TOE1 is an inhibitor of HIV-1 replication with cell-penetrating capability.

Target of Egr1 (TOE1) is a nuclear protein localized primarily in nucleoli and Cajal bodies that was identified as a downstream target of the immediate early gene Egr1. TOE1 displays a functional deadenylation domain and has been shown to participate in spliceosome assembly. We report here that TOE1 can function as an inhibitor of HIV-1 replication and show evidence that supports a direct interaction of TOE1 with the viral specific transactivator response element as part of the inhibitory mechanism. In addition, we show that TOE1 can be secreted by activated CD8(+) T lymphocytes and can be cleaved by the serine protease granzyme B, one of the main components of cytotoxic granules. Both full-length and cleaved TOE1 can spontaneously cross the plasma membrane and penetrate cells in culture, retaining HIV-1 inhibitory activity. Antiviral potency of TOE1 and its cell-penetrating capability have been identified to lie within a 35-amino-acid region containing the nuclear localization sequence.

Aberrant production of IL-13 by T cells promotes exocrinopathy in Id3 knockout mice.

Elevated levels of the cytokine IL-13 has been found to be associated with autoimmune diseases, including Sjögren's Syndrome. However, whether IL-13 plays a causative role in disease development is not known and cannot be easily studied in humans. Our previous work has shown that levels of IL-13 are elevated in Id3 knockout mice, which has been established as a model for primary Sjögren's Syndrome. Here, we utilized an IL-13 reporter to determine the source of the elevated IL-13 levels observed in Id3 knockout mice and assess its contribution to SS pathology. Our results indicate that T cells, notably CD4 and γδ T cells, in Id3 knockout mice acquire IL-13 competency at an elevated rate well before disease symptoms become apparent. We also show that T cells developing early in life are more predisposed to produce IL-13. Finally, analysis of Id3 and IL-13 double deficient mice demonstrated that IL-13 plays an essential role in the deterioration of gland function. Our study provides crucial genetic evidence that enhanced IL-13 production by T cells can play a causative role in the exocrinopathy observed in Id3 knockout mice.

Skeletal Muscle Is an Antigen Reservoir in Integrase-Defective Lentiviral Vector-Induced Long-Term Immunity.

We previously developed integrase-defective lentiviral vectors (IDLVs) as an antigen delivery system for inducing strong and prolonged immunity in animal models. Here, we examined the association between persistence of antigen expression and durability of immune response. Following a single intramuscular (i.m.) or subcutaneous (s.c.) injection of IDLV delivering GFP in mice, we evaluated antigen expression and inflammation at the site of injection and persistence of antigen-specific T cells at early and late time points. Durable antigen expression was detected up to 90 days only after i.m. immunization. Mononuclear inflammation was evident soon after IDLV injection in both i.m. and s.c. immunized mice, but remained detectable up to 30 days postinjection only in i.m. immunized mice. Similarly, GFP-specific T cells were more persistent in the i.m. immunized mice. Interestingly, GFP+ muscle fibers were co-expressing major histocompatibility complex (MHC) class I, suggesting that muscle cells are competent for presenting antigens to T cells in vivo. In in vitro experiments, we demonstrated that although both primary myoblasts and myocytes present the antigen to GFP-specific T cells through MHC class I, myoblasts are more resistant to Fas-dependent cytotoxic T lymphocyte (CTL) killing activity. Overall, these data indicate that muscle cells may serve as an antigen reservoir that contributes to the long-term immunity induced by IDLV vaccination.

The transcription factor Egr1 regulates the HIF-1alpha gene during hypoxia.

Using oligonucleotide expression microarrays we have examined the modulation of gene expression in the DU145 prostate cancer cell line. Our findings confirm that the Egr1 transcription factor is rapidly and transiently upregulated by hypoxia. Furthermore, we have demonstrated that HIF-1alpha mRNA is also transiently upregulated, as is its target gene VEGF. To elucidate the mechanism of the transcriptional upregulation of the HIF-1alpha gene, we have shown that Egr1 is able to directly bind to the HIF-1alpha promoter using chromatin immunoprecipitation. We also provide evidence that the binding of Egr1 is necessary for the trans-activation of the HIF-1alpha promoter. These studies highlight the importance for the Egr1 transcription factor in the hypoxic response in cultured prostate cancer cell lines, and indicate that the response of Egr1 is upstream of HIF-1 in these cells. These studies are the first demonstration that the HIF-1alpha transcription factor is targeted directly by Egr1 in hypoxia.

Limited allelic diversity of stimulatory two-domain killer cell immunoglobulin-like receptors.

Genomic sequencing was used to characterize most of the coding regions of the five two-domain stimulatory killer cell immunoglobulin-like receptor (KIR) loci from 80 unrelated, primarily Caucasian, individuals. Specific loci were present in from 26% (KIR2DS3) to 98% (KIR2DS4) of individuals. The number of known alleles present varied from one (KIR2DS1, KIR2DS5) to five (KIR2DS4). The frequencies of loci and alleles were similar to observations made in populations of European and Asian ethnicities. New alleles were found at 2DS1 (*00202, *00302, *005, *006, *007) and 2DS4 (*008) loci.

