H3K27 Hypermethylation Research Articles

PKA Mediated Phosphorylation of TAL1 Regulates Its Interaction with LSD1 During Hematopoiesis.

AbstractAbstract 2599TAL1 is a member of the basic helix-loop-helix (bHLH) family of transcription factors and is required for the development of all hematopoietic cell lineages. TAL1 is a phosphorylated protein and its activities are mediated by the corepressors and coactivators that associate with TAL1. However, the functional link between phosphorylation and the recruitment of co-regulators by TAL1 is currently unknown. We showed that TAL1 dynamically interacts with LSD1 complex containing both histone H3K4 demethylase and deacetylase activities during hematopoiesis (Proc. Natl. Acad. Sci. USA 106: 10141–10146). To further understand the molecular mechanism that regulates the TAL1 and LSD1 interaction during hematopoiesis, we determined whether TAL1 directly interacts with LSD1 and characterized the domains required for this interaction. TAL1 directly interacts with LSD1, and the interacting domain encompasses amino acids 142–185 proximal to the bHLH domain, which contains a serine 172 residue that becomes phosphorylated by Protein kinase A (PKA) during the transcriptional activation of TAL1. The PKA inhibitor, H89, stimulated TAL1 interaction with LSD1 in hematopoietic cells, while Forskolin, an activator of PKA, completely abolished TAL-LSD1 interaction. We further mutated serine 172 of TAL1 to Alanine (Ala) or to Aspartic acid (Asp) to mimic unphosphorylated or phosphorylated TAL1, respectively. While the TAL1Ser172Ala mutant remains interacted with LSD1, TAL1Ser172Asp specifically loses its interaction with LSD1 indicating that serine 172 phosphorylation of TAL1 by PKA destabilizes the TAL1 and LSD1 interaction. Our previous results indicate that LSD1 inhibits TAL1 mediated erythroid differentiation. To further test whether the activity of TAL1 is mediated through an interaction with LSD1, we expressed the TAL1 mutant that deleted the LSD1 interacting domain in erythroid progenitor cells and showed that the deletion of the LSD1 interacting domain of TAL1 leads to a promotion of erythroid differentiation and H3K4 hypermethylation of the P4.2 promoter. In contrast, the expression of the TAL1Ser172Ala mutant and TAL1-LSD1 chimerical fusion enhanced cellular proliferation and colony formation ability of the hematopoietic progenitor cells while these constructs inhibited erythroid differentiation. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and erythroid differentiation. The results suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs. Disclosures:No relevant conflicts of interest to declare.

Promoter-exon relationship of H3 lysine 9, 27, 36 and 79 methylation on pluripotency-associated genes

Evidence links pluripotency to a gene regulatory network organized by the transcription factors Oct4, Nanog and Sox2. Expression of these genes is controlled by epigenetic modifications on regulatory regions. However, little is known on profiles of trimethylated H3 lysine residues on coding regions of these genes in pluripotent and differentiated cells, and on the interdependence between promoter and exon occupancy of modified H3. Here, we determine how H3K9, H3K27, H3K36 and H3K79 methylation profiles on exons of OCT4, NANOG and SOX2 correlate with expression and promoter occupancy. Expression of OCT4, SOX2 and NANOG in embryonal carcinoma cells is associated with a looser chromatin configuration than mesenchymal progenitors or fibroblasts, determined by H3 occupancy. Promoter H3K27 trimethylation extends into the first exon of repressed OCT4, NANOG and SOX2, while H3K9me3 occupies the first exon of these genes irrespective of expression. Both H3K36me3 and H3K79me3 are enriched on exons of expressed genes, yet with a distinct pattern: H3K36me3 increases towards the 3′ end of genes, while H3K79me3 is preferentially enriched on first exons. Down-regulation of the H3K36 methyltransferase SetD2 by siRNA causes global and gene-specific H3K36 demethylation and global H3K27 hypermethylation; however it does not affect promoter levels of H3K27me3, suggesting for the genes examined independence of occupancy of H3K27me3 on promoters and H3K36me3 on exons. mRNA levels are however affected, raising the hypothesis of a role of SetD2 on transcription elongation and/or termination.

