Publications by authors named "Antonis Kirmizis"

24 Publications

  • Page 1 of 1

Microfluidics for single-cell lineage tracking over time to characterize transmission of phenotypes in .

STAR Protoc 2020 Dec 16;1(3):100228. Epub 2020 Dec 16.

Institute of Functional Epigenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany.

The budding yeast is an excellent model organism to dissect the maintenance and inheritance of phenotypes due to its asymmetric division. This requires following individual cells over time as they go through divisions to define pedigrees. Here, we provide a detailed protocol for collecting and analyzing time-lapse imaging data of yeast cells. The microfluidics protocol can achieve improved time resolution for single-cell tracking to enable characterization of maintenance and inheritance of phenotypes. For complete details on the use and execution of this protocol, please refer to Bheda et al. (2020a).
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http://dx.doi.org/10.1016/j.xpro.2020.100228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757727PMC
December 2020

N-Terminal Acetyltransferases Are Cancer-Essential Genes Prevalently Upregulated in Tumours.

Cancers (Basel) 2020 Sep 15;12(9). Epub 2020 Sep 15.

Department of Biological Sciences, University of Cyprus, 1678 Nicosia, Cyprus.

N-terminal acetylation (Nt-Ac) is an abundant eukaryotic protein modification, deposited in humans by one of seven N-terminal acetyltransferase (NAT) complexes composed of a catalytic and potentially auxiliary subunits. The involvement of NATs in cancers is being increasingly recognised, but a systematic cross-tumour assessment is currently lacking. To address this limitation, we conducted here a multi-omic data interrogation for NATs. We found that tumour genomic alterations of NATs or of their protein substrates are generally rare events, with some tumour-specific exceptions. In contrast, altered gene expression of NATs in cancers and their association with patient survival constitute a widespread cancer phenomenon. Examination of dependency screens revealed that (i), besides NAA60 and NAA80 and the NatA paralogues NAA11 and NAA16, the other ten NAT genes were within the top 80th percentile of the most dependent genes (ii); NATs act through distinct biological processes. NAA40 (NatD) emerged as a NAT with particularly interesting cancer biology and therapeutic potential, especially in liver cancer where a novel oncogenic role was supported by its increased expression in multiple studies and its association with patient survival. In conclusion, this study generated insights and data that will be of great assistance in guiding further research into the function and therapeutic potential of NATs in cancer.
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http://dx.doi.org/10.3390/cancers12092631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565035PMC
September 2020

The past determines the future: sugar source history and transcriptional memory.

Curr Genet 2020 Dec 19;66(6):1029-1035. Epub 2020 Jul 19.

Institute of Functional Epigenetics, Helmholtz Zentrum München, Neuherberg, Germany.

Transcriptional reinduction memory is a phenomenon whereby cells "remember" their transcriptional response to a previous stimulus such that subsequent encounters with the same stimulus can result in altered gene expression kinetics. Chromatin structure is thought to play a role in certain transcriptional memory mechanisms, leading to questions as to whether and how memory can be actively maintained and inherited to progeny through cell division. Here we summarize efforts towards dissecting chromatin-based transcriptional memory inheritance of GAL genes in Saccharomyces cerevisiae. We focus on methods and analyses of GAL (as well as MAL and INO) memory in single cells and discuss the challenges in unraveling the underlying mechanisms in yeast and higher eukaryotes.
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http://dx.doi.org/10.1007/s00294-020-01094-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599190PMC
December 2020

Histone N-alpha terminal modifications: genome regulation at the tip of the tail.

Epigenetics Chromatin 2020 07 17;13(1):29. Epub 2020 Jul 17.

Epigenetics Laboratory, Department of Biological Sciences, University of Cyprus, 2109, Nicosia, Cyprus.

Histone proteins are decorated with numerous post-(PTMs) or co-(CTMs) translational modifications mainly on their unstructured tails, but also on their globular domain. For many decades research on histone modifications has been focused almost solely on the biological role of modifications occurring at the side-chain of internal amino acid residues. In contrast, modifications on the terminal N-alpha amino group of histones-despite being highly abundant and evolutionarily conserved-have been largely overlooked. This oversight has been due to the fact that these marks were being considered inert until recently, serving no regulatory functions. However, during the past few years accumulating evidence has drawn attention towards the importance of chemical marks added at the very N-terminal tip of histones and unveiled their role in key biological processes including aging and carcinogenesis. Further elucidation of the molecular mechanisms through which these modifications are regulated and by which they act to influence chromatin dynamics and DNA-based processes like transcription is expected to enlighten our understanding of their emerging role in controlling cellular physiology and contribution to human disease. In this review, we clarify the difference between N-alpha terminal (Nt) and internal (In) histone modifications; provide an overview of the different types of known histone Nt-marks and the associated histone N-terminal transferases (NTTs); and explore how they function to shape gene expression, chromatin architecture and cellular phenotypes.
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http://dx.doi.org/10.1186/s13072-020-00352-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367250PMC
July 2020

Synthetic dosage lethal (SDL) interaction data of Hmt1 arginine methyltransferase.

