Publications by authors named "Jiazhi Hu"

22 Publications

  • Page 1 of 1

Transcription shapes DNA replication initiation to preserve genome integrity.

Genome Biol 2021 Jun 9;22(1):176. Epub 2021 Jun 9.

The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Genome Editing Research Center, Peking University, Beijing, 100871, China.

Background: Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage.

Results: We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage.

Conclusion: RNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.
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http://dx.doi.org/10.1186/s13059-021-02390-3DOI Listing
June 2021

FACT interacts with Set3 HDAC and fine-tunes GAL1 transcription in response to environmental stimulation.

Nucleic Acids Res 2021 Jun;49(10):5502-5519

State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.

The histone chaperone facilitates chromatin transactions (FACT) functions in various DNA transactions. How FACT performs these multiple functions remains largely unknown. Here, we found, for the first time, that the N-terminal domain of its Spt16 subunit interacts with the Set3 histone deacetylase complex (Set3C) and that FACT and Set3C function in the same pathway to regulate gene expression in some settings. We observed that Spt16-G132D mutant proteins show defects in binding to Set3C but not other reported FACT interactors. At the permissive temperature, induction of the GAL1 and GAL10 genes is reduced in both spt16-G132D and set3Δ cells, whereas transient upregulation of GAL10 noncoding RNA (ncRNA), which is transcribed from the 3' end of the GAL10 gene, is elevated. Mutations that inhibit GAL10 ncRNA transcription reverse the GAL1 and GAL10 induction defects in spt16-G132D and set3Δ mutant cells. Mechanistically, set3Δ and FACT (spt16-G132D) mutants show reduced histone acetylation and increased nucleosome occupancy at the GAL1 promoter under inducing conditions and inhibition of GAL10 ncRNA transcription also partially reverses these chromatin changes. These results indicate that FACT interacts with Set3C, which in turn prevents uncontrolled GAL10 ncRNA expression and fine-tunes the expression of GAL genes upon a change in carbon source.
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http://dx.doi.org/10.1093/nar/gkab312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191775PMC
June 2021

Structural insight into the assembly and conformational activation of human origin recognition complex.

Cell Discov 2020 Nov 24;6(1):88. Epub 2020 Nov 24.

State Key Laboratory of Membrane Biology, Peking-Tsinghua Joint Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China.

The function of the origin recognition complex (ORC) in DNA replication is highly conserved in recognizing and marking the initiation sites. The detailed molecular mechanisms by which human ORC is reconfigured into a state competent for origin association remain largely unknown. Here, we present structural characterizations of human ORC1-5 and ORC2-5 assemblies. ORC2-5 exhibits a tightly autoinhibited conformation with the winged-helix domain of ORC2 completely blocking the central DNA-binding channel. The binding of ORC1 partially relieves the autoinhibitory effect of ORC2-5 through remodeling ORC2-WHD, which makes ORC2-WHD away from the central channel creating a still autoinhibited but more dynamic structure. In particular, the AAA+ domain of ORC1 is highly flexible to sample a variety of conformations from inactive to potentially active states. These results provide insights into the detailed mechanisms regulating the autoinhibition of human ORC and its subsequent activation for DNA binding.
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http://dx.doi.org/10.1038/s41421-020-00232-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7684300PMC
November 2020

REV7 is required for processing AID initiated DNA lesions in activated B cells.

Nat Commun 2020 06 4;11(1):2812. Epub 2020 Jun 4.

State Key Laboratory of Molecular Biology, Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, 200031, China.

Activation-induced cytidine deaminase (AID) initiates both antibody class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. DNA double-strand break response (DSBR) factors promote rearrangement in CSR, while translesion synthesis (TLS) polymerases generate mutations in SHM. REV7, a component of TLS polymerase zeta, is also a downstream effector of 53BP1-RIF1 DSBR pathway. Here, we study the multi-functions of REV7 and find that REV7 is required for the B cell survival upon AID-deamination, which is independent of its roles in DSBR, G2/M transition or REV1-mediated TLS. The cell death in REV7-deficient activated B cells can be fully rescued by AID-deficiency in vivo. We further identify that REV7-depedent TLS across UNG-processed apurinic/apyrimidinic sites is required for cell survival upon AID/APOBEC deamination. This study dissects the multiple roles of Rev7 in antibody diversification, and discovers that TLS is not only required for sequence diversification but also B cell survival upon AID-initiated lesions.
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http://dx.doi.org/10.1038/s41467-020-16632-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272641PMC
June 2020

ERCC6L2 promotes DNA orientation-specific recombination in mammalian cells.

