ZEB1 Regulates Multiple Oncogenic Components Involved in Uveal Melanoma Progression.

Human uveal melanoma (UM) is a major ocular malignant tumor with high risk of metastasis and requires multiple oncogenic factors for progression. ZEB1 is a zinc finger E-box binding transcription factor known for participating epithelial-mesenchymal transition (EMT), a critical cellular event for metastasis of malignant tumors of epithelium origin. ZEB1 is also expressed in UM and high expression of ZEB1 correlates with UM advancement, but has little effect on cell morphology. We show that spindle UM cells can become epithelioid but not vice versa; and ZEB1 exerts its tumorigenic effects by promoting cell dedifferentiation, proliferation, invasiveness, and dissemination. We provide evidence that ZEB1 binds not only to repress critical genes involving in pigment synthesis, mitosis, adherent junctions, but also to transactivate genes involving in matrix degradation and cellular locomotion to propel UM progression towards metastasis. We conclude that ZEB1 is a major oncogenic factor required for UM progression and could be a potential therapeutic target for treating UM in the clinic.

LINE-1 silencing by retinoblastoma proteins is effected through the nucleosomal and remodeling deacetylase multiprotein complex.

BACKGROUND: Long Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms. We have previously shown that the regulatory region of human and murine L1s interact with retinoblastoma (RB) proteins to effect retroelement silencing. The present studies were conducted to identify the corepressor complex responsible for RB-mediated silencing of L1. METHODS: Chromatin immunoprecipitation and silencing RNA technology were used to identify the repressor complex that silences L1 in human and murine cells. RESULTS: Components of the Nucleosomal and Remodeling Deacetylase (NuRD) multiprotein complex specifically enriched the L1 5'-untranslated DNA sequence in human and murine cells. Genetic ablation of RB proteins in murine cells destabilized interactions within the NuRD macromolecular complex and mediated nuclear rearrangement of Mi2-ß, an ATP-dependent helicase subunit with nucleosome remodeling activity. Depletion of Mi2-ß, RbAP46 and HDAC2 reduced the repressor activity of the NuRD complex and reactivated a synthetic L1 reporter in human cells. Epigenetic reactivation of L1 in RB-null cells by DNA damage was markedly enhanced compared to wild type cells. CONCLUSIONS: RB proteins stabilize interactions of the NuRD corepressor complex within the L1 promoter to effect L1 silencing. L1 retroelements may serve as a scaffold on which RB builds heterochromatic regions that regulate chromatin function.

HPV E7 viral oncoprotein disrupts transcriptional regulation of L1Md retrotransposon.

Murine L1Md-A5 retrotransposon is a redox-inducible element regulated by Nrf-2/JunD and E2F/Rb-binding sites within its promoter (5'-UTR). Because the human papillomavirus (HPV) oncoprotein E7 interacts with retinoblastoma (pRb) and members of the AP1 family, studies were conducted to examine functional interactions between HPV E7, pRb, and histone deacetylase 2 (HDAC2) in the regulation of L1Md-A5. Using a transient heterologous transcription system we found that HPV E7 alone, or in combination with HDAC2, disrupted pRb-mediated L1MdA-5 transactivation. HPV E7 also ablated the transcriptional response of L1Md-A5 to genotoxic stress, but did not interfere with basal activity. We conclude that HPV E7 associates with proteins involved in the assembly of macromolecular complexes that regulate antioxidant and E2F/Rb sites within L1MdA-5 to regulate biological activity.

Epigenetic control of embryonic renal cell differentiation by L1 retrotransposon.

BACKGROUND: L1 retroelements may play a central role in morphogenesis through epigenetic mechanisms involving recruitment of chromatin modifying protein complexes. Retroelements are repressed in terminally differentiated cells, and highly active in embryonic, undifferentiated, and transformed cells. It is not clear if the modulation of differentiation by L1 is a "cause" or "effect". The purpose of this study was to determine if murine embryonic kidney cells of clonal origin (mK4 cells) harbor retrotransposition events upon ectopic expression of L1, and the impact of L1 on embryonic kidney cell differentiation. Given that L1 is reactivated by aryl hydrocarbon receptor (AHR) ligands, we also sought to investigate the effects of benzo(a)pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the genetic network of mK4 cells. METHODS: The mK4 cells overexpressing human L1(RP) were assessed for changes in proliferation and expression of molecular markers of cellular differentiation. RESULTS: L1(RP) increased proliferation rates and markedly downregulated differentiation programming in mK4 cells. These genetic alterations were recapitulated by exogenous activation of L1 by AHR ligands. CONCLUSION: L1 regulates nephrogenesis in vitro via both insertional and non-insertional mechanisms that disrupt mesenchymal to epithelial transition. Thus, a feedback loop involving L1, WT1, and AHR may play a role in regulation of kidney morphogenesis. Birth Defects Research (Part A), 2011. © 2011 Wiley-Liss, Inc.

