EZH2 is a key component of hepatoblastoma tumor cell growth.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2) catalyzes the trimethylation of histone H3 at lysine 27 via the polycomb recessive complex 2 (PRC2) and plays a time-specific role in normal fetal liver development. EZH2 is overexpressed in hepatoblastoma (HB), an embryonal tumor. EZH2 can also promote tumorigenesis via a noncanonical, PRC2-independent mechanism via proto-oncogenic, direct protein interaction, including ß-catenin. We hypothesize that the pathological activation of EZH2 contributes to HB propagation in a PRC2-independent manner. METHODS AND RESULTS: We demonstrate that EZH2 promotes proliferation in HB tumor-derived cell lines through interaction with ß-catenin. Although aberrant EZH2 expression occurs, we determine that both canonical and noncanonical EZH2 signaling occurs based on specific gene-expression patterns and interaction with SUZ12, a PRC2 component, and ß-catenin. Silencing and inhibition of EZH2 reduce primary HB cell proliferation. CONCLUSIONS: EZH2 overexpression promotes HB cell proliferation, with both canonical and noncanonical function detected. However, because EZH2 directly interacts with ß-catenin in human tumors and EZH2 overexpression is not equal to SUZ12, it seems that a noncanonical mechanism is contributing to HB pathogenesis. Further mechanistic studies are necessary to elucidate potential pathogenic downstream mechanisms and translational potential of EZH2 inhibitors for the treatment of HB.

Therapeutic Targeting of the GSK3ß-CUGBP1 Pathway in Myotonic Dystrophy.

Myotonic Dystrophy type 1 (DM1) is a neuromuscular disease associated with toxic RNA containing expanded CUG repeats. The developing therapeutic approaches to DM1 target mutant RNA or correct early toxic events downstream of the mutant RNA. We have previously described the benefits of the correction of the GSK3ß-CUGBP1 pathway in DM1 mice (HSALR model) expressing 250 CUG repeats using the GSK3 inhibitor tideglusib (TG). Here, we show that TG treatments corrected the expression of ~17% of genes misregulated in DM1 mice, including genes involved in cell transport, development and differentiation. The expression of chloride channel 1 (Clcn1), the key trigger of myotonia in DM1, was also corrected by TG. We found that correction of the GSK3ß-CUGBP1 pathway in mice expressing long CUG repeats (DMSXL model) is beneficial not only at the prenatal and postnatal stages, but also during adulthood. Using a mouse model with dysregulated CUGBP1, which mimics alterations in DM1, we showed that the dysregulated CUGBP1 contributes to the toxicity of expanded CUG repeats by changing gene expression and causing CNS abnormalities. These data show the critical role of the GSK3ß-CUGBP1 pathway in DM1 muscle and in CNS pathologies, suggesting the benefits of GSK3 inhibitors in patients with different forms of DM1.

Correction of GSK3ß at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a progressive neuromuscular disease caused by expanded CUG repeats, which misregulate RNA metabolism through several RNA-binding proteins, including CUG-binding protein/CUGBP1 elav-like factor 1 (CUGBP1/CELF1) and muscleblind 1 protein. Mutant CUG repeats elevate CUGBP1 and alter CUGBP1 activity via a glycogen synthase kinase 3ß (GSK3ß)-cyclin D3-cyclin D-dependent kinase 4 (CDK4) signaling pathway. Inhibition of GSK3ß corrects abnormal activity of CUGBP1 in DM1 mice [human skeletal actin mRNA, containing long repeats ( HSALR) model]. Here, we show that the inhibition of GSK3ß in young HSALR mice prevents development of DM1 muscle pathology. Skeletal muscle in 1-yr-old HSALR mice, treated at 1.5 mo for 6 wk with the inhibitors of GSK3, exhibits high fiber density, corrected atrophy, normal fiber size, with reduced central nuclei and normalized grip strength. Because CUG-GSK3ß-cyclin D3-CDK4 converts the active form of CUGBP1 into a form of translational repressor, we examined the contribution of CUGBP1 in myogenesis using Celf1 knockout mice. We found that a loss of CUGBP1 disrupts myogenesis, affecting genes that regulate differentiation and the extracellular matrix. Proteins of those pathways are also misregulated in young HSALR mice and in muscle biopsies of patients with congenital DM1. These findings suggest that the correction of GSK3ß-CUGBP1 pathway in young HSALR mice might have a positive effect on the myogenesis over time.-Wei, C., Stock, L., Valanejad, L., Zalewski, Z. A., Karns, R., Puymirat, J., Nelson, D., Witte, D., Woodgett, J., Timchenko, N. A., Timchenko, L. Correction of GSK3ß at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

