Zebrafish prmt3 negatively regulates antiviral responses.

Arginine methylation catalyzed by protein arginine methyltransferases (PRMT) is a common post-translational modification in histone and nonhistone proteins, which regulates many cellular functions. Protein arginine methyltransferase 3 (prmt3), a type I arginine methyltransferase, has been shown to carry out the formation of stable monomethylarginine as an intermediate before the establishment of asymmetric dimethylarginine. To date, however, the role of PRMT3 in antiviral innate immunity has not been elucidated. This study showed that zebrafish prmt3 was upregulated by virus infection and that the overexpression of prmt3 suppressed cellular antiviral response. The PRMT3 inhibitor, SGC707, enhanced antiviral capability. Consistently, prmt3-null zebrafish were more resistant to Spring Viremia of Carp Virus (SVCV) and Grass Carp Reovirus (GCRV) infection. Further assays showed that the overexpression of prmt3 diminished the phosphorylation of irf3 and prmt3 interacted with rig-i. In addition, both zinc-finger domain and catalytic domain of prmt3 were required for the suppressive function of prmt3 on IFN activation. Our findings suggested that zebrafish prmt3 negatively regulated the antiviral responses, implicating the vital role of prmt3-or even arginine methylation-in antiviral innate immunity.

A single C4 Zinc finger-containing protein from Litopenaeus vannamei involved in antibacterial responses.

The Zinc finger domains (ZnFs), which contain finger-like protrusions stabilized by zinc ions and function to bind DNA, RNA, protein and lipid substrates, are ubiquitously present in a large number of proteins. In this study, a novel protein containing a single C4 type Znf domain (SZnf) was identified from Pacific white shrimp, Litopenaeus vannamei and its role in immunity was further investigated. The ZnF domain of SZnF but not other regions shared high homology with those of fushi tarazu-factor 1 (FTZ-F1) proteins. The SZnF protein was mainly localized in the cytoplasm and was also present in the nucleus at a small level. SZnF was high expressed in the scape and muscle tissues of healthy shrimp and its expression in gill and heptopancreas was strongly up-regulated during bacterial infection. Silencing of SZnf in vivo could strongly increase the susceptibility of shrimp to infection with Vibrio parahaemolyticus but not white spot syndrome virus (WSSV), suggesting that SZnf could be mainly involved in antibacterial responses. Both dual luciferase reporter assays and real-time PCR analysis demonstrated that SZnf could positively regulate the expression of various antimicrobial peptides in vitro and in vivo, which could be part of the mechanism underlying its antibacterial effects. In summary, the current study could help learn more about the function of ZnF-containing proteins and the regulatory mechanisms of immune responses against pathogen infection in crustaceans.

A novel GATA-like zinc finger transcription factor involving in hematopoiesis of Eriocheir sinensis.

GATA transcription factor is a family of DNA-binding proteins that can recognize and bind to sequence of (A/T) GATA (A/G). In the present study, a GATA-like protein (named as EsGLP) was characterized from Eriocheir sinensis, including an 834 bp full length open reading frame of EsGLP, encoding a polypeptide of 277 amino acids. The deduced amino acid sequence of EsGLP contained one conserved GATA-type zinc finger of the form Cys-X2-Cys-X17-Cys-X2-Cys, with four cysteine sites. The EsGLP mRNA transcripts were mainly detected in the hematopoietic tissue, hepatopancreas and gonad. The recombinant EsGLP protein was prepared for the antibody production. The EsGLP protein was mainly distributed in the edge of lobules in the HPT and the cytoplasm of hemocytes. The mRNA transcripts of EsGLP in hemocytes were significantly decreased at 24 h (0.39-fold and 0.27-fold, p < .05) and 48 h (0.35-fold and 0.16-fold, p < .05) after LPS and Aeromonas hydrophila stimulation, respectively. However, one peak of EsGLP mRNA transcripts were recorded at 24 h (8.71-fold, p < .05) in HPT after A. hydrophila stimulation. The expression level of EsGLP mRNA in HPT was significantly up-regulated at 2 h, 2.5 h and 9 h (41.74-fold, 45.38-fold and 26.07-fold, p < .05) after exsanguination stimulation. When EsGLP gene expression was inhibited by the injection of double-stranded RNA, both the total hemocytes counts and the rate of EdU-positive hemocytes were significantly decreased (0.32-fold and 0.56-fold compared to that in control group, p < .05). All these results suggested that EsGLP was an important regulatory factor in E. sinensis which involved in the hemocytes generation and the immune response against invading pathogens.