Retinoblastoma family of proteins and chromatin epigenetics: a repetitive story in a few LINEs.

The retinoblastoma (RB) protein family in mammals is composed of three members: pRB (or RB1), p107, and p130. Although these proteins do not directly bind DNA, they associate with the E2F family of transcription factors which function as DNA sequence-specific transcription factors. RB proteins alter gene transcription via direct interference with E2F functions, as well as recruitment of transcriptional repressors and corepressors that silence gene expression through DNA and histone modifications. E2F/RB complexes shape the chromatin landscape through recruitment to CpG-rich regions in the genome, thus making E2F/RB complexes function as local and global regulators of gene expression and chromatin dynamics. Recruitment of E2F/pRB to the long interspersed nuclear element (LINE1) promoter enhances the role that RB proteins play in genome-wide regulation of heterochromatin. LINE1 elements are dispersed throughout the genome and therefore recruitment of RB to the LINE1 promoter suggests that LINE1 could serve as the scaffold on which RB builds up heterochromatic regions that silence and shape large stretches of chromatin. We suggest that mutations in RB function might lead to global rearrangement of heterochromatic domains with concomitant retrotransposon reactivation and increased genomic instability. These novel roles for RB proteins open the epigenetic-based way for new pharmacological treatments of RB-associated diseases, namely inhibitors of histone and DNA methylation, as well as histone deacetylase inhibitors.

Reactivation of L1 retrotransposon by benzo(a)pyrene involves complex genetic and epigenetic regulation.

Benzo(a)pyrene (BaP), is an environmental pollutant present in tobacco smoke and a byproduct of fossil fuel combustion which likely contributes to the tumorigenic processes in human cancers including lung and esophageal. Long Interspersed Nuclear Element-1 (LINE-1) or L1 is a mobile element within the mammalian genome that propagates via a "copy-and-paste" mechanism using reverse transcriptase and RNA intermediates. L1 is strongly expressed during early embryogenesis and then silenced as cells initiate differentiation programming. Although the complex transcriptional control mechanisms of L1 are not well understood, L1 reactivation has been described in several human cancers and following exposure of mouse or human cells to BaP. In this study we investigated the molecular mechanisms and epigenetic events that regulate L1 reactivation following BaP exposure. We show that challenge of HeLa cells with BaP induces early enrichment of the transcriptionally-active chromatin markers histone H3 trimethylated at lysine 4 (H3K4Me3) and histone H3 acetylated at lysine 9 (H3K9Ac), and reduces association of DNA methyltransferase-1 (DNMT1) with the L1 promoter. These changes are followed by proteasome-dependent decreases in cellular DNMT1 expression and sustained reduction of cytosine methylation within the L1 promoter CpG island. Pharmacological inhibition of the proteasome signaling pathway with the inhibitor MG132 blocks degradation of DNMT1 and alters BaP-mediated histone epigenetic modifications. We conclude that genetic reactivation of L1 by BaP involves an ordered cascade of epigenetic events that begin with nucleosomal histone modifications and is completed with alterations in DNMT1 recruitment to the L1 promoter and reduced DNA methylation of CpG islands.

Assessment of genetic variation for the LINE-1 retrotransposon from next generation sequence data.

BACKGROUND: In humans, copies of the Long Interspersed Nuclear Element 1 (LINE-1) retrotransposon comprise 21% of the reference genome, and have been shown to modulate expression and produce novel splice isoforms of transcripts from genes that span or neighbor the LINE-1 insertion site. RESULTS: In this work, newly released pilot data from the 1000 Genomes Project is analyzed to detect previously unreported full length insertions of the retrotransposon LINE-1. By direct analysis of the sequence data, we have identified 22 previously unreported LINE-1 insertion sites within the sequence data reported for a mother/father/daughter trio. CONCLUSIONS: It is demonstrated here that next generation sequencing data, as well as emerging high quality datasets from individual genome projects allow us to assess the amount of heterogeneity with respect to the LINE-1 retrotransposon amongst humans, and provide us with a wealth of testable hypotheses as to the impact that this diversity may have on the health of individuals and populations.

Epigenetic control of mammalian LINE-1 retrotransposon by retinoblastoma proteins.

