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Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations.

This work describes the set-up of a shared platform among the laboratories of the Alleanza Contro il Cancro (ACC) Italian Research Network for the identification of fusion transcripts in sarcomas by using Next Generation Sequencing (NGS). Different NGS approaches, including anchored multiplex PCR and hybrid capture-based panels, were employed to profile a large set of sarcomas of different histotypes. The analysis confirmed the reliability of NGS RNA-based approaches in detecting sarcoma-specific rearrangements. Overall, the anchored multiplex PCR assay proved to be a fast and easy-to-analyze approach for routine diagnostics laboratories.

Multiple myeloma (MM) is a hematologic malignancy produced by a clonal expansion of plasma cells and characterized by abnormal production and secretion of monoclonal antibodies. This pathology exhibits an enormous heterogeneity resulting not only from genetic alterations but also from several epigenetic dysregulations. Here we provide evidence that Che-1/AATF (Che-1), an interactor of RNA polymerase II, promotes MM proliferation by affecting chromatin structure and sustaining global gene expression. We found that Che-1 depletion leads to a reduction of “active chromatin” by inducing a global decrease of histone acetylation. In this context, Che-1 directly interacts with histones and displaces histone deacetylase class I members from them. Strikingly, transgenic mice expressing human Che-1 in plasma cells develop MM with clinical features resembling those observed in the human disease. Finally, Che-1 downregulation decreases BRD4 chromatin accumulation to further sensitize MM cells to bromodomain and external domain inhibitors. These findings identify Che-1 as a promising target for MM therapy, alone or in combination with bromodomain and external domain inhibitors.

We have previously shown that melatonin exerts tumor suppressor activities by inducing the p38-p53 axis. This occurred within a few hours while no data are available on how melatonin pathway can be sustained on the long term. Here we show that miR-24, which has been demonstrated to target genes involved in the DNA repair process, targets p38, p53, PML and H2AX simultaneously. We show that long-term treatment with melatonin can decrease miR-24 levels post-transcriptionally, which pairs with a long-wave regulation of genes involved in cell proliferation, DNA damage, RNA metabolism and cell shape and transformation. Moreover, we show that melatonin can inhibit cell proliferation and migration, at least in part, by downregulating miR-24. Furthermore, we propose the involvement of hnRNP A1, which is downregulated by melatonin and involved in miRNA processing, in the regulation of miR-24 levels by melatonin. We conclude showing that miR-24 is upregulated in colon, breast and head and neck datasets and its levels negatively correlate with overall survival.

Genomic technologies are reshaping the molecular landscape of colorectal cancer (CRC), revealing that oncogenic driver mutations (APC and TP53) coexist with still underappreciated genetic events. We hypothesized that mutational analysis of CRC-linked genes may provide novel information on the connection between genetically-deregulated pathways and clinical outcomes. We performed next-generation sequencing (NGS) analysis of 16 recurrently mutated genes in CRC exploiting tissue specimens from 98 advanced CRC patients. Multiple correspondence analysis (MCA) was used to identify gene sets characterizing negative and positive outliers (patients in the lowest and highest quartile of progression-free survival, PFS). Variables potentially affecting PFS and overall survival (OS) were tested in univariate and multivariate Cox proportional hazard models. Sensitivity analyses and resampling were used to assess the robustness of genomic predictors. MCA revealed that APC and TP53 mutations were close to the negative outlier group, whereas mutations in other WNT pathway genes were in proximity of the positive outliers. Reasoning that genetic alterations interact epistatically, producing greater or weaker consequences in combination than when individually considered, we tested whether patients whose tumors carried a genetic background characterized by APC and TP53 mutations without coexisting mutations in other WNT genes (AMER1, FBXW7, TCF7L2, CTNNB1, SOX9) had adverse survival outcomes. With this approach, we identified two oncodriver signatures (ODS1 and ODS2) associated with shorter PFS (ODS1 multivariate Cox for PFS: HR 2.16, 95%CI: 1.28–3.64, p = 0.004; ODS2 multivariate Cox for PFS: HR 2.61, 95%CI: 1.49–4.58, p = 0.001). Clinically-focused and molecularly-focused sensitivity analyses, resampling, and reclassification of mutations confirmed the stability of ODS1/2. Moreover, ODS1/2 negatively impacted OS. Collectively, our results point to co-occurring driver mutations as an adverse molecular factor in advanced CRC. This relationship depends on a broader genetic context highlighting the importance of genetic interactions.