Setdb1-mediated histone H3K9 hypermethylation in neurons worsens the neurological phenotype of Mecp2-deficient mice

Rett syndrome (RTT, OMIM # 312750), a neurodevelopmental disorder of early childhood, is primarily caused by mutations in the gene encoding methyl-CpG-binding protein 2 ( MECP2). Various molecular functions have been ascribed to MECP2, including the regulation of histone modifications associated with repressive chromatin remodeling, but the role of these mechanisms for the pathophysiology of RTT remains unclear. Here, we explore whether or not neuronal expression of the histone H3-lysine 9 specific methyl-transferase, Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e, which is normally present only at low levels in differentiated neurons, rescues the RTT-like phenotype of Mecp2-deficient mice. A myc-tagged Setdb1 cDNA was expressed through the tau locus for ubiquitous expression in CNS neurons, or under control of the calcium/calmodulin-dependent protein kinase II (CK) promoter to selectively target postmitotic neurons in forebrain. However, the CK-Setdb1 transgene lead to an enhanced neurological deficit, and the tauSetdb1 allele further shortened life span of mice with a brain-wide deletion of Mecp2 during prenatal development. In contrast, no neurological deficits or premature death was observed in CK-Setdb1 and tauSetdb1 mice expressing wildtype Mecp2. However, levels of trimethylated H3K9 at pericentromeric repeats were fully maintained in differentiated neurons from symptomatic Mecp2 null mutant mice. Based on these results, we draw two conclusions: First, neuronal chromatin in RTT brain is not affected by a generalized deficit in H3K9 trimethylation. Second, artificial up-regulation of this repressive chromatin mark via Setdb1 gene delivery specifically to neurons is harmful for the Mecp2-deficient brain. This article is part of a Special Issue entitled ‘Trends in Neuropharmacology: In Memory of Erminio Costa’.

Apoptosis-induced histone H3 methylation is targeted by autoantibodies in systemic lupus erythematosus

ObjectivesIn systemic lupus erythematosus (SLE) apoptotic chromatin is present extracellularly, which is most likely the result of disturbed apoptosis and/or insufficient removal. Released chromatin, modified during apoptosis, activates the immune...

AF17 Competes with AF9 for Binding to Dot1a to Up-regulate Transcription of Epithelial Na+ Channel α

We previously reported that Dot1a.AF9 complex represses transcription of the epithelial Na(+) channel subunit alpha (alpha-ENaC) gene in mouse inner medullary collecting duct mIMCD3 cells and mouse kidney. Aldosterone relieves this repression by down-regulating the complex through various mechanisms. Whether these mechanisms are sufficient and conserved in human cells or can be applied to other aldosterone-regulated genes remains largely unknown. Here we demonstrate that human embryonic kidney 293T cells express the three ENaC subunits and all of the ENaC transcriptional regulators examined. These cells respond to aldosterone and display benzamil-sensitive Na(+) currents, as measured by whole-cell patch clamping. We also show that AF17 and AF9 competitively bind to the same domain of Dot1a in multiple assays and have antagonistic effects on expression of an alpha-ENaC promoter-luciferase construct. Overexpression of Dot1a or AF9 decreased mRNA expression of the ENaC subunits and their transcriptional regulators and reduced benzamil-sensitive Na(+) currents. AF17 overexpression caused the opposite effects, accompanied by redirection of Dot1a from the nucleus to the cytoplasm and reduction in histone H3 K79 methylation. The nuclear export inhibitor leptomycin B blocked the effect of AF17 overexpression on H3 K79 hypomethylation. RNAi-mediated knockdown of AF17 yielded nuclear enrichment of Dot1a and histone H3 K79 hypermethylation. As with AF9, AF17 displays nuclear and cytoplasmic co-localization with Sgk1. Therefore, AF17 competes with AF9 to bind Dot1a, decreases Dot1a nuclear expression by possibly facilitating its nuclear export, and relieves Dot1a.AF9-mediated repression of alpha-ENaC and other target genes.

