Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors.

Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. Using two newly generated transgenic mice that report and trace the expression of terminal deoxynucleotidyl transferase (TdT), transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset that lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction on MPPs reflected a transcriptionally dynamic but uncommitted stage, characterized by low expression of lineage-associated genes. Single-cell CITE-seq indicated that multipotency in the TdT+ MPPs is associated with expression of the endothelial cell adhesion molecule ESAM. Stable and progressive upregulation of TdT defined the lymphoid developmental trajectory. Collectively, we here identify a new multipotent progenitor within the MPP4 compartment. Specification and commitment are defined by downregulation of ESAM which marks the progressive loss of alternative fates along all lineages.

Recording Binding Information Directly into DNA-Encoded Libraries Using Terminal Deoxynucleotidyl Transferase.

Terminal deoxynucleotidyl transferase (TdT) is an unusual DNA polymerase that adds untemplated dNTPs to 3'-ends of DNA. If a target protein is expressed as a TdT fusion and incubated with a DNA-encoded library (DEL) in the presence of dATP, the binders of the target induce proximity between TdT and the DNA, promoting the synthesis of a poly-adenine (polyA) tail. The polyA tail length is proportional to the binding affinity, effectively serving as a stable molecular record of binding events. The polyA tail is also a convenient handle to enrich binders with magnetic poly(dT)25 beads before sequencing. In a benchmarking system, we show that ligands spanning nanomolar to double-digit micromolar binding can be cleanly identified by TdT extension, whereas only the tightest binding ligands are identified by classical affinity selection. The method is simple to implement and can function on any DEL that bears a free 3'-end.

Absence of terminal deoxynucleotidyl transferase expression in T-ALL/LBL accumulates chromosomal abnormalities to induce drug resistance.

T-acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) is a malignant neoplasm of immature lymphoblasts. Terminal deoxynucleotidyl transferase (TDT) is a template-independent DNA polymerase that plays an essential role in generating diversity for immunoglobulin genes. T-ALL/LBL patients with TDT- have a worse prognosis. However, how TDT- promotes the disease progression of T-ALL/LBL remains unknown. Here we analyzed the prognosis of T-ALL/LBL patients in Shanghai Children's Medical Center (SCMC) and confirmed that TDT- patients had a higher rate of recurrence and remission failure and worse outcomes. Cellular experiments demonstrated that TDT was involved in DNA damage repair. TDT knockout delayed DNA repair, arrested the cell cycle and decreased apoptosis to induce the accumulation of chromosomal abnormalities and tolerance to abnormal karyotypes. Our study demonstrated that the poor outcomes in TDT- T-ALL/LBL might be due to the drug resistance (VP16 and MTX) induced by chromosomal abnormalities. Our findings revealed novel functions and mechanisms of TDT in T-ALL/LBL and supported that hematopoietic stem cell transplantation (HSCT) might be a better choice for these patients.

Detection of apoptotic cells based on in situ hybridization chain reaction using specific hairpins.

There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.

DNTT activation, TdT-aided gene length mutation, and better prognosis in ATG-based regimen allo-HSCT in AML.

This study aimed to investigate the relationship between anomalous DNA nucleotidylexotransferase (DNTT) activation and the mutagenesis of gene length mutations (LMs) in acute myeloid leukemia (AML), and the relevance of their prognosis in antithymocyte globulin (ATG)-based regimen allogeneic hematopoietic stem cell transplantation (allo-HSCT). A cohort of 578 AML cases was enrolled. Next-generation sequencing was performed to screen mutations of 86 leukemia driver genes. RNA-seq was used to analyze gene expression. Prognostic analysis was investigated in 239 AML cases who underwent ATG-based regimen allo-HSCT. We report a refined subtyping algorithm of LMs (type I-IV) based on sequence anatomy considering the TdT-aided mutagenesis mechanism. GC content adjacent to LM junctions, inserted nontemplate nucleotide bases, and DNTT expression analysis supported the DNTT activation and TdT-aided mutagenesis in type II/III LMs in the total AML cohort. Both single-variate and multivariate analyses showed a better overall survival of FLT3 type III compared to type I in a subset of ATG-based regimen allo-HSCT cases. The novel LM subtyping algorithm not only deciphers the etiology of the mutagenesis of LMs but also helps to fine-tune prognosis differentiation in AML. The possible prognostic versatility of this novel LM subtyping algorithm in terms of chemotherapy, targeted therapy, and allo-HSCT merits further investigation.

