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Biomedical subjects

E M Oltz

Publications and source records attributed to E M Oltz.

At least 19 recordsLinked to original sources
PubMed2000

A switch in distinct I kappa B alpha degradation mechanisms mediates constitutive NF-kappa B activation in mature B cells.

Inducible activation of cytoplasmic NF-kappa B/Rel transcription factors occurs via proteasome-dependent degradation of an associated inhibitor, termed I kappa B alpha. Mature B lymphocytes constitutively express nuclear NF-kappa B, which is important for their long-term survival. The intrinsic mechanisms by which B cells constitutively activate NF-kappa B are unknown. In this paper we demonstrate that maintenance of NF-kappa B activity in primary B cells is mediated by a novel calcium-dependent, but proteasome-independent, mechanism. Moreover, we show that differentiation of conditionally transformed pre-B cells is accompanied by a switch from proteasome-dependent to proteasome-independent degradation of I kappa B alpha. Our findings indicate that I kappa B alpha degradation mechanisms are dynamic during B cell development, and ultimately establish constitutive NF-kappa B activity in mature B lymphocytes.

Animals↗
PubMed2000

Fus deficiency in mice results in defective B-lymphocyte development and activation, high levels of chromosomal instability and perinatal death.

The gene FUS (also known as TLS (for translocated in liposarcoma) and hnRNP P2) is translocated with the gene encoding the transcription factor ERG-1 in human myeloid leukaemias. Although the functions of wild-type FUS are unknown, the protein contains an RNA-recognition motif and is a component of nuclear riboprotein complexes. FUS resembles a transcription factor in that it binds DNA, contributes a transcriptional activation domain to the FUS-ERG oncoprotein and interacts with several transcription factors in vitro. To better understand FUS function in vivo, we examined the consequences of disrupting Fus in mice. Our results indicate that Fus is essential for viability of neonatal animals, influences lymphocyte development in a non-cell-intrinsic manner, has an intrinsic role in the proliferative responses of B cells to specific mitogenic stimuli and is required for the maintenance of genomic stability. The involvement of a nuclear riboprotein in these processes in vivo indicates that Fus is important in genome maintenance.

Animals↗
PubMed2000

Cloning and functional characterization of the early-lymphocyte-specific Pb99 gene.

The Pb99 gene is specifically expressed in pre-B cells and thymocytes and not in mature B and T cells or nonlymphoid tissues, implying that it may function in early lymphoid development. We have previously described the cloning of an incomplete cDNA for Pb99. Here we report the isolation of full-length cDNAs and genomic clones for the murine Pb99 gene and the mapping of its location to mouse chromosome 8. Sequence analyses of different Pb99 cDNA clones suggest that there may be at least three forms of the Pb99 protein generated by differential processing of the Pb99 transcript. The cDNA with the longest open reading frame encodes a putative protein that has seven hydrophobic domains similar to those of seven membrane-spanning proteins, such as the classical G protein-coupled receptors. To directly address the role of the Pb99 protein in lymphoid development, Pb99-deficient mice were generated by gene targeting, and lymphocyte development in these mice was analyzed.

Amino Acid Sequence↗
PubMed1999

An inducible cell model for studies of V(D)J recombinational control.

Antigen receptor gene assembly is controlled by enhancer-directed changes in the accessibility of chromosomal gene segments to V(D)J recombinase. To dissect mechanisms that regulate rearrangement efficiencies, we developed a cell system (TDR19) in which recombination activating gene (RAG) expression is repressed by tetracycline. Under conditions of RAG repression, recombination substrates were consistently integrated into the TDR19 genome in an unrearranged form. Subsequent rearrangement of chromosomal substrates containing a transcriptional enhancer correlated inversely with tetracycline concentrations. Together, these features underscore the utility of TDR19 as a cell model for defining the molecular determinants of V(D)J recombinational accessibility.

Cell Line↗
PubMed1999

An intact NF-kappa B signaling pathway is required for maintenance of mature B cell subsets.