Moz and Retinoic Acid Coordinately Regulate H3K9 Acetylation, Hox Gene Expression, and Segment Identity

We report that embryos deficient in the histone acetyltransferase Moz (Myst3/Kat6a) show histone H3 lysine 9 (H3K9) hypoacetylation, corresponding H3K9 hypermethylation, and reduced transcription at Hox gene loci. Consistent with an observed caudal shift in Hox gene expression, segment identity is shifted anteriorly, such that Moz-deficient mice show a profound homeotic transformation of the axial skeleton and the nervous system. Intriguingly, histone acetylation defects are relatively specific to H3K9 at Hox loci, as neither Hox H3K14 acetylation nor bulk H3K9 acetylation levels throughout the genome are strongly affected; H4K16 acetylation actually increases in the absence of Moz. H3K9 hypoacetylation, Hox gene repression, and the homeotic transformation caused by lack of Moz are all reversed by treatment with retinoic acid (RA). In conclusion, our data show that Moz regulates H3K9 acetylation at Hox gene loci and that RA can act independently of Moz to establish specific Hox gene expression boundaries.

Promoter histone H3 lysine 9 di-methylation is associated with DNA methylation and aberrant expression of p16 in gastric cancer cells

In the course of gastric cancer development, gene silencing by DNA hypermethylation is an important mechanism. While DNA methylation often co-exists with histone modifications to regulate gene expression, the function of histone modifications in gene silencing in gastric cancer has not been evaluated in detail. p16, a well-known tumor suppressor gene, is frequently silenced in DNA hypermethylation manner in gastric cancer. Accordingly, we chose p16 to clarify whether there is a correlation among histone H3 lysine 9 (H3-K9) di-methylation, H3-K9 acetylation, DNA methylation and p16 expression in human gastric cancer. Three gastric cancer cells, MKN-45, SGC-7901 and BGC-823, were treated with 5-aza-2'-deoxycytidine (5-Aza-dC) and/or trichostatin A (TSA). We investigated p16 promoter DNA methylation status, p16 mRNA levels, regional and global levels of di-methyl-H3-K9 and acetyl-H3-K9 in four groups: i) 5-Aza-dC, ii) TSA, iii) the combination of 5-Aza-dC and TSA and iv) control group with no treatments. p16 silencing is characterized by DNA hypermethylation, H3-K9 hypoacetylation and H3-K9 hypermethylation at the promoter region. Treatment with TSA, increased H3-K9 acetylation at the hypermethylated promoter, but did not affect H3-K9 di-methylation or p16 expression. By contrast, treatment with 5-Aza-dC, reduced H3-K9 di-methylation, increased H3-K9 acetylation at the hypermethylated promoter and reactivated the expression of p16. Combined treatment restored the expression of p16 synergistically. In addition, 5-Aza-dC and the combined treatment did not result in global alteration of H3-K9 di-methylation. These results suggest that H3-K9 di-methylation, H3-K9 acetylation and DNA methylation work in combination to silence p16 in gastric cancer. The decreased H3-K9 di-methylation correlates with DNA demethylation and reactivation of p16. H3-K9 di-methylation as well as DNA methylation related to p16 silencing is limited to the promoter region. In addition to its effect on DNA methylation, 5-Aza-dC can act at histone modification levels to reactivate p16 expression in region-specific and DNA methylation-dependent manner.

Global reduction of the epigenetic H3K79 methylation mark and increased chromosomal instability in CALM-AF10–positive leukemias

AbstractChromosomal translocations generating fusion proteins are frequently found in human leukemias. The fusion proteins play an important role in leukemogenesis by subverting the function of one or both partner proteins. The leukemogenic CALM-AF10 fusion protein is capable of interacting with the histone H3 lysine 79 (H3K79)–specific methyltransferase hDOT1L through the fused AF10 moiety. This interaction leads to local H3K79 hypermethylation on Hoxa5 loci, which up-regulates the expression of Hoxa5 and contributes to leukemogenesis. However, the long latency of leukemogenesis of CALM-AF10 transgenic mice suggests that the direct effects of fusion oncogene are not sufficient for the induction of leukemia. In this study, we show that the CALM-AF10 fusion protein can also greatly reduce global H3K79 methylation in both human and murine leukemic cells by disrupting the AF10-mediated association of hDOT1L with chromatin. Cells with reduced H3K79 methylation are more sensitive to γ-irradiation and display increased chromosomal instability. Consistently, leukemia patients harboring CALM-AF10 fusion have more secondary chromosomal aberrations. These findings suggest that chromosomal instability associated with global epigenetic alteration contributes to malignant transformation in certain leukemias, and that leukemias with this type of epigenetic alteration might benefit from treatment regimens containing DNA-damaging agents. This study is registered with www.clinicaltrials.gov as NCT00266136.

