Binding of the Sp1 transcription factor by the human Harvey ras1 proto-oncogene promoter.

Members of the ras gene family encode proteins that when overproduced or mutated can transform immortalized mammalian cells. It is therefore important to understand the mechanisms by which the ras genes are regulated. The promoter region of the human Harvey ras proto-oncogene c-Ha-ras1 initiates RNA transcription at multiple sites and contains repeated copies of the hexanucleotide GGGCGG and its inverted complement CCGCCC, referred to as GC boxes. These GC boxes consist of sequences identical to those found in the SV40 early promoter, where the human cellular transcriptional factor Sp1 binds. Footprinting analysis with deoxyribonuclease I was used to show that Sp1 binds to six GC box sequences within the c-Ha-ras1 promoter. An in vivo transfection assay showed competition between the 21-base pair repeats of the SV40 promoter and the c-Ha-ras1 promoter for common regulatory factors. In this system the presence of Sp1 is apparently required for c-Ha-ras1 transcription. Analysis of deletions of the c-Ha-ras1 promoter region by means of a transient expression assay revealed that the three Sp1 binding sites closest to the RNA start sites were sufficient for full transcriptional activity.

Human T-cell leukemia virus type 1 Tax attenuates gamma-irradiation-induced apoptosis through physical interaction with Chk2.

Checkpoint kinase 2 (Chk2) is known to mediate diverse cellular responses to genotoxic stress. The fundamental role of Chk2 is to regulate the network of genome-surveillance pathways that coordinate cell-cycle progression with DNA repair and cell survival or death. Defects in Chk2 contribute to the development of both hereditary and sporadic human cancers. We now present evidence that the human T-cell leukemia virus type-1 (HTLV-1) Tax protein directly interacts with Chk2 and the kinase activity of Chk2 is inhibited by Tax. The physical interaction of Chk2 and Tax was observed by co-immunoprecipitation assays in HTLV-1-infected T cells (C81) as well as GST pull-down assays using purified proteins. Binding and kinase activity inhibition studies with Tax deletion mutants indicated that at least two domains of Tax mediate the interaction with Chk2. We have analysed the functional consequence of de novo expression of Tax upon the cellular DNA-damage-induced apoptosis, which is mediated by Chk2. Using transient transfection and TUNEL assay, we found that gamma-irradiation-induced apoptosis was decreased in 293T and HCT-116 (p53(-/-)) cells expressing HTLV-1 Tax. Our studies demonstrate an important potential target of Tax in cellular transformation.

The tax protein from the primate T-cell lymphotropic virus type 3 is expressed in vivo and is functionally related to HTLV-1 Tax rather than HTLV-2 Tax.

Human T-cell leukemia virus and simian T-cell leukemia virus (STLV) form the primate T-cell lymphotropic viruses group. Human T-cell leukemia virus type 1 and type 2 (HTLV-1 and HTLV-2) encode the Tax viral transactivator (Tax1 and Tax2, respectively). Tax1 possesses an oncogenic potential and is responsible for cell transformation both in vivo and in vitro. We and others have recently discovered the existence of human T-cell lymphotropic virus type 3. However, there is currently no evidence for the presence of a Tax protein in HTLV-3-infected individuals. We show that the serum of an HTLV-3 asymptomatic carrier and the sera of two STLV-3-infected monkeys contain specific anti-Tax3 antibodies. We also show that tax3 mRNA is present in the PBMCs obtained from an STLV-3-infected monkey, demonstrating that Tax3 is expressed in vivo. We further demonstrate that Tax3 intracellular localization is very similar to that of Tax1 and that Tax3 binds to both CBP and p300 coactivators. Using purified Tax3, we show that the protein increases transcription from a 4TxRE G-free cassette plasmid in an in vitro transcription assay. In all cell types tested, including transiently transfected lymphocytes, Tax3 activates its own promoter STLV-3 long terminal repeat (LTR), which contains only two Tax Responsive Elements (TREs), and activates also HTLV-1 and HTLV-2 LTRs. In addition, Tax3 also activates the NF-kappaB pathway. We also show that Tax3 possesses a PDZ-binding sequence at its C-terminal end. Our results demonstrate that Tax3 is a transactivator, and that its properties are more similar to that of Tax1, rather than of Tax2. This suggests the possible occurrence of lymphoproliferative disorders among HTLV-3-infected populations.

