Research Fellow, Boston Children's Hospital, Program in Cellular and Molecular Medicine
PhD, University of Washington, Biochemistry (2009)
BA, Vassar College, Biochemistry (2001)
Frederick W Alt, Richard L Frock, Jiazhi Hu, Robin M Meyers. " Patent WO2016081798 Methods relating to the detection of recurrent and non-specific double strand breaks in the genome", Nov 20, 2015
Current Research and Scholarly Interests
Mechanisms of DNA double-strand break repair and chromosomal translocations
Parp3 promotes long-range end joining in murine cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2018; 115 (40): 10076–81
Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class-switch recombination in primary B cells, and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.
View details for DOI 10.1073/pnas.1801591115
View details for Web of Science ID 000446078700075
View details for PubMedID 30213852
View details for PubMedCentralID PMC6176633
Mechanism of tandem duplication formation in BRCA1-mutant cells.
2017; 551 (7682): 590–95
Small, approximately 10-kilobase microhomology-mediated tandem duplications are abundant in the genomes of BRCA1-linked but not BRCA2-linked breast cancer. Here we define the mechanism underlying this rearrangement signature. We show that, in primary mammalian cells, BRCA1, but not BRCA2, suppresses the formation of tandem duplications at a site-specific chromosomal replication fork barrier imposed by the binding of Tus proteins to an array of Ter sites. BRCA1 has no equivalent role at chromosomal double-stranded DNA breaks, indicating that tandem duplications form specifically at stalled forks. Tandem duplications in BRCA1 mutant cells arise by a replication restart-bypass mechanism terminated by end joining or by microhomology-mediated template switching, the latter forming complex tandem duplication breakpoints. Solitary DNA ends form directly at Tus-Ter, implicating misrepair of these lesions in tandem duplication formation. Furthermore, BRCA1 inactivation is strongly associated with ~10 kilobase tandem duplications in ovarian cancer. This tandem duplicator phenotype may be a general signature of BRCA1-deficient cancer.
View details for PubMedID 29168504
Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR-Cas9 genome editing.
Nature biomedical engineering
2017; 1 (11): 878–88
Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end-joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumour protein p53-binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.
View details for PubMedID 31015609
PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (38): 10619-10624
Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation. The XRCC4-like-factor (XLF) C-NHEJ protein is dispensable for V(D)J recombination in normal cells, but because of functional redundancy, it is absolutely required for this process in cells deficient for the ataxia telangiectasia-mutated (ATM) DSB response factor. The recently identified paralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to ionizing radiation-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B-cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B-cell line, and dispensable for normal ionizing radiation resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to ionizing radiation exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested pro-B-cell lines. Finally, we also find that PAXX deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ.
View details for DOI 10.1073/pnas.1611882113
View details for Web of Science ID 000383622600051
View details for PubMedID 27601633
View details for PubMedCentralID PMC5035843
Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development.
journal of experimental medicine
2016; 213 (9): 1921-1936
T cell antigen receptor δ (Tcrd) variable region exons are assembled by RAG-initiated V(D)J recombination events in developing γδ thymocytes. Here, we use linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) to map hundreds of thousands of RAG-initiated Tcrd D segment (Trdd1 and Trdd2) rearrangements in CD4(-)CD8(-) double-negative thymocyte progenitors differentiated in vitro from bone marrow-derived hematopoietic stem cells. We find that Trdd2 joins directly to Trdv, Trdd1, and Trdj segments, whereas Trdd1 joining is ordered with joining to Trdd2, a prerequisite for further rearrangement. We also find frequent, previously unappreciated, Trdd1 and Trdd2 rearrangements that inactivate Tcrd, including sequential rearrangements from V(D)J recombination signal sequence fusions. Moreover, we find dozens of RAG off-target sequences that are generated via RAG tracking both upstream and downstream from the Trdd2 recombination center across the Tcrd loop domain that is bounded by the upstream INT1-2 and downstream TEA elements. Disruption of the upstream INT1-2 boundary of this loop domain allows spreading of RAG on- and off-target activity to the proximal Trdv domain and, correspondingly, shifts the Tcrd V(D)J recombination landscape by leading to predominant V(D)J joining to a proximal Trdv3 pseudogene that lies just upstream of the normal boundary.