Daxx represses expression of a subset of antiapoptotic genes regulated by nuclear factor-kappaB.

Daxx is a nuclear protein that localizes to PML oncogenic domains, sensitizes cells to apoptosis, and functions as a transcriptional repressor. We found that Daxx represses the expression of several antiapoptotic genes regulated by nuclear factor-kappaB, including cIAP2, in human tumor cell lines. Daxx interacts with RelB and inhibits RelB-mediated transcriptional activation of the human cIAP2 gene promoter. Daxx also forms complexes with RelB while bound to its target sites in the cIAP2 promoter, as shown by electrophoretic mobility shift assays and chromatin immunoprecipitation experiments. Using cells from daxx-/- mouse embryos, we observed that levels of the corresponding murine c-IAP mRNA and protein are increased in cells lacking Daxx. Conversely, c-IAP mRNA and protein levels were reduced in relB-/- cells. Taken together, these observations provide a mechanism that links two previously ascribed functions of Daxx: transcriptional repression and sensitization to apoptosis.

HLA haplotypes in Singapore: a study of mothers and their cord blood units.

During 2005, a total of 174 cord blood units with their paired maternal samples from the Singapore Cord Blood Bank were typed for HLA-A, -B, -C at intermediate resolution and DRB1 at allelic resolution. Analysis of allele segregation in mother and child assigned 185 different four locus (HLA-A, -B, -C, -DRB1) haplotypes in Chinese, 66 in Malays, and 34 in Asian Indians. Very few four locus haplotypes were shared among population groups. To evaluate the frequencies of four locus haplotypes, the Expectation Maximization algorithm was used with HLA assignments from 536 unrelated Chinese volunteers from the Singapore Bone Marrow Donor Program registry. The paired maternal and cord blood study identified 75 different B-C associations in Chinese, 52 in Malays, and 24 in Asian Indians. Common B-C associations may be shared among population groups; for example, B*4001g-Cw*0702g was common in Chinese and Malays, whereas B*1502g-Cw*0801g and B*3501g-Cw*0401g were found in all three groups. The high diversity of four locus haplotypes originates from multiple combinations of both HLA-A and -DRB1 alleles with each B-C haplotype.

"Promoter array" studies identify cohorts of genes directly regulated by methylation, copy number change, or transcription factor binding in human cancer cells.

DNA microarrays of promoter sequences have been developed in order to identify the profile of genes bound and activated by DNA regulatory proteins such as the transcription factors c-Jun and ATF2 as well as DNA-modifying methylases. The arrays contain 3083 unique human promoter sequences from +500 to -1000 nts from the transcription start site. Cisplatin-induced DNA damage rapidly leads to specific activation of the Jun kinase pathway leading to increased phosphorylation of c-Jun and ATF2-DNA complexes at hundreds of sites within 3 hours. Using three statistical criteria, approximately 269 most commonly phosphorylated c-Jun/ATF2-DNA complexes were identified and representative cases were verified by qPCR measurement of ChIP-captured DNA. Expression was correlated at the mRNA and protein levels. The largest functional cohort was 24 genes of known DNA repair function, most of which exhibited increased protein expression indicated coordinate gene regulation. In addition, cell lines of prostate cancer exhibit stable methylation or copy number changes that reflect the alterations of the corresponding primary tumors. 504 (18.5%) promoters showed differential hybridization between immortalized control prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, eight had previously been observed in prostate cancer, and 13 were previously determined methylation targets in other cancers. The vast majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes to study the role of DNA methylation in prostate tumors. Earlier studies using prototype promoter arrays examine approximately 7% of the proximal regulatory sequences while the current gene regulatory events surveyed here occur on a large scale and may rapidly effect the coordinated expression of a large number of genes.

Egr1 signaling in prostate cancer.

Egr1 is a multifunctional transcription factor regulating a remarkable spectrum of cellular responses from survival to apoptosis, growth to growth arrest, differentiation to transformation, senescence as well as memory and learning effects. In prostate cancer, Egr1 levels are constitutively high and closely linked to cancer development and progression. This zinc-finger protein is a short-lived, immediate early growth response gene known to be induced by a large number of extracellular stimuli such as irradiation (all wavelengths tested), hypoxia, hyperoxia, chemotherapy agents, and more. Therefore the target genes that Egr1 regulates in prostate cancer cells play an important role in generating many of the cellular responses that characterize these cells. After Egr1 binds to its binding sites on gene promoters, specificity of response is determined by whether Egr1 transcriptionally up- or downregulates the target genes. Expression microarray analyses combined with binding data promise new ways to identify stage specific cancer markers, to aid in patient risk assessment and in therapeutic choices.

E proteins in lymphocyte development and lymphoid diseases.