Intravenous miR-144 inhibits tumor growth in diethylnitrosamine-induced hepatocellular carcinoma in mice.

Previous in vitro studies have demonstrated that miR-144 inhibits hepatocellular carcinoma cell proliferation, invasion, and migration. We have shown that miR-144, injected intravenously, is taken up by the liver and induces endogenous hepatic synthesis of miR-144. We hypothesized that administered miR-144 has tumor-suppressive effects on liver tumor development in vivo. The effects of miR-144 on tumorigenesis and tumor growth were tested in a diethylnitrosamine-induced hepatocellular carcinoma mouse model. MiR-144 injection had no effect on body weight but significantly reduced diethylnitrosamine-induced liver enlargement compared with scrambled microRNA. MiR-144 had no effect on diethylnitrosamine-induced liver tumor number but reduced the tumor size above 50%, as evaluated by magnetic resonance imaging (scrambled microRNA 23.07 ± 5.67 vs miR-144 10.38 ± 2.62, p < 0.05) and histological analysis (scrambled microRNA 30.75 ± 5.41 vs miR-144 15.20 ± 3.41, p < 0.05). The levels of miR-144 was suppressed in tumor tissue compared with non-tumor tissue in all treatment groups (diethylnitrosamine-phosphate-buffered saline non-tumor 1.05 ± 0.09 vs tumor 0.54 ± 0.08, p < 0.01; diethylnitrosamine-scrambled microRNA non-tumor 1.23 ± 0.33 vs tumor 0.44 ± 0.10, p < 0.05; diethylnitrosamine-miR-144 non-tumor 54.72 ± 11.80 vs tumor 11.66 ± 2.75, p < 0.01), but injection of miR-144 greatly increased miR-144 levels both in tumor and non-tumor tissues. Mechanistic studies showed that miR-144 targets epidermal growth factor receptor and inhibits the downstream Src/AKT signaling pathway which has previously been implicated in hepatocellular carcinoma tumorigenesis. Exogenously delivered miR-144 may be a therapeutic strategy to suppress tumor growth in hepatocellular carcinoma.

Cooperation of C/EBP family proteins and chromatin remodeling proteins is essential for termination of liver regeneration.

UNLABELLED: Liver cancer is the fifth most common cancer. A highly invasive surgical resection of the liver tumor is the main approach used to eliminate the tumor. Mechanisms that terminate liver regeneration when the liver reaches the original size are not known. The aims of this work were to generate an animal model that fails to stop liver regeneration after surgical resections and elucidate mechanisms that are involved in termination of liver regeneration. Because epigenetic control of liver function has been previously implicated in the regulation of liver proliferation, we generated C/EBPα-S193A knockin mice, which have alterations in formation of complexes of C/EBP family proteins with chromatin remodeling proteins. The C/EBPα-S193A mice have altered liver morphology and altered liver function leading to changes of glucose metabolism and blood parameters. Examination of the proliferative capacity of C/EBPα-S193A livers showed that livers of S193A mice have a higher rate of proliferation after birth, but stop proliferation at the age of 2 months. These animals have increased liver proliferation in response to liver surgery as well as carbon tetrachloride (CCl4 )-mediated injury. Importantly, livers of C/EBPα-S193A mice fail to stop liver regeneration after surgery when livers reach the original, preresection, size. The failure of S193A livers to stop regeneration correlates with the epigenetic repression of key regulators of liver proliferation C/EBPα, p53, FXR, SIRT1, PGC1α, and TERT by C/EBPß-HDAC1 complexes. The C/EBPß-HDAC1 complexes also repress promoters of enzymes of glucose synthesis PEPCK and G6Pase. CONCLUSION: Proper cooperation of C/EBP and chromatin remodeling proteins is essential for the termination of liver regeneration after surgery and for maintenance of liver functions.

