Holger Karsunky

Features of systemic lupus erythematosus in Dnase1-deficient mice.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that affects over one million people in the United States. SLE is characterized by the presence of anti-nuclear antibodies (ANA) directed against naked DNA and entire nucleosomes. It is thought that the resulting immune complexes accumulate in vessel walls, glomeruli and joints and cause a hypersensitivity reaction type III, which manifests as glomerulonephritis, arthritis and general vasculitis. The aetiology of SLE is unknown, but several studies suggest that increased liberation or disturbed clearance of nuclear DNA-protein complexes after cell death may initiate and propagate the disease. Consequently, Dnase1, which is the major nuclease present in serum, urine and secreta, may be responsible for the removal of DNA from nuclear antigens at sites of high cell turnover and thus for the prevention of SLE (refs 7–11). To test this hypothesis, we have generated Dnase1-deficient mice by gene targeting. We report here that these animals show the classical symptoms of SLE, namely the presence of ANA, the deposition of immune complexes in glomeruli and full-blown glomerulonephritis in a Dnase1-dose-dependent manner. Moreover, in agreement with earlier reports, we found Dnase1 activities in serum to be lower in SLE patients than in normal subjects. Our findings suggest that lack or reduction of Dnase1 is a critical factor in the initiation of human SLE.

Repression of p15INK4b expression by Myc through association with Miz-1

Deregulated expression of c-myc can induce cell proliferation in established cell lines and in primary mouse embryonic fibroblasts (MEFs), through a combination of both transcriptional activation and repression by Myc. Here we show that a Myc-associated transcription factor, Miz-1, arrests cells in G1 phase and inhibits cyclin D-associated kinase activity. Miz-1 upregulates expression of the cyclin-dependent kinases (CDK) inhibitor p15INK4b by binding to the initiator element of the p15INK4b promoter. Myc and Max form a complex with Miz-1 at the p15 initiator and inhibit transcriptional activation by Miz-1. Expression of Myc in primary cells inhibits the accumulation of p15INK4b that is associated with cellular senescence; conversely, deletion of c-myc in an established cell line activates p15INK4b expression. Alleles of c-myc that are unable to bind to Miz-1 fail to inhibit accumulation of p15INK4b messenger RNA in primary cells and are, as a consequence, deficient in immortalization.

The zinc finger protein Gfi-1 can enhance STAT3 signaling by interacting with the STAT3 inhibitor PIAS3

STAT factors act as signal transducers of cytokine receptors and transcriptionally activate specific target genes. The recently discovered protein PIAS3 binds directly to STAT3 and blocks transcriptional activation. Here, we present experimental evidence implementing the zinc finger protein Gfi‐1 as a new regulatory factor in STAT3‐mediated signal transduction. The interaction between the two proteins first became evident in a yeast two‐hybrid screen but was also seen in coprecipitation experiments from eukaryotic cells. Moreover, we found that both Gfi‐1 and PIAS3 colocalize in a characteristic nuclear dot structure. While PIAS3 exerts a profound inhibitory effect on STAT3‐mediated transcription of target promoters, Gfi‐1 can overcome the PIAS3 block and significantly enhances STAT3‐mediated transcriptional activation. In primary T cells, Gfi‐1 was able to amplify IL‐6‐dependent T‐cell activation. As Gfi‐1 is a known, dominant proto‐oncogene, our findings bear particular importance for the recently described ability of STAT3 to transform cells malignantly and offer an explanation of the oncogenic potential of Gfi‐1 in T lymphocytes.

Zinc finger protein GFI-1 has low oncogenic potential but cooperates strongly with pim and myc genes in T-cell lymphomagenesis

