Development of a zona-free method of nuclear transfer in the mouse.

In the present study, a zona-free nuclear transfer (NT) technique, which had been originally developed in cattle, was modified for the mouse. Steps involved in this approach include removing the zona pellucida and enucleating without a holding pipette; sticking donor cells to the cytoplast before electric pulses are applied to fuse them and culturing reconstructed embryos individually in single droplets, to prevent aggregation. Control zona-free and zona-intact embryos from mated donors showed no significant difference in development to blastocyst, but did show reduced development to term. Removal of the zona pellucida affected the response to activation by strontium in the absence of calcium as a significant proportion of zona-free control oocytes and embryos reconstructed by NT lysed during this treatment. A comparison between cumulus and ES cells as donor cells revealed significant differences in fusion efficiency (58.1 +/- 4.0%, n = 573 vs. 42.9 +/- 2.2%, n = 2064, respectively, p < 0.001), cleavage (77.2 +/- 3.4%, n = 334 vs. 40.8 +/- 2.7%, n = 903, respectively, p < 0.001) but not for development to morula/blastocyst (8.7 +/- 2.1%, n = 334 vs. 13.9 +/- 1.8%, n = 903, respectively, p < 0.1). The stage at which embryo development arrested was also affected by donor cell type. A majority of embryos reconstructed from cumulus cells arrested at two-cell stage, usually with two nuclei, whereas those reconstructed from ES cells arrested at one-cell stage, usually with two pseudo-pronuclei. After transfer of ES cell-derived NT embryos, a viable cloned mouse was produced (3.0% of transferred embryos developed to term). These observations establish that a zona-free cloning approach is possible in the mouse, although further research is required to increase the efficiency.

TCRA gene rearrangement in immature thymocytes in absence of CD3, pre-TCR, and TCR signaling.

During thymocyte differentiation, TCRA genes are massively rearranged only after productively rearranged TCRB genes are expressed in association with pTalpha and CD3 complex molecules within a pre-TCR. Signaling from the pre-TCR via the CD3 complex is thought to be required to promote TCRA gene accessibility and recombination. However, alphabeta(+) thymocytes do develop in pTalpha-deficient mice, showing that TCRalpha-chain genes are rearranged, either in CD4(-)CD8(-) or CD4(+)CD8(+) thymocytes, in the absence of pre-TCR expression. In this study, we analyzed the TCRA gene recombination status of early immature thymocytes in mutant mice with arrested thymocyte development, deficient for either CD3 or pTalpha and gammac expression. ADV genes belonging to different families were found rearranged to multiple AJ segments in both cases. Thus, TCRA gene rearrangement is independent of CD3 and gammac signaling. However, CD3 expression was found to play a role in transcription of rearranged TCRalpha-chain genes in CD4(-)CD8(-) thymocytes. Taken together, these results provide new insights into the molecular control of early T cell differentiation.

Location analysis of DNA-bound proteins at the whole-genome level: untangling transcriptional regulatory networks.

In this post-sequencing era, geneticists can focus on functional genomics on a much larger scale than ever before. One goal is the discovery and elucidation of the intricate genetic networks that co-ordinate transcriptional activation in different regulatory circuitries. High-throughput gene expression measurement using DNA arrays has thus become routine strategy. This approach, however, does not directly identify gene loci that belong to the same regulatory group; e.g., those that are bound by a common (set of) transcription factor(s). Working in yeast, two groups have recently published an elegant method that could circumvent this problem, by combining chromatin immunoprecipitation and DNA microarrays.(1,2) The method is likely to provide a powerful tool for the dissection of global regulatory networks in eukaryotic cells.

Novel CD28-responsive enhancer activated by CREB/ATF and AP-1 families in the human interleukin-2 receptor alpha-chain locus.

The interaction of interleukin-2 (IL-2) with its receptor (IL-2R) critically regulates the T-cell immune response, and the alpha chain CD25/IL-2Ralpha is required for the formation of the high-affinity receptor. Tissue-specific, inducible expression of the IL-2Ralpha gene is regulated by at least three positive regulatory regions (PRRI, PRRII, and PRRIII), but none responded to CD28 engagement in gene reporter assays although CD28 costimulation strongly amplifies IL-2Ralpha gene transcription. By DNase I hypersensitivity analysis, we have identified a novel TCR-CD3- and CD28-responsive enhancer (CD28rE) located 8.5 kb 5' of the IL-2Ralpha gene. PRRIV/CD28rE contains a functional CRE/TRE element required for CD28 signaling. The T-cell-specific, CD28-responsive expression of the IL-2Ralpha gene appears controlled through PRRIV/CD28rE by cooperation of CREB/ATF and AP-1 family transcription factors.