The zinc finger protein CG12744 is essential for differentiation and regeneration after infection in the adult Drosophila midgut.

Pluripotent stem cell activity is essential to maintain regeneration and homeostasis in the Drosophila midgut following environmental challenges. Although multiple pathways have been implicated in epithelial renewal, the underlying regulatory mechanisms and correlations between relevant genes and pathways remain elusive. In this study, we show that the zinc finger protein CG12744 plays an important role in the differentiation and regeneration of epithelial cells in response to oral infection with Erwinia carotovora carotovora 15. Knocking down CG12744 in enteroblasts decreased the post-infection proportion of enteroblasts and enterocytes and increased the post-infection number of enteroendocrine cells. In addition, in precursors, CG12744 affected the Osa, jun-N-terminal kinase and bone morphogenetic protein signaling pathways to control enterocyte differentiation. Finally, CG12744 maintained epithelial architecture and cell fate in enterocytes following an acute infectious challenge.

RNA-binding proteins in immune regulation: a focus on CCCH zinc finger proteins.

Nearly 60 CCCH zinc finger proteins have been identified in humans and mice. These proteins are involved in the regulation of multiple steps of RNA metabolism, including mRNA splicing, polyadenylation, transportation, translation and decay. Several CCCH zinc finger proteins, such as tristetraprolin (TTP), roquin 1 and MCPIP1 (also known as regnase 1), are crucial for many aspects of immune regulation by targeting mRNAs for degradation and modulation of signalling pathways. In this Review, we focus on the emerging roles of CCCH zinc finger proteins in the regulation of immune responses through their effects on cytokine production, immune cell activation and immune homeostasis.

Epigenetic regulation of the expression of Il12 and Il23 and autoimmune inflammation by the deubiquitinase Trabid.

The proinflammatory cytokines interleukin 12 (IL-12) and IL-23 connect innate responses and adaptive immune responses and are also involved in autoimmune and inflammatory diseases. Here we describe an epigenetic mechanism for regulation of the genes encoding IL-12 (Il12a and Il12b; collectively called 'Il12' here) and IL-23 (Il23a and Il12b; collectively called 'Il23' here) involving the deubiquitinase Trabid. Deletion of Zranb1 (which encodes Trabid) in dendritic cells inhibited induction of the expression of Il12 and Il23 by Toll-like receptors (TLRs), which impaired the differentiation of inflammatory T cells and protected mice from autoimmune inflammation. Trabid facilitated TLR-induced histone modifications at the promoters of Il12 and Il23, which involved deubiqutination and stabilization of the histone demethylase Jmjd2d. Our findings highlight an epigenetic mechanism for the regulation of Il12 and Il23 and establish Trabid as an innate immunological regulator of inflammatory T cell responses.

Identification of Barramundi (Lates calcarifer) DC-SCRIPT, a Specific Molecular Marker for Dendritic Cells in Fish.

Antigen presentation is a critical step bridging innate immune recognition and specific immune memory. In mammals, the process is orchestrated by dendritic cells (DCs) in the lymphatic system, which initiate clonal proliferation of antigen-specific lymphocytes. However, fish lack a classical lymphatic system and there are currently no cellular markers for DCs in fish, thus antigen-presentation in fish is poorly understood. Recently, antigen-presenting cells similar in structure and function to mammalian DCs were identified in various fish, including rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). The present study aimed to identify a potential molecular marker for DCs in fish and therefore targeted DC-SCRIPT, a well-conserved zinc finger protein that is preferentially expressed in all sub-types of human DCs. Putative dendritic cells were obtained in culture by maturation of spleen and pronephros-derived monocytes. DC-SCRIPT was identified in barramundi by homology using RACE PCR and genome walking. Specific expression of DC-SCRIPT was detected in barramundi cells by Stellaris mRNA FISH, in combination with MHCII expression when exposed to bacterial derived peptidoglycan, suggesting the presence of DCs in L. calcarifer. Moreover, morphological identification was achieved by light microscopy of cytospins prepared from these cultures. The cultured cells were morphologically similar to mammalian and trout DCs. Migration assays determined that these cells have the ability to move towards pathogens and pathogen associated molecular patterns, with a preference for peptidoglycans over lipopolysaccharides. The cells were also strongly phagocytic, engulfing bacteria and rapidly breaking them down. Barramundi DCs induced significant proliferation of responder populations of T-lymphocytes, supporting their role as antigen presenting cells. DC-SCRIPT expression in head kidney was higher 6 and 24 h following intraperitoneal challenge with peptidoglycan and lipopolysaccharide and declined after 3 days relative to PBS-injected controls. Relative expression was also lower in the spleen at 3 days post challenge but increased again at 7 days. As DC-SCRIPT is a constitutively expressed nuclear receptor, independent of immune activation, this may indicate initial migration of immature DCs from head kidney and spleen to the injection site, followed by return to the spleen for maturation and antigen presentation. DC-SCRIPT may be a valuable tool in the investigation of antigen presentation in fish and facilitate optimisation of vaccines and adjuvants for aquaculture.