Cell Res 2020 09 30;30(9):732-744. Epub 2020 Apr 30.

State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China.

Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role with the XLF end-joining factor in V(D)J recombination. Strikingly, ERCC6L2 controls orientation-specific joining of broken ends during CSR, which relies on its helicase activity. Thus, ERCC6L2 facilitates programmed recombination through directional repair of distant breaks.
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http://dx.doi.org/10.1038/s41422-020-0328-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608219PMC
September 2020

The Deubiquitinase USP38 Promotes NHEJ Repair through Regulation of HDAC1 Activity and Regulates Cancer Cell Response to Genotoxic Insults.

Cancer Res 2020 02 24;80(4):719-731. Epub 2019 Dec 24.

State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.

The DNA damage response (DDR) is essential for maintaining genome integrity. Mounting evidence reveals that protein modifications play vital roles in the DDR. Here, we show that USP38 is involved in the DDR by regulating the activity of HDAC1. In response to DNA damage, USP38 interacted with HDAC1 and specifically removed the K63-linked ubiquitin chain promoting the deacetylase activity of HDAC1. As a result, HDAC1 was able to deacetylate H3K56. USP38 deletion resulted in persistent focal accumulation of nonhomologous end joining (NHEJ) factors at DNA damage sites and impaired NHEJ efficiency, causing genome instability and sensitizing cancer cells to genotoxic insults. Knockout of USP38 rendered mice hypersensitive to irradiation and shortened survival. In addition, USP38 was expressed at low levels in certain types of cancers including renal cell carcinoma, indicating dysregulation of USP38 expression contributes to genomic instability and may lead to tumorigenesis. In summary, this study identifies a critical role of USP38 in modulating genome integrity and cancer cell resistance to genotoxic insults by deubiquitinating HDAC1 and regulating its deacetylation activity. SIGNIFICANCE: This study demonstrates that USP38 regulates genome stability and mediates cancer cell resistance to DNA-damaging therapy, providing insight into tumorigenesis and implicating USP38 as a potential target for cancer diagnosis.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-2149DOI Listing
February 2020

Improved HTGTS for CRISPR/Cas9 off-target detection.

Bio Protoc 2019 May 5;9(9):e3229. Epub 2019 May 5.

The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking University, Beijing, 100871, China.

Precise genome editing is essential for scientific research and clinical application. At present, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is one of most effective methods to evaluate the off-target activity of CRISPR-Cas9, which is based on chromosomal translocation and employs a "bait" DNA double-stranded break (DSB) to capture genome-wide "prey" DNA DSBs. Here, we described an improved HTGTS (iHTGTS) method, in which size-selection beads were used to enhance reaction efficiency and a new primer system was designed to be compatible with Illumina Hiseq sequencing. Compared with LAM-HTGTS, iHTGTS is lower cost and has much higher sensitivity for off-target detection in HEK293T, K562, U2OS and HCT116 cell lines. So we believe that iHTGTS is a powerful method for comprehensively assessing Cas9 off-target effect.
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http://dx.doi.org/10.21769/BioProtoc.3229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854062PMC
May 2019

Optimizing genome editing strategy by primer-extension-mediated sequencing.

Cell Discov 2019 26;5:18. Epub 2019 Mar 26.

1The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking University, Beijing, 100871 China.

