Lysine Residues Of Histone H3 Research Articles

Histone Deacetylase 2 (HDAC2) Regulates Chromosome Segregation and Kinetochore Function via H4K16 Deacetylation during Oocyte Maturation in Mouse

Changes in histone acetylation occur during oocyte development and maturation, but the role of specific histone deacetylases in these processes is poorly defined. We report here that mice harboring Hdac1 −/+/Hdac2 −/− or Hdac2 −/− oocytes are infertile or sub-fertile, respectively. Depleting maternal HDAC2 results in hyperacetylation of H4K16 as determined by immunocytochemistry—normal deacetylation of other lysine residues of histone H3 or H4 is observed—and defective chromosome condensation and segregation during oocyte maturation occurs in a sub-population of oocytes. The resulting increased incidence of aneuploidy likely accounts for the observed sub-fertility of mice harboring Hdac2 −/− oocytes. The infertility of mice harboring Hdac1 −/+/Hdac2 −/−oocytes is attributed to failure of those few eggs that properly mature to metaphase II to initiate DNA replication following fertilization. The increased amount of acetylated H4K16 likely impairs kinetochore function in oocytes lacking HDAC2 because kinetochores in mutant oocytes are less able to form cold-stable microtubule attachments and less CENP-A is located at the centromere. These results implicate HDAC2 as the major HDAC that regulates global histone acetylation during oocyte development and, furthermore, suggest HDAC2 is largely responsible for the deacetylation of H4K16 during maturation. In addition, the results provide additional support that histone deacetylation that occurs during oocyte maturation is critical for proper chromosome segregation.

Overexpression of Sharp-1 in MLL-AF6-Positive Acute Myeloid Leukemia

AbstractAbstract 3493Patients with acute myeloid leukemia (AML) carry various chromosomal translocations and/or mutations. Although t(6;11)(q27;q23) is observed in only less than 0.5 % of adult AML patients, this translocation is reported to be one of the independent poor prognostic factors in AML. This translocation induces expression of MLL-AF6 fusion protein, but the precise mechanism of leukemogenesis by MLL-AF6 has not yet been elucidated compared to other MLL fusion proteins. During the analysis of gene expression profiling of >500 AML patients registered in Erasmus University Medical Center in Rotterdam, we happened to find that all of five patients carrying t(6;11) translocation exhibited overexpression of Sharp-1/Dec2, which is involved in the circadian rhythm. AML cell lines carrying this translocation, ML-2 and CTS, also showed more than 10-fold upregulation of Sharp-1 expression. Knockdown of MLL-AF6 expression to 10 % in ML-2 cells by shRNA suppressed expression of Sharp-1 to less than 1 % through the change in the methylation status of histone H3 lysine residues in the Sharp-1 gene promoter region confirmed by chromatin immunoprecipitation. In addition, this knockdown induced growth suppression of ML-2 cells to 40–50 %. Proliferation of ML-2 cells was also suppressed by the knockdown of Sharp-1. Growth suppression of ML-2 cells is neither due to increased apoptosis nor increased differentiation, but due to increased number of cells undergoing cellular senescence together with the upregulation of p21. Furthermore, exogenous expression of Sharp-1 in murine bone marrow cells induced the proliferation of myeloid cells with the reduction of lymphoid cells in the bone marrow transplantation model. These results suggested that Sharp-1 plays a crucial role in the pathogenesis of AML with t(6;11)(q27;q23) and that targeting of Sharp-1 in leukemic stem cells would be a novel therapy for this poor outcome AML. Disclosures:No relevant conflicts of interest to declare.

