TIF1gamma, a novel member of the transcriptional intermediary factor 1 family.

We report the cloning and characterization of a novel member of the Transcriptional Intermediary Factor 1 (TIF1) gene family, human TIF1gamma. Similar to TIF1alpha and TIF1beta, the structure of TIF1beta is characterized by multiple domains: RING finger, B boxes, Coiled coil, PHD/TTC, and bromodomain. Although structurally related to TIF1alpha and TIF1beta, TIF1gamma presents several functional differences. In contrast to TIF1alpha, but like TIF1beta, TIF1 does not interact with nuclear receptors in yeast two-hybrid or GST pull-down assays and does not interfere with retinoic acid response in transfected mammalian cells. Whereas TIF1alpha and TIF1beta were previously found to interact with the KRAB silencing domain of KOX1 and with the HP1alpha, MODI (HP1beta) and MOD2 (HP1gamma) heterochromatinic proteins, suggesting that they may participate in a complex involved in heterochromatin-induced gene repression, TIF1gamma does not interact with either the KRAB domain of KOX1 or the HP1 proteins. Nevertheless, TIF1gamma, like TIF1alpha and TIF1beta, exhibits a strong silencing activity when tethered to a promoter. Since deletion of a novel motif unique to the three TIF1 proteins, called TIF1 signature sequence (TSS), abrogates transcriptional repression by TIF1gamma, this motif likely participates in TIF1 dependent repression.

Estrogen receptor impairs interleukin-6 expression by preventing protein binding on the NF-kappaB site.

Interleukin-6 (IL-6) is a multifunctional cytokine thought to be a key factor in post-menopausal osteoporosis, given its ability to induce osteoclast maturation and its down regulation by estrogens. We have previously shown that the effects of TNFalphaand estradiol on the human IL-6 promoter were dependent on a region of the promoter containing a C/EBP site and a NF-kappaB site. To define the molecular mode of action of estrogens, we performed gel shift assays with this DNA fragment as a probe, and nuclear extracts from TNFalpha-induced HeLa, MCF7 and Saos2 cells. Several induced complexes specifically bound the probe. The use of various competitor DNA suggested that most of the complexes detected contained NF-kappaB factors, and that C/EBP site binding factors were important for the overall binding to the probe. Addition of in vitro translated human estrogen receptor (hER) impaired the binding of three complexes in HeLa cells and two complexes in MCF7 and Saos2 cells. Competition experiments suggested that the NF-kappaB site was necessary for the effect of hER. The use of antisera against NF-kappaB and C/EBP proteins showed that the target complexes of hER contained the c-rel proto-oncogene product and to a lesser extent, the RelA protein. Taken together, these data show that hER impairs TNFalphainduction of IL-6 by preventing c-rel and, to a lesser extent, RelA proteins binding to the NF-kappaB site of the IL-6 promoter.

Involvement of CCAAT/enhancer-binding protein and nuclear factor-kappa B binding sites in interleukin-6 promoter inhibition by estrogens.

Bone loss observed in postmenopausal women is clearly associated with a decrease in estrogen levels. Interleukin 6 (IL-6), a multifunctional cytokine involved in osteoclast differentiation, is secreted by osteoblasts and appears to be a key molecule in the osteoporotic process. As previous reports have shown that the human IL-6 promoter is inhibited by estradiol, we investigated the mechanism of estradiol (E2)-mediated IL-6 inhibition in human cells. Analysis of the IL-6 secretion as a function of time in osteoblastoma Saos-2 cells, using an IL-6 ELISA test, showed that a maximal E2 inhibition of tumor necrosis factor-alpha (TNF alpha) induction could be monitored between 2 and 24 h of treatment. IL-6 inhibition was clearly estrogen agonist-specific in Saos-2 and MCF7 cells. Transient transfections of HeLa cells with a pIL-6/CAT plasmid and an estrogen receptor (human ER) expression vector, confirmed the role of the human ER in inhibition of the IL-6 promoter. Deletion and mutational analysis of the promoter highlighted the role of the -185/-60 region and showed that in both MCF7 and HeLa cells, the nuclear factor-IL 6 (NF-IL6) site cooperates with the nuclear factor-kappa B (NF-kappa B) motif to produce maximal induction by TNF alpha, whereas the CCAAT/enhancer-binding protein (C/EBP) site displayed different cooperative effects toward NF-kappa B depending on the cell line used. In HeLa cells, but not in MCF7 cells, we defined an essential role for the C/EBP site by showing that the E2 sensitivity was clearly dependent on its integrity. In these cell lines, the NF-kappa B site mutation abrogated both the TNF alpha-and E2- sensitivity of the construct.

Differential effects of ras and jun family members on complex retrovirus promoter activities.

Infection by complex retroviruses such as Visna-maedi, HIV1, HTLV-I and HFV is generally not followed by particle production, but rather by a more or less prolonged latency period. Knowledge of the mechanisms triggering an infectious cycle from the latent provirus(es) is of great importance in comprehending the appearance of the disease. It is currently admitted that cellular factors regulate viral expression. In this paper, we report the ability of ras, c-jun, jun-B and jun-D to diversely stimulate the LTR promoter activity of these retroviruses. Transient transfection assays using a luciferase reporter gene linked to LTR show that the Visna-maedi virus LTR, despite high intrinsic activity, is stimulated by Ha-ras and c-jun. The HTLV-I and HIV1 LTR were identically stimulated by ras, but differently by c-jun. In contrast, jun-B and jun-D were weaker activators, since they respectively stimulated only HTLV-I LTR and HIV1 LTR. HFV LTR remains unresponsive to either of these factors.

Differential effects of c-jun and CREB on c-AMP response element activation by Ha-ras.

We have previously shown that a Long Terminal Repeat (LTR) of the Intracisternal A-type Particle (IAP) element was activated by ras oncogenes. Here we show that, like the somatostatin CRE (som CRE) and the collagenase TPA Response Element (coll TRE), the IAP CRE is activated by c-jun and that Val 12 Ha-ras cooperates with c-jun to activate these motifs. Neither jun-B nor jun-D activated the IAP CRE, although they were able to act on the som CRE and the coll TRE and to synergize with ras. The CREB factor activated both CREs and modestly inhibited the coll TRE, but diminished the effect of ras on the coll TRE. Finally, forskolin was shown to cooperate with Ha-ras to activate the CRE and the coll TRE. Taken together, these results show that CREB is not involved in ras activation of the CRE and suggest that c-jun is at least one of the elements implicated in this phenomenon.

ras oncogene activates the intracisternal A particle long terminal repeat promoter through a c-AMP response element.

To understand the mechanism responsible for the high expression of Intracisternal A Particles (IAP) in murine cells transformed by the Kirsten Mouse Sarcoma Virus (Ki-MSV), we have investigated the effect of p21ras on IAP transcription. By transient cotransfections of IAP LTR-CAT plasmids with v-Ki-ras and c-Ki-ras expression vectors, we have found that p21v-Ki-ras, and the p21c-Ki-ras to a lesser extent, stimulate the promoter activity of the 5' IAP LTR. We constructed several plasmids containing the CAT gene under control of IAP LTRs deleted in different regions. CAT assays demonstrate that the ras responsive sequence is a 16 bp oligonucleotide containing an effective c-AMP Response Element (CRE) TGACGTCA, which is located in the U3 region, 20 bp upstream of the CAAT box.