Diagnostic potential of zinc finger protein-specific autoantibodies and associated linear B-cell epitopes in colorectal cancer.

Colorectal cancer is one of the most common cancers worldwide with almost 700,000 deaths every year. Detection of colorectal cancer at an early stage significantly improves patient survival. Cancer-specific autoantibodies found in sera of cancer patients can be used for pre-symptomatic detection of the disease. In this study we assess the zinc finger proteins ZNF346, ZNF638, ZNF700 and ZNF768 as capture antigens for the detection of autoantibodies in colorectal cancer. Sera from 96 patients with colorectal cancer and 35 control patients with no evidence of cancer on colonoscopy were analysed for the presence of ZNF-specific autoantibodies using an indirect ELISA. Autoantibodies to individual ZNF proteins were detected in 10-20% of colorectal cancer patients and in 0-5.7% of controls. A panel of all four ZNF proteins resulted in an assay specificity of 91.4% and sensitivity of 41.7% for the detection of cancer patients in a cohort of non-cancer controls and colorectal cancer patients. Clinicopathological and survival analysis revealed that ZNF autoantibodies were independent of disease stage and did not correlate with disease outcome. Since ZNF autoantibodies were shared between patients and corresponding ZNF proteins showed similarities in their zinc finger motifs, we performed an in silico epitope sequence analysis. Zinc finger proteins ZNF700 and ZNF768 showed the highest sequence similarity with a bl2seq score of 262 (E-value 1E-81) and their classical C2H2 ZNF motifs were identified as potential epitopes contributing to their elevated immunogenic potential. Our findings show an enhanced and specific immunogenicity to zinc finger proteins, thereby providing a multiplexed autoantibody assay for minimally invasive detection of colorectal cancer.

Fatal autoimmunity results from the conditional deletion of Snai2 and Snai3.

Transcriptional regulation of gene expression is a key component of orchestrating proper immune cell development and function. One strategy for maintaining these transcriptional programs has been the evolution of transcription factor families with members possessing overlapping functions. Using the germ line deletion of Snai2 combined with the hematopoietic specific deletion of Snai3, we report that these factors function redundantly to preserve the development of B and T cells. Such animals display severe lymphopenia, alopecia and dermatitis as well as profound autoimmunity manifested by the production of high levels of autoantibodies as early as 3 weeks of age and die by 30 days after birth. Autoantibodies included both IgM and IgG isotypes and were reactive against cytoplasmic and membranous components. A regulatory T cell defect contributed to the autoimmune response in that adoptive transfer of wild type regulatory T cells alleviated symptoms of autoimmunity. Additionally, transplantation of Snai2/Snai3 double deficient bone marrow into Snai2 sufficient Rag2(-/-) recipients resulted in autoantibody generation. The results demonstrated that appropriate expression of Snai2 and Snai3 in cells of hematopoietic derivation plays an important role in development and maintenance of immune tolerance.

Proteomic profiling reveals autoimmune targets in sarcoidosis.