Dynamic Link between Histone H3 Acetylation and an Increase in the Functional Characteristics of Human ESC/iPSC-Derived Cardiomyocytes

Cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are functionally heterogeneous, display insufficient biological efficacy and generally possess the electrophysiological properties seen in fetal CMs. However, a homogenous population of hESC/hiPSC-CMs, with properties similar to those of adult human ventricular cells, is required for use in drug cardiotoxicity screening. Unfortunately, despite the requirement for the functional characteristics of post-mitotic beating cell aggregates to mimic the behavior of mature cardiomyocytes in vitro, few technological improvements have been made in this field to date. Previously, we showed that culturing hESC-CMs under low-adhesion conditions with cyclic replating confers continuous contractility on the cells, leading to a functional increase in cardiac gene expression and electrophysiological properties over time. The current study reveals that culturing hESC/hiPSC-CMs under non-adhesive culture conditions enhances the electrophysiological properties of the CMs through an increase in the acetylation of histone H3 lysine residues, as confirmed by western blot analyses. Histone H3 acetylation was induced chemically by treating primitive hESC/hiPSC-CMs with Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, resulting in an immediate increase in global cardiac gene expression. In functional analyses using multi-electrode array (MEA) recordings, TSA-treated hESC/hiPSC-CM colonies showed appropriate responses to particular concentrations of known potassium ion channel inhibitors. Thus, the combination of a cell-autonomous functional increase in response to non-adhesive culture and short-term TSA treatment of hESC/hiPSC-CM colonies cultured on MEA electrodes will help to make cardiac toxicity tests more accurate and reproducible via genome-wide chromatin activation.

Small RNA-Mediated Epigenetic Myostatin Silencing

Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

High-Throughput TR-FRET Assays for Identifying Inhibitors of LSD1 and JMJD2C Histone Lysine Demethylases

Lysine demethylase 1 (LSD1) and Jumonji C domain–containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z′ > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 µL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent Km of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.

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Histone Deacetylase 9 Deficiency Protects against Effector T Cell-mediated Systemic Autoimmunity

Co-repressor histone deacetylase 9 (HDAC9) plays a key role in the development and differentiation of many types of cells, including regulatory T cells. However, the biological function of HDAC9 in T effector cells is unknown. Systemic autoimmune diseases like lupus, diabetes, and rheumatoid arthritis have dysfunctional effector T cells. To determine the role of HDAC9 in systemic autoimmunity, we created MRL/lpr mice with HDAC9 deficiency that have aberrant effector T cell function. HDAC9 deficiency led to decreased lympho-proliferation, inflammation, autoantibody production, and increased survival in MRL/lpr mice. HDAC9-deficient mice manifested Th2 polarization, decreased T effector follicular cells positive for inducible co-stimulator, and activated T cells in vivo compared with HDAC9-intact MRL/lpr mice. HDAC9 deficiency also resulted in increased GATA3 and roquin and decreased BCL6 gene expression. HDAC9 deficiency was associated with increased site-specific lysine histone acetylation at H3 (H3K9, H3K14, and H3K18) globally that was localized to IL-4, roquin, and peroxisome proliferator-activated receptor-γ promoters with increased gene expression, respectively. In kidney and spleen, HDAC9 deficiency decreased inflammation and cytokine and chemokine production due to peroxisome proliferator-activated receptor γ overexpression. These findings suggest that HDAC9 acts as an epigenetic switch in effector T cell-mediated systemic autoimmunity.

Identification of mutations of the SWI/SNF complex gene PBRM1 by exome sequencing in renal carcinoma.

4571 Background: The genetics of renal cancer is dominated by inactivation of the VHL tumour suppressor gene in clear cell carcinoma (ccRCC), the commonest histological subtype. Methods: A recent large-scale screen of ~3500 genes by PCR-based exon re-sequencing identified several new cancer genes in ccRCC including UTX (KDM6A), JARID1C (KDM5C)and SETD2. These genes encode enzymes that demethylate (UTX, JARID1C) or methylate (SETD2) key lysine residues of histone H3. Results: Modification of the methylation state of these lysine residues of histone H3 regulates chromatin structure and is implicated in transcriptional control. However, together these mutations are present in fewer than 15% of ccRCC, suggesting the existence of additional, currently unidentified cancer genes. Here, we have sequenced the protein coding exome in a series of primary ccRCC and report the identification of the SWI/SNF chromatin remodeling complex gene PBRM1 as a second major ccRCC cancer gene, with truncating mutations in 41% (92/227) of cases. Conclusions: These data further elucidate the somatic genetic architecture of ccRCC and emphasize the marked contribution of aberrant chromatin biology.

