Juliane Lüscher-Firzlaff

Stimulation of c‐MYC transcriptional activity and acetylation by recruitment of the cofactor CBP

The c‐MYC oncoprotein regulates various aspects of cell behaviour by modulating gene expression. Here, we report the identification of the cAMP‐response‐element‐binding protein (CBP) as a novel c‐MYC binding partner. The two proteins interact both in vitro and in cells, and CBP binds to the carboxy‐terminal region of c‐MYC. Importantly, CBP, as well as p300, is associated with E‐box‐containing promoter regions of genes that are regulated by c‐MYC. Furthermore, c‐MYC and CBP/p300 function synergistically in the activation of reporter‐gene constructs. Thus, CBP and p300 function as positive cofactors for c‐MYC. In addition, c‐MYC is acetylated in cells. This modification does not require MYC box II, suggesting that it is independent of TRRAP complexes. Instead, CBP acetylates c‐MYC in vitro, and co‐expression of CBP with c‐MYC stimulates in vivo acetylation. Functionally, this results in a decrease in ubiquitination and stabilization of c‐MYC proteins. Thus, CBP and p300 are novel functional binding partners of c‐MYC.

The Ins and Outs of MYC Regulation by Posttranslational Mechanisms

The proteins of the MYC family are key regulators of cell behavior. MYC, originally identified as an oncoprotein, affects growth, proliferation, differentiation, and apoptosis of cells through its ability to regulate a significant number of genes. In addition MYC governs events associated with tumor progression, including genetic stability, migration, and angiogenesis. The pleiotropic activities attributed to MYC and their balanced control requires that the expression and function of MYC is tightly controlled. Indeed many different pathways and factors have been identified that impinge on MYC gene expression and protein function. In particular the protein is subject to different posttranslational modifications, including phosphorylation, ubiquitinylation, and acetylation. Here we discuss the latest developments regarding these modifications that control various aspects of MYC function, including its stability, the interaction with partner proteins, and the transcriptional potential.

IL-31 regulates differentiation and filaggrin expression in human organotypic skin models

BACKGROUND Atopic dermatitis (AD) is an inflammatory skin disease affecting 10% to 20% of children and 1% to 3% of adults in industrialized countries. Enhanced expression of IL-31 is detected in skin samples of patients with AD, but its physiological relevance is not known. OBJECTIVE We sought to determine the role of IL-31 in skin differentiation. METHODS We used human 3-dimensional organotypic skin models with either primary keratinocytes or HaCaT keratinocytes with inducible IL-31 receptor α to evaluate the effect of IL-31. The consequences were studied by using histology, the expression of markers analyzed by immunofluoresence and quantitative RT-PCR, and gene expression arrays. RESULTS We observed that IL-31 interferes with keratinocyte differentiation. Gene expression analysis revealed a limited set of genes deregulated in response to IL-31, including IL20 and IL24. In HaCaT keratinocytes with inducible IL-31 receptor α, IL-31 inhibited proliferation upon induction of IL-31 receptor α by inducing cell cycle arrest. As in primary cells, IL-31-treated HaCaT cells elicited a differentiation defect in organotypic skin models, associated with reduced epidermal thickness, disturbed epidermal constitution, altered alignment of the stratum basale, and poor development of the stratum granulosum. The differentiation defect was associated with a profound repression of terminal differentiation markers, including filaggrin, an essential factor for skin barrier formation, and a reduced lipid envelope. The highly induced proinflammatory cytokines IL-20 and IL-24 were responsible for part of the effect on FLG expression and thus for terminal differentiation. CONCLUSION Our study suggests that IL-31 is an important regulator of keratinocyte differentiation and demonstrates a link between the presence of IL-31 in skin, as found in patients with AD, and filaggrin expression.