Chromatin remodeling by the T cell receptor (TCR)-beta gene enhancer during early T cell development: Implications for the control of TCR-beta locus recombination.

Gene targeting studies have shown that T cell receptor (TCR)-beta gene expression and recombination are inhibited after deletion of an enhancer (Ebeta) located at the 3' end of the approximately 500-kb TCR-beta locus. Using knockout mouse models, we have measured, at different regions throughout the TCR-beta locus, the effects of Ebeta deletion on molecular parameters believed to reflect epigenetic changes associated with the control of gene activation, including restriction endonuclease access to chromosomal DNA, germline transcription, DNA methylation, and histone H3 acetylation. Our results demonstrate that, in early developing thymocytes, Ebeta contributes to major chromatin remodeling directed to an approximately 25-kb upstream domain comprised of the Dbeta-Jbeta locus regions. Accordingly, treatment of Ebeta-deleted thymocytes with the histone deacetylase inhibitor trichostatin A relieved the block in TCR-beta gene expression and promoted recombination within the Dbeta-Jbeta loci. Unexpectedly, however, epigenetic processes at distal Vbeta genes on the 5' side of the locus and at the 3' proximal Vbeta14 gene appear to be less dependent on Ebeta, suggesting that Ebeta activity is confined to a discrete region of the TCR-beta locus. These findings have implications with respect to the developmental control of TCR-beta gene recombination, and the process of allelic exclusion at this locus.

The Pim-1 kinase stimulates maturation of TCRbeta-deficient T cell progenitors: implications for the mechanism of Pim-1 action.

We demonstrate that overexpression of Pim-1, a cytoplasmic serine/threonine kinase of poorly defined function, results in the development of substantial numbers of CD4(+)CD8(+) double-positive thymocytes in two independent knock-out mouse models (i.e. the RAG-1-deficient and TCRbeta gene enhancer-deleted mice) in which production of a functionally rearranged TCRbeta gene (hence the pre-TCR) is impaired. This activity of Pim-1, however, does not affect signaling through the Ras/Raf/MAP kinase cascade nor signaling which mediates suppression of TCRbeta gene recombination (i.e. allelic exclusion). While overexpression of Pim-1 positively affects cell cycle progression in selected CD4(-)CD8(-) double-negative precursors, it did not affect expression of components of the cell cycle machinery, with the exception of the G(1)-specific phosphatase Cdc25A upon antigen receptor stimulation. We propose that Pim-1 acts downstream, or in parallel, to pre-TCR-mediated selection as one factor involved in the proliferative expansion of beta-selected pre-T cells.

T cell development in TCR beta enhancer-deleted mice: implications for alpha beta T cell lineage commitment and differentiation.

T cell differentiation in the mouse thymus is an intricate, highly coordinated process that requires the assembly of TCR complexes from individual components, including those produced by the precisely timed V(D)J recombination of TCR genes. Mice carrying a homozygous deletion of the TCR beta transcriptional enhancer (E beta) demonstrate an inhibition of V(D)J recombination at the targeted TCR beta locus and a block in alpha beta T cell differentiation. In this study, we have characterized the T cell developmental defects resulting from the E beta-/- mutation, in light of previously reported results of the analyses of TCR beta-deficient (TCR beta-/-) mice. Similar to the latter mice, production of TCR beta-chains is abolished in the E beta-/- animals, and under these conditions differentiation into cell-surface TCR-, CD4+CD8+ double positive (DP) thymocytes depends essentially on the cell-autonomous expression of TCR delta-chains and, most likely, TCR gamma-chains. However, contrary to previous reports using TCR beta-/- mice, a minor population of TCR gamma delta+ DP thymocytes was found within the E beta-/- thymi, which differ in terms of T cell-specific gene expression and V(D)J recombinase activity, from the majority of TCR-, alpha beta lineage-committed DP thymocytes. We discuss these data with respect to the functional role of E beta in driving alpha beta T cell differentiation and the mechanism of alpha beta T lineage commitment.

TCRalpha enhancer activation occurs via a conformational change of a pre-assembled nucleo-protein complex.