The gfi-1 gene encodes a zinc finger containing protein that is specifically expressed in T-lymphocytes and is a frequent target of proviral insertion in T-cell lymphoma provoked by infection with MoMuLV–a non acute transforming retrovirus. Expression of a gfi-1 transgene targeted to T-cells by the lck proximal promoter provokes a reduction of peripheral CD4 and CD8 positive T-cells but nevertheless weakly predisposes transgenic animals for the development of T-cell lymphoma. Forced coexpression of the serine/threonine kinase Pim-1 can partially restore normal T-cell numbers in double pim-1/gfi-1 transgenic mice. Moreover, the combinatorial expression of Pim-1 and Gfi-1 leads to accelerated development of T-cell lymphoma with a mean latency period of 114 days. A similar accelerated rate of lymphoma development was observed when lck-gfi-1 mice were crossed with mice that carry a L-myc gene targeted to be expressed at high levels in T-cells. The results show that gfi-1 can act with low activity as a dominant oncogene when overexpressed but also demonstrate that it is most efficient only in the presence of a cooperative partner protein as for example Pim-1 or L-Myc. In addition, the results suggest that Pim-1 and Gfi-1 are acting synergistically in both T-cell lymphomagenesis and T-cell development.

Induction of cyclin E-cdk2 kinase activity, E2F-dependent transcription and cell growth by Myc are genetically separable events.

The c‐myc gene has been implicated in three distinct genetic programs regulating cell proliferation: control of cyclin E–cdk2 kinase activity, E2F‐dependent transcription and cell growth. We have now used p27−/− fibroblasts to dissect these downstream signalling pathways. In these cells, activation of Myc stimulates transcription of E2F target genes, S‐phase entry and cell growth without affecting cyclin E–cdk2 kinase activity. Both cyclin D2 and E2F2, potential direct target genes of Myc, are induced in p27−/− MycER cells. Ectopic expression of E2F2, but not of cyclin D2, induces S‐phase entry, but, in contrast to Myc, does not stimulate cell growth. Our results show that stimulation of cyclin E–cdk2 kinase, of E2F‐dependent transcription and of cell growth by Myc can be genetically separated from each other.

EVIDENCE IMPLICATING GFI-1 AND PIM-1 IN PRE-T-CELL DIFFERENTIATION STEPS ASSOCIATED WITH BETA -SELECTION

After rearrangement of the T‐cell receptor (TCR) β‐locus, early CD4−/CD8− double negative (DN) thymic T‐cells undergo a process termed ‘β‐selection’ that allows the preferential expansion of cells with a functional TCR β‐chain. This process leads to the formation of a rapidly cycling subset of DN cells that subsequently develop into CD4+/CD8+ double positive (DP) cells. Using transgenic mice that constitutively express the zinc finger protein Gfi‐1 and the serine/threonine kinase Pim‐1, we found that the levels of both proteins are important for the correct development of DP cells from DN precursors at the stage where ‘β‐selection’ occurs. Analysis of the CD25+/CD44−,lo DN subpopulation from these animals revealed that Gfi‐1 inhibits and Pim‐1 promotes the development of larger β‐selected cycling cells (‘L subset’) from smaller resting cells (‘E subset’) within this subpopulation. We conclude from our data that both proteins, Pim‐1 and Gfi‐1, participate in the regulation of β‐selection‐associated pre‐T‐cell differentiation in opposite directions and that the ratio of both proteins is important for pre‐T‐cells to pass the ‘E’ to ‘L’ transition correctly during β‐selection.

Miz1 Is Required for Early Embryonic Development during Gastrulation

ABSTRACT Miz1 is a member of the POZ domain/zinc finger transcription factor family. In vivo, Miz1 forms a complex with the Myc oncoprotein and recruits Myc to core promoter elements. Myc represses transcription through Miz1 binding sites. We now show that the Miz1 gene is ubiquitously expressed during mouse embryogenesis. In order to elucidate the physiological function of Miz1, we have deleted the mouse Miz1 gene by homologous recombination. Miz1+/− mice are indistinguishable from wild-type animals; in contrast, Miz1−/− embryos are not viable. They are severely retarded in early embryonic development and do not undergo normal gastrulation. Expression of Goosecoid and Brachyury is detectable in Miz1−/− embryos, suggesting that Miz1 is not required for signal transduction by Nodal. Expression of p21Cip1, a target gene of Miz1 is unaltered; in contrast, expression of p57Kip2, another target gene of Miz1 is absent in Miz1−/− embryos. Miz1−/− embryos succumb to massive apoptosis of ectodermal cells around day 7.5 of embryonic development. Our results show that Miz1 is required for early embryonic development during gastrulation.