Tight correlation between expression of the Forkhead transcription factor FOXM1 and HER2 in human breast cancer

BackgroundFOXM1 regulates expression of cell cycle related genes that are essential for progression into DNA replication and mitosis. Consistent with its role in proliferation, elevated expression of FOXM1 has been reported in a variety of human tumour entities. FOXM1 is a gene of interest because recently chemical inhibitors of FOXM1 were described to limit proliferation and induce apoptosis in cancer cells in vitro, indicating that FOXM1 inhibitors could represent useful anticancer therapeutics.MethodsUsing immunohistochemistry (IHC) we systematically analysed FOXM1 expression in human invasive breast carcinomas (n = 204) and normal breast tissues (n = 46) on a tissue microarray. Additionally, using semiquantitative realtime PCR, a collection of paraffin embedded normal (n = 12) and cancerous (n = 25) breast tissue specimens as well as benign (n = 3) and malignant mammary cell lines (n = 8) were investigated for FOXM1 expression. SPSS version 14.0 was used for statistical analysis.ResultsFOXM1 was found to be overexpressed in breast cancer in comparison to normal breast tissue both on the RNA and protein level (e.g. 8.7 fold as measured by realtime PCR). We found a significant correlation between FOXM1 expression and the HER2 status determined by HER2 immunohistochemistry (P < 0.05). Univariate survival analysis showed a tendency between FOXM1 protein expression and unfavourable prognosis (P = 0.110).ConclusionFOXM1 may represent a novel breast tumour marker with prognostic significance that could be included into multi-marker panels for breast cancer. Interestingly, we found a positive correlation between FOXM1 expression and HER2 status, pointing to a potential role of FOXM1 as a new drug target in HER2 resistant breast tumour, as FOXM1 inhibitors for cancer treatment were described recently. Further studies are underway to analyse the potential interaction between FOXM1 and HER2, especially whether FOXM1 directly activates the HER2 promoter.

Signaling by IL-31 and functional consequences

Cytokines are key to control cellular communication. Interleukin-31 (IL-31) was recently discovered as a new member of the IL-6 family of cytokines. IL-31 signals through a heterodimeric receptor composed of OSMR and IL-31RA, a complex that stimulates the JAK-STAT, the RAS/ERK and the PI3K/AKT signal transduction pathways. The available data suggests that IL-31 is important for both innate and adaptive immunity in tissues that are in close contact with the environment, i.e. the skin, the airways and the lung, and the lining of the intestine. Enhanced expression of IL-31 is associated with a number of diseases, including pruritic diseases such as atopic dermatitis, but also in allergy and inflammatory bowel disease. In these tissues IL-31 coordinates the interaction of different immune cells, including T-cells, mast cells, and eosinophils, with epithelial cells. In this review we have summarized the available data on IL-31 and its receptor, their expression pattern and how they are regulated. We describe the current state of knowledge of the involvement of IL-31 in diseases, both in humans and in mouse models. From these studies it is becoming clear that IL-31 plays an important role in the proper functioning of the skin and of airway and intestinal epithelia. The findings available suggest that IL-31 might be an interesting target for directed drug therapy.

Regulation of the transcription factor FOXM1c by Cyclin E/CDK2

The FOXM1 forkhead proteins, originally identified as M‐phase phosphoproteins, are proliferation‐associated transcriptional regulators involved in cell cycle progression, genetic stability and tumorigenesis. Here we demonstrate that Cyclin‐dependent kinases regulate the transcriptional activity of FOXM1c. This is independent of an N‐terminal negative regulatory domain and of the forkhead DNA binding domain. Instead we mapped the responsive sites in the transactivation domain. A combination of three phosphorylation sites mediates the Cyclin E and Cyclin A/CDK2 effects. Our findings provide evidence for a novel Cyclin E/CDK2 substrate that functions in cell cycle control.

