Survival of naive T cells is dependent upon IL-7, which is present in vivo in limiting amounts with the result that naive T cells must compete for IL-7-mediated survival signals. It would seem imperative during T cell homeostasis that limiting IL-7 be shared by the greatest possible number of T cells. We now describe a novel regulatory mechanism that specifically suppresses IL7Ralpha transcription in response to IL-7 and other prosurvival cytokines (IL-2, IL-4, IL-6, and IL-15). Consequently, IL7R expression is reduced on T cells that have received cytokine-mediated survival signals so they do not compete with unsignaled T cells for remaining IL-7. Interestingly, cytokine-mediated suppression of IL7Ralpha transcription involves different molecular mechanisms in CD4+ and CD8+ T cells, as CD8+ T cells utilize the transcriptional repressor GFI1 while CD4+ T cells do not. We suggest that this homeostatic regulatory mechanism promotes survival of the maximum possible number of T cells for the amount of IL-7 available.

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The growth factor independent 1 (Gfi1) transcriptional regulator oncoprotein plays a crucial role in hematopoietic, inner ear, and pulmonary neuroendocrine cell development and governs cell processes as diverse as selfrenewal of hematopoietic stem cells, proliferation, apoptosis, differentiation, cell fate specification, and oncogenesis. However, the molecular basis of its transcriptional functions has remained elusive. Here we show that Gfi1 recruits the histone lysine methyltransferase G9a and the histone deacetylase 1 (HDAC1) in order to modify the chromatin of genes targeted for repression by Gfi1. G9a and HDAC1 are both in a repressive complex assembled by Gfi1. Endogenous Gfi1 colocalizes with G9a, HDAC1, and K9-dimethylated histone H3. Gfi1 associates with G9a and HDAC1 on the promoter of the cell cycle regulator p21 Cip/WAF1, resulting in an increase in K9 dimethylation at histone H3. Silencing of Gfi1 expression in myeloid cells reverses G9a and HDAC1 recruitment to p21Cip/WAF1 and elevates its expression. These findings highlight the role of epigenetics in the regulation of development and oncogenesis by Gfi1.

Apoptosis or programmed cell death is a series of events in a cell that leads to its death. Human polymorphonuclear leukocytes (PMN) 3 take part in host defense mechanisms against infection and inflammatory diseases. Inappropriate termination of PMN activation or failure to remove apoptotic PMNs results in inflammation. This apoptotic process has been suggested to represent an in vivo mechanism limiting oxidant-induced tissue injury caused by PMNs at the sites of inflammation. Although PMNs are constitutively committed to apoptosis from the time they enter circulation, the rate of apoptosis is not fixed. We reported that interleukin-8, granulocytemacrophage colony-stimulating factor, LTB 4 , and bacterial lipopolysaccharide (LPS) delay constitutive PMN apoptosis through the activation of the serine/threonine kinase Akt (1, 2). We demonstrated that p38 mitogen-activated protein kinase (MAPK) activity is required for Akt phosphorylation and activation (3). Additionally, we showed that Akt exists in a signaling module with p38 MAPK, MAPK-activated protein kinase-2 (MK2), and heat shock protein 27 (Hsp27) (3).Heat shock proteins represent a group of chaperone proteins that protect the cells against a variety of stresses. Besides being involved in functioning as a chaperone, Hsp27 has also been shown to regulate stability of the cytoskeleton, cell motility (4 -7), and apoptosis (8 -13). When overexpressed in tumor cells, Hsp27 increases their tumorigenicity by overexpressing MMP-9 expression and down-regulating Src tyrosine kinase Yes expression (14 -16) and protects against apoptotic cell death triggered by various stimuli, including cytotoxic drugs and ligation of the Fas/Apo-1/CD95 death receptor (17)(18)(19). Mice overexpressing Hsp27 were protected from lethal ischemia/reperfusion injury compared with their negative littermates (20). Possible mechanisms of Hsp27 anti-apoptotic activity are proposed to result from its activity as a molecular chaperone. Hsp27 binds to and inactivates the pro-apoptotic molecules Smac, caspase 3, caspase 9, and cytochrome c (21-25). Hsp27-mediated suppression of Bid translocation to the mitochondria correlates with an inhibition of cytochrome c release (25). Hsp27 has also been shown to promote survival * This work was supported by American Heart Association-Scientist Development Grant 0335278N (to M. J. R.) and National Institutes of Health Grant 1R56AI059165-01A2 (to M. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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. ChIP 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, 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 co-repressor protein recruitment by Rb, loss of histone epigenetic marks, heterochromatin formation, and increased histone H3 acetylation.

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.

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.

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 Ajuba⅐Gfi1⅐HDAC 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.