Transcriptional and translational regulation of C/EBPß-HDAC1 protein complexes controls different levels of p53, SIRT1, and PGC1α proteins at the early and late stages of liver cancer.

Cancer changes biological processes in the liver by altering gene expression at the levels of transcription, translation, and protein modification. The RNA binding protein CUGBP1 is a key regulator of translation of CCAAT enhancer binding protein ß and histone deacetylase 1 (HDAC1). These proteins form complexes that are involved in the regulation of liver biology. Here we show a critical role of the translational activation of CCAAT/enhancer binding protein ß-HDAC1 complexes in the development of liver cancer mediated by diethylnitrosamine. We found that diethylnitrosamine increases the levels of CUGBP1 and activates CUGBP1 by phosphorylation, leading to the formation of the CUGBP1-eIF2 complex, which is an activator of translation of CUGBP1-dependent mRNAs. The elevation of the CUGBP1-eIF2 complex increases translation of C/EBPß and HDAC1, resulting in an increase of C/EBPß-HDAC1 complexes at later stages of liver cancer. We found that C/EBPß-HDAC1 complexes repress promoters of three key regulators of liver functions: p53, SIRT1, and PGC1α. As the result of this suppression, the p53-, SIRT1-, and PGC1α-dependent downstream pathways are reduced, leading to increased liver proliferation. We also found that the proper regulation of C/EBPß-HDAC1 complexes is required for the maintenance of biological levels of p53, SIRT1, and PGC1α in quiescent livers and at early stages of liver cancer. Taken together, these studies showed that the development of liver cancer includes a tight regulation of levels of C/EBPß-HDAC1 complexes on the levels of transcription, translation, and posttranslational modifications.

Farnesoid X receptor inhibits gankyrin in mouse livers and prevents development of liver cancer.

UNLABELLED: One of the early events in the development of liver cancer is a neutralization of tumor suppressor proteins Rb, p53, hepatocyte nuclear factor 4α (HNF4α), and CCAAT/enhancer binding protein (C/EBP) α. The elimination of these proteins is mediated by a small subunit of proteasome, gankyrin, which is activated by cancer. The aim of this study was to determine the mechanisms that repress gankyrin in quiescent livers and mechanisms of activation of gankyrin in liver cancer. We found that farnesoid X receptor (FXR) inhibits expression of gankyrin in quiescent livers by silencing the gankyrin promoter through HDAC1-C/EBPß complexes. C/EBPß is a key transcription factor that delivers HDAC1 to gankyrin promoter and causes epigenetic silencing of the promoter. We show that down-regulation of C/EBPß in mouse hepatoma cells and in mouse livers reduces C/EBPß-HDAC1 complexes and activates the gankyrin promoter. Deletion of FXR signaling in mice leads to de-repression of the gankyrin promoter and to spontaneous development of liver cancer at 12 months of age. Diethylnitrosoamine (DEN)-mediated liver cancer in wild-type mice also involves the reduction of FXR and activation of gankyrin. Examination of liver cancer in old mice and liver cancer in human patients revealed that FXR is reduced, while gankyrin is elevated during spontaneous development of liver cancer. Searching for animal models with altered levels of FXR, we found that long-lived Little mice have high levels of FXR and do not develop liver cancer with age and after DEN injections due to failure to activate gankyrin and eliminate Rb, p53, HNF4α and C/EBPα proteins. CONCLUSION: FXR prevents liver cancer by inhibiting the gankyrin promoter via C/EBPß-HDAC1 complexes, leading to subsequent protection of tumor suppressor proteins from degradation.