Dynamic subcellular localization of the mono-ADP-ribosyltransferase ARTD10 and interaction with the ubiquitin receptor p62

BackgroundADP-ribosylation is a posttranslational modification catalyzed in cells by ADP-ribosyltransferases (ARTD or PARP enzymes). The ARTD family consists of 17 members. Some ARTDs modify their substrates by adding ADP-ribose in an iterative process, thereby synthesizing ADP-ribose polymers, the best-studied example being ARTD1/PARP1. Other ARTDs appear to mono-ADP-ribosylate their substrates and are unable to form polymers. The founding member of this latter subclass is ARTD10/PARP10, which we identified as an interaction partner of the nuclear oncoprotein MYC. Biochemically ARTD10 uses substrate-assisted catalysis to modify its substrates. Our previous studies indicated that ARTD10 may shuttle between the nuclear and cytoplasmic compartments. We have now addressed this in more detail.ResultsWe have characterized the subcellular localization of ARTD10 using live-cell imaging techniques. ARTD10 shuttles between the cytoplasmic and nuclear compartments. When nuclear, ARTD10 can interact with MYC as measured by bimolecular fluorescence complementation. The shuttling is controlled by a Crm1-dependent nuclear export sequence and a central ARTD10 region that promotes nuclear localization. The latter lacks a classical nuclear localization sequence and does not promote full nuclear localization. Rather this non-conventional nuclear localization sequence results in an equal distribution of ARTD10 between the cytoplasmic and the nuclear compartments. ARTD10 forms discrete and dynamic bodies primarily in the cytoplasm but also in the nucleus. These contain poly-ubiquitin and co-localize in part with structures containing the poly-ubiquitin receptor p62/SQSTM1. The co-localization depends on the ubiquitin-associated domain of p62, which mediates interaction with poly-ubiquitin.ConclusionsOur findings demonstrate that ARTD10 is a highly dynamic protein. It shuttles between the nuclear and cytosolic compartments dependent on a classical nuclear export sequence and a domain that mediates nuclear uptake. Moreover ARTD10 forms discrete bodies that exchange subunits rapidly. These bodies associate at least in part with the poly-ubiquitin receptor p62. Because this protein is involved in the uptake of cargo into autophagosomes, our results suggest a link between the formation of ARTD10 bodies and autophagy.Lay abstractPost-translational modifications refer to changes in the chemical appearance of proteins and occur, as the name implies, after proteins have been synthesized. These modifications frequently affect the behavior of proteins, including alterations in their activity or their subcellular localization. One of these modifications is the addition of ADP-ribose to a substrate from the cofactor NAD+. The enzymes responsible for this reaction are ADP-ribosyltransferases (ARTDs or previously named PARPs). Presently we know very little about the role of mono-ADP-ribosylation of proteins that occurs in cells. We identified ARTD10, a mono-ADP-ribosyltransferase, as an interaction partner of the oncoprotein MYC. In this study we have analyzed how ARTD10 moves within a cell. By using different live-cell imaging technologies that allow us to follow the position of ARTD10 molecules over time, we found that ARTD10 shuttles constantly in and out of the nucleus. In the cytosol ARTD10 forms distinct structures or bodies that themselves are moving within the cell and that exchange ARTD10 subunits rapidly. We have identified the regions within ARTD10 that are required for these movements. Moreover we defined these bodies as structures that interact with p62. This protein is known to play a role in bringing other proteins to a structure referred to as the autophagosome, which is involved in eliminating debris in cells. Thus our work suggests that ARTD10 might be involved in and is regulated by ADP-riboslyation autophagosomal processes.