Members of the NF-kappaB/Rel transcription factor family are expressed constitutively during B cell development and are further induced by mitogen activation. Mice harboring germline disruptions in individual NF-kappaB subunits exhibit distinct defects in B lymphocyte activation and survival. However, the role of NF-kappaB in the production and maintenance of B cell subsets has been difficult to dissect in these knockout animals due to functional impairment of other immune cells. To directly address the cell autonomous requirements for NF-kappaB in humoral immune compartments, transgenic mice were generated that express a transdominant form of Ikappa-Balpha in B lineage cells. Whereas expression of the inhibitor had only modest effects on basal or LPS-induced levels of NF-kappaB, transgenic B cells were significantly impaired for cellular proliferation and NF-kappaB induction in response to B cell receptor (BCR) crosslinking. Furthermore, the trans-dominant inhibitor produced a dose-dependent reduction in the population of mature splenic B cells. This cellular defect was more pronounced in long-lived B lymphocyte subsets that recirculate to the adult bone marrow. Together, these results indicate that BCR-mediated signaling must maintain NF-kappaB levels above a stringent threshold for proper regulation of B cell homeostasis.

Animals↗
PubMed1999

Regulation of V(D)J recombination by transcriptional promoters.

Enhancer elements potentiate the rearrangement of antigen receptor loci via changes in the accessibility of gene segment clusters to V(D)J recombinase. Here, we show that enhancer activity per se is insufficient to target T-cell receptor beta miniloci for DbetaJbeta recombination. Instead, a promoter situated 5' to Dbeta1 (PDbeta) was required for efficient rearrangement of chromosomal substrates. A critical function for promoters in regulating gene segment accessibility was further supported by the ability of heterologous promoters to direct rearrangement of enhancer-containing substrates. Importantly, activation of a synthetic tetracycline-inducible promoter (Ptet) positioned upstream from the Dbeta gene segment was sufficient to target recombination of miniloci lacking a distal enhancer element. The latter result suggests that DNA loops, generated by interactions between flanking promoter and enhancer elements, are not required for efficient recognition of chromosomal gene segments by V(D)J recombinase. Unexpectedly, the Ptet substrate exhibited normal levels of rearrangement despite its retention of a hypermethylated DNA status within the DbetaJbeta cluster. Together, our findings support a model in which promoter activation, rather than intrinsic properties of enhancers, is the primary determinant for regulating recombinational accessibility within antigen receptor loci.

DNA Methylation↗
PubMed1998

A developmental stage-specific promoter directs germline transcription of D beta J beta gene segments in precursor T lymphocytes.

The tissue- and stage-specific assembly of Ag receptor genes is regulated by transcriptional control elements positioned within Ig and TCR loci. To further understand the role of cis-acting elements in these regulatory mechanisms, we have characterized a transcriptional promoter that drives germline expression of TCR beta gene segments in vivo. The activity of this promoter, termed PD beta, is restricted to a highly conserved 400-bp region located directly upstream from D beta 1-coding sequences. Maximal PD beta activity requires a TATA element situated within the D beta 1 recombination signal sequences and consensus binding sites for the ubiquitous SP1 and the T cell-specific GATA-3 transcription factors. When linked to active enhancer elements, PD beta directs transcription in most cell types; however, the TCR beta enhancer (E beta) stimulates PD beta function specifically in precursor T lymphocytes. These findings suggest that PD beta/E beta interactions may contribute to differential regulation of regions within the TCR beta locus during thymocyte development.

Animals↗
PubMed1997

Transcription factor NF-kappaB regulates inducible Oct-2 gene expression in precursor B lymphocytes.

The POU transcription factors Oct-1 and Oct-2 regulate the activity of octamer-dependent promoters, including those that direct transcription from rearranged immunoglobulin genes. Unlike Oct-1, which is constitutively expressed in many cell types, Oct-2 expression is restricted primarily to B lymphocytes and can be induced in precursor B cells by stimulation with bacterial lipopolysaccharide (LPS). However, the precise factors that mediate this induction mechanism remain unknown. In the present study, we monitored Oct-2 expression in cells arrested for the activation of NF-kappaB, an LPS-responsive member of the Rel transcription factor family. Despite stimulation with LPS, disruption of the NF-kappaB signaling pathway in precursor B cells led to the loss of inducible Oct-2 DNA binding activity in vitro and the suppression of Oct-2-directed transcription in vivo. This biochemical defect correlated with a specific block to Oct-2 gene expression at the level of transcription, whereas the expression of Oct-1 was unaffected. The finding that Oct-2 is under NF-kappaB control highlights an important cross-talk mechanism involving two distinct transcription factor families that regulate B lymphocyte function.