Transcriptional regulation of the mouse alpha A-crystallin gene: activation dependent on a cyclic AMP-responsive element (DE1/CRE) and a Pax-6-binding site.

Two cis-acting promoter elements (-108 to -100 and -49 to -33) of the mouse alpha A-crystallin gene, which is highly expressed in the ocular lens, were studied. Here we show that DE1 (-108 to -100; 5'TGACGGTG3'), which resembles the consensus cyclic AMP (cAMP)-responsive element sequence (CRE; 5'TGACGT[A/C][A/G]3'), behaves like a functional CRE site. Transfection experiments and electrophoretic mobility shift assays (EMSAs) using site-specific mutations correlated a loss of function with deviations from the CRE consensus sequence. Results of EMSAs in the presence of antisera against CREB, delta CREB, and CREM were consistent with the binding of CREB-like proteins to the DE1 sequence. Stimulation of alpha A-crystallin promoter activity via 8-bromo-cAMP, forskolin, or human T-cell leukemia virus type I Tax1 in transfections and reduction of activity of this site in cell-free transcription tests by competition with the somatostatin CRE supported the idea that DE1 is a functional CRE. Finally, Pax-6, a member of the paired-box family of transcription factors, activated the mouse alpha A-crystallin promoter in cotransfected COP-8 fibroblasts and bound to the -59 to -29 promoter sequence in EMSAs. These data provide evidence for a synergistic role of Pax-6 and CREB-like proteins for high expression of the mouse alpha A-crystallin gene in the lens.

Physical and functional interaction between the human T-cell lymphotropic virus type 1 Tax1 protein and the CCAAT binding protein NF-Y.

Tax1, a potent activator of human T-cell lymphotropic virus type 1 (HTLV-1) transcription, has been shown to modulate expression of many cellular genes. Tax1 does not bind DNA directly but regulates transcription through protein-protein interactions with sequence-specific transcription factors. Using the yeast two-hybrid system to screen for proteins which interact with Tax1, we isolated the B subunit of the CCAAT binding protein NF-Y from a HeLa cDNA library. The interaction of Tax1 with NF-YB was specific in that NF-YB did not interact with a variety of other transcription factors, including human immunodeficiency virus Tat, human papillomavirus E6, and Bicoid, or with the M7 (amino acids 29CP-AS) Tax1 mutant. However, NF-YB did interact with the C-terminal Tax1 mutants M22 (130TL-AS) and M47 (319LL-RS). We also show that in vitro-translated NF-YB specifically bound to a glutathione S-transferase-Tax1 fusion protein. Further, Tax1 coimmunoprecipitated with NF-Y from nuclear extracts of HTLV-1-transformed cells, providing evidence for in vivo interaction of Tax1 and NF-YB. We further demonstrate that Tax1 specifically activated the NF-Y-responsive DQbeta promoter, as well as a minimal promoter which contains only the Y-box element. In addition, mutation of the Y-box element alone abrogated Tax1-mediated activation. Taken together, these data indicate that Tax1 interacts with NF-Y through the B subunit and that this interaction results in activation of the major histocompatibility complex class II promoter. Through activation of this and other NF-Y driven promoters, the Tax1-NF-Y interaction may play a critical role in causing cellular transformation and HTLV-1 pathogenesis.

Indirect binding of human T-cell leukemia virus type I tax1 to a responsive element in the viral long terminal repeat.

