Sichuan Xi

Lsh is involved in de novo methylation of DNA

Deletion of Lsh perturbs DNA methylation patterns in mice yet it is unknown whether Lsh plays a direct role in the methylation process. Two types of methylation pathways have been distinguished: maintenance methylation by Dnmt1 occurring at the replication fork, and de novo methylation established by the methyltransferases Dnmt3a and Dnmt3b. Using an episomal vector in Lsh−/− embryonic fibroblasts, we demonstrate that the acquisition of DNA methylation depends on the presence of Lsh. In contrast, maintenance of previously methylated episomes does not require Lsh, implying a functional role for Lsh in the establishment of novel methylation patterns. Lsh affects Dnmt3a as well as Dnmt3b directed methylation suggesting that Lsh can cooperate with both enzymatic activities. Furthermore, we demonstrate that embryonic stem cells with reduced Lsh protein levels show a decreased ability to silence retroviral vector or to methylate endogenous genes. Finally, we demonstrate that Lsh associates with Dnmt3a or Dnmt3b but not with Dnmt1 in embryonic cells. These results suggest that the epigenetic regulator, Lsh, is directly involved in the control of de novo methylation of DNA.

Cigarette Smoke Induces C/EBP-β-Mediated Activation of miR-31 in Normal Human Respiratory Epithelia and Lung Cancer Cells

Background Limited information is available regarding mechanisms by which miRNAs contribute to pulmonary carcinogenesis. The present study was undertaken to examine expression and function of miRNAs induced by cigarette smoke condensate (CSC) in normal human respiratory epithelia and lung cancer cells. Methodology Micro-array and quantitative RT-PCR (qRT-PCR) techniques were used to assess miRNA and host gene expression in cultured cells, and surgical specimens. Software-guided analysis, RNA cross-link immunoprecipitation (CLIP), 3′ UTR luciferase reporter assays, qRT-PCR, focused super-arrays and western blot techniques were used to identify and confirm targets of miR-31. Chromatin immunoprecipitation (ChIP) techniques were used to evaluate histone marks and transcription factors within the LOC554202 promoter. Cell count and xenograft experiments were used to assess effects of miR-31 on proliferation and tumorigenicity of lung cancer cells. Results CSC significantly increased miR-31 expression and activated LOC554202 in normal respiratory epithelia and lung cancer cells; miR-31 and LOC554202 expression persisted following discontinuation of CSC exposure. miR-31 and LOC554202 expression levels were significantly elevated in lung cancer specimens relative to adjacent normal lung tissues. CLIP and reporter assays demonstrated direct interaction of miR-31 with Dickkopf-1 (Dkk-1) and DACT-3. Over-expression of miR-31 markedly diminished Dkk-1 and DACT3 expression levels in normal respiratory epithelia and lung cancer cells. Knock-down of miR-31 increased Dkk-1 and DACT3 levels, and abrogated CSC-mediated decreases in Dkk-1 and DACT-3 expression. Furthermore, over-expression of miR-31 diminished SFRP1, SFRP4, and WIF-1, and increased Wnt-5a expression. CSC increased H3K4Me3, H3K9/14Ac and C/EBP-β levels within the LOC554202 promoter. Knock-down of C/EBP-β abrogated CSC-mediated activation of LOC554202. Over-expression of miR-31 significantly enhanced proliferation and tumorigenicity of lung cancer cells; knock-down of miR-31 inhibited growth of these cells. Conclusions Cigarette smoke induces expression of miR-31 targeting several antagonists of cancer stem cell signaling in normal respiratory epithelia and lung cancer cells. miR-31 functions as an oncomir during human pulmonary carcinogenesis.

The pivotal role of IKKα in the development of spontaneous lung squamous cell carcinomas.