The TCR alpha enhancer (Ealpha) has served as a paradigm for studying how enhancers organize trans-activators into nucleo-protein complexes thought to recruit and synergistically stimulate the transcriptional machinery. Little is known, however, of either the extent or dynamics of Ealpha occupancy by nuclear factors during T cell development. Using dimethyl sulfate (DMS) in vivo footprinting, we demonstrate extensive Ealpha occupancy, encompassing both previously identified and novel sites, not only in T cells representing a developmental stage where Ealpha is known to be active (CD4(+)CD8(+)-DP cells), but surprisingly, also in cells at an earlier developmental stage where Ealpha is not active (CD4(-)CD8(-)-DN cells). Partial occupancy was also established in B-lymphoid but not non-lymphoid cells. In vivo DNase I footprinting, however, implied developmentally induced changes in nucleo-protein complex topography. Stage-specific differences in factor composition at Ealpha sequences were also suggested by EMSA analysis. These results, which indicate that alterations in the structure of a pre-assembled nucleo-protein complex correlate with the onset of Ealpha activity, may exemplify one mechanism by which enhancers can rapidly respond to incoming stimuli.

Definition of a T-cell receptor beta gene core enhancer of V(D)J recombination by transgenic mapping.

V(D)J recombination in differentiating lymphocytes is a highly regulated process in terms of both cell lineage and the stage of cell development. Transgenic and knockout mouse studies have demonstrated that transcriptional enhancers from antigen receptor genes play an important role in this regulation by activating cis-recombination events. A striking example is the T-cell receptor beta-chain (TCRbeta) gene enhancer (Ebeta), which in the mouse consists of at least seven nuclear factor binding motifs (betaE1 to betaE7). Here, using a well-characterized transgenic recombination substrate approach, we define the sequences within Ebeta required for recombination enhancer activity. The Ebeta core is comprised of a limited set of motifs (betaE3 and betaE4) and an additional previously uncharacterized 20-bp sequence 3' of the betaE4 motif. This core element confers cell lineage- and stage-specific recombination within the transgenic substrates, although it cannot bypass the suppressive effects resulting from transgene integration in heterochromatic centromeres. Strikingly, the core enhancer is heavily occupied by nuclear factors in immature thymocytes, as shown by in vivo footprinting analyses. A larger enhancer fragment including the betaE1 through betaE4 motifs but not the 3' sequences, although active in inducing germ line transcription within the transgenic array, did not retain the Ebeta recombinational activity. Our results emphasize the multifunctionality of the TCRbeta enhancer and shed some light on the molecular mechanisms by which transcriptional enhancers and associated nuclear factors may impact on cis recombination, gene expression, and lymphoid cell differentiation.

The lack of chromosomal protein Hmg1 does not disrupt cell growth but causes lethal hypoglycaemia in newborn mice.

High mobility group 1 (HMG1) protein is an abundant component of all mammalian nuclei, and related proteins exist in all eukaryotes. HMG1 binds linear DNA with moderate affinity and no sequence specificity, but bends the double helix significantly on binding through the minor groove. It binds with high affinity to DNA that is already sharply bent, such as linker DNA at the entry and exit of nucleosomes; thus, it is considered a structural protein of chromatin. HMG1 is also recruited to DNA by interactions with proteins required for basal and regulated transcriptions and V(D)J recombination. Here we generate mice harbouring deleted Hmg1. Hmg1-/- pups are born alive, but die within 24 hours due to hypoglycaemia. Hmg1-deficient mice survive for several days if given glucose parenterally, then waste away with pleiotropic defects (but no alteration in the immune repertoire). Cell lines lacking Hmg1 grow normally, but the activation of gene expression by the glucocorticoid receptor (GR, encoded by the gene Grl1) is impaired. Thus, Hmg1 is not essential for the overall organization of chromatin in the cell nucleus, but is critical for proper transcriptional control by specific transcription factors.

Differential gene expression in CD3epsilon- and RAG1-deficient thymuses: definition of a set of genes potentially involved in thymocyte maturation.

A set of 3000 mouse thymus cDNAs was analyzed by extensive measurement of expression using complex-probe hybridization of DNA arrays ("quantitative differential screening"). The complex probes were initially prepared using total thymus RNA isolated from C57BL/6 wild-type (WT), CD3epsilon- and RAG1-deficient mice. Over 100 clones displaying over- or under-expression by at least a factor of two between WT and knockout (KO) thymuses were further analyzed by measuring hybridization signatures with probes from a wide range of KO thymuses, cell types, organs, and embryonic thymuses. A restricted set of clones was selected by virtue of their expression spectra (modulation in KO thymuses and thymocytes, lymphoid cell specificity, and differential expression during embryonic thymus development), sequenced at one extremity, and compared to sequences in databases. Clones corresponding to previously identified genes (e.g., Tcrbeta, Tcf1 or CD25) showed expression patterns that were consistent with existing data. Ten distinct clones corresponding to new genes were subjected to further study: Northern blot hybridization, in situ hybridization on thymus sections, and partial or complete mRNA sequence determination. Among these genes, we report a new serine peptidase highly expressed in cortical epithelial cells that we have named thymus-specific serine peptidase (TSSP), and an acidic protein expressed in thymocytes and of unknown function that we have named thymus-expressed acidic protein (TEAP). This approach identifies new molecules likely to be involved in thymocyte differentiation and function.