Histone H3 lysine 9 methylation is associated with the expression of hMLH1 and DNA methylation in gastric cancer cells

To identify the association of his tone H3 lysine 9 (H3-K9) methylation with DNA methylation and the expression of the mismatch repair gene hMLH1 in human gastric cancer cells. Gastric cancer cells of the lines BGC-823 and MGC-803 were cultured and treated with 5-Aza-2'-deoxycytidine (5-Aza-dC), a demethylation agent, for 72 hour. Chromatin immunoprecipitation (ChIP) assay was used to assess the status of histone H3 lysine 9 methylation in the promoter regions of hMLH1 gene. Methylation-specific PCR (MSP) was used to evaluate the effect of 5-Aza-dC on DNA methylation status. RT-PCR was used to examine the hMLH1 gene expression. In the MGC-803 cells, silenced hMLH1 gene was characterized by DNA methylation and histone H3-K9 hypermethylation; 5-Aza-dC demethylated the DNA and reduced the histone H3-K9 methylation at silenced loci and resulted in reactivation of hMLH1 gene therein. Contrary to the MGC-803 cells, BGC-823 cells expressed hMLH1 gene with DNA demethylation and histone H3-K9 hypomethylation; and 5-Aza-dC had no effects on the gene expression, DNA methylation, and histone H3-K9 methylation therein. Hypermethylation of DNA in the promoter region is related to transcriptional silencing of hMLH1 gene. Histone H3-K9 methylation in different regions of the promoter studied correlates with DNA methylation status of hMLH1 gene in gastric cancer cells. Alteration of DNA methylation affects histone H3-K9 methylation. 5-Aza-dC can control hMLH1 expression, DNA methylation, and histone H3-K9 methylation in the promoter.

Role of hMOF-Dependent Histone H4 Lysine 16 Acetylation in the Maintenance of TMS1/ASC Gene Activity

Epigenetic silencing of tumor suppressor genes in human cancers is associated with aberrant methylation of promoter region CpG islands and local alterations in histone modifications. However, the mechanisms that drive these events remain unclear. Here, we establish an important role for histone H4 lysine 16 acetylation (H4K16Ac) and the histone acetyltransferase hMOF in the regulation of TMS1/ASC, a proapoptotic gene that undergoes epigenetic silencing in human cancers. In the unmethylated and active state, the TMS1 CpG island is spanned by positioned nucleosomes and marked by histone H3K4 methylation. H4K16Ac was uniquely localized to two sharp peaks that flanked the unmethylated CpG island and corresponded to strongly positioned nucleosomes. Aberrant methylation and silencing of TMS1 was accompanied by loss of the H4K16Ac peaks, loss of nucleosome positioning, hypomethylation of H3K4, and hypermethylation of H3K9. In addition, a single peak of histone H4 lysine 20 trimethylation was observed near the transcription start site. Down-regulation of hMOF or another component of the MSL complex resulted in a gene-specific decrease in H4K16Ac, loss of nucleosome positioning, and silencing of TMS1. Gene silencing induced by H4K16 deacetylation occurred independently of changes in histone methylation and DNA methylation and was reversed on hMOF reexpression. These results indicate that the selective marking of nucleosomes flanking the CpG island by hMOF is required to maintain TMS1 gene activity and suggest that the loss of H4K16Ac, mobilization of nucleosomes, and transcriptional down-regulation may be important events in the epigenetic silencing of certain tumor suppressor genes in cancer.

Long‐lasting depression‐like behavior and epigenetic changes of BDNF gene expression induced by perinatal exposure to methylmercury

Substantial evidence indicates that predisposition to diseases can be acquired during early stages of development and interactions between environmental and genetic factors may be implicated in the onset of many pathological conditions. Data collected over several decades have shown that chemicals are among the relevant factors that can endanger CNS. We previously showed that perinatal exposure to methylmercury (MeHg) causes persistent changes in learning and motivational behavior in mice. In this study, we report that the depression-like behavior in MeHg-exposed male mice is reversed by chronic treatment with the antidepressant fluoxetine. Behavioral alterations are associated with a decrease in brain-derived neurotrophic factor (BDNF) mRNA in the hippocampal dentate gyrus and fluoxetine treatment restores BDNF mRNA expression. We also show that MeHg-exposure induces long-lasting repressive state of the chromatin structure at the BDNF promoter region, in particular DNA hypermethylation, an increase in histone H3-K27 tri-methylation and a decrease in H3 acetylation at the promoter IV. While fluoxetine treatment does not alter hypermethylation of H3-K27, it significantly up-regulates H3 acetylation at the BDNF promoter IV in MeHg-exposed mice. Our study shows that developmental exposure to low levels of MeHg predisposes mice to depression and induces epigenetic suppression of BDNF gene expression in the hippocampus.