Structure and Histone Binding Properties of the Vps75-Rtt109 Chaperone-Lysine Acetyltransferase Complex

The histone chaperone Vps75 presents the remarkable property of stimulating the Rtt109-dependent acetylation of several histone H3 lysine residues within (H3-H4)(2) tetramers. To investigate this activation mechanism, we determined x-ray structures of full-length Vps75 in complex with full-length Rtt109 in two crystal forms. Both structures show similar asymmetric assemblies of a Vps75 dimer bound to an Rtt109 monomer. In the Vps75-Rtt109 complexes, the catalytic site of Rtt109 is confined to an enclosed space that can accommodate the N-terminal tail of histone H3 in (H3-H4)(2). Investigation of Vps75-Rtt109-(H3-H4)(2) and Vps75-(H3-H4)(2) complexes by NMR spectroscopy-probed hydrogen/deuterium exchange suggests that Vps75 guides histone H3 in the catalytic enclosure. These findings clarify the basis for the enhanced acetylation of histone H3 tail residues by Vps75-Rtt109.

히스톤 H3K27 변형과 유전자 전사

진핵세포의 크로마틴에서 히스톤 단백질 H3와 H4의 라이신 잔기는 공유결합에 의해 변형된다. 히스톤 H3에서 27번 라이신은 아세틸화되거나(H3K27ac) 세 가지 단계로 메틸화가 될 수 있으며(H3K27me1, H3K27me2, H3K27me3), 이러한 H3K27의 변형들은 각각 독특한 형태로 유전자 전사 및 크로마틴 구조와 관련된다. 일반적으로 H3K27ac과 H3K27me1은 좌위조절부위나 활발히 전사되는 유전자처럼 활성 크로마틴에서 나타나고, 이에 반해 전사가 일어나지 않은 유전자는 높은 수준의 H3K27me2과 H3K27me3이 관찰된다. 이러한 변형들은 각각 다른 종류의 변형효소에 의해 촉매된다. 최근 연구들은 유전자 전사 및 크로마틴 구조 형성에서 H3K27의 네 가지 변형들 사이에 상관 관계가 있음을 제시하고 있다. Lysine residues of histone H3 and H4 are covalently modified in the chromatin of eukaryotic cells. Lysine 27 in histone H3 was acetylated (H3K27ac) or methylated at three levels; mono-, di-, and trimethylation (H3K27me1, H3K27me2, and H3K27me3). These modifications at H3K27 were related with gene transcription and/or chromatin structure in distinct patterns. Generally, H3K27ac and H3K27me1 were enriched in active chromatin, such as the locus control region or transcriptionally active genes, while transcriptionally inactive genes were highly marked by H3K27me2 and H3K27me3. These modifications appear to have been catalyzed by distinct histone-modifying enzymes. Recent studies suggest that the four kinds of modifications at H3K27 have inter-correlation in gene transcription or chromatin structure formation.

Abstract 2805: Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma

Abstract The genetics of renal cancer is dominated by inactivation of the VHL tumour suppressor gene in clear cell carcinoma (ccRCC), the commonest histological subtype. A recent large-scale screen of ∼3500 genes by PCR-based exon re-sequencing identified several new cancer genes in ccRCC including UTX (KDM6A), JARID1C (KDM5C) and SETD2. These genes encode enzymes that demethylate (UTX, JARID1C) or methylate (SETD2) key lysine residues of histone H3. Modification of the methylation state of these lysine residues of histone H3 regulates chromatin structure and is implicated in transcriptional control. However, together these mutations are present in fewer than 15% of ccRCC, suggesting the existence of additional, currently unidentified cancer genes. Here, we have sequenced the protein coding exome in a series of primary ccRCC and report the identification of the SWI/SNF chromatin remodeling complex gene PBRM1 as a second major ccRCC cancer gene, with truncating mutations in 41% (92/227) of cases. These data further elucidate the somatic genetic architecture of ccRCC and emphasize the marked contribution of aberrant chromatin biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2805. doi:10.1158/1538-7445.AM2011-2805

Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma.