Enhancer control of V(D)J recombination at the TCRbeta locus: differential effects on DNA cleavage and joining.

Deletion of the TCRbeta transcriptional enhancer (Ebeta) results in nearly complete inhibition of V(D)J recombination at the TCRbeta locus and a block in alpha beta T cell development. This result, along with previous work from many laboratories, has led to the hypothesis that transcriptional enhancers affect V(D)J recombination by regulating the accessibility of the locus to the recombinase. Here we test this hypothesis by performing a detailed analysis of the recombination defect in Ebeta-deleted (Ebeta-/-) mice using assays that detect various reaction intermediates and products. We found double-strand DNA breaks at recombination signal sequences flanking Dbeta and Jbeta gene segments in Ebeta-/- thymuses at about one-third to one-thirtieth the level found in thymuses with an unaltered TCRbeta locus. These sites are also subject to in vitro cleavage by the V(D)J recombinase in both Ebeta-/- and Ebeta+/+ thymocyte nuclei. However, the corresponding Dbeta-to-Jbeta coding joints are further reduced (by 100- to 300-fold) in Ebeta-/- thymuses. Formation of extrachromosomal Dbeta-to-Jbeta signal joints appears to be intermediately affected and nonstandard Dbeta-to-Dbeta joining occurs at the Ebeta-deleted alleles. These data indicate that, unexpectedly, loss of accessibility alone cannot explain the loss of TCRbeta recombination in the absence of the Ebeta element and suggest an additional function for Ebeta in the process of DNA repair at specific TCRbeta sites during the late phase of the recombination reaction.

Transcriptional control during T-cell development.

During the past few years, the essential role of distinct transcription factors in specifying cell-fate decisions in a stepwise fashion during T-cell differentiation has been revealed. One striking feature is that a single factor can act at several sites throughout T-cell development, possibly through interactions with different partners. The challenge is now to understand how these interactions can account for the co-ordination of complex extracellular signals and gene expression programs, such as those involved in T-cell receptor gene recombination and expression.

Natural killer cell acceptance of H-2 mismatch bone marrow grafts in transgenic mice expressing HLA-Cw3 specific killer cell inhibitory receptor.

Natural killer (NK) cells express killer cell inhibitory receptors (KIRs) for major histocompatibility complex class I molecules. Engagement of these surface receptors inhibits NK cell cytotoxic programs. KIR can also be expressed on T cell subsets, and their engagement similarly results in inhibition of effector functions initiated by the CD3/T cell receptor complex. KIR genes belong to two distinct families: the immunoglobulin superfamily (IgSF KIRs) and dimeric C2 lectins (lectin-like KIRs). Whereas both IgSF (p58: CD158, p70, and p140) and lectin-like KIRs (CD94/NKG2A heterodimers) have been found in human, only lectin-like KIRs (all members of the Ly-49 family) have been described in the mouse. We have generated transgenic mice expressing an IgSF KIR, CD158b (p58.2), which recognizes HLA-Cw3. Our data show that CD158b is necessary and sufficient to confer specificity to NK cells, as well as to modulate T cell activation programs in vitro. In addition, we did not detect any adaptation of CD158b cell surface expression to that of HLA class I ligands in the CD158b x HLA-Cw3 double transgenic mice, in contrast to observations with Ly-49 in the mouse. Therefore, distinct strategies of selection/calibration appear to be used by IgSF and lectin-like KIRs. Finally, the transgenic expression of CD158b KIR prevents the in vivo rejection of H-2 mismatch bone marrow grafts, which express the cognate major histocompatibility class I HLA-Cw3 allele, demonstrating for the first time the in vivo implication of human IgSF KIRs in the negative regulation of NK cell function.

A dominant-negative mutant of the Rab5 GTPase enhances T cell signaling by interfering with TCR down-modulation in transgenic mice.