Several laboratories have demonstrated that tandem copies of the human T-cell leukemia virus type I 21-base-pair (bp) repeat cloned upstream of either a homologous or heterologous promoter increase transcription in the presence of tax1 protein. In this report, we provide evidence for a second tax1-responsive sequence in the viral long terminal repeat. Analysis of human T-cell leukemia virus type I promoter deletion mutants and plasmids containing cloned oligonucleotide motifs demonstrated that this 47-bp sequence, located between -117 and -163, confers responsiveness to tax1. We further demonstrated that proteins present in HeLa nuclear extracts bind specifically to this tax1-responsive sequence. Mutants that affected in vivo activity also decreased in vitro binding. Using an in vitro binding assay, we demonstrated that tax1 interacts indirectly with the 47-bp sequence, most likely through protein-protein interaction. Thus, while tax1 does not bind directly to DNA to enhance transcription, it may influence sequence-specific responses by interacting with the primary DNA-protein complex.

Simian virus 40 guanine-cytosine-rich sequences function as independent transcriptional control elements in vitro.

We have recently shown that DNA sequences located within the simian virus 40 (SV40) G-C-rich, 21-base-pair repeats constitute an important transcriptional control element of the SV40 late promoter (Brady et al., Mol. Cell. Biol. 4:133-141, 1984). To gain further insight into the mechanism by which the SV40 G-C-rich repeats function, we have analyzed the transcriptional properties of several recombinant DNAs. The results presented in this report suggest that the SV40 G-C-rich sequences can function as independent RNA polymerase II transcriptional-control elements. In vitro competition studies demonstrated that sequences within the G-C-rich, 21-base-pair repeats, in the absence of either the SV40 early or late -25 transcriptional-control signals or the major RNA initiation sites, efficiently competed for transcription factors required for SV40 early and late RNA synthesis. Our transcription studies also demonstrated that in the absence of contiguous SV40 transcription control sequences, G-C-rich sequences stimulated initiation of transcription in a bidirectional manner, from proximally located sequences. Finally, we demonstrated that the 21-base-pair-repeat region can stimulate in vitro transcription from the heterologous adenovirus 2 major late promoter.

Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription.

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of carboxyl-terminal domain (CTD) kinases to the HIV-1 promoter. Using an immobilized DNA template assay, we have analyzed the effect of Tat on kinase activity during the initiation and elongation phases of HIV-1 transcription. Our results demonstrate that cyclin-dependent kinase 7 (CDK7) (TFIIH) and CDK9 (P-TEFb) both associate with the HIV-1 preinitiation complex. Hyperphosphorylation of the RNA polymerase II (RNAP II) CTD in the HIV-1 preinitiation complex, in the absence of Tat, takes place at CTD serine 2 and serine 5. Analysis of preinitiation complexes formed in immunodepleted extracts suggests that CDK9 phosphorylates serine 2, while CDK7 phosphorylates serine 5. Remarkably, in the presence of Tat, the substrate specificity of CDK9 is altered, such that the kinase phosphorylates both serine 2 and serine 5. Tat-induced CTD phosphorylation by CDK9 is strongly inhibited by low concentrations of 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of transcription elongation by RNAP II. Analysis of stalled transcription elongation complexes demonstrates that CDK7 is released from the transcription complex between positions +14 and +36, prior to the synthesis of transactivation response (TAR) RNA. In contrast, CDK9 stays associated with the complex through +79. Analysis of CTD phosphorylation indicates a biphasic modification pattern, one in the preinitiation complex and the other between +36 and +79. The second phase of CTD phosphorylation is Tat-dependent and TAR-dependent. These studies suggest that the ability of Tat to increase transcriptional elongation may be due to its ability to modify the substrate specificity of the CDK9 complex.

Interaction of the human T-cell lymphotropic virus type 1 tax transactivator with transcription factor IIA.