RATIONALE: There is a need to further characterize the antibody repertoire in relation to sarcoidosis and potentially related autoantigens. OBJECTIVES: We investigated bronchoalveolar lavage (BAL) and serum samples from patients with sarcoidosis and healthy and diseased control subjects to discover sarcoidosis-associated autoantigens. METHODS: Antigen microarrays built on 3,072 protein fragments were used to screen for IgG reactivity in 73 BAL samples from subjects with sarcoidosis, subjects with asthma, and healthy subjects. A set of 131 targets were selected for subsequent verification on suspension bead arrays using 272 additional BAL samples and 141 paired sera. Reactivity to four antigens was furthermore analyzed in 22 unprocessed BAL samples from patients with fibrosis and 269 plasma samples from patients diagnosed with myositis. MEASUREMENTS AND MAIN RESULTS: Reactivity toward zinc finger protein 688 and mitochondrial ribosomal protein L43 were discovered with higher frequencies in patients with sarcoidosis, for mitochondrial ribosomal protein L43 especially in patients with non-Löfgren syndrome. Increased reactivity toward nuclear receptor coactivator 2 was also observed in patients with non-Löfgren syndrome as compared with patients with Löfgren syndrome. The antigen representing adenosine diphosphate-ribosylation factor GTPase activating protein 1 revealed high reactivity frequency in all sample groups but with significantly higher level of IgG reactivities in patients with sarcoidosis. CONCLUSIONS: Autoantigen reactivity was present in most BAL and serum samples analyzed, and the results revealed high interindividual heterogeneity, with most of the reactivities observed in single individuals only. Four proteins are here proposed as sarcoidosis-associated autoimmune targets and of interest for further validation in independent cohorts.

Zinc finger protein Zfp335 is required for the formation of the naïve T cell compartment.

The generation of naïve T lymphocytes is critical for immune function yet the mechanisms governing their maturation remain incompletely understood. We have identified a mouse mutant, bloto, that harbors a hypomorphic mutation in the zinc finger protein Zfp335. Zfp335(bloto/bloto) mice exhibit a naïve T cell deficiency due to an intrinsic developmental defect that begins to manifest in the thymus and continues into the periphery, affecting T cells that have recently undergone thymic egress. The effects of Zfp335(bloto) are multigenic and cannot be attributed to altered thymic selection, proliferation or Bcl2-dependent survival. Zfp335 binds to promoter regions via a consensus motif, and its target genes are enriched in categories related to protein metabolism, mitochondrial function, and transcriptional regulation. Restoring the expression of one target, Ankle2, partially rescues T cell maturation. These findings identify Zfp335 as a transcription factor and essential regulator of late-stage intrathymic and post-thymic T cell maturation.

Early biodistribution and persistence of a protective live attenuated SIV vaccine elicits localised innate responses in multiple lymphoid tissues.

Vaccination of Mauritian cynomolgus macaques with the attenuated nef-truncated C8 variant of SIVmac251/32H (SIVmacC8) induces early, potent protection against pathogenic, heterologous challenge before the maturation of cognate immunity. To identify processes that contribute to early protection in this model the pathogenesis, anatomical distribution and viral vaccine kinetics were determined in relation to localised innate responses triggered by vaccination. The early biodistribution of SIVmacC8 was defined by rapid, widespread dissemination amongst multiple lymphoid tissues, detectable after 3 days. Cell-associated viral RNA dynamics identified mesenteric lymph nodes (MLN) and spleen, as well as the gut mucosae, as early major contributors of systemic virus burden. Rapid, localised infection was populated by discrete foci of persisting virus-infected cells. Localised productive infection triggered a broad innate response, with type-1 interferon sensitive IRF-7, STAT-1, TRIM5α and ApoBEC3G genes all upregulated during the acute phase but induction did not prevent viral persistence. Profound changes in vaccine-induced cell-surface markers of immune activation were detected on macrophages, B-cells and dendritic cells (DC-SIGN, S-100, CD40, CD11c, CD123 and CD86). Notably, high DC-SIGN and S100 staining for follicular and interdigitating DCs respectively, in MLN and spleen were detected by 3 days, persisting 20 weeks post-vaccination. Although not formally evaluated, the early biodistribution of SIVmacC8 simultaneously targets multiple lymphoid tissues to induce strong innate immune responses coincident at the same sites critical for early protection from wild-type viruses. HIV vaccines which stimulate appropriate innate, as well as adaptive responses, akin to those generated by live attenuated SIV vaccines, may prove the most efficacious.

Molecular signatures and transcriptional regulatory networks of human immature decidual NK and mature peripheral NK cells.