Genetic Ablation of C/EBPα-p300 Pathway Blocks Development of Obese Pregnancy Associated Liver Disorders in Offspring.

BACKGROUND & AIMS: The obesity-associated nonalcoholic fatty liver disease represents a common cause of pediatric liver diseases, including the pediatric liver cancer hepatoblastoma. The mechanisms behind the development of fatty liver in children are not yet known. We examined the role of the C/EBPα-p300 pathway in the development of maternal obesity-associated fatty liver phenotype in offspring. METHODS: Because the ability of C/EBPα to promote fatty liver phenotype is enhanced by CDK4-mediated phosphorylation of C/EBPα at Ser193 and subsequent formation of C/EBPα-p300 complexes, we used wild-type (WT) and C/EBPα-S193D and C/EBPα-S193A mutant mice to study the effects of maternal high-fat diet (HFD) on the liver health of offspring. The females of these mouse lines were fed an HFD before mating, and the pups were further subjected to either an HFD or a normal diet for 12 weeks. RESULTS: WT female mice on the HFD before and during pregnancy and their subsequent offspring on the HFD had severe fatty liver, fibrosis, and an increased rate of liver proliferation. However, the HFD in C/EBPα-S193A mice did not cause development of these disorders. In HFD-HFD treated WT mice, C/EBPα is phosphorylated at Ser193 and forms complexes with p300, which activate expression of genes involved in development of fatty liver, fibrosis, and proliferation. However, S193A-C/EBPα mice do not have complexes of C/EBPα-S193A with p300, leading to a lack of activation of genes of fatty liver, fibrosis, and proliferation. The mutant C/EBPα-S193D mice have accelerated cdk4-dependent pathway and have developed steatosis at early stages. CONCLUSIONS: These studies identified the epigenetic cause of obese pregnancy-associated liver diseases and suggest a potential therapy based on inhibition of cdk4-ph-S193-C/EBPα-p300 pathway.

Cellular origin and molecular mechanisms of lung metastases in patients with aggressive hepatoblastoma.

BACKGROUND AND AIMS: Lung metastases are the most threatening signs for patients with aggressive hepatoblastoma (HBL). Despite intensive studies, the cellular origin and molecular mechanisms of lung metastases in patients with aggressive HBL are not known. The aims of these studies were to identify metastasis-initiating cells in primary liver tumors and to determine if these cells are secreted in the blood, reach the lung, and form lung metastases. APPROACH: We have examined mechanisms of activation of key oncogenes in primary liver tumors and lung metastases and the role of these mechanisms in the appearance of metastasis-initiating cells in patients with aggressive HBL by RNA-Seq, immunostaining, chromatin immunoprecipitation, Real-Time Quantitative Reverse Transcription PCR and western blot approaches. Using a protocol that mimics the exit of metastasis-initiating cells from tumors, we generated 16 cell lines from liver tumors and 2 lines from lung metastases of patients with HBL. RESULTS: We found that primary HBL liver tumors have a dramatic elevation of neuron-like cells and cancer-associated fibroblasts and that these cells are released into the bloodstream of patients with HBL and found in lung metastases. In the primary liver tumors, the ph-S675-ß-catenin pathway activates the expression of markers of cancer-associated fibroblasts; while the ZBTB3-SRCAP pathway activates the expression of markers of neurons via cancer-enhancing genomic regions/aggressive liver cancer domains leading to a dramatic increase of cancer-associated fibroblasts and neuron-like cells. Studies of generated metastasis-initiating cells showed that these cells proliferate rapidly, engage in intense cell-cell interactions, and form tumor clusters. The inhibition of ß-catenin in HBL/lung metastases-released cells suppresses the formation of tumor clusters. CONCLUSIONS: The inhibition of the ß-catenin-cancer-enhancing genomic regions/aggressive liver cancer domains axis could be considered as a therapeutic approach to treat/prevent lung metastases in patients with HBL.

Intracellular signaling and hepatocellular carcinoma.