View details for DOI 10.1084/jem.20160670
View details for PubMedID 27526713
View details for PubMedCentralID PMC4995090
Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing
2016; 11 (5): 853-871
Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of the mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as those to evaluate the on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for the detection of genome-wide 'prey' DSBs via their translocation in cultured mammalian cells to a fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide-level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis is necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable and straightforward to implement with a turnaround time of <1 week.
View details for DOI 10.1038/nprot.2016.043
View details for Web of Science ID 000374445900002
View details for PubMedID 27031497
View details for PubMedCentralID PMC4895203
Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes
2015; 163 (4): 947-959
RAG initiates antibody V(D)J recombination in developing lymphocytes by generating "on-target" DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically inserted RSS pairs to identify huge numbers of RAG "off-target" breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them.
View details for DOI 10.1016/j.cell.2015.10.016
View details for Web of Science ID 000364829700019
View details for PubMedID 26593423
View details for PubMedCentralID PMC4660266
Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
2015; 33 (2): 179-186
Although great progress has been made in the characterization of the off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification-mediated modification of a previously published high-throughput, genome-wide, translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases more than tenfold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described previously. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.
View details for DOI 10.1038/nbt.3101
View details for Web of Science ID 000349198800024
View details for PubMedID 25503383
View details for PubMedCentralID PMC4320661
Mechanisms of Recurrent Chromosomal Translocations
Chromosomal Translocations and Genome Rearrangements in Cancer
Springer International Publishing. 2015; 1: 27–51
View details for DOI 10.1007/978-3-319-19983-2_3
Cardiomyocyte-Specific Expression of Lamin A Improves Cardiac Function in Lmna(-/-) Mice
2012; 7 (8)
Lmna(-/-) mice display multiple tissue defects and die by 6-8 weeks of age reportedly from dilated cardiomyopathy with associated conduction defects. We sought to determine whether restoration of lamin A in cardiomyocytes improves cardiac function and extends the survival of Lmna(-/-) mice. We observed increased total desmin protein levels and disorganization of the cytoplasmic desmin network in ~20% of Lmna(-/-) ventricular myocytes, rescued in a cell-autonomous manner in Lmna(-/-) mice expressing a cardiac-specific lamin A transgene (Lmna(-/-); Tg). Lmna(-/-); Tg mice displayed significantly increased contractility and preservation of myocardial performance compared to Lmna(-/-) mice. Lmna(-/-); Tg mice attenuated ERK1/2 phosphorylation relative to Lmna(-/-) mice, potentially underlying the improved localization of connexin43 to the intercalated disc. Electrocardiographic recordings from Lmna(-/-) mice revealed arrhythmic events and increased frequency of PR interval prolongation, which is partially rescued in Lmna(-/-); Tg mice. These findings support our observation that Lmna(-/-); Tg mice have a 12% median extension in lifespan compared to Lmna(-/-) mice. While significant, Lmna(-/-); Tg mice only have modest improvement in cardiac function and survival likely stemming from the observation that only 40% of Lmna(-/-); Tg cardiomyocytes have detectable lamin A expression. Cardiomyocyte-specific restoration of lamin A in Lmna(-/-) mice improves heart-specific pathology and extends lifespan, demonstrating that the cardiac pathology of Lmna(-/-) mice limits survival. The expression of lamin A is sufficient to rescue certain cellular defects associated with loss of A-type lamins in cardiomyocytes in a cell-autonomous fashion.