The Tax protein of human T-cell lymphotropic virus type 1 (HTLV-1) is a 40-kDa transcriptional activator which is critical for HTLV-1 gene regulation and virus-induced cellular transformation. Tax is localized to the DNA through its interaction with the site-specific activators cyclic AMP-responsive element-binding protein, NF-kappaB, and serum response factor. It has been suggested that the recruitment of Tax to the DNA positions Tax for interaction with the basal transcriptional machinery. On the basis of several independent assays, we now report a physical and functional interaction between Tax and the transcription factor, TFIIA. First, Tax was found to interact with the 35-kDa (alpha) subunit of TFIIA in the yeast two-hybrid interaction system. Importantly, two previously characterized mutants with point mutations in Tax, M32 (Y196A, K197S) and M41 (H287A, P288S), which were shown to be defective in Tax-activated transcription were unable to interact with TFIIA in this assay. Second, a glutathione-S-transferase (GST) affinity-binding assay showed that the interaction of holo-TFIIA with GST-Tax was 20-fold higher than that observed with either the GST-Tax M32 activation mutant or the GST control. Third, a coimmunoprecipitation assay showed that in HTLV-1-infected human T lymphocytes, Tax and TFIIA were associated. Finally, TFIIA facilitates Tax transactivation in vitro and in vivo. In vitro transcription studies showed reduced levels of Tax-activated transcription in cell extracts depleted of TFIIA. In addition, transfection of human T lymphocytes with TFIIA expression vectors enhanced Tax-activated transcription of an HTLV-1 long terminal repeat-chloramphenicol acetyltransferase reporter construct. Our study suggests that the interaction of Tax with the transcription factor TFIIA may play a role in Tax-mediated transcriptional activation.

A 36-kilodalton cellular transcription factor mediates an indirect interaction of human T-cell leukemia/lymphoma virus type I TAX1 with a responsive element in the viral long terminal repeat.

The human T-cell leukemia/lymphoma virus type I (HTLV-I) trans activator, TAX1, interacts indirectly with a TAX1-responsive element, TRE-2, located at positions -117 to -163 in the viral long terminal repeat. This report describes the characterization of a 36-kilodalton (kDa) protein identified in HeLa nuclear extract which mediates the interaction of TAX1 with TRE-2. Purification of the protein was achieved by zinc chelate chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The renatured 36-kDa protein bound specifically to a TRE-2 oligonucleotide but not to nonfunctional base substitution mutant probes in a gel retardation assay. Renatured proteins of differing molecular weights were unable to form this complex. In addition, the 36-kDa protein specifically activated transcription from the HTLV-I promoter in vitro. Purified TAX1 protein formed a complex with the TRE-2 oligonucleotide in the presence of the 36-kDa protein, suggesting that indirect interaction of TAX1 with the viral long terminal repeat may be one of the mechanisms by which HTLV-I transcription is regulated.

PCAF interacts with tax and stimulates tax transactivation in a histone acetyltransferase-independent manner.

Recent studies have shown that the p300/CREB binding protein (CBP)-associated factor (PCAF) is involved in transcriptional activation. PCAF activity has been shown strongly associated with histone acetyltransferase (HAT) activity. In this report, we present evidence for a HAT-independent transcription function that is activated in the presence of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein. In vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrate that there is a direct interaction between Tax and PCAF, independent of p300/CBP. PCAF can be recruited to the HTLV-1 Tax responsive element in the presence of Tax, and PCAF cooperates with Tax in vivo to activate transcription from the HTLV-1 LTR over 10-fold. Point mutations at Tax amino acid 318 (TaxS318A) or 319 to 320 (Tax M47), which have decreased or no activity on the HTLV-1 promoter, are defective for PCAF binding. Strikingly, the ability of PCAF to stimulate Tax transactivation is not solely dependent on the PCAF HAT domain. Two independent PCAF HAT mutants, which knock out acetyltransferase enzyme activity, activate Tax transactivation to approximately the same level as wild-type PCAF. In contrast, p300 stimulation of Tax transactivation is HAT dependent. These studies provide experimental evidence that PCAF contains a coactivator transcription function independent of the HAT activity on the viral long terminal repeat.

