Kristina Heyne

HERV-K(HML-2) rec and np9 transcripts not restricted to disease but present in many normal human tissues

BackgroundHuman endogenous retroviruses of the HERV-K(HML-2) group have been associated with the development of tumor diseases. Various HERV-K(HML-2) loci encode retrovirus-like proteins, and expression of such proteins is upregulated in certain tumor types. HERV-K(HML-2)-encoded Rec and Np9 proteins interact with functionally important cellular proteins and may contribute to tumor development. Though, the biological role of HERV-K(HML-2) transcription and encoded proteins in health and disease is less understood. We therefore investigated transcription specifically of HERV-K(HML-2) rec and np9 mRNAs in a panel of normal human tissues.ResultsWe obtained evidence for rec and np9 mRNA being present in all examined 16 normal tissue types. A total of 18 different HERV-K(HML-2) loci were identified as generating rec or np9 mRNA, among them loci not present in the human reference genome and several of the loci harboring open reading frames for Rec or Np9 proteins. Our analysis identified additional alternative splicing events of HERV-K(HML-2) transcripts, some of them encoding variant Rec/Np9 proteins. We also identified a second HERV-K(HML-2) locus formed by L1-mediated retrotransposition that is likewise transcribed in various human tissues.ConclusionsHERV-K(HML-2) rec and np9 transcripts from different HERV-K(HML-2) loci appear to be present in various normal human tissues. It is conceivable that Rec and Np9 proteins and variants of those proteins are part of the proteome of normal human tissues and thus various cell types. Transcription of HERV-K(HML-2) may thus also have functional relevance in normal human cell physiology.

Resistance of mitochondrial p53 to dominant inhibition.

BackgroundMutation of a tumor suppressor allele leaves the second as backup. Not necessarily so with p53. This homo-tetrameric transcription factor can become contaminated with mutant p53 through hetero-tetramerization. In addition, it can be out-competed by the binding to p53 DNA recognition motifs of transactivation-incompetent isoforms (ΔN and ΔTA-isoforms) of the p53/p63/p73 family of proteins. Countermeasures against such dominant-negative or dominant-inhibitory action might include the evolutionary gain of novel, transactivation-independent tumor suppressor functions by the wild-type monomer.ResultsHere we have studied, mostly in human HCT116 colon adenocarcinoma cells with an intact p53 pathway, the effects of dominant-inhibitory p53 mutants and of Δex2/3p73, a tumor-associated ΔTA-competitor of wild-type p53, on the nuclear transactivation-dependent and extra-nuclear transactivation-independent functions of wild-type p53. We report that mutant p53 and Δex2/3p73, expressed from a single gene copy per cell, interfere with the stress-induced expression of p53-responsive genes but leave the extra-nuclear apoptosis by mitochondrial p53 largely unaffected, although both wild-type and mutant p53 associate with the mitochondria. In accord with these observations, we present evidence that in contrast to nuclear p53 the vast majority of mitochondrial p53, be it wild-type or mutant, is consisting of monomeric protein.ConclusionThe extra-nuclear p53-dependent apoptosis may constitute a fail-safe mechanism against dominant inhibition.

Tumor suppression in skin and other tissues via cross-talk between vitamin D- and p53-signaling.

P53 and its family members have been implicated in the direct regulation of the vitamin D receptor (VDR). Vitamin D- and p53-signaling pathways have a significant impact on spontaneous or carcinogen-induced malignant transformation of cells, with VDR and p53 representing important tumor suppressors. VDR and the p53/p63/p73 proteins all function typically as receptors or sensors that turn into transcriptional regulators upon stimulus, with the main difference being that the nuclear VDR is activated as a transcription factor after binding its naturally occurring ligand 1,25-dihydroxyvitamin D with high affinity while the p53 family of transcription factors, mostly in the nucleoplasm, responds to a large number of alterations in cell homeostasis commonly referred to as stress. An increasing body of evidence now convincingly demonstrates a cross-talk between vitamin D- and p53-signaling that occurs at different levels, has genome-wide implications and that should be of high importance for many malignancies, including non-melanoma skin cancer. One interaction involves the ability of p53 to increase skin pigmentation via POMC derivatives including alpha-MSH and ACTH. Pigmentation protects the skin against UV-induced DNA damage and skin carcinogenesis, yet on the other hand reduces cutaneous synthesis of vitamin D. A second level of interaction may be through the ability of 1,25-dihydroxyvitamin D to increase the survival of skin cells after UV irradiation. UV irradiation-surviving cells show significant reductions in thymine dimers in the presence of 1,25-dihydroxyvitamin D that are associated with increased nuclear p53 protein expression, and significantly reduced NO products. A third level of interaction is documented by the ability of vitamin D compounds to regulate the expression of the murine double minute 2 (MDM2) gene in dependence of the presence of wild-type p53. MDM2 has a well-established role as a key negative regulator of p53 activity. Finally, p53 and family members have been implicated in the direct regulation of VDR. This overview summarizes some of the implications of the cross-talk between vitamin D- and p53-signaling for carcinogenesis in the skin and other tissues.

