Ag receptor genes and can alter the configuration of Ag receptor loci in ways that limit subsequent productive rearrangements. The formation of these nonfunctional hybrid joints occurs rarely in wild type lymphocytes, demonstrating that mechanisms exist to limit both the NHEJ-dependent and the NHEJ-independent joining of a signal end to a coding end. In contrast to wild-type cells, hybrid joint formation occurs at high levels in ataxia telangiectasia mutated (Atm)-deficient lymphocytes, suggesting that Atm functions to limit the formation of these aberrant joints. In this study, we show that hybrid joint formation in Atm-deficient cells requires the NHEJ proteins Artemis, DNA-PKcs, and Ku70, demonstrating that Atm functions primarily by modulating the NHEJ-dependent, and not the NHEJ-independent, joining of coding ends to signal ends.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3598579PMC
http://dx.doi.org/10.4049/jimmunol.181.4.2620DOI Listing
August 2008

Dynamic regulation of c-Myc proto-oncogene expression during lymphocyte development revealed by a GFP-c-Myc knock-in mouse.

Eur J Immunol 2008 Feb;38(2):342-9

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA.

c-Myc induces widely varying cellular effects, including cell proliferation and cell death. These different cellular effects are determined, in part, by c-Myc protein expression levels, which are regulated through several transcriptional and post-transcriptional pathways. c-Myc transcripts can be detected in cells at all stages of B and T lymphocyte development. However, little is known about c-Myc protein expression, and how it varies, in developing lymphocytes. Here mice have been generated in which the endogenous c-Myc locus has been modified (c-Myc(G)) so that it encodes a GFP-c-Myc fusion protein. c-Myc(G/G) mice are viable, appear normal and exhibit grossly normal lymphocyte development. Flow cytometric analyses revealed significant heterogeneity in c-Myc protein expression levels in developing c-Myc(G/G) B and T lymphocytes. GFP-c-Myc expression levels were highest in proliferating lymphocytes, suggesting that c-Myc up-regulation is important for promoting lymphocyte cell division, and demonstrating that GFP-c-Myc expression is a marker of proliferating lymphocytes in vivo.
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http://dx.doi.org/10.1002/eji.200737972DOI Listing
February 2008

SWI-SNF: promoter of accessibility.

Nat Immunol 2007 Aug;8(8):795-6

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http://dx.doi.org/10.1038/ni0807-795DOI Listing
August 2007