Inactivation of p53 by human T-cell lymphotropic virus type 1 Tax requires activation of the NF-kappaB pathway and is dependent on p53 phosphorylation.

p53 plays a key role in guarding cells against DNA damage and transformation. We previously demonstrated that the human T-cell lymphotropic virus type 1 (HTLV-1) Tax can inactivate p53 transactivation function in lymphocytes. The present study demonstrates that in T cells, Tax-induced p53 inactivation is dependent upon NF-kappaB activation. Analysis of Tax mutants demonstrated that Tax inactivation of p53 function correlates with the ability of Tax to induce NF-kappaB but not p300 binding or CREB transactivation. The Tax-induced p53 inactivation can be overcome by overexpression of a dominant IkappaB mutant. Tax-NF-kappaB-induced p53 inactivation is not due to p300 squelching, since overexpression of p300 does not recover p53 activity in the presence of Tax. Further, using wild-type and p65 knockout mouse embryo fibroblasts (MEFs), we demonstrate that the p65 subunit of NF-kappaB is critical for Tax-induced p53 inactivation. While Tax can inactivate endogenous p53 function in wild-type MEFs, it fails to inactivate p53 function in p65 knockout MEFs. Importantly, Tax-induced p53 inactivation can be restored by expression of p65 in the knockout MEFs. Finally, we present evidence that phosphorylation of serines 15 and 392 correlates with inactivation of p53 by Tax in T cells. This study provides evidence that the divergent NF-kappaB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels, including posttranslational modification of p53.

Regulation of insulin-like growth factor II P3 promotor by p53: a potential mechanism for tumorigenesis.

Human insulin-like growth factor (IGF)-II mRNA has been shown to be expressed at high levels in a variety of tumors, including rhabdomyosarcomas. In addition, many tumors have alterations in p53 expression. To investigate whether p53 regulates IGF-II gene expression, we transfected wild-type p53 expression vectors and luciferase constructs driven by IGF-II P3 promotors into multiple cell lines. We found that p53 reduced, in a dose-dependent manner, both endogenous IGF-II P3 transcripts and transfected P3 luciferase expression. The inhibition of P3 luciferase expression by p53 was more pronounced in the two cell lines that expressed mutant p53 protein, RD, and HTB114. The element responsible for this inhibition was mapped to the minimal promoter region. We also transfected an HPV-16 E6 expression plasmid into CCL13 cells containing functional p53 and found that E6 up-regulated IGF-II P3 activity. Wild-type, but not mutant, p53 interfered with the binding of TATA-binding protein to the TATA motif of P3, although both could directly associate with human TATA-binding protein. Our results suggest that p53 may play a role in regulation of IGF-II gene expression.

Activation of interleukin-2 receptor alpha expression by extracellular HTLV-I Tax1 protein: a potential role in HTLV-I pathogenesis.

The Tax1 protein of human T cell leukemia/lymphoma virus type I (HTLV-I) has been shown to stimulate the proliferation of human lymphocytes. Here we report that lymphocyte proliferation can be induced at extracellular Tax1 concentrations as low as 25 pM. The proliferative response induced by extracellular Tax1 is accompanied by an activation of endogenous interleukin-2 receptor alpha-chain (IL-2R alpha) expression in human lymphocytes. Functional activation of IL-2R alpha expression in peripheral blood lymphocytes treated with Tax1 was demonstrated using an [125I]IL-2-binding assay. In addition, an enzyme-linked immunosorbent assay demonstrated that soluble IL-2R alpha in the medium of IL-2- and Tax1-treated cells was over 13-fold greater than in the medium of control treated cells. Overexpression of IL-2R alpha is a common clinical feature of some patients with adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I-associated myopathy (TSP/HAM). The ability of extracellular Tax1 protein to activate expression of IL-2R alpha in both infected and uninfected lymphocytes may contribute to the abnormal lymphocyte proliferation observed in both ATL and TSP/HAM.

Stabilization of wild-type p53 in human T-lymphocytes transformed by HTLV-I.