Many differences exist between human immature and mature natural killer (NK) cells, but their respective molecular signatures and transcriptional regulators are relatively unknown. To gain new insights into the diversity and developmental regulation of human NK cells, we used data from high-resolution microarrays with independent verification to describe a comprehensive comparative analysis between immature decidual NK (idNK) cells with a CD56(bright) CD16(-) T-bet(-) phenotype and mature peripheral NK (mpNK) cells with a CD56(dim) CD16(+) T-bet(+) phenotype. This study shows that many novel growth factors, cytokines, and chemokines are expressed by NK cells, and they may regulate NK-cell development or function in an autocrine manner. Notably, we present that idNK and mpNK cells are enriched for homeobox and zinc-finger transcription factors (TFs), respectively. Additionally, many novel candidate transcriptional regulators are common to both idNK and mpNK cells. We further describe the transcriptional regulatory networks of NK cells and show that the endogenous growth factors, cytokines, and TFs enriched in idNK cells regulate each other and may contribute to idNK-cell immaturity. Together, these findings provide novel molecular signatures for immature and mature NK cells, and the novel candidate regulators identified here can be used to describe and further understand NK-cell differentiation and function.

BTB-ZF transcription factors, a growing family of regulators of early and late B-cell development.

The differentiation of early B-cell precursors in the bone marrow into the variety of mature and effector B-cell subsets of the periphery is a complex process that requires tight regulation at the transcriptional level. Different members of the broad complex, tramtrack, bric-à-brac and zinc finger (BTB-ZF) family of transcription factors have recently been shown to have key roles in many phases of B-cell development, including early B-cell development in the bone marrow, peripheral B-cell maturation and specialization into effector cells during an immune response. This review highlights the critical functions mediated by BTB-ZF transcription factors within the B-cell lineage and emphasizes how the deregulation of these transcription factors can lead to B-cell malignancies.

Gene targeting in NOD mouse embryos using zinc-finger nucleases.

Studies in NOD mice have provided important insight into the genetics and pathogenesis of type 1 diabetes (T1D). Our goal was to further explore novel methods of genetic manipulation in this mouse model. We tested the feasibility of using zinc-finger nucleases (ZFNs) to knock out a gene directly in a pure NOD background, bypassing the need of embryonic stem cells. We report here the successful application of ZFN pairs to specifically and efficiently knock out Tnfrsf9 (encoding CD137/4-1BB) directly in the NOD mouse by embryo microinjection. Histology and T1D incidence studies indicated that CD137 was dispensable for the development of insulitis but played a role to promote progression to overt diabetes in NOD mice. We also demonstrated that CD137-deficient T-cells were less diabetogenic than their wild-type counterpart when adoptively transferred into NOD.Rag1(-/-) recipients, even when CD25(+) cells were predepleted. In vitro assays suggested that CD137 deficiency had a limited effect on the suppressive function of CD4(+)CD25(+) regulatory T-cells (Tregs). Therefore, CD137 deficiency predominately affected effector T-cells rather than Tregs. Our study demonstrates the ability to generate gene-targeted knockouts in a pure NOD background by using ZFNs without potential confounding factors introduced by contaminating genetic materials obtained from other strains.

An anti-inflammatory role of A20 zinc finger protein during trauma combined with endotoxin challenge.

OBJECTIVE: To investigate the anti-inflammatory role of A20 zinc finger protein during trauma combined with bacterial endotoxin challenge and explore the molecular mechanism underlying this process. METHODS: Traumatic bone impact injury was induced in the hind limbs of mice. One hour after injury, mice were challenged with purified gram-negative bacterial endotoxins, lipopolysaccharides (LPSs), by tail vein injection. Effects on A20 messenger RNA and protein expressions were assessed by reverse transcription-polymerase chain reaction and Western blotting, respectively. A20 recombinant adenoviruses, full-length (pAdA20 1-775) and N-terminal mutant (pAdA20 1-367), were constructed and used to infect RAW264.7 macrophage cells or mice. Responses in the tumor necrosis factor α (TNF-α)-nuclear factor κB (NF-κB) signaling pathway were evaluated by enzyme-linked immunosorbent assay (for TNF-α) and electrophoretic mobility shift assay (for NF-κB). RESULTS: Trauma combined with LPS challenge and LPS challenge alone dramatically promoted A20 expression in mouse liver tissues. LPS challenge increased A20 messenger RNA levels appreciably in RAW264.7 cells within 1 h. Full-length A20 recombinant adenoviruses (pAdA20 1-775) suppressed NF-κB activity and TNF-α expression and protected against liver damage and animal death otherwise induced by trauma combined with LPS challenge. CONCLUSIONS: A20 zinc finger protein plays an anti-inflammatory role and protects against liver injury associated with trauma combined with LPS challenge.

The E3-ligase TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors.