Molecular roulette: nucleophosmin mutations in AML are orchestrated through N-nucleotide addition by TdT.

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML). AML with mutated NPM1 is recognized as a separate entity in the World Health Organization 2016 classification and carries a relatively favorable prognosis. NPM1 mutations are predominantly 4-bp duplications or insertions in the terminal exon that arise through an unknown mechanism. Here we analyze 2430 NPM1 mutations from 2329 adult and 101 pediatric patients to address their origin. We show that NPM1 mutations display the hallmarks of replication slippage, but lack suitable germline microhomology available for priming. Insertion mutations display G/C-rich N-nucleotide tracts, with a significant bias toward polypurine and polypyrimidine stacking (P < .001). These features suggest terminal deoxynucleotidyl transferase (TdT) primes replication slippage through N-nucleotide addition, with longer syntheses manifesting as N-regions. The recurrent type A, type D, and type B mutations require 1, 2, and 3 N-nucleotide extensions of T, CC, and CAT, respectively, with the last nucleotide used as occult microhomology. This TdT-mutator model successfully predicts the relative incidence of the 256 potential 4-bp insertion/duplication mutations at position c.863_864 over 4 orders of magnitude (ρ = 0.484, P < .0001). Children have a different NPM1 mutation spectrum to adults, including a shift away from type A mutations and toward longer N-regions, consistent with higher TdT activity in pediatric myeloid stem cells. These findings complement our FLT3-ITD data, suggesting illegitimate TdT activity contributes to around one-half of AMLs. AML may therefore reflect the price for adaptive immunity.

Terminal deoxynucleotidyl transferase promotes acute myeloid leukemia by priming FLT3-ITD replication slippage.

FLT3-internal tandem duplications (FLT3-ITDs) are prognostic driver mutations found in acute myeloid leukemia (AML). Although these short duplications occur in 25% of AML patients, little is known about the molecular mechanism underlying their formation. Understanding the origin of FLT3-ITDs would advance our understanding of the genesis of AML. We analyzed the sequence and molecular anatomy of 300 FLT3-ITDs to address this issue, including 114 ITDs with additional nucleotides of unknown origin located between the 2 copies of the repeat. We observed anatomy consistent with replication slippage, but could only identify the germline microhomology (1-6 bp) anticipated to prime such slippage in one-third of FLT3-ITDs. We explain the paradox of the "missing" microhomology in the majority of FLT3-ITDs through occult microhomology: specifically, by priming through use of nontemplated nucleotides (N-nucleotides) added by terminal deoxynucleotidyl transferase (TdT). We suggest that TdT-mediated nucleotide addition in excess of that required for priming creates N-regions at the duplication junctions, explaining the additional nucleotides observed at this position. FLT3-ITD N-regions have a G/C content (66.9%), dinucleotide composition (P < .001), and length characteristics consistent with synthesis by TdT. AML types with high TdT show an increased incidence of FLT3-ITDs (M0; P = .0017). These results point to an unexpected role for the lymphoid enzyme TdT in priming FLT3-ITDs. Although the physiological role of TdT is to increase antigenic diversity through N-nucleotide addition during V(D)J recombination of IG/TCR genes, here we propose that illegitimate TdT activity makes a significant contribution to the genesis of AML.

A case of primary cutaneous B-cell lymphoma with immature features in an old man. Diffuse large B-cell lymphoma with immature features or B-cell lymphoblastic lymphoma?