A novel mechanism of crosstalk between the p53 and NFκB pathways: MDM2 binds and inhibits p65RelA.

The inflammation regulating transcription factor NFκB and the tumor-suppressing transcription factor p53 can act as functional antagonists. Chronic inflammation (NFκB activity) may contribute to the development of cancer through the inhibition of p53 function, while, conversely, p53 activity may dampen inflammation. Here we report that the E3 ubiquitin ligase MDM2, whose gene is transcriptionally activated by both NFκB and p53, can bind and inhibit the p65RelA subunit of NFκB. The interaction is mediated through the N-terminal and the acidic/zinc finger domains of MDM2 on the one hand and through the N-terminal Rel homology domain of p65RelA on the other hand. Co-expression of MDM2 and p65RelA caused ubiquitination of the latter in the nucleus, and this modification was dependent of a functional MDM2 RING domain. Conversely, inhibition of endogenous MDM2 by small-molecule inhibitors or siRNA significantly reduced the ubiquitination of ectopic and endogenous p65RelA. MDM2 was able to equip p65RelA with mutated ubiquitin moieties capable of multiple monoubiquitination but incapable of polyubiquitination; moreover, MDM2 failed to destabilize p65RelA detectably, suggesting that the ubiquitin modification of p65RelA by MDM2 was mostly regulatory rather than stability-determining. MDM2 inhibited the NFκB-mediated transactivation of a reporter gene and the binding of NFκB to its DNA binding motif in vitro. Finally, knockdown of endogenous MDM2 increased the activity of endogenous NFκB as a transactivator. Thus, MDM2 can act as a direct negative regulator of NFκB by binding and inhibiting p65RelA.

NIR, an inhibitor of histone acetyltransferases, regulates transcription factor TAp63 and is controlled by the cell cycle

p63 is a sequence-specific transcription factor that regulates epithelial stem cell maintenance and epithelial differentiation. In addition, the TAp63 isoform with an N-terminal transactivation domain functions as an inducer of apoptosis during the development of sympathetic neurons. Previous work has indicated that the co-activator and histone acetyltransferase (HAT), p300, can bind to TAp63 and stimulate TAp63-dependent transcription of the p21Cip1 gene. Novel INHAT Repressor (NIR) is an inhibitor of HAT. Here, we report that the central portion of NIR binds to the transactivation domain and the C-terminal oligomerization domain of TAp63. NIR is highly expressed in G2/M phase of the cell cycle and only weakly expressed in G1/S. Furthermore, except during mitosis, NIR is predominantly localized in the nucleolus; only a small portion co-localizes with TAp63 in the nucleoplasm and at the p21 gene promoter. Consistent with NIR acting as a repressor, the induced translocation of NIR from the nucleolus into the nucleoplasm resulted in the inhibition of TAp63-dependent transactivation of p21. Conversely, knockdown of NIR by RNAi stimulated p21 transcription in the presence of TAp63. Thus, NIR is a cell-cycle-controlled, novel negative regulator of TAp63. The low levels of nucleoplasmic NIR might act as a buffer toward potentially toxic TAp63.