Whether PD-L1 expression is associated with survival outcomes in gastric cancer (GC) is controversial. The inhibition of the PD-1/PD-L1 pathway is effective against genomically unstable tumors. Hypothesizing that also the clinical significance of PD-L1 might be dependent on the activation of molecular circuits ensuring genomic stability, we evaluated PD-L1 expression in tissue samples from 72 advanced GC patients treated with first-line chemotherapy. Samples were already characterized for DNA damage repair (DDR) component expression (pATM, pChk1, pWee1, γ-H2AX and pRPA2) along with mutations in DDR-linked genes (TP53 and ARID1A). Overall, PD-L1 expression was not associated with progression-free survival (PFS) and overall survival (OS), independently on whether we considered its expression in tumor cells (PD-L1-TCs) or in the immune infiltrate (PD-L1-TILs). In subgroup analysis, positive PD-L1-TC immunostaining was associated with better PFS in patients whose tumors did not carry DDR activation (multivariate Cox: HR 0.34, 95%CI: 0.15–0.76, p = 0.008). This subset (DDR(off)) was characterized by negative pATM expression or the presence of ARID1A mutations. Conversely, the relationship between PD-L1-TC expression and PFS was lost in a molecular scenario denoting DDR activation (DDR(on)), as defined by concomitant pATM expression and ARID1A wild-type form. Surprisingly, while PD-L1-TC expression was associated with better OS in the DDR(off) subset (multivariate Cox: HR 0.41, 95% CI: 0.17–0.96, p = 0.039), in the DDR(on) subgroup we observed an opposite impact on OS (multivariate Cox: HR 2.56, 95%CI: 1.06–6.16, p = 0.036). Thus, PD-L1-TC expression may impact survival outcomes in GC on the basis of the activation/inactivation of genome-safeguarding pathways.

Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates cell growth, metabolism, and autophagy to maintain cellular homeostasis. Its activity is inhibited by adverse conditions, including nutrient limitation, hypoxia, and DNA damage. In this study, we demonstrate that Che-1, a RNA polymerase II-binding protein activated by the DNA damage response, inhibits mTOR activity in response to stress conditions. We found that, under stress, Che-1 induces the expression of two important mTOR inhibitors, Redd1 and Deptor, and that this activity is required for sustaining stress-induced autophagy. Strikingly, Che-1 expression correlates with the progression of multiple myeloma and is required for cell growth and survival, a malignancy characterized by high autophagy response.

Background Solid tumours are less oxygenated than normal tissues. Consequently, cancer cells acquire to be adapted to a hypoxic environment. The poor oxygenation of solid tumours is also a major indicator of an adverse cancer prognosis and leads to resistance to conventional anticancer treatments. We previously showed the involvement of Che-1/AATF (Che-1) in cancer cell survival under stress conditions. Herein we hypothesized that Che-1 plays a role in the response of cancer cells to hypoxia. Methods The human colon adenocarcinoma HCT116 and HT29 cell lines undepleted or depleted for Che-1 expression by siRNA, were treated under normoxic and hypoxic conditions to perform studies regarding the role of this protein in metabolic adaptation and cell proliferation. Che-1 expression was detected using western blot assays; cell metabolism was assessed by NMR spectroscopy and functional assays. Additional molecular studies were performed by RNA seq, qRT-PCR and ChIP analyses. Results Here we report that Che-1 expression is required for the adaptation of cells to hypoxia, playing an important role in metabolic modulation. Indeed, Che-1 depletion impacted on HIF-1α stabilization, thus downregulating the expression of several genes involved in the response to hypoxia and affecting glucose metabolism. Conclusions We show that Che-1 a novel player in the regulation of HIF-1α in response to hypoxia. Notably, we found that Che-1 is required for SIAH-2 expression, a member of E3 ubiquitin ligase family that is involved in the degradation of the hydroxylase PHD3, the master regulator of HIF-1α stability. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0497-1) contains supplementary material, which is available to authorized users.