Primary cutaneous B-cell lymphomas are a heterogeneous group of lymphoid neoplasms primarily occurring in the skin. Although most cases are represented by primary cutaneous follicle center cell lymphoma, primary cutaneous marginal zone lymphoma and leg-type diffuse large B-cell lymphoma, other diffuse large B-cell lymphomas and B-cell lymphoblastic lymphoma may rarely present primarily in the skin. In this setting, the presence of histopathologic and immunohistochemical features of cellular immaturity is exceedingly rare and may represent a diagnostic challenge. We present the first case of a primary cutaneous diffuse large B-cell lymphoma characterized by diminished expression of CD45, expression of TdT and rearrangement of MYC gene. The differential diagnosis mainly included B-cell lymphoblastic lymphoma, and required the genetic analysis of heavy chain (IGH) gene rearrangements.

Highly efficient single-stranded DNA ligation technique improves low-input whole-genome bisulfite sequencing by post-bisulfite adaptor tagging.

Whole-genome bisulfite sequencing (WGBS) is the current gold standard of methylome analysis. Post-bisulfite adaptor tagging (PBAT) is an increasingly popular WGBS protocol because of high sensitivity and low bias. PBAT originally relied on two rounds of random priming for adaptor-tagging of single-stranded DNA (ssDNA) to attain high efficiency but at a cost of library insert length. To overcome this limitation, we developed terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation as an alternative to random priming. In this method, TdT attaches adenylates to the 3'-end of input ssDNA, which are then utilized by RNA ligase as an efficient connector to the ssDNA adaptor. A protocol that uses TACS ligation instead of the second random priming step substantially increased the lengths of PBAT library fragments. Moreover, we devised a dual-library strategy that splits the input DNA to prepare two libraries with reciprocal adaptor polarity, combining them prior to sequencing. This strategy ensured an ideal base-color balance to eliminate the need for DNA spike-in for color compensation, further improving the throughput and quality of WGBS. Adopting the above strategies to the HiSeq X Ten and NovaSeq 6000 platforms, we established a cost-effective, high-quality WGBS, which should accelerate various methylome analyses.

Mimivirus encodes a multifunctional primase with DNA/RNA polymerase, terminal transferase and translesion synthesis activities.

Acanthamoeba polyphaga mimivirus is an amoeba-infecting giant virus with over 1000 genes including several involved in DNA replication and repair. Here, we report the biochemical characterization of gene product 577 (gp577), a hypothetical protein (product of L537 gene) encoded by mimivirus. Sequence analysis and phylogeny suggested gp577 to be a primase-polymerase (PrimPol)-the first PrimPol to be identified in a nucleocytoplasmic large DNA virus (NCLDV). Recombinant gp577 protein purified as a homodimer and exhibited de novo RNA as well as DNA synthesis on circular and linear single-stranded DNA templates. Further, gp577 extends a DNA/RNA primer annealed to a DNA or RNA template using deoxyribonucleoties (dNTPs) or ribonucleotides (NTPs) demonstrating its DNA/RNA polymerase and reverse transcriptase activity. We also show that gp577 possesses terminal transferase activity and is capable of extending ssDNA and dsDNA with NTPs and dNTPs. Mutation of the conserved primase motif residues of gp577 resulted in the loss of primase, polymerase, reverse transcriptase and terminal transferase activities. Additionally, we show that gp577 possesses translesion synthesis (TLS) activity. Mimiviral gp577 represents the first protein from an NCLDV endowed with primase, polymerase, reverse transcriptase, terminal transferase and TLS activities.

Molecular characteristics of terminal deoxynucleotidyl transferase negative precursor B-cell phenotype Burkitt leukemia with IGH-MYC rearrangement.