View details for DOI 10.1371/journal.pone.0042918
View details for Web of Science ID 000307823600034
View details for PubMedID 22905185
View details for PubMedCentralID PMC3419749
Genome-wide Translocation Sequencing Reveals Mechanisms of Chromosome Breaks and Rearrangements in B Cells
2011; 147 (1): 107-119
Whereas chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high-throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double-strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for activation-induced cytidine deaminase (AID)-dependent IgH class switching. DSBs translocated widely across the genome but were preferentially targeted to transcribed chromosomal regions. Additionally, numerous AID-dependent and AID-independent hot spots were targeted, with the latter comprising mainly cryptic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short microhomologies. Our findings have implications for diverse fields, including gene therapy and cancer genomics.
View details for DOI 10.1016/j.cell.2011.07.049
View details for Web of Science ID 000295396700019
View details for PubMedID 21962511
View details for PubMedCentralID PMC3186939
Cell-Extrinsic Defective Lymphocyte Development in Lmna(-/-) Mice
2010; 5 (4)
Mutations in the LMNA gene, which encodes all A-type lamins, result in a variety of human diseases termed laminopathies. Lmna(-/-) mice appear normal at birth but become runted as early as 2 weeks of age and develop multiple tissue defects that mimic some aspects of human laminopathies. Lmna(-/-) mice also display smaller spleens and thymuses. In this study, we investigated whether altered lymphoid organ sizes are correlated with specific defects in lymphocyte development.Lmna(-/-) mice displayed severe age-dependent defects in T and B cell development which coincided with runting. Lmna(-/-) bone marrow reconstituted normal T and B cell development in irradiated wild-type recipients, driving generation of functional and self-MHC restricted CD4(+) and CD8(+) T cells. Transplantation of Lmna(-/-) neonatal thymus lobes into syngeneic wild-type recipients resulted in good engraftment of thymic tissue and normal thymocyte development.Collectively, these data demonstrate that the severe defects in lymphocyte development that characterize Lmna(-/-) mice do not result directly from the loss of A-type lamin function in lymphocytes or thymic stroma. Instead, the immune defects in Lmna(-/-) mice likely reflect indirect damage, perhaps resulting from prolonged stress due to the striated muscle dystrophies that occur in these mice.
View details for DOI 10.1371/journal.pone.0010127
View details for Web of Science ID 000276705900011
View details for PubMedID 20405040
View details for PubMedCentralID PMC2853576
Lamin A/C and emerin are critical for skeletal muscle satellite cell differentiation
GENES & DEVELOPMENT
2006; 20 (4): 486-500
Mutations within LMNA, encoding A-type nuclear lamins, are associated with multiple tissue-specific diseases, including Emery-Dreifuss (EDMD2/3) and Limb-Girdle muscular dystrophy (LGMD1B). X-linked EDMD results from mutations in emerin, a lamin A-associated protein. The mechanisms through which these mutations cause muscular dystrophy are not understood. Here we show that most, but not all, cultured muscle cells from lamin A/C knockout mice exhibit impaired differentiation kinetics and reduced differentiation potential. Similarly, normal muscle cells that have been RNA interference (RNAi) down-regulated for either A-type lamins or emerin have impaired differentiation potentials. Replicative myoblasts lacking A-type lamins or emerin also have decreased levels of proteins important for muscle differentiation including pRB, MyoD, desmin, and M-cadherin; up-regulated Myf5; but no changes in Pax3, Pax7, MEF2C, MEF2D, c-met, and beta-catenin. To determine whether impaired myogenesis is linked to reduced MyoD or desmin levels, these proteins were individually expressed in Lmna(-/-) myoblasts that were then induced to undergo myogenesis. Expression of either MyoD or, more surprisingly, desmin in Lmna(-/-) myoblasts resulted in increased differentiation potential. These studies indicate roles for A-type lamins and emerin in myogenic differentiation and also suggest that these effects are at least in part due to decreased endogenous levels of other critical myoblast proteins. The delayed differentiation kinetics and decreased differentiation potential of lamin A/C-deficient and emerin-deficient myoblasts may in part underlie the dystrophic phenotypes observed in patients with EDMD.