Here, we report that kinase-dead IKKα knockin mice develop spontaneous lung squamous cell carcinomas (SCCs) associated with IKKα downregulation and marked pulmonary inflammation. IKKα reduction upregulated the expression of p63, Trim29, and keratin 5 (K5), which serve as diagnostic markers for human lung SCCs. IKKα(low)K5(+)p63(hi) cell expansion and SCC formation were accompanied by inflammation-associated deregulation of oncogenes, tumor suppressors, and stem cell regulators. Reintroducing transgenic K5.IKKα, depleting macrophages, and reconstituting irradiated mutant animals with wild-type bone marrow (BM) prevented SCC development, suggesting that BM-derived IKKα mutant macrophages promote the transition of IKKα(low)K5(+)p63(hi) cells to tumor cells. This mouse model resembles human lung SCCs, sheds light on the mechanisms underlying lung malignancy development, and identifies targets for therapy of lung SCCs.

Inhibition of Histone Lysine Methylation Enhances Cancer-Testis Antigen Expression in Lung Cancer Cells: Implications for Adoptive Immunotherapy of Cancer

Cancer-testis antigens (CTA), such as NY-ESO-1, MAGE-A1, and MAGE-A3, are immunogenic proteins encoded by genes, which are normally expressed only in male germ cells but are activated by ill-defined epigenetic mechanisms in human tumors, including lung cancers. Previously, we reported induction of these CTAs in cancer cells, but not normal cells, by DNA-demethylating agents and histone deacetylase inhibitors using clinically achievable exposure conditions. In the present study, we evaluated chromatin alterations associated with repression/activation of cancer-testis genes in lung cancer cells to further develop gene-induction regimens for cancer immunotherapy. Repression of NY-ESO-1, MAGE-A1, and MAGE-A3 coincided with DNA hypermethylation, recruitment, and binding of polycomb-group proteins, and histone heterochromatin modifications within the promoters of these genes. Derepression coincided with DNA demethylation, dissociation of polycomb proteins, and presence of euchromatin marks within the respective promoters. Short hairpin RNAs were used to inhibit several histone methyltransferases (KMT) and histone demethylases (KDM) that mediate histone methylation and repress gene expression. Knockdown of KMT6, KDM1, or KDM5B markedly enhanced deoxyazacytidine (DAC)-mediated activation of these cancer-testis genes in lung cancer cells. DZNep, a pharmacologic inhibitor of KMT6 expression, recapitulated the effects of KMT6 knockdown. Following DAC-DZNep exposure, lung cancer cells were specifically recognized and lysed by allogeneic lymphocytes expressing recombinant T-cell receptors recognizing NY-ESO-1 and MAGE-A3. Combining DNA-demethylating agents with compounds, such as DZNep, that modulate histone lysine methylation may provide a novel epigenetic strategy to augment cancer-testis gene expression as an adjunct to adoptive cancer immunotherapy.

Lsh controls Hox gene silencing during development

Polycomb-mediated repression and DNA methylation are important epigenetic mechanisms of gene silencing. Recent evidence suggests a functional link between the polycomb repressive complex (PRC) and Dnmts in cancer cells. Here we provide evidence that Lsh, a regulator of DNA methylation, is also involved in normal control of PRC-mediated silencing during embryogenesis. We demonstrate that Lsh, a SNF2 homolog, can associate with some Hox genes and regulates Dnmt3b binding, DNA methylation, and silencing of Hox genes during development. Moreover, Lsh can associate with PRC1 components and influence PRC-mediated histone modifications. Thus Lsh is part of a physiological feedback loop that reinforces DNA methylation and silencing of PRC targets.

Lsh Participates in DNA Methylation and Silencing of Stem Cell Genes

Transcriptional control of stem cell genes is a critical step in differentiation of embryonic stem cells and in reprogramming of somatic cells into stem cells. Here we report that Lsh, a regulator of repressive chromatin at retrotransposons, also plays an important role in silencing of stem cell‐specific genes such as Oct4. We found that CpG methylation is gained during in vitro differentiation of several stem cell‐specific genes (in 11 of 12 promoter regions) and thus appears to be a common epigenetic mark. Lsh depletion prevents complete silencing of stem cell gene expression and moreover promotes the maintenance of stem cell characteristics in culture. Lsh is required for establishment of DNA methylation patterns at stem cell genes during differentiation, in part by regulating access of Dnmt3b to its genomic targets. Our results indicate that Lsh is involved in the control of stem cell genes and suggest that Lsh is an important epigenetic modulator during early stem cell differentiation. STEM CELLS 2009;27:2691–2702

Lsh, chromatin remodeling family member, modulates genome-wide cytosine methylation patterns at nonrepeat sequences.