Efficient and precise genome editing is essential for clinical applications and generating animal models, which requires engineered nucleases with high editing ability while low off-target activity. Here we present a high-throughput sequencing method, primer-extension-mediated sequencing (PEM-seq), to comprehensively assess both editing ability and specificity of engineered nucleases. We showed CRISPR/Cas9-generated breaks could lead to chromosomal translocations and large deletions by PEM-seq. We also found that Cas9 nickase possessed lower off-target activity while with some loss of target cleavage ability. However, high-fidelity Cas9 variants, including both eCas9 and the new FeCas9, could significantly reduce the Cas9 off-target activity with no obvious editing retardation. Moreover, we found AcrIIA4 inhibitor could greatly reduce the activities of Cas9, but off-target loci were not so effectively suppressed as the on-target sites. Therefore, PEM-seq fully evaluating engineered nucleases could help choose better genome editing strategy at given loci than other methods detecting only off-target activity.
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http://dx.doi.org/10.1038/s41421-019-0088-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434046PMC
March 2019

Improving Ionic Conductivity with Bimodal-Sized LiLaZrO Fillers for Composite Polymer Electrolytes.

ACS Appl Mater Interfaces 2019 Apr 20;11(13):12467-12475. Epub 2019 Mar 20.

Department of Chemical and Materials Engineering , University of Kentucky , Lexington 40506 , United States.

Ceramic-polymer composite electrolytes (CPEs) are being explored to achieve both high ionic conductivity and mechanical flexibility. Here, we show that, by incorporating 10 wt % (3 vol %) mixed-sized fillers of LiLaZrO (LLZO) doped with Nb/Al, the room-temperature ionic conductivity of a polyvinylidene fluoride (PVDF)-LiClO-based composite can be as high as 2.6 × 10 S/cm, which is 1 order of magnitude higher than that with nano- or micrometer-sized LLZO particles as fillers. The CPE also shows a high lithium-ion transference number of 0.682, a stable and low Li/CPE interfacial resistance, and good mechanical properties favorable for all-solid-state lithium-ion battery applications. X-ray photoelectron spectroscopy and Raman analysis demonstrate that the LLZO fillers of all sizes interact with PVDF and LiClO. High packing density (i.e., lower porosity) and long conducting pathways are believed responsible for the excellent performance of the composite electrolyte filled with mixed-sized ionically conducting ceramic particles.
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http://dx.doi.org/10.1021/acsami.8b21770DOI Listing
April 2019

CRISPR/Cas9-mediated targeted chromosome elimination.

Genome Biol 2017 11 24;18(1):224. Epub 2017 Nov 24.

Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.

Background: The CRISPR/Cas9 system has become an efficient gene editing method for generating cells carrying precise gene mutations, including the rearrangement and deletion of chromosomal segments. However, whether an entire chromosome could be eliminated by this technology is still unknown.

Results: Here we demonstrate the use of the CRISPR/Cas9 system to eliminate targeted chromosomes. Using either multiple cleavages induced by a single-guide RNA (sgRNA) that targets multiple chromosome-specific sites or a cocktail of multiple sgRNAs, each targeting one specific site, we found that a sex chromosome could be selectively eliminated in cultured cells, embryos, and tissues in vivo. Furthermore, this approach was able to produce a targeted autosome loss in aneuploid mouse embryonic stem cells with an extra human chromosome and human induced pluripotent stem cells with trisomy 21, as well as cancer cells.

Conclusions: CRISPR/Cas9-mediated targeted chromosome elimination offers a new approach to develop animal models with chromosome deletions, and a potential therapeutic strategy for human aneuploidy diseases involving additional chromosomes.
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http://dx.doi.org/10.1186/s13059-017-1354-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701507PMC
November 2017

Sap1 is a replication-initiation factor essential for the assembly of pre-replicative complex in the fission yeast .

J Biol Chem 2017 04 21;292(15):6056-6075. Epub 2017 Feb 21.

From the Peking-Tsinghua Center for Life Sciences, National Laboratory of Protein and Plant Gene Research, College of Life Sciences,