B-Lymphocytes↗
PubMed1997

Coordinate transcription and V(D)J recombination of the kappa immunoglobulin light-chain locus: NF-kappaB-dependent and -independent pathways of activation.

To further elucidate the potential role of mitogens and cytokines in regulation of the kappa immunoglobulin light-chain locus, we have characterized the activation of transcription factor binding, kappa germ line transcription, DNase I hypersensitivity, and Vkappa-to-Jkappa recombination upon induction of model pre-B-cell lines. We find that both lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) are capable of activating germ line transcription, DNase I hypersensitivity, and recombination of the kappa locus. We also find that transforming growth factor beta is capable of completely inhibiting LPS activation of transcription and recombination but has no apparent effect on activation of transcription factor binding, including activation of NF-kappaB. To address the functional role of NF-kappaB in LPS and IFN-gamma induction of these events, we blocked the nuclear translocation of NF-kappaB by overexpression of a dominant negative mutant of IkappaB-alpha (IkappaB deltaN). Overexpression of the IkappaB deltaN protein results in an inhibition of LPS but not IFN-gamma activation of germ line transcription, DNase I hypersensitivity, and Vkappa-to-Jkappa recombination. Our results demonstrate that activation of NF-kappaB is necessary but not sufficient for LPS activation of transcription and recombination at kappa. These results also suggest that NF-kappaB is not required for IFN-gamma activation of transcription or recombination. These results are important in establishing that there are multiple independent pathways of activation of both transcription and recombination.

B-Lymphocytes↗
PubMed1996

Corepression of RelA and c-rel inhibits immunoglobulin kappa gene transcription and rearrangement in precursor B lymphocytes.

Multiple members of the NF-kappa B/Rel protein family are induced during B cell differentiation and have been implicated in transcriptional activation of the immunoglobulin kappa (Ig kappa) locus. Despite these findings, normal numbers of Ig kappa + B lymphocytes are produced by mice bearing targeted mutations in individual NF-kappa B/Rel genes. In the present study, precursor B lymphocytes were engineered to express a trans-dominant form of I kappa B alpha that simultaneously impairs the c-Rel and RelA transactivating subunits of NF-kappa B. This dual block in NF-kappa B/Rel signaling led to potent inhibition of germline Ig kappa transcription and rearrangement, whereas recombinase activity was unaffected. These findings suggest that c-Rel and RelA serve compensatory functional roles in the developmental mechanisms that govern Ig kappa gene assembly.

Animals↗
PubMed1995

Promotion of V(D)J recombinational accessibility by the intronic E kappa element: role of the kappa B motif.

The accessibility of a chromosomally integrated TCR beta minilocus recombination substrate in a V(D)J recombinase-inducible cell line (HDR37) depends on incorporation of transcriptional enhancer elements such as the Ig kappa light chain intronic enhancer (E kappa). The E kappa element contains several functional motifs including the kappa B motif, which binds the NF-kappa B transcription factor. To assess molecular mechanisms by which E kappa promotes V(D)J recombinational accessibility, we compared the abilities of the wild-type E kappa, a corresponding E kappa sequence with a mutant kappa B motif (E kappa-kappa B-) and a kappa B motif dimer (kappa B2) to function in the context of the TCR beta minilocus/HDR37 system. The E kappa-containing minilocus underwent demethylation, transcription and V(D)J recombination, independently of copy number of integration site. Transfectants containing low copy numbers (one or two) of the E kappa-kappa B(-)-containing minilocus, like enhancerless or kappa B2-containing miniloci at any copy number, were inactive with respect to all three processes. In contrast, high-copy-number integrants of the E kappa-kappa B- substrates showed an integration-site dependent activation of all three processes. Together these data show that the kappa B motif plays a critical role in the ability of E kappa to confer V(D)J recombinational accessibility, but that it is not sufficient to mediate this process by itself.