The activity of the murine Bax promoter is regulated by Sp1/3 and E-box binding proteins but not by p53

In human cells the expression of the pro-apoptotic protein Bax appears to be regulated through p53-dependent transcriptional activation. However, in the mouse, p53-deficiency does not affect Bax expression. To shed more light on the transcriptional regulation of the bax gene we have analyzed the murine bax promoter. We find several E-box and Sp1/Sp3 binding sites as well as three putative p53 binding sites that are conserved in the human promoter sequence. We can show that both the Sp1 and the E-box binding sites are necessary for proper regulation of bax transcription and show by genomic DMS footprinting that all these sites are occupied in vivo. In contrast, the putative p53 binding sites were not occupied by protein in vivo in primary murine thymocytes either before or after induction of p53 by DNA damage. Moreover, p53 was unable to regulate the transcription of bax promoter fragments up to 6.5 kb in length. Further, steady state levels of bax mRNA did not correlate with Bax protein expression levels in DNA damage-induced cell death. Our findings exclude a direct transcriptional transactivation of the bax gene by p53 in murine cells suggesting a dominance of p53 independent mechanisms for the regulation of Bax protein expression.

Malignant transformation by cyclin E and Ha-Ras correlates with lower sensitivity towards induction of cell death but requires functional Myc and CDK4

We demonstrate in this paper that the G1 phase specific cell cycle regulator cyclin E is able to provoke focus formation when cotransfected with activated Ha-ras into primary rat embryo fibroblasts (REFs). Cyclin E/Ha-ras transformed cells are highly tumorigenic in synergeneic rats, are able to form colonies in soft agar and show protection towards apoptosis upon serum starvation or DNA damage compared to cells transformed by the combination of Myc, cyclin D1 or SV40 large T-antigen and Ha-ras. Lines that were established after cyclin E/Ha-ras or cyclin D1/Ha-ras transformation contain a large percentage of polyploid cells. This was not observed in cells transformed with other oncoproteins and Ha-ras pointing to an involvement of D- and E type cyclins in genomic instability. The cyclin dependent kinase inhibitors p21 and p27 but also p16 completely abrogate focus formation by cyclin E and Ha-ras suggesting that the oncogenic activity of cyclin E still requires functional G1 specific cyclin/CDK complexes. Moreover, inhibition of Myc function also blocks the oncogenic activity of cyclin E indicating a requirement of Myc for cyclin E function. The findings presented here demonstrate that cyclin E can act as an oncoprotein with a potential involvement in genomic instability and the prevention of cell death. Our data also present more evidence for a strict functional interdependency between G1 cyclin/CDK complexes and c-Myc.

Oncogenic potential of cyclin E in T-cell lymphomagenesis in transgenic mice: evidence for cooperation between cyclin E and Ras but not Myc

To study the oncogenic activity of cyclin E in an in vivo system we generated transgenic mice expressing high levels of cyclin E in T-lymphocytes by using a construct containing the CD2 locus control region. These animals were neither predisposed to develop any tumors spontaneously nor showed an increased incidence when crossbred with Eμ L-myc transgenic mice but developed hyperplasia in peripheral lymphoid organs at later age with an incidence of 27%. When treated with the DNA methylating carcinogen N-methylnitrosourea (MNU) that provokes the development of T-cell lymphomas, CD2-cyclin E transgenic animals came down with T-cell neoplasia showing a significant higher incidence (54%) than normal non transgenic controls (31%). In one of eight tumors that arose in normal MNU treated mice we could find an expected activating point mutation in the Ki-ras gene (12.5%). In contrast, the same mutation occurred in five of 16 tumors from CD2-cyclin E transgenic mice (31.2%). Whereas cyclin E overexpression alone did not lead to an increased CDK2 activity we observed in all tumors that emerged from either MNU treated normal mice or treated CD2-cyclin E transgenics a downregulation of p27KIP1 and a higher histone H1 kinase activity in CDK2 immunoprecipitates compared to normal tissue. These findings demonstrate that high level expression of cyclin E can predispose T-cells for hyperplasia and malignant transformation. However, the results also suggest that this activity of cyclin E is manifest only when other cooperating oncogenes in particular ras genes are present and activated. This would be consistent with our previous finding that cyclin E and Ha-Ras cooperate in focus formation assays in rat embryo fibroblasts.