As members of the basic helix-loop-helix (bHLH) family of transcription factors, E proteins function in the immune system by directing and maintaining a vast transcriptional network that regulates cell survival, proliferation, differentiation, and function. Proper activity of this network is essential to the functionality of the immune system. Aberrations in E protein expression or function can cause numerous defects, ranging from impaired lymphocyte development and immunodeficiency to aberrant function, cancer, and autoimmunity. Additionally, disruption of inhibitor of DNA-binding (Id) proteins, natural inhibitors of E proteins, can induce additional defects in development and function. Although E proteins have been investigated for several decades, their study continues to yield novel and exciting insights into the workings of the immune system. The goal of this chapter is to discuss the various classical roles of E proteins in lymphocyte development and highlight new and ongoing research into how these roles, if compromised, can lead to disease.

TOE1 interacts with p53 to modulate its transactivation potential.

The TOE1 gene was discovered as a target of the Egr1 transcription factor that participates in cell growth regulation through the upregulation of p21 and a cell cycle delay at the G2/M phase. We report here that TOE1 is able to bind to the p53 tumor suppressor protein, specifically interacting with the C terminal tetramerization domain of p53. We have further characterized this interaction through determination of binding kinetics using nanoporous optical interferometry and demonstrated that this interaction is capable of enhancing the transcriptional activity of p53-dependent gene targets. These results suggest a mechanistic role for TOE1 as a co-regulator of p53.

Promoter variants of KIR2DL5 add to diversity and may impact gene expression.

Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA encompassing the putative proximal promoter and the coding region was used to identify KIR2DL5 alleles from 77 unrelated Caucasian individuals. PCR and sequencing were used to link each new allele to its neighboring KIR locus to identify 2DL5A or 2DL5B loci. Allele 2DL5A*001 was found in 24 of the 37 2DL5 positive individuals; 2DL5B*0020101 and 2DL5A*0050101 were also observed. Two new alleles, 2DL5B*008 and 2DL5B*009, contained substitutions altering the amino acid sequence of the leader and transmembrane region, respectively. Two other novel alleles, 2DL5B*0020102 and 2DL5A*0050102, contained alterations of the 5' upstream region, bringing the number of unique promoter sequences to six. Promoter activity of the alleles was compared using luciferase reporter assays. Our results support those recently published, in which the promoter of 2DL5B*0020101 was shown to be more active in vitro compared to 2DL5A*001, and also provide additional information about the transcriptional activity of the promoters of the newly characterized alleles related to two altered transcription factor binding sites.

Coactivating factors p300 and CBP are transcriptionally crossregulated by Egr1 in prostate cells, leading to divergent responses.

Related coactivators p300 and CBP affect the transcriptional activities of many transcription factors (TF), producing multiple downstream effects. Here we show that immediate early response TF, Egr1, acts upstream of p300/CBP to induce or to repress transcription, depending on the stimulus. Cells induced with serum to increase endogenous Egr1 increase the transcription of p300/CBP only when Egr1 binding sites in the promoter are not mutated, causing the expression of downstream targets of Egr1 which leads to survival and growth. Induction of p300/CBP by Egr1 results in acetylation and stabilization of Egr1 and transactivation of survival genes but repression of Egr1 and p300/CBP in negative feedback loops. In contrast, induction of Egr1 by UV-C irradiation leads to repression of p300/CBP transcription: Egr1 is preferentially phosphorylated, leading to regulation of target genes that cause cell death. This complex balance of opposing effects appears to finely modulate important cellular life and death responses.

In vivo cloning and characterization of a new growth suppressor protein TOE1 as a direct target gene of Egr1.

Egr1, an immediate early transcription factor, responds to diverse stimuli and affects gene transcription to accomplish its biological effects. One important effect of Egr1 expression is to decrease the growth and tumorigenic potential of several tumor cell types. To identify important Egr1 target genes, we have adapted a methodology involving formaldehyde-induced protein-DNA cross-linking, chromatin immunoprecipitation, and multiplex PCR. Using this approach, we report the cloning of a new Egr1 target gene that is able to account, at least in part, for the growth inhibitory activity of Egr1. We have named this new protein TOE1 for target of Egr1.

Insulin-like growth factor-II regulates PTEN expression in the mammary gland.

The tumor suppressor PTEN is altered in many cancers, including breast cancer, but only a handful of factors are known to control its expression. PTEN plays a vital role in cell survival and proliferation by regulating Akt phosphorylation, a key component of the phosphatidylinositol 3 kinase (PI3K) pathway. Here we show that insulin-like growth factor-II (IGF-II), which signals through PI3K, regulates PTEN expression in the mammary gland. IGF-II injection into mouse mammary gland significantly increased PTEN expression. Transgenic IGF-II expression also increased mammary PTEN protein, leading to reductions in Akt phosphorylation, epithelial proliferation, and mammary morphogenesis. IGF-II induced PTEN promoter activity and protein levels and this involved the immediate early gene egr-1. Thus, we have identified a novel negative feedback loop within the PI3K pathway where IGF-II induces PTEN expression to modulate its physiologic effects.