Epigenetic regulation of Nanog gene in embryonic stem and trophoblast stem cells

The Nanog and Oct-4 genes are essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that DNA methylation and chromatin remodeling underlie the cell type-specific mechanism of Oct-4 gene expression. In the present study, we found that there is a tissue-dependent and differentially methylated region (T-DMR) in the Nanog up-stream region. The T-DMR is hypomethylated in ES cells, but is heavily methylated in trophoblast stem (TS) cells and NIH/3T3 cells, in which the Nanog gene is repressed. Furthermore, in vitro methylation of T-DMR suppressed Nanog promoter activity in reporter assay. Chromatin immunoprecipitation assay revealed that histone H3 and H4 are highly acetylated, and H3 lysine (K) 4 is hypermethylated at the Nanog locus in ES cells. Conversely, histone deacetylation and H3-K4 demethylation occurred in TS cells. Importantly, in TS cells, hypermethylation of H3-K9 and -K27 is found only at the Nanog locus, not the Oct-4 locus, indicating that the combination of histone modifications associated with the Nanog gene is distinct from that of the Oct-4 gene. In conclusion, the Nanog gene is regulated by epigenetic mechanisms involving DNA methylation and histone modifications.

Activation of the chicken Ig- locus by the collaboration of scattered regulatory regions through changes in chromatin structure

A total of 10 B-lymphocyte-specific DNase I hypersensitive sites located in the chicken Ig-β locus were divided into four regions and combinations of deletions of these regions were carried out. A decrease in transcription of the Ig-β gene to <3% was demonstrated in cells with deletions in all four regions. The Ig-β chromatin was resistant to DNase I digestion in these cells. Thus, the collaboration is shown to convert the Ig-β chromatin from the condensed state to a relaxed state. H3 and H4 acetylation decreased to <8% but H3K4 hypermethylation was observed at the Ig-β promoter and exon 3. The collaboration of four regions had virtually no effect on CG hypomethylation in the region upstream the transcriptional start site. Accordingly, neither the DNase I general sensitive state in the Ig-β chromatin nor hyperacetylation of H3 and H4 histones in the promoter proximal region causes H3K4 di-methylation or CG hypomethylation in the promoter. From these analyses, a chromatin situation was found in which both an active state, such as enhanced H3K4 methylation, or CG hypomethylation, and an inactive state, such as DNase I resistance in the Ig-β chromatin or hypoacetylation of H3 and H4 histones in the Ig-β locus, coexist.

Histone modification and the control of heterochromatic gene silencing in Drosophila

Covalent modifications of histones index structurally and functionally distinct chromatin domains in eukaryotic nuclei. Drosophila with its polytene chromosomes and developed genetics allows detailed cytological as well as functional analysis of epigenetic histone modifications involved in the control of gene expression pattern during development. All H3K9 mono- and dimethylation together with all H3K27 methylation states and H4K20 trimethylation are predominant marks of pericentric heterochromatin. In euchromatin, bands and interbands are differentially indexed. H3K4 and H3K36 methylation together with H3S10 phosphorylation are predominant marks of interband regions whereas in bands different H3K27 and H4K20 methylation states are combined with acetylation of H3K9 and H3K14. Genetic dissection of heterochromatic gene silencing in position-effect variegation (PEV) by Su(var) and E(var) mutations allowed identification and functional analysis of key factors controlling the formation of heterochromatin. SU(VAR)3-9 association with heterochromatic sequences followed by H3K9 methylation initiates the establishment of repressive SU(VAR)3-9/HP1/SU(VAR)3-7 protein complexes. Differential enzymatic activities of novel point mutants demonstrate that the silencing potential of SU(VAR)3-9 is mainly determined by the kinetic properties of the HMTase reaction. In Su(var)3-9ptn a significantly enhanced enzymatic activity results in H3K9 hypermethylation, enhanced gene silencing and extensive chromatin compaction. Mutations in factors controlling active histone modification marks revealed the dynamic balance between euchromatin and heterochromatin. Further analysis and definition of Su(var) and E(var) genes in Drosophila will increase our understanding of the molecular hierarchy of processes controlling higher-order structures in chromatin.