Precursor B cell phenotype Burkitt lymphoma/leukemia with IGH-MYC is a rare subtype of Burkitt lymphoma (BL). BL and B lymphoblastic leukemia/lymphoma (B-ALL/LBL) differ as regards treatment and the distinction between these two entities is crucial. Patients demonstrating a terminal deoxynucleotidyl transferase (TdT)-positive precursor B cell phenotype with IGH-MYC rearrangement have been reported to be molecularly distinct from BL and closer to B-ALL/LBL. We investigated the molecular characteristics of two cases of a rare but distinct TdT-negative precursor B cell phenotype BL. Both patients showed FAB L3 morphology with IGH-MYC translocation, but had precursor B cell immunophenotypes including dim to moderate expression of CD45 and absence of BCL6, CD20, monoclonal kappa, and lambda light chain expression. To characterize the molecular features, we performed exome sequencing and analyzed the breakpoint junction of the IGH-MYC rearrangement. We detected KMT2D mutations in both cases, a rarely acquired chromatin modifying gene mutation in BL. The breakpoint analysis revealed that the IGH-MYC rearrangement occurred due to an aberrant VDJ recombination in one case. The treatment protocols differed, including high-grade lymphoma treatment and standard B-ALL treatment. Complete remission was achieved in the patient who received B-ALL treatment. The degree of resemblance of BL and B-ALL differed between two cases, but the molecular pathogenesis and manifesting features of both TdT-negative precursor B cell phenotype BL case were distinct from classic BL, which indicates the need for a better understanding of this uncommon entity that does not fit in current diagnostic and classification categories.

[Expression of terminal deoxynucleotidyl transferase in the pharyngeal and palatine tonsils in local infectious and inflammatory diseases of the upper respiratory tract and pharynx in childhood]. / Ekspressiya terminal'noi dezoksinukleotidiltransferazy v glotochnoi i nebnykh mindalinakh pri mestnykh infektsionno-vospalitel'nykh zabolevaniyakh verkhnikh dykhatel'nykh putei i glotki v detskom vozraste.

Along with central immune organs, the peripheral lymphoepithelial organs of the pharynx are actively involved in protecting the body from infections. Adaptive, or induced, immunity occurs during the postnatal ontogenesis of immunocompetent lymphocytes, which includes the secondary somatic recombination of the V genes with the participation of recombination-activating gene (RAG) and terminal deoxynucleotidyl transferase (Tdt) proteins. This publication discusses the results of detection of Tdt-positive cells in the pharyngeal and palatine tonsils of children of different ages, who had been operated on for adenoid vegetations and chronic tonsillitis. Attention is drawn to the localization of Tdt+ cells, the level of Tdt expression, an attempt to clarify the phenotype, destination, and place in the diagnostic arrays of functional markers when an adaptive immunity is generated in children.

Structural evidence for an in trans base selection mechanism involving Loop1 in polymerase µ at an NHEJ double-strand break junction.

Eukaryotic DNA polymerase (Pol) X family members such as Pol µ and terminal deoxynucleotidyl transferase (TdT) are important components for the nonhomologous DNA end-joining (NHEJ) pathway. TdT participates in a specialized version of NHEJ, V(D)J recombination. It has primarily nontemplated polymerase activity but can take instructions across strands from the downstream dsDNA, and both activities are highly dependent on a structural element called Loop1. However, it is unclear whether Pol µ follows the same mechanism, because the structure of its Loop1 is disordered in available structures. Here, we used a chimeric TdT harboring Loop1 of Pol µ that recapitulated the functional properties of Pol µ in ligation experiments. We solved three crystal structures of this TdT chimera bound to several DNA substrates at 1.96-2.55 Å resolutions, including a full DNA double-strand break (DSB) synapsis. We then modeled the full Pol µ sequence in the context of one these complexes. The atomic structure of an NHEJ junction with a Pol X construct that mimics Pol µ in a reconstituted system explained the distinctive properties of Pol µ compared with TdT. The structure suggested a mechanism of base selection relying on Loop1 and taking instructions via the in trans templating base independently of the primer strand. We conclude that our atomic-level structural observations represent a paradigm shift for the mechanism of base selection in the Pol X family of DNA polymerases.