Innate immunity conferred by the type I interferon is critical for antiviral defense. To date only a limited number of tripartite motif (TRIM) proteins have been implicated in modulation of innate immunity and anti-microbial activity. Here we report the complementary DNA cloning and systematic analysis of all known 75 human TRIMs. We demonstrate that roughly half of the 75 TRIM-family members enhanced the innate immune response and that they do this at multiple levels in signaling pathways. Moreover, messenger RNA levels and localization of most of these TRIMs were found to be altered during viral infection, suggesting that their regulatory activities are highly controlled at both pre- and posttranscriptional levels. Taken together, our data demonstrate a very considerable dedication of this large protein family to the positive regulation of the antiviral response, which supports the notion that this family of proteins evolved as a component of innate immunity.

EKLF/KLF1, a tissue-restricted integrator of transcriptional control, chromatin remodeling, and lineage determination.

Erythroid Krüppel-like factor (EKLF or KLF1) is a transcriptional regulator that plays a critical role in lineage-restricted control of gene expression. KLF1 expression and activity are tightly controlled in a temporal and differentiation stage-specific manner. The mechanisms by which KLF1 is regulated encompass a range of biological processes, including control of KLF1 RNA transcription, protein stability, localization, and posttranslational modifications. Intact KLF1 regulation is essential to correctly regulate erythroid function by gene transcription and to maintain hematopoietic lineage homeostasis by ensuring a proper balance of erythroid/megakaryocytic differentiation. In turn, KLF1 regulates erythroid biology by a wide variety of mechanisms, including gene activation and repression by regulation of chromatin configuration, transcriptional initiation and elongation, and localization of gene loci to transcription factories in the nucleus. An extensive series of biochemical, molecular, and genetic analyses has uncovered some of the secrets of its success, and recent studies are highlighted here. These reveal a multilayered set of control mechanisms that enable efficient and specific integration of transcriptional and epigenetic controls and that pave the way for proper lineage commitment and differentiation.

ZNF385B is characteristically expressed in germinal center B cells and involved in B-cell apoptosis.

We previously identified zinc finger (ZF) protein ZNF385B as a molecule specifically expressed in Burkitt's lymphoma (BL) among hematologic malignancies. Here, we investigated ZNF385B expression in healthy B cells in a variety of hematological tissues by RT-PCR and immunohistochemistry. ZNF385B expression was found to be limited to a subset of GC B cells, the healthy counterpart to BL B cells. To elucidate the function of ZNF385B in healthy B cells, we established a tetracycline-controlled protein-inducible system in B-cell lines and observed that ectopic expression of the longest transcript variant of ZNF385B, possessing four ZF domains, induced upregulation of PERP and FAS/CD95, a downstream target of p53, and activation of caspase, resulting in apoptosis induction. However, a ZNF385B deletion mutant with three ZF domains corresponding to shorter isoforms, did not induce upregulation; rather it inhibited apoptosis induced by CD20 cross-linking and BCR stimulation. The direct binding of ZNF385B with p53 has suggested the involvement of ZNF385B in B-cell apoptosis via modulation of p53 transactivation; our data indicate that ZNF385B characteristically expressed in GC B cells has both proapoptotic and antiapoptotic activities depending on the type of isoform and should be a novel player in GC B-cell selection.

Transcription factor zinc finger and BTB domain 1 is essential for lymphocyte development.

Absent T lymphocytes were unexpectedly found in homozygotes of a transgenic mouse from an unrelated project. T cell development did not progress beyond double-negative stage 1 thymocytes, resulting in a hypocellular, vestigial thymus. B cells were present, but NK cell number and B cell isotype switching were reduced. Transplantation of wild-type hematopoietic cells corrected the defect, which was traced to a deletion involving five contiguous genes at the transgene insertion site on chromosome 12C3. Complementation using bacterial artificial chromosome transgenesis implicated zinc finger BTB-POZ domain protein 1 (Zbtb1) in the immunodeficiency, confirming its role in T cell development and suggesting involvement in B and NK cell differentiation. Targeted disruption of Zbtb1 recapitulated the T(-)B(+)NK(-) SCID phenotype of the original transgenic animal. Knockouts for Zbtb1 had expanded populations of bone marrow hematopoietic stem cells and also multipotent and early lymphoid lineages, suggesting a differentiation bottleneck for common lymphoid progenitors. Expression of mRNA encoding Zbtb1, a predicted transcription repressor, was greatest in hematopoietic stem cells, thymocytes, and pre-B cells, highlighting its essential role in lymphoid development.