Adult T-cell leukemia/lymphoma is an aggressive malignancy associated with infection by the human T-lymphotropic virus type-I (HTLV-I). We now demonstrate that p53 expression is elevated in the HTLV-I-transformed T-lymphocyte lines C81, MT-2, MT-4 and HUT 102. In pulse-chase experiments, the p53 protein demonstrated a prolonged half-life of 2 to 8 h in HTLV-I-transformed cells compared with 0.5 to 1.0 h for wild-type p53 in primary human and murine fibroblasts, or human peripheral blood lymphocytes. In cell lines C81 and HUT 102, which exhibited the longest p53 protein half-life, the wild-type-related PAb1620 epitope was detected at reduced levels. The PAb240 mutant-related p53 epitope was not detected in any of the transformed cell lines. By direct sequence analysis of RT-PCR products, the entire p53 cDNA coding sequence was determined to be wild-type in all four cell lines. Stabilization of wild-type p53 may represent its functional inactivation and contribute to lymphocyte transformation by HTLV-I.

HTLV-I Tax transrepresses the human c-Myb promoter independently of its interaction with CBP or p300.

The c-Myb proto-oncogene is preferentially expressed in hematopoietic lineages, and highly expressed in several leukemia types. The Human T-cell Leukemia Virus Type I (HTLV-I) is the etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL). A previous report suggested that Tax, the viral transactivator, is able to suppress the transactivation potential of c-Myb protein by competing for recruitment of CBP. We tested whether such a competition could affect transcription from the c-Myb promoter in Tax expressing T-cells. Using several c-Myb promoter reporter constructs carrying mutations in various regions, we demonstrate that Tax suppression of c-Myb transactivation results in transrepression of the c-Myb promoter through the Myb responsive elements in Jurkat T-cells. The ability of Tax mutants M22, M47 and V89A to interact with the full-length CBP and p300 proteins in vitro, and their ability to repress the c-Myb promoter, was then evaluated. Although both M47 and M22 bind to CBP and p300 to a similar extent, only M47 was able to repress the c-Myb promoter, suggesting that competition for CBP/p300 binding was not the mechanism underlying Tax's effect. This concept was further supported by the fact that the Tax mutant V89A transrepresses the c-Myb promoter efficiently in spite of an impaired binding to CBP and p300. Therefore, Tax-mediated repression of the c-Myb promoter appears to be independent from a direct competition between c-Myb and Tax for recruitment of CBP/p300. Interestingly, a decreased transcription from the endogenous c-Myb promoter was observed in several HTLV-I transformed T-cell lines. Finally, the ability of Tax to directly repress the endogenous c-Myb promoter was demonstrated in a Jurkat cell line stably transfected with a tax gene driven by a cadmium-inducible promoter.

Human herpesvirus 6 (HHV-6) ORF-1 transactivating gene exhibits malignant transforming activity and its protein binds to p53.

The 357 amino acid open reading frame 1 (ORF-1), also designated DR7, within the SalI-L fragment of human herpesvirus 6 (HHV-6) exhibited transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter and increased HIV-1 replication (Kashanchi et al., Virology, 201, 95-106, 1994). In the current study, the SalI-L transforming region was localized to the SalI-L-SH subfragment. Several ORFs identified in SalI-L-SH by sequence analysis were cloned into a selectable mammalian expression vector, pBK-CMV. Only pBK/ORF1 transformed NIH3T3 cells. Furthermore, cells expressing ORF-1 protein produced fibrosarcomas when injected into nude mice, whereas control cells, expressing either no ORF-1 protein or C-terminal truncated (after residue 172) ORF-1 protein, were not tumorigenic. Western blot analysis of proteins extracted from the tumors revealed ORF-1 protein. Additional studies indicated that ORF-1 was expressed in HHV-6-infected human T-cells by 18 h. Co-immunoprecipitation experiments showed that ORF-1 protein bound to tumor suppressor protein p53, and the ORF-1 binding domain on p53 was located between residues 28 and 187 of p53, overlapping with the specific DNA binding domain. Functional studies showed that p53-activated transcription was inhibited in ORF-1, but not in truncated ORF-1, expressing cells. Importantly, the truncated ORF-1 mutant also failed to cause transformation. Analysis of several human tumors by PCR revealed ORF-1 DNA sequences in some angioimmunoblastic lymphadenopathies, Hodgkin's and non-Hodgkin's lymphomas and glioblastomas. The detection of ORF-1 sequences in human tumors, while not proof per se, is a prerequisite for establishing its role in tumor development. Taken together, the results demonstrate that ORF-1 is an HHV-6 oncogene that binds to and affects p53. The identification of both transforming and transactivating activities within ORF-1 is a characteristic of other viral oncogenes and is the first reported for HHV-6.