SummaryThe genetics of renal cancer is dominated by inactivation of the VHL tumour suppressor gene in clear cell carcinoma (ccRCC), the commonest histological subtype. A recent large-scale screen of ~3500 genes by PCR-based exon re-sequencing identified several new cancer genes in ccRCC including UTX (KDM6A)1, JARID1C (KDM5C) and SETD22. These genes encode enzymes that demethylate (UTX, JARID1C) or methylate (SETD2) key lysine residues of histone H3. Modification of the methylation state of these lysine residues of histone H3 regulates chromatin structure and is implicated in transcriptional control3. However, together these mutations are present in fewer than 15% of ccRCC, suggesting the existence of additional, currently unidentified cancer genes. Here, we have sequenced the protein coding exome in a series of primary ccRCC and report the identification of the SWI/SNF chromatin remodeling complex gene PBRM14 as a second major ccRCC cancer gene, with truncating mutations in 41% (92/227) of cases. These data further elucidate the somatic genetic architecture of ccRCC and emphasize the marked contribution of aberrant chromatin biology.

Functional characterization of the zebrafish WHSC1-related gene, a homolog of human NSD2

Methylation of specific lysine residues of histone H3 and H4 has been reported to be important in the structuring of chromatin and for the transcription of certain genes. Proteins with SET domains have been shown to methylate specific lysine residues of histone H3 and H4.We isolated a SET domain-containing gene from the zebrafish (Danio rerio). The gene has the highest sequence similarity to human NSD2, also known as Wolf–Hirschhorn syndrome candidate 1 or WHSC1, and therefore, was named DrWhsc1. DrWhsc1 mRNA is expressed in various tissues with the highest level in testis. Morpholino oligonucleotides for the DrWhsc1 gene affected early embryogenesis in zebrafish, such as endbrain enlargement, abnormal cartilage, marked reduction of bone, and incomplete motor neuron formation. Such developmental abnormalities are also observed in Wolf–Hirschhorn syndrome patients and Whsc1-deficient mice. In addition, suppression of the DrWhsc1 gene or defect in the SET domain of DrWhsc1 resulted in impairment of di-methylation of histone H3K36 at early embryogenesis. These results indicate that DrWhsc1 is a functional homolog of WHSC1 and that the SET domain of DrWhsc1 is essential for di-methylation of histone H3K36 in zebrafish.

Ornithine Decarboxylase Antizyme Induces Hypomethylation of Genome DNA and Histone H3 Lysine 9 Dimethylation (H3K9me2) in Human Oral Cancer Cell Line

BackgroundMethylation of CpG islands of genome DNA and lysine residues of histone H3 and H4 tails regulates gene transcription. Inhibition of polyamine synthesis by ornithine decarboxylase antizyme-1 (OAZ) in human oral cancer cell line resulted in accumulation of decarboxylated S-adenosylmethionine (dcSAM), which acts as a competitive inhibitor of methylation reactions. We anticipated that accumulation of dcSAM impaired methylation reactions and resulted in hypomethylation of genome DNA and histone tails.Methodology/Principal FindingsGlobal methylation state of genome DNA and lysine residues of histone H3 and H4 tails were assayed by Methylation by Isoschizomers (MIAMI) method and western blotting, respectively, in the presence or absence of OAZ expression. Ectopic expression of OAZ mediated hypomethylation of CpG islands of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2). Protein level of DNA methyltransferase 3B (DNMT3B) and histone H3K9me specific methyltransferase G9a were down-regulated in OAZ transfectant.Conclusions/SignificanceOAZ induced hypomethylation of CpG islands of global genome DNA and H3K9me2 by down-regulating DNMT3B and G9a protein level. Hypomethylation of CpG islands of genome DNA and histone H3K9me2 is a potent mechanism of induction of the genes related to tumor suppression and DNA double strand break repair.