Nucleotide Composition of Human Ig Nontemplated Regions Depends on Trimming of the Flanking Gene Segments, and Terminal Deoxynucleotidyl Transferase Favors Adding Cytosine, Not Guanosine, in Most VDJ Rearrangements.

The formation of nontemplated (N) regions during Ig gene rearrangement is a major contributor to Ab diversity. To gain insights into the mechanisms behind this, we studied the nucleotide composition of N regions within 29,962 unique human VHDJH rearrangements and 8728 unique human DJH rearrangements containing exactly one identifiable D gene segment and thus two N regions, N1 and N2. We found a distinct decreasing content of cytosine (C) and increasing content of guanine (G) across each N region, suggesting that N regions are typically generated by concatenation of two 3' overhangs synthesized by addition of nucleoside triphosphates with a preference for dCTP. This challenges the general assumption that the terminal deoxynucleotidyl transferase favors dGTP in vivo. Furthermore, we found that the G and C gradients depended strongly on whether the germline gene segments were trimmed or not. Our data show that C-enriched N addition preferentially happens at trimmed 3' ends of VH, D, and JH gene segments, indicating a dependency of the transferase mechanism upon the nuclease mechanism.

Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines against Streptococcus pneumoniae and Salmonella enterica Serovar Typhi.

B cell antigen receptor (BCR) diversity increases by several orders of magnitude due to the action of terminal deoxynucleotidyl transferase (TdT) during V(D)J recombination. Unlike adults, infants have limited BCR diversity, in part due to reduced expression of TdT. Since human infants and young mice respond poorly to polysaccharide vaccines, such as the pneumococcal polysaccharide vaccine Pneumovax23 and Vi polysaccharide (ViPS) of Salmonella enterica serovar Typhi, we tested the contribution of TdT-mediated BCR diversity in response to these vaccines. We found that TdT+/- and TdT-/- mice generated comparable antibody responses to Pneumovax23 and survived Streptococcus pneumoniae challenge. Moreover, passive immunization of B cell-deficient mice with serum from Pneumovax23-immunized TdT+/- or TdT-/- mice conferred protection. TdT+/- and TdT-/- mice generated comparable levels of anti-ViPS antibodies and antibody-dependent, complement-mediated bactericidal activity against S Typhi in vitro To test the protective immunity conferred by ViPS immunization in vivo, TdT+/- and TdT-/- mice were challenged with a chimeric Salmonella enterica serovar Typhimurium strain expressing ViPS, since mice are nonpermissive hosts for S Typhi infection. Compared to their unimmunized counterparts, immunized TdT+/- and TdT-/- mice challenged with ViPS-expressing S Typhimurium exhibited a significant reduction in the bacterial burden and liver pathology. These data suggest that the impaired antibody response to the Pneumovax23 and ViPS vaccines in the young is not due to limited TdT-mediated BCR diversification.

Identification and expression analysis of terminal deoxynucleotidyl transferase in humphead snapper Lutjanus sanguineus.

A tdt gene was identified successfully from humphead snapper Lutjanus sanguineus, which contained 1710 bp encoding a protein of 463 amino acids. Results of quantitative real-time polymerase chain reaction (qRT-PCR) indicated that tdt mainly expressed in thymus and head kidney and the transcripts of tdt in these tissues were up-regulated significantly at 36 and 48 h after Vibrio harveyi infection. Meanwhile Tdt-producing cells were found in thymus and head kidney.

Pediatric B-Cell Lymphoma With Lymphoblastic Morphology, TdT Expression, MYC Rearrangement, and Features Overlapping With Burkitt Lymphoma.

Burkitt lymphoma (BL) and B-lymphoblastic lymphoma are subtypes of pediatric non-Hodgkin lymphoma with different presenting features, treatment, and outcomes. This case report documents a 5-year-old female who presented with B-cell lymphoma with lymphoblastic morphology, terminal deoxynucleotidyl transferase expression, MYC rearrangement, and features overlapping with BL. Genomic microarray analysis identified a gain on the long arm of chromosome 1 without other definitive changes. She was treated according to a BL protocol and remains in remission 16-months after initial diagnosis.