Animals↗
PubMed1993

LH-2: a LIM/homeodomain gene expressed in developing lymphocytes and neural cells.

A screen for early markers of B-lymphocyte differentiation has identified a homeobox gene, denoted LH-2, that has a pattern of expression distinct from that of other related genes. The LH-2 cDNA sequence encodes a polypeptide of 426 amino acids that contains a homeodomain and two repeats of a cysteine-rich domain referred to as a LIM domain. The homeodomain of the LH-2 protein is related to that of other LIM/homeodomain proteins, most strikingly with that of the Drosophila apterous protein. Expression of LH-2 was found in B- and T-lymphoid cell lines. Expression in B-cell lines was highest in lines that represent early stages of differentiation, whereas in T-cell lines there was no clear correlation with the stage of differentiation. In embryonic and adult tissues, the highest level of LH-2 expression was found in discrete regions of the developing central nervous system, primarily in diencephalic and telencephalic structures, and in a subset of lymphoid tissues. The expression pattern and structural characteristics of the LH-2 gene suggest that it encodes a transcriptional regulatory protein involved in the control of cell differentiation in developing lymphoid and neural cell types.

Aging↗
PubMed1993

Comparison of RAG gene expression in normal and transformed precursor lymphocytes.

Analyses of mechanisms that regulate V(D)J recombination have relied heavily on the use of transformed precursor lymphocyte cell lines. We now show that such lines have highly variable and frequently low levels of recombination activating genes (RAG)-1 and -2 gene expression. We also show that expression levels of the RAG genes can vary > 100-fold between different subcloned cells of an individual pre-B line. We discuss these findings in the context of normal regulation of RAG gene expression and the implication for the use of transformed pre-B cell lines as models for studying control of V(D)J recombination activity.

Animals↗
PubMed1993

A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination.

Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.

Animals↗
PubMed1992

RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement.

We have generated mice that carry a germline mutation in which a large portion of the RAG-2 coding region is deleted. Homozygous mutants are viable but fail to produce mature B or T lymphocytes. Very immature lymphoid cells were present in primary lymphoid organs of mutant animals as defined by surface marker analyses and Abelson murine leukemia virus (A-MuLV) transformation assays. However, these cells did not rearrange their immunoglobulin or T cell receptor loci. Lack of V(D)J recombination activity in mutant pre-B cell lines could be restored by introduction of a functional RAG-2 expression vector. Therefore, loss of RAG-2 function in vivo results in total inability to initiate V(D)J rearrangement, leading to a novel severe combined immune deficient (SCID) phenotype. Because the SCID phenotype was the only obvious abnormality detected in RAG-2 mutant mice, RAG-2 function and V(D)J recombinase activity, per se, are not required for development of cells other than lymphocytes.

Animals↗
PubMed1992

A novel regulatory myosin light chain gene distinguishes pre-B cell subsets and is IL-7 inducible.

We describe a novel regulatory myosin light chain gene (termed precursor lymphocyte-specific regulatory light chain or PLRLC) that is expressed specifically in precursor B and T lymphocytes. PLRLC is the first example of a regulatory myosin light chain gene which displays specific expression in non-muscle cells. PLRLC is expressed in adult bone marrow derived normal and transformed pre-B cells; in the former, PLRLC expression levels are induced by the pre-B cell specific growth factor interleukin-7 (IL-7). PLRLC is not expressed in either transformed pre-B cells derived from fetal liver or in normal fetal liver pre-B clones grown in the presence of IL-7. Therefore this gene provides the first marker that clearly distinguishes these two pre-B subsets. Finally, several of the different PLRLC transcripts potentially encode regulatory myosin light chains with unique structural features. The unique distribution, regulation and structural features of the PLRLC gene products suggest an important role for PLRLC during lymphocyte development.

Amino Acid Sequence↗
PubMed1992

VDJ recombination.

The ability of lymphocyte receptor V, D and J gene segments to rearrange generates much of the receptor diversity that is the hallmark of the immune system. Naturally, the mechanisms of immunoglobulin and T-cell receptor gene recombination are of enormous interest. Here, Fred Alt and colleagues review current understanding of the process and speculate on future findings.

Animals↗