A central step in the initiation of chromosomal DNA replication in eukaryotes is the assembly of pre-replicative complex (pre-RC) at late M and early G phase of the cell cycles. Since 1973, four proteins or protein complexes, including cell division control protein 6 (Cdc6)/Cdc18, minichromosome maintenance protein complex, origin recognition complex (ORC), and Cdt1, are known components of the pre-RC. Previously, we reported that a non-ORC protein binds to the essential element Δ9 of the DNA-replication origin ARS3001. In this study, we identified that the non-ORC protein is Sap1. Like ORC, Sap1 binds to DNA origins during cell growth cycles. But unlike ORC, which binds to asymmetric AT-rich sequences through its nine AT-hook motifs, Sap1 preferentially binds to a DNA sequence of 5'-(A/T) (C/G)(A/T)(G/C)(A/T) -3' ( ≥ 1). We also found that Sap1 and ORC physically interact. We further demonstrated that Sap1 is required for the assembly of the pre-RC because of its essential role in recruiting Cdc18 to DNA origins. Thus, we conclude that Sap1 is a replication-initiation factor that directly participates in the assembly of the pre-RC. DNA-replication origins in fission yeast are defined by possessing two essential elements with one bound by ORC and the other by Sap1.
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http://dx.doi.org/10.1074/jbc.M116.767806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391739PMC
April 2017

Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells.

J Biol Chem 2017 03 3;292(12):4777-4788. Epub 2017 Feb 3.

From the Peking-Tsinghua Center for Life Sciences, The National Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China

During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast (). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing The mean and median lengths of the flaps in wild-type cells were ∼51 and ∼41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided direct evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish a new strategy for interrogation of this fascinating process.
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http://dx.doi.org/10.1074/jbc.M116.758599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377794PMC
March 2017

Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development.

J Exp Med 2016 08 15;213(9):1921-36. Epub 2016 Aug 15.

Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine Children's Hospital Boston, Department of Genetics, Harvard Medical School, Boston, MA 02115

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.
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http://dx.doi.org/10.1084/jem.20160670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4995090PMC
August 2016

Highly sensitive and unbiased approach for elucidating antibody repertoires.

Proc Natl Acad Sci U S A 2016 07 27;113(28):7846-51. Epub 2016 Jun 27.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115; Department of Genetics, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes.
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http://dx.doi.org/10.1073/pnas.1608649113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948367PMC
July 2016

Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing.

Nat Protoc 2016 May 31;11(5):853-71. Epub 2016 Mar 31.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

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.
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http://dx.doi.org/10.1038/nprot.2016.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4895203PMC
May 2016

Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes.

Cell 2015 Nov 22;163(4):947-59. Epub 2015 Oct 22.

Howard Hughes Medical Institute; Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2015.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4660266PMC
November 2015

Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching.

Nature 2015 Sep 26;525(7567):134-139. Epub 2015 Aug 26.

Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

During B-cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200 kilobases downstream in the same transcriptional orientation as V(D)J and Cμ exons. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH (ref. 2). Activation-induced cytidine deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region; these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S-region DSB. However, the relative frequency of deletional to inversional CSR junctions has not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved is unknown. To address this question, we adapt high-throughput genome-wide translocation sequencing into a highly sensitive DSB end-joining assay and apply it to endogenous AID-initiated S-region DSBs in mouse B cells. We show that CSR is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis Igh organizational features in combination with frequent S-region DSBs initiated by AID. We further implicate ATM-dependent DSB-response factors in enforcing this mechanism and provide an explanation of why CSR is so reliant on the 53BP1 DSB-response factor.
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http://dx.doi.org/10.1038/nature14970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4592165PMC
September 2015

Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases.

Nat Biotechnol 2015 Feb 15;33(2):179-86. Epub 2014 Dec 15.

1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. [3] Howard Hughes Medical Institute, Boston, Massachusetts, USA.

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.
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http://dx.doi.org/10.1038/nbt.3101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320661PMC
February 2015

Convergent transcription at intragenic super-enhancers targets AID-initiated genomic instability.

Cell 2014 Dec 4;159(7):1538-48. Epub 2014 Dec 4.

Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single-stranded DNA targets. Though largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting "convergent" transcription arises from antisense transcription that emanates from super-enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells.
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http://dx.doi.org/10.1016/j.cell.2014.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4322776PMC
December 2014

Developmental propagation of V(D)J recombination-associated DNA breaks and translocations in mature B cells via dicentric chromosomes.

Proc Natl Acad Sci U S A 2014 Jul 30;111(28):10269-74. Epub 2014 Jun 30.

Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115

Mature IgM(+) B-cell lymphomas that arise in certain ataxia telangiectasia-mutated (ATM)-deficient compound mutant mice harbor translocations that fuse V(D)J recombination-initiated IgH double-strand breaks (DSBs) on chromosome 12 to sequences downstream of c-myc on chromosome 15, generating dicentric chromosomes and c-myc amplification via a breakage-fusion-bridge mechanism. As V(D)J recombination DSBs occur in developing progenitor B cells in the bone marrow, we sought to elucidate a mechanism by which such DSBs contribute to oncogenic translocations/amplifications in mature B cells. For this purpose, we applied high-throughput genome-wide translocation sequencing to study the fate of introduced c-myc DSBs in splenic IgM(+) B cells stimulated for activation-induced cytidine deaminase (AID)-dependent IgH class switch recombination (CSR). We found frequent translocations of c-myc DSBs to AID-initiated DSBs in IgH switch regions in wild-type and ATM-deficient B cells. However, c-myc also translocated frequently to newly generated DSBs within a 35-Mb region downstream of IgH in ATM-deficient, but not wild-type, CSR-activated B cells. Moreover, we found such DSBs and translocations in activated B cells that did not express AID or undergo CSR. Our findings indicate that ATM deficiency leads to formation of chromosome 12 dicentrics via recombination-activating gene-initiated IgH DSBs in progenitor B cells and that these dicentrics can be propagated developmentally into mature B cells where they generate new DSBs downstream of IgH via breakage-fusion-bridge cycles. We propose that dicentrics formed by joining V(D)J recombination-associated IgH DSBs to DSBs downstream of c-myc in ATM-deficient B lineage cells similarly contribute to c-myc amplification and mature B-cell lymphomas.
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http://dx.doi.org/10.1073/pnas.1410112111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104897PMC
July 2014

Mechanisms that can promote peripheral B-cell lymphoma in ATM-deficient mice.

Cancer Immunol Res 2014 Sep 9;2(9):857-66. Epub 2014 Jun 9.

Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital; and Department of Genetics, Harvard Medical School, Boston, Massachusetts

The Ataxia Telangiectasia-mutated (ATM) kinase senses DNA double-strand breaks (DSB) and facilitates their repair. In humans, ATM deficiency predisposes to B- and T-cell lymphomas, but in mice it leads only to thymic lymphomas. We tested the hypothesis that increased DSB frequency at a cellular oncogene could promote B-cell lymphoma by generating ATM-deficient mice with a V(D)J recombination target (DJβ cassette) within c-myc intron 1 ("DA" mice). We also generated ATM-deficient mice carrying an Eμ-Bcl-2 transgene (AB mice) to test whether enhanced cellular survival could promote B-cell lymphomas. About 30% of DA or AB mice and nearly 100% of mice harboring the combined genotypes (DAB mice) developed mature B-cell lymphomas. In all genotypes, B-cell tumors harbored oncogenic c-myc amplification generated by breakage-fusion-bridge (BFB) from dicentric chromosomes formed through fusion of IgH V(D)J recombination-associated DSBs on chromosome 12 to sequences downstream of c-myc on chromosome 15. AB tumors demonstrate that B lineage cells harboring spontaneous DSBs leading to IgH/c-myc dicentrics are blocked from progressing to B-cell lymphomas by cellular apoptotic responses. DA and DAB tumor translocations were strictly linked to the cassette, but occurred downstream, frequently in a 6-kb region adjacent to c-myc that harbors multiple cryptic V(D)J recombination targets, suggesting that bona fide V(D)J target sequences may activate linked cryptic targets. Our findings indicate that ATM deficiency allows IgH V(D)J recombination DSBs in developing B cells to generate dicentric translocations that, via BFB cycles, lead to c-myc-activating oncogenic translocations and amplifications in mature B cells.
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http://dx.doi.org/10.1158/2326-6066.CIR-14-0090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156541PMC
September 2014

The intra-S phase checkpoint targets Dna2 to prevent stalled replication forks from reversing.

Cell 2012 Jun;149(6):1221-32

The National Laboratory of Protein Engineering and Plant Genetic Engineering, The College of Life Sciences, Peking University, Beijing, China.

When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ∼10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.
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http://dx.doi.org/10.1016/j.cell.2012.04.030DOI Listing
June 2012