Long interspersed nuclear elements (LINEs or L1 elements) are targeted for epigenetic silencing during early embryonic development and remain inactive in most cells and tissues. Here we show that E2F-Rb family complexes participate in L1 elements epigenetic regulation via nucleosomal histone modifications and recruitment of histone deacetylases (HDACs) HDAC1 and HDAC2. Our experiments demonstrated that (i) Rb and E2F interact with human and mouse L1 elements, (ii) L1 elements are deficient in both heterochromatin-associated histone marks H3 tri methyl K9 and H4 tri methyl K20 in Rb family triple knock out (Rb, p107, and p130) fibroblasts (TKO), (iii) L1 promoter exhibits increased histone H3 acetylation in the absence of HDAC1 and HDAC2 recruitment, (iv) L1 expression in TKO fibroblasts is upregulated compared to wild type counterparts, (v) L1 expression increases in the presence of the HDAC inhibitor TSA. On the basis of these findings we propose a model in which L1 sequences throughout the genome serve as centers for heterochromatin formation in an Rb family-dependent manner. As such, Rb proteins and L1 elements may play key roles in heterochromatin formation beyond pericentromeric chromosomal regions. These findings describe a novel mechanism of L1 reactivation in mammalian cells mediated by failure of corepressor protein recruitment by Rb, loss of histone epigenetic marks, heterochromatin formation, and increased histone H3 acetylation.

Ajuba functions as a histone deacetylase-dependent co-repressor for autoregulation of the growth factor-independent-1 transcription factor.

Growth factor independent-1 (Gfi1) is a zinc finger protein with a SNAG-transcriptional repressor domain. Ajuba is a LIM domain protein that shuttles between the cytoplasm and the nucleus. Ajuba functions as a co-repressor for synthetic Gfi1 SNAG-repressor domain-containing constructs, but a role for Ajuba co-repression of the cognate DNA bound Gfi1 protein has not been defined. Co-immunoprecipitation of synthetic and endogenous proteins and co-elution with gel filtration suggest that an endogenous Ajuba.Gfi1.HDAC multiprotein complex is possible. Active histone deacetylase activity co-immunoprecipitates with Ajuba or Gfi1, and both proteins depend upon histone deacetylases for full transcriptional repression activity. Ajuba LIM domains directly bind to Gfi1, but the association is not SNAG domain-dependent. ChIP analysis and reciprocal knockdown experiments suggest that Ajuba selectively functions as a co-repressor for Gfi1 autoregulation. The data suggest that Ajuba is utilized as a corepressor selectively on Gfi1 target genes.

Hsp27 regulates Akt activation and polymorphonuclear leukocyte apoptosis by scaffolding MK2 to Akt signal complex.

We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPK-activated protein kinase-2 (MK2), and heat shock protein 27 (Hsp27) and MK2 phosphorylates Akt on Ser-473. Additionally, dissociation of Hsp27 from Akt, prior to Akt activation, induced polymorphonuclear leukocyte (PMN) apoptosis. However, the role of Hsp27 in regulating Akt activation was not examined. This study tested the hypothesis that Hsp27 regulates Akt activation and promotes cell survival by scaffolding MK2 to the Akt signal complex. Here we show that loss of Akt/Hsp27 interaction by anti-Hsp27 antibody treatment resulted in loss of Akt/MK2 interaction, loss of Akt-Ser-473 phosphorylation, and induced PMN apoptosis. Transfection of myristoylated Akt (AktCA) in HK-11 cells induced Akt-Ser-473 phosphorylation, activation, and Hsp27-Ser-82 phosphorylation. Cotransfection of AktCA with Hsp27 short interfering RNA, but not scrambled short interfering RNA, silenced Hsp27 expression, without altering Akt expression in HK-11 cells. Silencing Hsp27 expression inhibited Akt/MK2 interaction, inhibited Akt phosphorylation and Akt activation, and induced HK-11 cell death. Deletion mutagenesis studies identified acidic linker region (amino acids 117-128) on Akt as an Hsp27 binding region. Deletion of amino acids 117-128 on Akt resulted in loss of its interaction with Hsp27 and MK2 but not with Hsp90 as demonstrated by immunoprecipitation and glutathione S-transferase pulldown studies. Co-transfection studies demonstrated that constitutively active MK2 (MK2EE) phosphorylated Aktwt (wild type) on Ser-473 but failed to phosphorylate Akt(Delta117-128) mutant in transfixed cells. These studies collectively define a novel role of Hsp27 in regulating Akt activation and cellular apoptosis by mediating interaction between Akt and its upstream activator MK2.

Computational and biological inference of gene regulatory networks of the LINE-1 retrotransposon.

Computational approaches were used to define structural and functional determinants of a putative genetic regulatory network of murine LINE-1 (long interspersed nuclear element-1), an active mammalian retrotransposon that uses RNA intermediates to populate new sites throughout the genome. Polymerase (RNA) II polypeptide E AI845735 and mouse DNA homologous to Drosophila per fragment M12039 were identified as primary attractors. siRNA knockdown of the aryl hydrocarbon receptor NM_013464 modulated gene expression within the network, including LINE-1, Sgpl1, Sdcbp, and Mgst1. Genes within the network did not exhibit physical proximity and instead were dispersed throughout the genome. The potential impact of individual members of the network on the global dynamical behavior of LINE-1 was examined from a theoretical and empirical framework.