DNA methylation is critical for normal development and plays important roles in genome organization and transcriptional regulation. Although DNA methyltransferases have been identified, the factors that establish and contribute to genome-wide methylation patterns remain elusive. Here, we report a high-resolution cytosine methylation map of the murine genome modulated by Lsh, a chromatin remodeling family member that has previously been shown to regulate CpG methylation at repetitive sequences. We provide evidence that Lsh also controls genome-wide cytosine methylation at nonrepeat sequences and relate those changes to alterations in H4K4me3 modification and gene expression. Deletion of Lsh alters the allocation of cytosine methylation in chromosomal regions of 50 kb to 2 Mb and, in addition, leads to changes in the methylation profile at the 5′ end of genes. Furthermore, we demonstrate that loss of Lsh promotes—as well as prevents—cytosine methylation. Our data indicate that Lsh is an epigenetic modulator that is critical for normal distribution of cytosine methylation throughout the murine genome.

Lsh Mediated RNA Polymerase II Stalling at HoxC6 and HoxC8 Involves DNA Methylation

DNA cytosine methylation is an important epigenetic mechanism that is involved in transcriptional silencing of developmental genes. Several molecular pathways have been described that interfere with Pol II initiation, but at individual genes the molecular mechanism of repression remains uncertain. Here, we study the molecular mechanism of transcriptional regulation at Hox genes in dependence of the epigenetic regulator Lsh that controls CpG methylation at selected Hox genes. Wild type cells show a nucleosomal deprived region around the transcriptional start site at methylated Hox genes and mediate gene silencing via Pol II stalling. Hypomethylation in Lsh−/− cells is associated with efficient transcriptional elongation and splicing, in part mediated by the chromodomain protein Chd1. Dynamic modulation of DNA methylation in Lsh−/− and wild type cells demonstrates that catalytically active DNA methyltransferase activity is required for Pol II stalling. Taken together, the data suggests that DNA methylation can be compatible with Pol II binding at selected genes and Pol II stalling can act as alternate mechanism to explain transcriptional silencing associated with DNA methylation.

DNA hypomethylation caused by Lsh deletion promotes erythroleukemia development

Hematopoietic malignancies are frequently associated with DNA hypomethylation but the molecular mechanisms involved in tumor formation remain poorly understood. Here we report that mice lacking Lsh develop leukemia associated with DNA hypomethylation and oncogene activation. Lsh is a member of the SNF2 chromatin remodeling family and is required for de novo methylation of genomic DNA. Mice that received Lsh deficient hematopoietic progenitors showed severe impairment of hematopoiesis, suggesting that Lsh is necessary for normal hematopoiesis. A subset of mice developed erythroleukemia, a tumor that does not spontaneously occur in mice. Tumor tissues were CpG hypomethylated and showed a modest elevation of the transcription factor PU.1, an oncogene that is crucial for Friend virus induced erythroleukemia. Analysis of Lsh-/-hematopoietic progenitors revealed widespread DNA hypomethylation at repetitive sequences and hypomethylation at specific retroviral elements within the PU.1 gene. Wild type cells showed Lsh and Dnmt3b binding at the retroviral elements located within the PU.1 gene. On the other hand, Lsh deficient cells had no detectable Dnmt3b association suggesting that Lsh is necessary for recruitment of Dnmt3b to its target. Furthermore, Lsh-/- hematopoietic precursors showed impaired suppression of retroviral elements in the PU.1 gene, an increase of PU.1 transcripts and protein levels. Thus DNA hypomethylation caused by Lsh depletion is linked to transcriptional upregulation of retroviral elements and oncogenes such as PU.1 which in turn may promote the development of erythroleukemia in mice.

Lymphoid-Specific Helicase (HELLS) Is Essential for Meiotic Progression in Mouse Spermatocytes

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells−/− to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells−/−, Hells+/−, and Hells+/+ mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells−/− than from Hells+/+ mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells−/− mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells−/− spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.