Liver cancer is the fifth most common cancer and the third most common cause of cancer related death in the world. The recent development of new techniques for the investigations of global change in the gene expression, signaling pathways and wide genome binding has provided novel information for the mechanisms underlying liver cancer progression. Although these studies identified gene expression signatures in hepatocellular carcinoma, the early steps of the development of hepatocellular carcinomas (HCC) are not well understood. The development of HCC is a multistep process which includes the progressive alterations of gene expression leading to the increased proliferation and to liver cancer. This review summarizes recent progress in the identification of the key steps of the development of HCC with the focus on early events of carcinogenesis and on the role of translational and epigenetic alterations in the development of HCC. Quiescent stage of the liver is supported by several tumor suppressor proteins including p53, Rb and C/EBPα. Studies with chemical models of liver carcinogenesis and with human HCC have shown that the elevation of gankyrin is responsible for the elimination of these three proteins at early steps of carcinogenesis. Later stages of progression of the liver cancer are associated with alterations in many signaling pathways including translation which leads to epigenetic silencing/activation of many genes. Particularly, recent reports suggest a critical role of histone deacetylase 1, HDAC1, in the development of HCC through the interactions with transcription factors such as C/EBP family proteins.

The reduction of SIRT1 in livers of old mice leads to impaired body homeostasis and to inhibition of liver proliferation.

Age declines liver functions, leading to the development of age-associated diseases. A member of the sirtuins family, SIRT1, is involved in the control of glucose homeostasis and fat metabolism. Because aging livers have alterations in glucose and fat metabolism, we examined a possible role of SIRT1 in these alterations. We found that aged livers have a reduced expression of SIRT1 and have lost proper control of the regulation of SIRT1 after partial hepatectomy (PH). Down-regulation of SIRT1 in the liver of old mice is mediated by CCAAT/Enhancer Binding Protein/histone deacetylase 1 (C/EBPß-HDAC1) complexes, which bind to and repress the SIRT1 promoter. In the livers of young mice, SIRT1 is activated after PH and supports high levels of glucose and triglycerides during liver regeneration. In old mice, however, C/EBPß-HDAC1-mediated repression of the SIRT1 promoter blocks activation of SIRT1, leading to low levels of glucose and triglycerides during liver regeneration. Down-regulation of SIRT1 in the livers of young mice resulted in alterations similar to those observed in the livers of old mice, whereas the normalization of SIRT1 in the livers of old mice corrects the levels of glucose and triglycerides after PH. The normalization of SIRT1 in old mice also improves liver regeneration via the elimination of the C/EBPα-Brm complex. These studies showed a critical role of the reduction of SIRT1 in age-associated liver dysfunctions and provide a potential tool for the correction of liver functions in old patients after surgical resections.

Calmodulin controls liver proliferation via interactions with C/EBPbeta-LAP and C/EBPbeta-LIP.

A truncated isoform of C/EBPbeta, C/EBPbeta-LIP, is required for liver proliferation. This isoform is expressed at high levels in proliferating liver and in liver tumors. However, high levels of C/EBPbeta-LIP are also observed in non-proliferating livers during acute phase response (APR). In this paper we present mechanisms by which liver regulates activities of C/EBPbeta-LIP. We found that calmodulin (CaM) inhibits the ability of C/EBPbeta-LIP to promote liver proliferation during APR through direct interactions. This activity of CaM is under negative control of Ca(2+), which is reduced in nuclei of livers with APR, whereas it is increased in nuclei of proliferating livers. A mutant CaM, which does not interact with C/EBPbeta-LIP, also fails to inhibit the growth promotion activity of C/EBPbeta-LIP. Down-regulation of CaM in livers of LPS-treated mice causes liver proliferation via activation of C/EBPbeta-LIP. Overexpression of C/EBPbeta-LIP above levels of CaM also initiates liver proliferation in LPS-treated mice. In addition, CaM regulates transcriptional activity of another isoform of C/EBPbeta, C/EBPbeta-LAP, and might control liver biology through the regulation of both isoforms of C/EBPbeta. In searching for molecular mechanisms by which C/EBPbeta-LIP promotes cell proliferation, we found that C/EBPbeta-LIP releases E2F.Rb-dependent repression of cell cycle genes by a disruption of E2F1.Rb complexes and by a direct interaction with E2F-dependent promoters. CaM inhibits these growth promotion activities of C/EBPbeta-LIP and, therefore, supports liver quiescence. Thus, our findings discover a new pathway of the regulation of liver proliferation that involves calcium-CaM signaling.