View details for DOI 10.1101/gad.1364906
View details for Web of Science ID 000235428600011
View details for PubMedID 16481476
View details for PubMedCentralID PMC1369050
A-type nuclear lamins, progerias and other degenerative disorders
MECHANISMS OF AGEING AND DEVELOPMENT
2005; 126 (4): 447-460
Nuclear lamins were identified as core nuclear matrix constituents over 20 years ago. They have been ascribed structural roles such as maintaining nuclear integrity and assisting in nuclear envelope formation after mitosis, and have also been linked to nuclear activities including DNA replication and transcription. Recently, A-type lamin mutations have been linked to a variety of rare human diseases including muscular dystrophy, lipodystrophy, cardiomyopathy, neuropathy and progeroid syndromes (collectively termed laminopathies). Most diseases arise from dominant, missense mutations, leading to speculation as to how different mutations in the same gene can give rise to such a diverse set of diseases, some of which share little phenotypic overlap. Understanding the cellular dysfunctions that lead to laminopathies will almost certainly provide insight into specific roles of A-type lamins in nuclear organization. Here, we compare and contrast the LMNA mutations leading to laminopathies with emphasis on progerias, and discuss possible functional roles for A-type lamins in the maintenance of healthy tissues.
View details for DOI 10.1016/j.mad.2004.10.006
View details for Web of Science ID 000227605900001
View details for PubMedID 15722103
A-type lamins regulate retinoblastoma protein function by promoting subnuclear localization and preventing proteasomal degradation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2004; 101 (26): 9677-9682
The retinoblastoma protein (pRB) is a critical regulator of cell proliferation and differentiation and an important tumor suppressor. In the G(1) phase of the cell cycle, pRB localizes to perinucleolar sites associated with lamin A/C intranuclear foci. Here, we examine pRB function in cells lacking lamin A/C, finding that pRB levels are dramatically decreased and that the remaining pRB is mislocalized. We demonstrate that A-type lamins protect pRB from proteasomal degradation. Both pRB levels and localization are restored upon reintroduction of lamin A. Lmna(-/-) cells resemble Rb(-/-) cells, exhibiting altered cell-cycle properties and reduced capacity to undergo cell-cycle arrest in response to DNA damage. These findings establish a functional link between a core nuclear structural component and an important cell-cycle regulator. They further raise the possibility that altered pRB function may be a contributing factor in dystrophic syndromes arising from LMNA mutation.
View details for DOI 10.1073/pnas.0403250101
View details for Web of Science ID 000222405600030
View details for PubMedID 15210943
View details for PubMedCentralID PMC470734
Nuclear reorganization of mammalian DNA synthesis prior to cell cycle exit
MOLECULAR AND CELLULAR BIOLOGY
2004; 24 (2): 595-607
In primary mammalian cells, DNA replication initiates in a small number of perinucleolar, lamin A/C-associated foci. During S-phase progression in proliferating cells, replication foci distribute to hundreds of sites throughout the nucleus. In contrast, we find that the limited perinucleolar replication sites persist throughout S phase as cells prepare to exit the cell cycle in response to contact inhibition, serum starvation, or replicative senescence. Proteins known to be involved in DNA synthesis, such as PCNA and DNA polymerase delta, are concentrated in perinucleolar foci throughout S phase under these conditions. Moreover, chromosomal loci are redirected toward the nucleolus and overlap with the perinucleolar replication foci in cells poised to undergo cell cycle exit. These same loci remain in the periphery of the nucleus during replication under highly proliferative conditions. These results suggest that mammalian cells undergo a large-scale reorganization of chromatin during the rounds of DNA replication that precede cell cycle exit.
View details for DOI 10.1128/MCB.24.2.595-607.2004
View details for Web of Science ID 000188211200010
View details for PubMedID 14701733
View details for PubMedCentralID PMC343811
Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila
2003; 130 (1): 173-184
The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power of Drosophila molecular genetics.
View details for DOI 10.1242/dev.00199
View details for Web of Science ID 000180537900016
View details for PubMedID 12441301