TCR triggering results in the down-modulation of engaged receptors by endocytosis. As a result of this process, Ag-binding sites are depleted from the surface and signaling responses should be attenuated. To test the importance of TCR down-regulation on T cell signaling, we generated mice expressing a dominant-negative form of Rab5 (Rab5N133I) in T cells. Rab5, a monomeric GTPase of the Ras superfamily, has been implicated in the regulation of early steps in the endocytic pathway. In Rab5N133I mice, mature thymocytes developed, but the absolute number of CD4+CD8+ double positive thymocytes was reduced. Fluid phase endocytosis was severely impaired in the transgenic thymocytes. In peripheral T cells, the kinetics and rate of ligand-induced TCR down-modulation were delayed and reduced. These effects were correlated with enhanced early and late signaling responses. Analysis of thymocyte development in doubly transgenic mice for Rab5N133I and a lymphocytic choriomeningitis virus (LCMV) peptide-specific TCR demonstrated that TCR signaling was enhanced by dominant inhibition of Rab5 function, resulting in altered thymic selection. These findings suggest that TCR endocytosis is an important regulatory component of TCR signaling and that defects in this regulation can result in prolonged signaling and alter thymic development.

Defect in rearrangement of the most 5' TCR-J alpha following targeted deletion of T early alpha (TEA): implications for TCR alpha locus accessibility.

To address the role of the TEA germline transcription, which initiates upstream of the TCR-J alpha S, in the regulation of TCR-J alpha locus accessibility, we created a mouse in which this region has been removed by homologous recombination. Normal development of T alpha beta cells and the expression of other TCR alpha germline transcripts in TEA-/- mice ruled out an exclusive role for TEA in the overall accessibility of the J alpha cluster. However, the rearrangement of the most 5' J alpha (J alpha 61 to J alpha 53) was severely impaired, indicating that TEA may control the DNA accessibility of a particular J alpha window. Moreover, the relative usage of every J alpha segment was affected. These results are consistent with TEA acting as a "rearrangement-focusing" element, targeting the primary waves of V alpha-J alpha recombination to the most 5' J alpha S in an ongoing TCR-J alpha rearrangement model.

Deletion of the mouse T-cell receptor beta gene enhancer blocks alphabeta T-cell development.

Intrathymic T-cell development requires temporally regulated rearrangement and expression of T-cell receptor (TCR) genes. To assess the role of the TCR beta gene transcriptional enhancer (Ebeta) in this process, mouse strains in which Ebeta is deleted were generated using homologous recombination techniques. We report that mice homozygous for the Ebeta deletion, whether a selectable marker gene is present or not, show a block in alphabeta T-cell development at the CD4-CD8- double-negative cell stage, whereas the number of gammadelta+ T cells is normal, few CD4+CD8+ double-positive thymocytes and no alphabeta+ T cells are produced. DNA-PCR and RNA-PCR analyses of thymic cells from homozygous mutants showed no evidence of TCR beta gene rearrangement although germ-line Vbeta transcripts were detected at a low level, in heterozygous T cells, the targeted allele is not rearranged. Thus, deletion of Ebeta totally prevents rearrangement, but not transcription, of the targeted beta locus. These data formally establish the critical role played by Ebeta in cis-activation of the TCR beta locus for V(D)J recombination during alphabeta T-cell development.

Altered T cell development in mice with a targeted mutation of the CD3-epsilon gene.

To determine which CD3 components are required for early T cell development, we generated mice with a targeted mutation of the CD3-epsilon gene and characterized their T cell populations relative to those found in CD3-zeta/eta-and recombinase activating gene (RAG)-deficient mice. In the absence of intact CD3-epsilon subunit, thymocytes do not progress beyond the CD44-/lowCD25+ triple-negative stage and appear to be arrested at the very same developmental control point as RAG-deficient thymocytes. In contrast, the disruption of the CD3-epsilon/eta gene does not totally abrogate the progression through this control point. CD3-epsilon-deficient thymocytes do rearrange their T cell receptor (TCR) beta gene segments and produce low levels of full-length TCR beta transcripts. Taken together, these results establish an essential role for the CD3-epsilon gene products during T cell development and further suggest that the CD3-epsilon polypeptides start to exert their function as part of a pre-TCR through which CD44-/lowCD25+ triple-negative cells monitor the occurrence of productive TCR beta gene rearrangements. Finally, the absence of intact CD3-epsilon polypeptides had no discernible effect on the completion of TCR gamma and TCR delta gene rearrangements, emphasizing that they are probably not subjected to the same epigenetic controls as those operating on the expression of TCR alpha and beta genes.