Unfolded protein response suppresses CEBPA by induction of calreticulin in acute myeloid leukaemia.

The unfolded protein response (UPR) is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). The role of the UPR during leukemogenesis is unknown so far. Here, we studied the induction of mediators of the UPR in leukaemic cells of AML patients. Increased expression of the spliced variant of the X-box binding protein 1 (XBP1s) was detected in 17.4% (16 of 92) of AML patients. Consistent with activated UPR, this group also had increased expression of ER-resident chaperones such as the 78 kD glucose-regulated protein (GRP78) and of calreticulin. Conditional expression of calreticulin in leukaemic U937 cells was found to increase calreticulin binding to the CEBPA mRNA thereby efficiently blocking translation of the myeloid key transcription factor CEBPA and ultimately affecting myeloid differentiation. Consequently, leukaemic cells from AML patients with activated UPR and thus increased calreticulin levels showed in fact suppressed CEBPA protein expression. We identified two functional ER stress response elements (ERSE) in the calreticulin promoter. The presence of NFY and ATF6, as well as an intact binding site for YY1 within these ERSE motifs were essential for mediating sensitivity to ER stress and activation of calreticulin. Thus, we propose a model of the UPR being activated in a considerable subset of AML patients through induction of calreticulin along the ATF6 pathway, thereby ultimately suppressing CEBPA translation and contributing to the block in myeloid differentiation.

Expression of RNA CCUG repeats dysregulates translation and degradation of proteins in myotonic dystrophy 2 patients.

Myotonic dystrophy 2 (DM2) is a multisystem skeletal muscle disease caused by an expansion of tetranucleotide CCTG repeats, the transcription of which results in the accumulation of untranslated CCUG RNA. In this study, we report that CCUG repeats both bind to and misregulate the biological functions of cytoplasmic multiprotein complexes. Two CCUG-interacting complexes were subsequently purified and analyzed. A major component of one of the complexes was found to be the 20S catalytic core complex of the proteasome. The second complex was found to contain CUG triplet repeat RNA-binding protein 1 (CUGBP1) and the translation initiation factor eIF2. Consistent with the biological functions of the 20S proteasome and the CUGBP1-eIF2 complexes, the stability of short-lived proteins and the levels of the translational targets of CUGBP1 were shown to be elevated in DM2 myoblasts. We found that the overexpression of CCUG repeats in human myoblasts from unaffected patients, in C2C12 myoblasts, and in a DM2 mouse model alters protein translation and degradation, similar to the alterations observed in DM2 patients. Taken together, these findings show that RNA CCUG repeats misregulate protein turnover on both the levels of translation and proteasome-mediated protein degradation.

Cyclin D3 maintains growth-inhibitory activity of C/EBPalpha by stabilizing C/EBPalpha-cdk2 and C/EBPalpha-Brm complexes.