The guardian of the genome p53 is embedded in a fine-spun network of MicroRNAs. p53 is able to activate or repress directly the transcription of MicroRNAs that are participating in the tumor-suppressive mission of p53. On the other hand, the expression of p53 is under tight control of MicroRNAs that are either targeting directly p53 or factors that are modifying its protein level or activity. Although the most important function of p53 is suggested to be transcriptional regulation, there are several nontranscriptional functions described. One of those regards the modulation of MicroRNA biogenesis. Wild-type p53 is increasing the maturation of selected MicroRNAs from the primary transcript to the precursor MiRNA by interacting with the Microprocessor complex. Furthermore, p53 is modulating the mRNA accessibility for certain MicroRNAs by association with the RISC complex and transcriptional regulation of RNA-binding proteins. In this way p53 is able to remodel the MiRNA-mRNA interaction network. As wild-type p53 is employing MicroRNAs to suppress cancer development, gain-of-function mutant p53 proteins use MicroRNAs to confer oncogenic properties like chemoresistance and the ability to drive metastasis. Like its wild-type counterpart mutant p53 is able to regulate MicroRNAs transcriptionally and posttranscriptionally. Mutant p53 affects the MiRNA processing at two cleavage steps through interfering with the Microprocessor complex and by downregulating Dicer and KSRP, a modulator of MiRNA biogenesis. Thus, MicroRNAs are essential components in the p53 pathway, contributing substantially to combat or enhance tumor development depending on the wild-type or mutant p53 context.

Growing evidence shows that mutant p53 proteins, which are present in many human tumors, gain oncogenic activities that can actively contribute to tumorigenesis. Mutant p53 proteins have been extensively shown to affect the expression of several genes involved in various aspects of cancer biology. We show here the ChIP-on-chip analysis of mutant p53 binding to a set of 154 promoters, composed of both validated and putative mutant p53 target genes. By using the chromatin obtained from mutant p53R175H-immunoprecipitation in proliferating SKBr3 breast cancer cells, we found that mutant p53 binds to 40 of the 154 promoters analyzed. siRNA-mediated mutant p53 knock-down modulates the transcript abundance of some of these target genes. Two-thirds of the mutant p53-bound promoters were also engaged by either p300 or PCAF acetyl-transferases, strongly indicating the presence of transcriptionally active complexes. We also found that NF-kB binding sites are overrepresented among the mutant p53-bound promoters; a ChIP-on-chip analysis confirmed that NF-kB p65 binds to 27 of the mutant p53-bound promoters, indicating that mutant p53 could influence the transcriptional output of these NF-kB target genes.

Tumor transformation is the result of genetic and epigenetic modifications that alter gene transcription, consequently producing a specific oncogenic program. Multiple myeloma (MM) is a neoplasia characterized by the accumulation of proliferating antibodies producing plasma cells in the bone marrow. This disease is the second most frequent haematological malignancy in the US and Europe. In this study we demonstrate that Che-1 plays a crucial role in the control of transcription and cellular proliferation by regulating the state of the chromatin and by increasing its accessibility, highlighting Che-1 as an essential component of the transcription machinery in MM. Che-1 loss induces a global transcription shut-off, by reducing RNA Pol II recruitment onto the DNA. Most importantly, transcriptional inhibition in Che-1 depleted cells could already be detected at pre-mRNA levels, leading to the hypothesis that Che-1 could regulate RNA pol II activity directly. Our study shows that Che-1 is required for the maintenance of open chromatin in MM cells, and is involved in general histone acetylation. Morever, we found that Che-1 downregulation further sensitizes MM cells to bromodomain and extra-terminal (BET) inhibitors. Indeed Che-1 silencing increased JQ1 sensitivity of MM cells. In summary, our findings identify Che-1 as a key player for maintaining the open chromatin structure required for sustaining MM growth. These findings support Che-1 as a possible target for MM therapy, alone or in combination with BET inhibitors. Citation Format: Tiziana Bruno, Francesca De Nicola, Frauke Goeman, Matteo Pallocca, Cristina Sorino, Valeria Catena, Gianluca Bossi, Bruno Amadio, Giovanni Cigliana, Enrico Spugnini, Maria Rosaria Ricciardi, Maria Teresa Petrucci, Alfonso Baldi, Mario Cioce, Giancarlo Cortese, Elisabetta Mattei, Roberta Merola, Umberto Gianelli, Francesco Pisani, Svitlana Gumenyuk, Andrea Mengarelli, Katja Hopker, Thomas Benzing, Aristide Floridi, Claudio Passananti, Giovanni Blandino, Simona Iezzi, Maurizio Fanciulli. Che-1/aatf-induced transcriptionally active chromatin promotes cell growth in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 350.