Aldosterone-sensitive repression of ENaCα transcription by a histone H3 lysine-79 methyltransferase

Aldosterone is a major regulator of epithelial Na(+) absorption. One of its principal targets is the epithelial Na(+) channel alpha-subunit (ENaCalpha), principally expressed in the kidney collecting duct, lung, and colon. Models of aldosterone-mediated trans-activation of the ENaCalpha gene have focused primarily on interactions of liganded nuclear receptors with the ENaCalpha gene promoter. Herein, we demonstrate that the murine histone H3 lysine-79 methyltransferase, murine disruptor of telomeric silencing alternative splice variant "a" (mDot1a), is a novel component in the aldosterone signaling network controlling transcription of the ENaCalpha gene. Aldosterone downregulated mDot1a mRNA levels in murine inner medullary collecting ducts cells, which was associated with histone H3 K79 hypomethylation in bulk histones and at specific sites in the ENaCalpha 5'-flanking region, and trans-activation of ENaCalpha. Knockdown of mDot1a by RNA interference increased activity of a stably integrated ENaCalpha promoter-luciferase construct and expression of endogenous ENaCalpha mRNA. Conversely, overexpression of EGFP-tagged mDot1a resulted in hypermethylation of histone H3 K79 at the endogenous ENaCalpha promoter, repression of endogenous ENaCalpha mRNA expression, and decreased activity of the ENaCalpha promoter-luciferase construct. mDot1a-mediated histone H3 K79 hypermethylation and repression of ENaCalpha promoter activity was abolished by mDot1a mutations that eliminate its methyltransferase activity. Collectively, our data identify mDot1a as a novel aldosterone-regulated histone modification enzyme, and, through binding the ENaCalpha promoter and hypermethylating histone H3 K79 associated with the ENaCalpha promoter, a negative regulator of ENaCalpha transcription.

Altered nucleosome occupancy and histone H3K4 methylation in response to ‘transcriptional stress’

We report that under 'transcriptional stress' in budding yeast, when most pol II activity is acutely inhibited, rapid deposition of nucleosomes occurs within genes, particularly at 3' positions. Whereas histone H3K4 trimethylation normally marks 5' ends of highly transcribed genes, under 'transcriptional stress' induced by 6-azauracil (6-AU) and inactivation of pol II, TFIIE or CTD kinases Kin28 and Ctk1, this mark shifted to the 3' end of the TEF1 gene. H3K4Me3 at 3' positions was dynamic and could be rapidly removed when transcription recovered. Set1 and Chd1 are required for H3K4 trimethylation at 3' positions when transcription is inhibited by 6-AU. Furthermore, Deltachd1 suppressed the growth defect of Deltaset1. We suggest that a 'transcriptional stress' signal sensed through Set1, Chd1, and possibly other factors, causes H3K4 hypermethylation of newly deposited nucleosomes at downstream positions within a gene. This response identifies a new role for H3K4 trimethylation at the 3' end of the gene, as a chromatin mark associated with impaired pol II transcription.

HDOT1L Links Histone Methylation to Leukemogenesis

Epigenetic modifications play an important role in human cancer. One such modification, histone methylation, contributes to human cancer through deregulation of cancer-relevant genes. The yeast Dot1 and its human counterpart, hDOT1L, methylate lysine 79 located within the globular domain of histone H3. Here we report that hDOT1L interacts with AF10, an MLL (mixed lineage leukemia) fusion partner involved in acute myeloid leukemia, through the OM-LZ region of AF10 required for MLL-AF10-mediated leukemogenesis. We demonstrate that direct fusion of hDOT1L to MLL results in leukemic transformation in an hDOT1L methyltransferase activity-dependent manner. Transformation by MLL-hDOT1L and MLL-AF10 results in upregulation of a number of leukemia-relevant genes, such as Hoxa9, concomitant with hypermethylation of H3-K79. Our studies thus establish that mistargeting of hDOT1L to Hoxa9 plays an important role in MLL-AF10-mediated leukemogenesis and suggests that the enzymatic activity of hDOT1L may provide a potential target for therapeutic intervention.