Sequential changes in chromatin structure during transcriptional activation in the β globin LCR and its target gene

Chromatin structure is modulated during transcriptional activation. The changes include the association of transcriptional activators, formation of hypersensitive sites and covalent modifications of histones. To understand the order of the various changes accompanying transcriptional activation, we analyzed the mouse β globin gene, which is transcriptionally inducible in erythroid MEL cells over a time course of HMBA treatment. Transcription of the globin genes requires the locus control region (LCR) consisting of several hypersensitive sites (HSs). Erythroid specific transcriptional activators such as NF-E2, GATA-1, TAL1 and EKLF were associated with the LCR in the uninduced state before transcriptional activation. The HSs of the LCR were formed in this state as revealed by high sensitivity to DNase I and MNase attack. However the binding of transcriptional activators and the depletion of histones were observed in the promoter of the β globin gene only after transcriptional activation. In addition, various covalent histone modifications were sequentially detected in lysine residues of histone H3 during the activation. Acetylation of K9, K36 and K27 was notable in both LCR HSs and gene after induction but before transcriptional initiation. Inactive histone marks such as K9me2, K36me2 and K27me2 were removed coincident with transcriptional initiation in the gene region. Taken together, these results indicate that LCR has a substantially active structure in the uninduced state while transcriptional activation serially adds active marks, including histone modifications, and removes inactive marks in the target gene of the LCR.

Phosphorylation of histone H3T6 by PKCβI controls demethylation at histone H3K4

Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4). In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9). Yet, the mechanisms that control this dual specificity of demethylation are unknown. Here we show that phosphorylation of histone H3 at threonine 6 (H3T6) by protein kinase C beta I (PKCbeta(I), also known as PRKCbeta) is the key event that prevents LSD1 from demethylating H3K4 during AR-dependent gene activation. In vitro, histone H3 peptides methylated at lysine 4 and phosphorylated at threonine 6 are no longer LSD1 substrates. In vivo, PKCbeta(I) co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression. RNA interference (RNAi)-mediated knockdown of PKCbeta(I) abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKCbeta(I) requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1). Notably, increased levels of PKCbeta(I) and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCbeta(I) blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xenografts in vivo. Together, our data establish that androgen-dependent kinase signalling leads to the writing of the new chromatin mark H3T6ph, which in consequence prevents removal of active methyl marks from H3K4 during AR-stimulated gene expression.

Genome-wide Analysis of Histone Methylation Reveals Chromatin State-Based Regulation of Gene Transcription and Function of Memory CD8+ T Cells

Memory lymphocytes are characterized by their ability to exhibit a rapid response to the recall antigen, in which differential transcription is important, yet the underlying mechanism is not understood. We report here a genome-wide analysis of histone methylation on two histone H3 lysine residues (H3K4me3 and H3K27me3) and gene expression profiles in naive and memory CD8(+) T cells. We found that specific correlation exists between gene expression and the amounts of H3K4me3 (positive correlation) and H3K27me3 (negative correlation) across the gene body. These correlations displayed four distinct modes (repressive, active, poised, and bivalent), reflecting different functions of these genes in CD8(+) T cells. Furthermore, a permissive chromatin state of each gene was established by a combination of different histone modifications. Our findings reveal a complex regulation by histone methylation in differential gene expression and suggest that histone methylation may be responsible for memory CD8(+) T cell function.