C/EBPalpha arrests proliferation of young livers by inhibition of cdk2. In old mice, C/EBPalpha inhibits growth by repression of E2F-dependent promoters through the C/EBPalpha-Brm complex. In this paper, we show that cyclin D3-cdk4/cdk6 supports the ability of C/EBPalpha to inhibit liver proliferation in both age groups. Although cyclin D3-cdk4/cdk6 kinases are involved in the promotion of growth, they are expressed in terminally differentiated cells, suggesting that they have additional functions in these settings. We demonstrate that C/EBPalpha represents a target for phosphorylation by cyclin D3-cdk4/cdk6 complexes in differentiated liver cells and in differentiated adipocytes. Cyclin D3-cdk4/cdk6 specifically phosphorylate C/EBPalpha at Ser193 in vitro and in the liver and support growth-inhibitory C/EBPalpha-cdk2 and C/EBPalpha-Brm complexes. We found that cyclin D3 is increased in old livers and activates cdk4/cdk6, resulting in stabilization of the C/EBPalpha-Brm complex. Old livers fail to reduce the activity of cyclin D3-cdk4/cdk6 after partial hepatectomy, leading to high levels of C/EBPalpha-Brm complexes after partial hepatectomy, which correlate with weak proliferation. We examined the role of cyclin D3 in the stabilization of C/EBPalpha-cdk2 and C/EBPalpha-Brm by using 3T3-L1 differentiated cells. In these cells, cyclin D3 is increased during differentiation and phosphorylates C/EBPalpha at Ser193, leading to the formation of growth-inhibitory C/EBPalpha-cdk2 and C/EBPalpha-Brm complexes. The inhibition of cyclin D3 blocks the formation of these complexes. Thus, these studies provide a new function of cyclin D3, which is to support the growth-inhibitory activity of C/EBPalpha.

Dynamic assembly of chromatin complexes during cellular senescence: implications for the growth arrest of human melanocytic nevi.

The retinoblastoma (RB)/p16(INK4a) pathway regulates senescence of human melanocytes in culture and oncogene-induced senescence of melanocytic nevi in vivo. This senescence response is likely due to chromatin modifications because RB complexes from senescent melanocytes contain increased levels of histone deacetylase (HDAC) activity and tethered HDAC1. Here we show that HDAC1 is prominently detected in p16(INK4a)-positive, senescent intradermal melanocytic nevi but not in proliferating, recurrent nevus cells that localize to the epidermal/dermal junction. To assess the role of HDAC1 in the senescence of melanocytes and nevi, we used tetracycline-based inducible expression systems in cultured melanocytic cells. We found that HDAC1 drives a sequential and cooperative activity of chromatin remodeling effectors, including transient recruitment of Brahma (Brm1) into RB/HDAC1 mega-complexes, formation of heterochromatin protein 1 beta (HP1 beta)/SUV39H1 foci, methylation of H3-K9, stable association of RB with chromatin and significant global heterochromatinization. These chromatin changes coincide with expression of typical markers of senescence, including the senescent-associated beta-galactosidase marker. Notably, formation of RB/HP1 beta foci and early tethering of RB to chromatin depends on intact Brm1 ATPase activity. As cells reached senescence, ejection of Brm1 from chromatin coincided with its dissociation from HP1 beta/RB and relocalization to protein complexes of lower molecular weight. These results provide new insights into the role of the RB pathway in regulating cellular senescence and implicate HDAC1 as a likely mediator of early chromatin remodeling events.

Dephosphorylated C/EBPalpha accelerates cell proliferation through sequestering retinoblastoma protein.

CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been previously considered a strong inhibitor of cell proliferation which uses multiple pathways to cause growth arrest. In this paper, we describe a new function of C/EBPalpha, which is an acceleration of cell proliferation. This new function of C/EBPalpha is created in proliferating livers by protein phosphatase 2A-mediated dephosphorylation of C/EBPalpha at Ser193. The Ser193-dephosphorylated C/EBPalpha interacts with retinoblastoma protein (Rb) independently on E2Fs and sequesters Rb, leading to a reduction of E2F-Rb repressors and to acceleration of proliferation. This new function of C/EBPalpha requires Rb, since the dephosphorylated C/EBPalpha does not promote proliferation in Rb-negative cells. We also show that a balance of Rb and Ser193-dephosphorylated C/EBPalpha determines if the cells are growth arrested or have an increased rate of proliferation. Consistently with these findings, a significant portion of Rb is sequestered into Rb-C/EBPalpha complexes in proliferating livers, and E2F-Rb complexes are not detectable in these livers. Our data demonstrate a new pathway by which the phosphorylation-dependent switch of biological functions of C/EBPalpha promotes liver proliferation.