Isolation of a cDNA clone, TRX encoding a human T-cell lymphotrophic virus type-I Tax1 binding protein.

Tax1 is essential for human T-cell lymphotropic virus type I (HTLV-I) virus replication and transformation. We have identified and characterized a Tax1 binding protein, TRX, by cDNA screening of a Jurkat T-cell cDNA library. TRX mRNA is ubiquitously expressed in human tissues tested and cell lines analyzed.

An Ets site in the whey acidic protein gene promoter mediates transcriptional activation in the mammary gland of pregnant mice but is dispensable during lactation.

The whey acidic protein (WAP) gene is specifically expressed in mammary tissue, and its transcription is induced several thousand-fold during pregnancy and remains high throughout lactation. A purine-rich sequence (PRS) located around -110 of the WAP gene promoter is conserved between mice, rats, and rabbits, suggesting that it features a regulatory element. This PRS contains an invariant GGAA/T core motif characteristic of the binding site for Ets transcription factors. Electromobility shift assays demonstrate that Ets1 binds specifically to the PRS. Experiments in transgenic mice further demonstrate that this PRS/Ets site plays a critical role in the activation of WAP transgenes during pregnancy, but that its presence is not required for high expression throughout lactation. Transgenes with an intact PRS/Ets site are expressed at high levels at day 13 of pregnancy, with little further increase during late pregnancy and lactation. In contrast, WAP transgenes with a mutation in the PRS/Ets site, which abrogates the binding of Ets1, are not expressed at midpregnancy, but their transcriptional activity is not affected during lactation. These results demonstrate that Ets-signaling pathways can function as stage-specific transcriptional activators of milk protein genes in the developing mammary gland. In addition, this work extends earlier findings that gene activation during pregnancy and lactation is mediated, in part, by different mechanisms.

Transcription of the HIV-1 LTR is regulated by the density of DNA CpG methylation.

Transcription from the HIV-1 long terminal repeat (LTR) was shown to be inhibited by DNA CpG methylation both in vivo and in vitro. Enzymatic methylation of CpG sites localized within the LTR decreased the transcription of the CAT reporter gene, chloramphenicol acetyltransferase, as assayed by the transient expression of this gene in tissue culture. The inhibitory effect could be initially overcome, in trans, by the transactivator tat. As a function of time, the presence of tat had no observable effect on transcription, within the limits of detection sensitivity, suggesting that the level of basal transcription was reduced to very low levels. This effect is suggestive of the involvement of cellular CpG methylation-dependent inhibitory factors which have been characterized by other laboratories. These data imply that transactivation is reduced to low levels after longer periods of time when the DNA template is sparsely methylated. The transcriptional inhibitory process may involve proteins such as MeCP which may interact with methylated DNA more slowly and/or weakly. Conversely, densely methylated DNA was transcriptionally repressed immediately which suggests the rapid/strong association of the cellular inhibitory factor(s). The transcriptional inhibitory effect was also observed in an in vitro transcription run-off system. These data suggest that the methylation-mediated inhibition of transcription is directly affected by CpG methylation density and may involve other factors.