Tal1/SCL Binding to Pericentromeric DNA Represses Transcription

Tal1/SCL is a basic helix-loop-helix transcription factor critical for normal hematopoiesis. To understand the mechanisms underlying transcriptional regulation by Tal1/SCL, we combined an in vitro DNA binding strategy and an in vivo chromatin immunoprecipitation analysis to search for Tal1/SCL target regions in K562 erythroleukemia cells. A 0.4-kb genomic DNA clone containing two Tal1/SCL binding E-boxes and GATA- and SATB1-binding motifs (EEGS) was identified that localized to the pericentromeric region with high homology to satellite 2 DNA. Pericentric DNA is related to heterochromatin and gene inactivation. We found that Tal1/SCL could complex with the histone H3 lysine 9 (H3K9)-specific methyltransferase Suv39H1. Binding of Tal1/SCL to EEGS chromatin correlated with hypermethylation of H3K9 and the association of heterochromatin protein HP1 to this region. In Rep4 reporter gene assays, EEGS affected repression in a manner dependent on the expression level of Tal1/SCL that was accompanied by increased H3K9 methylation in chromatin associated with EEGS and a linked promoter. A specific histone deacetylase inhibitor, trichostatin A, relieved Tal1/SCL-mediated repression by EEGS. In addition, SATB1 bound EEGS chromatin and promoted Tal1/SCL EEGS-dependent repression. We expand the list of potential interacting partners for Tal1/SCL by demonstrating direct associations of Tal1/SCL with SATB1 and with Suv39H1. These results reveal a novel mechanism of action for Tal1/SCL and implicate heterochromatin-like silencing via a cis-acting binding motif for transcriptional repression.

Epigenetic Marking Correlates with Developmental Potential in Cloned Bovine Preimplantation Embryos

During differentiation, somatic nuclei acquire highly specialized DNA and chromatin modifications, which are thought to result in cellular memory of the differentiated state [1]. Upon somatic nuclear transfer into oocytes, the donor nucleus may have to undergo reprogramming of these epigenetic marks in order to achieve totipotency. This may involve changes in epigenetic features similar to those that occur in normal embryos during early development [2–6]. However, there is accumulating evidence that epigenetic reprogramming is severely deficient in cloned embryos [7–12]. Several reports reveal inefficient demethylation and inappropriate reestablishment of DNA methylation in quantitative and qualitative patterns on somatic nuclear transfer [7–12]. Here we examine histone H3 lysine 9 (H3-K9) methylation and acetylation in normal embryos and in those created by somatic nuclear transfer. We find that H3-K9 methylation is reprogrammed in parallel with DNA methylation in normal embryos. However, the majority of cloned embryos exhibit H3-K9 hypermethylation associated with DNA hypermethylation, suggesting a genome-wide failure of reprogramming. Strikingly, the precise epigenotype in cloned embryos depends on the donor cell type, and the proportion of embryos with normal epigenotypes correlates closely with the proportion developing to the blastocyst stage. These results suggest a mechanistic link between DNA and histone methylation in the mammalian embryo and reveal an association between epigenetic marks and developmental potential of cloned embryos.

Targeted Recruitment of Set1 Histone Methylase by Elongating Pol II Provides a Localized Mark and Memory of Recent Transcriptional Activity

Set1, the yeast histone H3-lysine 4 (H3-K4) methylase, is recruited by the Pol II elongation machinery to a highly localized domain at the 5' portion of active mRNA coding regions. Set1 association depends upon the TFIIH-associated kinase that phosphorylates the Pol II C-terminal domain (CTD) and mediates the transition between initiation and elongation, and Set1 interacts with the form of Pol II whose CTD is phosphorylated at serine 5 but not serine 2. The Rtf1 and Paf1 components of the Pol II-associated Paf1 complex are also important for Set1 recruitment. Although the level of dimethylated H3-K4 is fairly uniform throughout the genome, the pattern of trimethylated H3-K4 strongly correlates with Set1 occupancy. Hypermethylated H3-K4 within the mRNA coding region persists for considerable time after transcriptional inactivation and Set1 dissociation from the chromatin, indicating that H3-K4 hypermethylation provides a molecular memory of recent transcriptional activity.