Lysine-Specific Demethylase 1 Is Strongly Expressed in Poorly Differentiated Neuroblastoma: Implications for Therapy

Aberrant epigenetic changes in DNA methylation and histone acetylation are hallmarks of most cancers, whereas histone methylation was previously considered to be irreversible and less versatile. Recently, several histone demethylases were identified catalyzing the removal of methyl groups from histone H3 lysine residues and thereby influencing gene expression. Neuroblastomas continue to remain a clinical challenge despite advances in multimodal therapy. Here, we address the functional significance of the chromatin-modifying enzyme lysine-specific demethylase 1 (LSD1) in neuroblastoma. LSD1 expression correlated with adverse outcome and was inversely correlated with differentiation in neuroblastic tumors. Differentiation of neuroblastoma cells resulted in down-regulation of LSD1. Small interfering RNA-mediated knockdown of LSD1 decreased cellular growth, induced expression of differentiation-associated genes, and increased target gene-specific H3K4 methylation. Moreover, LSD1 inhibition using monoamine oxidase inhibitors resulted in an increase of global H3K4 methylation and growth inhibition of neuroblastoma cells in vitro. Finally, targeting LSD1 reduced neuroblastoma xenograft growth in vivo. Here, we provide the first evidence that a histone demethylase, LSD1, is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells. We show that inhibition of LSD1 reprograms the transcriptome of neuroblastoma cells and inhibits neuroblastoma xenograft growth. Our results suggest that targeting histone demethylases may provide a novel option for cancer therapy.

Lysine-Specific Demethylase 1 Is Strongly Expressed in Poorly Differentiated Neuroblastoma: Implications for Therapy

Aberrant epigenetic changes are hallmarks of most cancers. Recently, a new class of histone modifying enzymes, demethylases, was identified catalyzing the removal of methyl groups from histone H3 lysine residues. Here, we address the functional significance of lysine-specific demethylase 1 (LSD1) in neuroblastoma. LSD1 expression correlated with adverse outcome and was inversely correlated with differentiation in neuroblastic tumors. Differentiation of neuroblastoma cells resulted in down-regulation of LSD1. Small interfering RNA-mediated knockdown of LSD1 decreased cellular growth, induced expression of differentiation-associated genes, and increased target gene-specific H3K4 methylation. LSD1 inhibition using monoamine oxidase inhibitors resulted in an increase of global H3K4 methylation, growth inhibition of neuroblastoma cells in vitro and reduced xenograft growth in vivo. To prove specificity of LSD1 targeting therapy in vivo, a transgenic mouse for tetracycline inducible expression of a shRNA against LSD1 was generated. This model is now used to simulate therapeutic LSD1 inhibition in vivo. Here, we provide first evidence that a histone demethylase, LSD1, is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells. Our results suggest that targeting histone demethylases may provide a novel option for cancer therapy.

Genetic and Genomewide Analysis of Simultaneous Mutations in Acetylated and Methylated Lysine Residues in Histone H3 inSaccharomyces cerevisiae

Acetylated and methylated lysine residues in histone H3 play important roles in regulating yeast gene expression and other cellular processes. Previous studies have suggested that histone H3 acetylated and methylated lysine residues may functionally interact through interdependent pathways to regulate gene transcription. A common genetic test for functional interdependence is to characterize the phenotype of a double mutant. Using this strategy, we tested the genetic interaction between histone H3 mutant alleles that simultaneously eliminate acetylated or methylated lysine residues. Our results indicate that mutation of histone H3 acetylated lysine residues alleviates growth phenotypes exhibited by the H3 methylated lysine mutant. In contrast, histone H3 acetylated and methylated lysine mutants display largely independent effects on yeast gene expression. Intriguingly, these expression changes are preferentially associated with chromosomal regions in which histone H3 lysine residues are hypoacetylated and hypomethylated. Finally, we show that the acetylated and methylated lysine mutants have strikingly different effects on the binding of Sir4 to yeast telomeres, suggesting that histone H3 acetylated lysine residues regulate yeast silencing through a mechanism independent of SIR binding.