Introduction PTEN is a protein that restrains cells from growing unchecked; when it is absent or not functioning (as it often happens in prostate and colorectal cancer - CRC), cancer develops more easily and grows in a more aggressive and therapy-resistant fashion. However, just measuring PTEN mutational status or gene/protein levels in clinical samples may not be sufficient to assess its function, as its regulation is complex. Therefore, we analysed miRNA, transcriptomic, and proteomic profiles of isogenic CRC models differing for PTEN status, in order to identify a set of genes or proteins that are stably modulated by the presence or absence of a functional PTEN. Material and methods To identify biomarkers of PTEN loss of function and functional downstream effectors of PTEN activity, the isogenic CRC cell lines HCT116 (PTEN+/+) and HCT116 PTEN-/-(Horizon Discovery Ltd) were subjected to comprehensive miRNA profiling (Human miRNA Microarray Release 21, Agilent), transcriptomic analysis (NextSeq 500, Illumina), and semi-quantitative phosphoproteomic analysis (Antibody Microarray Service, KinexTM). Results and discussions miRNA profiling found 18 miRNAsdifferentially expressed in HCT116 PTEN+/+ and their HCT116 PTEN-/- counterpart: only miR-196a-5p is up-regulated in HCT116 PTEN-/-, whereas other 17 miRNAs were all down-regulated; several of these target miRNAs have been validated by qRT-PCR. RNA-Seq data were analysed via the RAP pipeline available at CINECA that relies on the Tuxedo suite (Tophat-Cufflinks-Cuffdiff) for read mapping, transcript abundance estimation and transcript or gene-based differential expression. RNA-Seq analysis showed 247 differentially expressed genes in HCT116 PTEN+/+ cells as compared to HCT116 PTEN-/- (159 up-regulated and 88 down-regulated in HCT116 PTEN-/-) and clustering into 9 subgroups (including: tissue morphogenesis; negative regulation of phosphatase metabolic process; regulation of sequence-specific DNA binding transcription factor activity). qRT-PCR assay of 10 dysregulated genes was performed to validate the RNA-Seq data set. Proteomic analysis showed that 92 proteins (25 total proteins and 67 phospho-proteins) are differentially expressed in HCT116 PTEN+/+ and their HCT116 PTEN-/- counterpart, some of which have been validated by WB. Conclusion Our data identify differentially expressed sets of miRNA/genes/proteins between HCT116 PTEN+/+ and HCT116 PTEN-/-. Bioinformatics analysis to cluster these sets into functional PTEN-loss ‘signatures’ is ongoing.

RNA-sequencing is a revolutionary tool to follow differential expression after treatment with cancer chemopreventive agents. It allows a real genome-wide screening independent of prior assumptions and is well suited for analyzing coding but also long noncoding RNAs. It still consents the discovery of new genes and isoforms and increased our knowledge of antisense and other noncoding RNAs in a tremendous manner. Moreover, it permits to detect low-abundance and biologically critical isoforms and reveals genetic variants and gene fusions in one single assay. Here, we provide a detailed protocol for stranded RNA-sequencing.

Malignant pleural mesothelioma is a poorly treated neoplasia arising from the pleural mesothelial lining. Here we document that the leaf extract of Cynara scolymus exerts broad antitumoral effects both in vitro and in vivo on mesothelioma cell lines. We found that Cynara scolymus treatment affects strongly cell growth, migration and tumor engraftment of mesothelioma cell lines. Strikingly, dietary feeding with Cynara scolymus leaf extract reduces the growth of mesothelioma xenografted tumors similarly to pemetrexed, a commonly employed drug in the treatment of mesothelioma. In aggregate our findings suggest that leaf extract of Cynara scolymus holds therapeutic potential for the treatment of mesothelioma.

Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D) and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954) human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative). These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression.

There is growing evidence that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) plays a role in breast cancer prevention and survival. It elicits a variety of antitumor activities like controlling cellular differentiation, proliferation and angiogenesis. Most of its biological effects are exerted via its nuclear receptor which acts as a transcriptional regulator. Here, we carried out a genome-wide investigation of the primary transcriptional targets of 1α,25(OH)2D3 in breast epithelial cancer cells using RNA-Seq technology. We identified early transcriptional targets of 1α,25(OH)2D3 involved in adhesion, growth regulation, angiogenesis, actin cytoskeleton regulation, hexose transport, inflammation and immunomodulation, apoptosis, endocytosis and signaling. Furthermore, we found several transcription factors to be regulated by 1α,25(OH)2D3 that subsequently amplify and diversify the transcriptional output driven by 1α,25(OH)2D3 leading finally to a growth arrest of the cells. Moreover, we could show that 1α,25(OH)2D3 elevates the trimethylation of histone H3 lysine 4 at several target gene promoters. Our present transcriptomic analysis of differential expression after 1α,25(OH)2D3 treatment provides a resource of primary 1α,25(OH)2D3 targets that might drive the antiproliferative action in breast cancer epithelial cells.

Despite the overall progress in treatment of B-ALL, relapse occurs across the whole spectrum of all subtypes and has a dismal prognosis with an overall survival of 30%. This disease is typically characterized by genetic lesions resulting in oncogene activation, loss of tumor suppressor gene function and concurrent activation of pro-survival signaling pathways. The oncogene c-Myc is a master transcription factor governing many critical cell functions such as metabolism, proliferation and survival and it is involved in up to 70% of cancers. In Burkitt's Lymphoma, c-Myc gene duplications or translocations have been described as leading to its constitutive transcriptional deregulation. Che-1/AATF is an important RNA polymerase II binding protein involved in the regulation of gene transcription. Che-1 is required for proliferation in early embryogenesis, and exhibits an anti-apoptotic activity in different tumour contests. Previous findings showed that Che-1 is over-expressed in different leukemic cell lines correlated with c-myc gene over-amplification, but, although it has been shown that Che-1 controls different mechanisms of tumorigenesis in several tumour contests, its role in haematological malignancies has not been deeply explored. With this aim we found a complete correlation of Che-1 and c-Myc expression in 80 B-ALL patients compared with 15 bone marrows from healthy donors lacking the expression of both molecules. These results are in agreement with public data derived from an RNAseq experiment on non-transgenic (control) and Eµ-myc transgenic littermates (pre-tumour), and in lymphomas arising in adult Eµ-myc animals (tumour), in which our analysis demonstrated that Che-1 mRNA levels increase during lymphomagenesis. In addition we observed a total ablation of Che-1 and c-Myc in the remission status, whereas samples from resistant or relapsed patients maintained high levels of both proteins. At the same time we observed that the down-regulation of c-Myc strongly reduces Che-1 mRNA and protein levels. Chip analysis revealed the ability of c-Myc to bind Che-1 promoter. Moreover, the two proteins also physically interact as demonstrated by co-immunoprecipitation experiments. We observed that Che-1 down-regulation induced growth arrest of B-ALL cell lines and affected the amount of ribosomal RNA by inhibiting the cooperation with UBF and RNA pol I. Based on these findings, by RNAseq analysis we compared the effect of Che-1 versus c-Myc downregulation finding a strong sharing of the controlled pathways. We demonstrated that Che-1 may be considered as a useful prognostic factor, able to early evaluate the aggressiveness of B-ALL. Furthermore the modulation of Che-1 expression may represent a strategy to improve treatment outcomes particularly in high-risk patient subgroups, wherein failure of conventional chemotherapy and relapse are common. Disclosures No relevant conflicts of interest to declare.

Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (on-chip) is a technique well suited for a comprehensive analysis of transcription factor binding sites, histone modification patterns, and nucleosome occupancy. It can be restricted to a subset of genes or regions but also expanded up to a genome-wide range yielding insight into the functional elements of gene regulatory networks. Mutant p53 proteins have lost their capacity to bind to its cognate binding sites, but it is well established that it has retained the ability to bind indirectly to DNA via other transcription factors and therefore change the expression of several target genes. The identification of those transcription factors and binding regions sheds light on how mutant p53 is able to exert oncogenic functions.

p33ING2 belongs to the ING-gene family that is involved in tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. Most functions are dependent on the tumor suppressor p53. p33ING2 was also shown to bind to trimethylated lysine 4 of histone H3. Here, we show that p33ING2 contains a transferable silencing function, which is independent of p53. p33ING2-mediated gene silencing is resistant to the HDAC-inhibitor trichostatin A indicating that p33ING2 uses a non-HDAC class I or II pathway for gene repression in reporter assays. In line with that we show that p33ING2 is associated with histone methyltransferase (HMT) activity in vitro and in vivo, methylating specifically histone H3. Interestingly, the specificity is distinct from the MeCP2-recruited HMT. Mutation or methylation of lysine 9, a mark well known for repression, abrogates histone methylation by MeCP2 but not by the p33ING2 complex. Instead, the ING2-associated HMT shows an increased methylation activity if lysine 9 is methylated. In contrast, mutation or methylation of lysine 4, a methylation preferentially detected at active genes, led to a reduction of the ING2-associated HMT. Notably, also p33ING1 recruits HMT activity suggesting a more general biochemical interaction between members of p33ING family and HMT activity. Deletion analyses revealed that the ING2 C-terminus recruits HMT activity, which correlates with silencing function.

10 pages, 7 figures.-- et al., ING1 was identified as an inhibitor of growth and has been described as a tumor suppressor. Furthermore, the expression of ING1 is induced in senescent cells and antisense ING1 extends the proliferative life span of primary human fibroblasts. Cooperation of p33ING1 with p53 has been suggested to be an important function of ING1 in cell cycle control. Intriguingly, it has been shown that p33ING1 is associated with histone acetylation as well as with histone deacetylation function. Here we show that p33ING1 is a potent transcriptional silencer in various cell types. However, the silencing function is independent of the presence of p53. By use of deletion mutants two potent autonomous and transferable silencing domains were identified, but no evidence of an activation domain was found. The amino (N)-terminal silencing domain is sensitive to the histone deacetylase inhibitor trichostatin A (TSA) whereas the carboxy-terminal silencing function is resistant to TSA, suggesting that p33ING1 confers gene silencing through both HDAC-dependent and -independent mechanisms. Interestingly, the presence of oncogenic Ras, which is able to induce premature senescence, increases the p33ING1-mediated silencing function. Moreover, ING1-mediated silencing was reduced by coexpressing dominant-negative Ras or by treatment with the mitogen-activated protein kinase inhibitor PD98059 but not by treatment with SB203580, an inhibitor of the p38 pathway. In addition, we show that both silencing domains of ING1 are involved in cell cycle control, as measured by inhibition of colony formation of immortalized cells and by thymidine incorporation of primary human diploid fibroblasts (HDF). Interestingly, p33ING1 expression induces features of cellular senescence in HDFs., Part of this work was supported by the Graduiertenkolleg 370 (F.G.). This work has been supported by grants from the Spanish Ministry of Education (SAF03-00801) and the Cooperative Cancer Network of the Spanish Ministry of Health.

The highly conserved zinc-finger protein, CTCF, is a candidate tumor suppressor protein that binds to highly divergent DNA sequences. CTCF has been connected to multiple functions in chromatin organization and gene regulation including chromatin insulator activity and transcriptional enhancement and silencing. Here we show that CTCF harbors several autonomous repression domains. One of these domains, the zinc-finger cluster, silences transcription in all cell types tested and binds directly to the co-repressor SIN3A. Two distinct regions of SIN3A, the PAH3 domain and the extreme C-terminal region, bind independently to this zinc-finger cluster. Analysis of nuclear extract from HeLa cells revealed that CTCF is also capable of retaining functional histone deacetylase activity. Furthermore, the ability of regions of CTCF to retain deacetylase activity correlates with the ability to bind to SIN3A and to repress gene activity. We suggest that CTCF driven repression is mediated in part by the recruitment of histone deacetylase activity by SIN3A.