MDM2 binds and inhibits vitamin D receptor

The E3 ubiquitin ligase and transcriptional repressor MDM2 is a potent inhibitor of the p53 family of transcription factors and tumor suppressors. Herein, we report that vitamin D receptor (VDR), another transcriptional regulator and probably, tumor suppressor, is also bound and inhibited by MDM2. This interaction was not affected by vitamin D ligand. VDR was ubiquitylated in the cell and its steady-state level was controlled by the proteasome. Strikingly, overproduced MDM2 reduced the level of VDR whereas knockdown of endogenous MDM2 increased the level of VDR. In addition to ubiquitin-marking proteins for degradation, MDM2, once recruited to promoters by DNA-binding interaction partners, can inhibit the transactivation of genes. Transient transfections with a VDR-responsive luciferase reporter revealed that low levels of MDM2 potently suppress VDR-mediated transactivation. Conversely, knockdown of MDM2 resulted in a significant increase of transcript from the CYP24A1 and p21 genes, noted cellular targets of transactivation by liganded VDR. Our findings suggest that MDM2 negatively regulates VDR in some analogy to p53.

Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2

NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator.

Np9, a cellular protein of retroviral ancestry restricted to human, chimpanzee and gorilla, binds and regulates ubiquitin ligase MDM2.

Humans and primates are long-lived animals with long reproductive phases. One factor that appears to contribute to longevity and fertility in humans, as well as to cancer-free survival, is the transcription factor and tumor suppressor p53, controlled by its main negative regulator MDM2. However, p53 and MDM2 homologs are found throughout the metazoan kingdom from Trichoplacidae to Hominidae. Therefore the question arises, if p53/MDM2 contributes to the shaping of primate features, then through which mechanisms. Previous findings have indicated that the appearances of novel p53-regulated genes and wild-type p53 variants during primate evolution are important in this context. Here, we report on another mechanism of potential relevance. Human endogenous retrovirus K subgroup HML-2 (HERV-K(HML-2)) type 1 proviral sequences were formed in the genomes of the predecessors of contemporary Hominoidea and can be identified in the genomes of Nomascus leucogenys (gibbon) up to Homo sapiens. We previously reported on an alternative splicing event in HERV-K(HML-2) type 1 proviruses that can give rise to nuclear protein of 9 kDa (Np9). We document here the evolution of Np9-coding capacity in human, chimpanzee and gorilla, and show that the C-terminal half of Np9 binds directly to MDM2, through a domain of MDM2 that is known to be contacted by various cellular proteins in response to stress. Np9 can inhibit the MDM2 ubiquitin ligase activity toward p53 in the cell nucleus, and can support the transactivation of genes by p53. Our findings point to the possibility that endogenous retrovirus protein Np9 contributes to the regulation of the p53-MDM2 pathway specifically in humans, chimpanzees and gorillas.

Differential basal levels of MDM-2 transcription induced by p53-P72 and p53-R72.

4028 Cell Cycle Volume 9 issue 19 Human wild-type p53 comes in two common variants: a primordial one with a proline residue at position 72 within the regulatory proline-rich domain, and a more recent one with an arginine residue instead. The two p53s differ in their ability to regulate basal functions of the human organism. For example, p53R72 is a better transactivator of the LIF gene. Since LIF is crucial for the efficient implantation of the embryo into the uterus, increased fertility among p53-R72 females may have been the primary force behind the rapid fixation of the R72allele in the human population. On the other hand, p53-R72 individuals have a reduced life expectancy compared to p53P72 individuals for unknown reasons not related to tumor suppression. MDM-2 is the major negative regulator of p53; its gene is transactivated by p53. Resting unstressed primary cells express p53 at steady, very low basal levels. Cycling unstressed cells, in contrast, exhibit brief pulses of p53-overexpression in the G 1 and S/G 2 phases of cell cycle that are, however, non-productive with respect to transactivation of p53 target genes. One notable exception is the MDM-2 gene; it is transactivated in response to unstressed p53 pulses in cycling cells. To study differences between the two wild-type p53 variants in unstressed primary human cells and to avoid imponderability due to the G 1 and S/G 2 -pulses in asynchronously cycling cultures, we analyzed the expression of p53 target genes by RT-qPCR in resting contact-inhibited cultures of human diploid fibroblasts Differential basal levels of MDM-2 transcription induced by p53-P72 and p53-R72

Caspase-3 joins the p53 interactome.

Commentary to: Wild-type and mutant p53 proteins interact with mitochondrial caspase-3 Amanda K. Frank, E. Christine Pietsch, Patrick Dumont, Joy Tao and Maureen E. Murphy