The interaction of MYC with the trithorax protein ASH2L promotes gene transcription by regulating H3K27 modification

The appropriate expression of the roughly 30,000 human genes requires multiple layers of control. The oncoprotein MYC, a transcriptional regulator, contributes to many of the identified control mechanisms, including the regulation of chromatin, RNA polymerases, and RNA processing. Moreover, MYC recruits core histone-modifying enzymes to DNA. We identified an additional transcriptional cofactor complex that interacts with MYC and that is important for gene transcription. We found that the trithorax protein ASH2L and MYC interact directly in vitro and co-localize in cells and on chromatin. ASH2L is a core subunit of KMT2 methyltransferase complexes that target histone H3 lysine 4 (H3K4), a mark associated with open chromatin. Indeed, MYC associates with H3K4 methyltransferase activity, dependent on the presence of ASH2L. MYC does not regulate this methyltransferase activity but stimulates demethylation and subsequently acetylation of H3K27. KMT2 complexes have been reported to associate with histone H3K27-specific demethylases, while CBP/p300, which interact with MYC, acetylate H3K27. Finally WDR5, another core subunit of KMT2 complexes, also binds directly to MYC and in genome-wide analyses MYC and WDR5 are associated with transcribed promoters. Thus, our findings suggest that MYC and ASH2L–KMT2 complexes cooperate in gene transcription by controlling H3K27 modifications and thereby regulate bivalent chromatin.

Regulation of the MAD1 promoter by G-CSF.

MAD family proteins are transcriptional repressors that antagonize the functions of MYC oncoproteins. In particular, MAD1 has been demonstrated to interfere with MYC-induced proliferation, transformation and apoptosis. The MAD1 gene is expressed in distinct patterns, mainly associated with differentiation and quiescence. We observed that MAD1 is directly activated by G-CSF in promyelocytic cell lines. To investigate the transcriptional regulation of the human MAD1 gene, we have cloned and characterized its promoter. A region of high homology between the MAD1 orthologs of human, mouse and rat contains the core promoter, marked by open chromatin, high GC content and the lack of a TATA box. Using deletion constructs we identified two CCAAT-boxes occupied by C/EBPα and β in the homology region that mediate responsiveness to G-CSF receptor signaling. The necessary signals include the activation of STAT3 and the RAS/RAF/ERK pathway. STAT3 does not bind directly to promoter DNA, but is recruited by C/EBPβ. In summary, our studies provide a first analysis of the MAD1 promoter and suggest STAT3 functions as a C/EBPβ cofactor in the regulation of the MAD1 gene. Our findings provide the base for the characterization of additional signal transduction pathways that control the expression of MAD1.

Inhibition of apoptosis by MAD1 is mediated by repression of the PTEN tumor suppressor gene

The MYC/MAX/MAD network of transcriptional regulators controls distinct aspects of cell physiology, including cell proliferation and apoptosis. Within the network MAD proteins antagonize the functions of MYC oncoproteins, and the latter are deregulated in the majority of human cancers. While MYC sensitizes cells to proapoptotic signals, the transcriptional repressor MAD1 inhibits apoptosis in response to a broad range of stimuli, including oncoproteins. The molecular targets of MAD1 that mediate inhibition of apoptosis are not known. Here we describe the phos‐phatase and tensin homologue deleted on chromosome ten (PTEN) tumor suppressor gene as a target of MAD1. By binding to the proximal promoter region, MAD1 downregulated PTEN expression. PTEN functions as a lipid phosphatase that regulates the phospha‐tidylinositol 3‐kinase/AKT pathway. Indeed MAD1‐de‐pendent repression of PTEN led to activation of AKT and subsequent stimulation of the antiapoptotic NF‐κB pathway. Interfering with AKT function affected the control of Fas‐induced apoptosis by MAD1. In addition, knockdown of PTEN using small interfering RNA (siRNA) or the lack of PTEN rendered cells insensitive to inhibition of apoptosis by MAD1. These findings identify the PTEN gene as a target of the MYC‐antagonist MAD1 and provide a molecular framework critical for the ability of MAD1 to inhibit apoptosis. Rottmann S., Speckgens, S., Luscher‐Firzlaff, J., Lüscher B. Inhibition of apoptosis by MAD1 is mediated by repression of the PTENtumor suppressor gene. FASEB J. 22, 1124–1134 (2008)