Decision-making during NHEJ: a network of interactions in human Polµ implicated in substrate recognition and end-bridging.

Human Polµ is a DNA polymerase belonging to the X family that has been implicated in the non-homologous end-joining (NHEJ) pathway during repair of double-strand breaks in DNA. Loop1 is a flexible piece of Polµ which has a critical role during terminal transferase and end-joining activities: it acts as a pseudo-template when the template strand is discontinuous or unavailable, whilst diffusing away if present to avoid steric clashes. Mutational analysis and inspection of the 3D structures available allowed us to identify a network of residues in charge of sensing the presence or absence of discontinuities in the template strand, which will in turn determine the final position adopted by Loop1. This network is formed by the previously uncharacterized thumb mini-loop (NSH motif) and the positively charged helix N, which contribute to the correct positioning of Loop1 and to juxtapose the discontinuous template strand during NHEJ of incompatible ends. Accordingly, single mutation of specific conserved residues in these motifs, whilst irrelevant in most of the cases for gap filling, largely affected terminal transferase and end-joining activities. Other point mutations in the 'hinges' of Loop1, such as residues Phe385 or Phe389, corroborated the flexibility requirements of this motif.

Functional analyses of polymorphic variants of human terminal deoxynucleotidyl transferase.

Human terminal deoxynucleotidyl transferase (hTdT) is a DNA polymerase that functions to generate diversity in the adaptive immune system. Here, we focus on the function of naturally occurring single-nucleotide polymorphisms (SNPs) of hTdT to evaluate their role in genetic-generated immune variation. The data demonstrate that the genetic variations generated by the hTdT SNPs will vary the human immune repertoire and thus its responses. Human TdT catalyzes template-independent addition of nucleotides (N-additions) during coding joint formation in V(D)J recombination. Its activity is crucial to the diversity of the antigen receptors of B and T lymphocytes. We used in vitro polymerase assays and in vivo human cell V(D)J recombination assays to evaluate the activity and the N-addition levels of six natural (SNP) variants of hTdT. In vitro, the variants differed from wild-type hTdT in polymerization ability with four having significantly lower activity. In vivo, the presence of TdT varied both the efficiency of recombination and N-addition, with two variants generating coding joints with significantly fewer N-additions. Although likely heterozygous, individuals possessing these genetic changes may have less diverse B- and T-cell receptors that would particularly effect individuals prone to adaptive immune disorders, including autoimmunity.

Predominant role for activation-induced cytidine deaminase in generating IgG anti-nucleosomal antibodies of murine SLE.

Serum IgG anti-nuclear antibodies (ANA) directed to complexes of DNA and histones are a hallmark of systemic lupus erythematosus (SLE) and reflect a failure in lymphocyte self-tolerance. A prior study utilizing spontaneously autoimmune B6.Nba2 mice deficient in terminal deoxynucleotidyl transferase (TdT) and with heterozygous deficiencies in Jh and Igk loci underscored the importance of somatic hypermutation (SHM) as a major generator of SLE-associated ANA. This interpretation had to be qualified because of severely limited opportunities for receptor editing and restricted VHCDR3 diversity. Therefore, we performed the converse study using mice that carried functional Tdt genes and wild type Jh and Igk loci but that could not undergo SHM. Analyses of ANA and ANA-producing hybridomas from B6.Nba2 Aicda(-/-) mice revealed that few animals produced high titers of the prototypical ANA directed to complexes of histones and DNA, that this response was delayed and that those cells that did produce such antibody exhibited limited clonal expansion, unusual Jk use and only infrequent dual receptor expression. This, together with the additional finding of an intrinsic propensity for SHM to generate Arg codons selectively in CDRs, reinforce the view that most IgG autoimmune clones producing prototypical anti-nucleosome antibodies in wild type mice are created by SHM.