Data Brief 2020 Aug 21;31:105885. Epub 2020 Jun 21.

Department of Biological Sciences, University of Cyprus, 1 University Ave, Nicosia, Aglantzia 2109, Cyprus.

The introduction of methyl groups on arginine residues is catalysed by Protein Arginine Methyltransferases (PRMTs). However, the regulatory mechanisms that dictate the levels of protein arginine methylation within cells are still not completely understood. We employed Synthetic Dosage Lethality (SDL) screening in , for the identification of putative regulators of arginine methylation mediated by Hmt1 (HnRNP methyltransferase 1), ortholog of human PRMT1. We developed an SDL array of 4548 yeast strains in which each strain contained a single non-essential gene deletion, in combination with a galactose-inducible construct overexpressing wild-type (WT) Hmt1-HZ tagged protein. We identified 129 consistent SDL interactions for WT Hmt1-HZ which represented genes whose deletion displayed significant growth reduction when combined with WT Hmt1 overexpression. To identify among the SDL interactions those that were dependent on the methyltransferase activity of Hmt1, SDL screens were repeated using an array overexpressing a catalytically inactive Hmt1(G68R)-HZ protein. Furthermore, an additional SDL control screen was performed using an array overexpressing only the protein tag HZ (His-HA-ZZ) to eliminate false-positive SDL interactions. This analysis has led to a dataset of 50 high-confidence SDL interactions of WT Hmt1 which enrich eight Gene Ontology biological process terms. This dataset can be further exploited in biochemical and functional studies to illuminate which of the SDL interactors of Hmt1 correspond to factors implicated in the regulation of Hmt1-mediated arginine methylation and reveal the underlying molecular mechanisms.
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http://dx.doi.org/10.1016/j.dib.2020.105885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330151PMC
August 2020

Single-Cell Tracing Dissects Regulation of Maintenance and Inheritance of Transcriptional Reinduction Memory.

Mol Cell 2020 06 8;78(5):915-925.e7. Epub 2020 May 8.

Institute of Functional Epigenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany; Faculty of Biology, Ludwig-Maximilians Universität München, Munich, Germany. Electronic address:

Transcriptional memory of gene expression enables adaptation to repeated stimuli across many organisms. However, the regulation and heritability of transcriptional memory in single cells and through divisions remains poorly understood. Here, we combined microfluidics with single-cell live imaging to monitor Saccharomyces cerevisiae galactokinase 1 (GAL1) expression over multiple generations. By applying pedigree analysis, we dissected and quantified the maintenance and inheritance of transcriptional reinduction memory in individual cells through multiple divisions. We systematically screened for loss- and gain-of-memory knockouts to identify memory regulators in thousands of single cells. We identified new loss-of-memory mutants, which affect memory inheritance into progeny. We also unveiled a gain-of-memory mutant, elp6Δ, and suggest that this new phenotype can be mediated through decreased histone occupancy at the GAL1 promoter. Our work uncovers principles of maintenance and inheritance of gene expression states and their regulators at the single-cell level.
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http://dx.doi.org/10.1016/j.molcel.2020.04.016DOI Listing
June 2020

Histone Modifications as an Intersection Between Diet and Longevity.

Front Genet 2019 12;10:192. Epub 2019 Mar 12.

Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus.

Histone modifications are key epigenetic regulators that control chromatin structure and gene transcription, thereby impacting on various important cellular phenotypes. Over the past decade, a growing number of studies have indicated that changes in various histone modifications have a significant influence on the aging process. Furthermore, it has been revealed that the abundance and localization of histone modifications are responsive to various environmental stimuli, such as diet, which can also affect gene expression and lifespan. This supports the notion that histone modifications can serve as a main cellular platform for signal integration. Hence, in this review we focus on the role of histone modifications during aging, report the data indicating that diet affects histone modification levels and explore the idea that histone modifications may function as an intersection through which diet regulates lifespan. A greater understanding of the epigenetic mechanisms that link environmental signals to longevity may provide new strategies for therapeutic intervention in age-related diseases and for promoting healthy aging.
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http://dx.doi.org/10.3389/fgene.2019.00192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422915PMC
March 2019

NAA40 contributes to colorectal cancer growth by controlling PRMT5 expression.

Cell Death Dis 2019 03 11;10(3):236. Epub 2019 Mar 11.

Epigenetics Laboratory, Department of Biological Sciences, University of Cyprus, 2109, Nicosia, Cyprus.

N-alpha-acetyltransferase 40 (NAA40) catalyzes the transfer of an acetyl moiety to the alpha-amino group of serine 1 (S1) on histones H4 and H2A. Our previous studies linked NAA40 and its corresponding N-terminal acetylation of histone H4 (N-acH4) to colorectal cancer (CRC). However, the role of NAA40 in CRC development was not investigated. Here, we show that NAA40 protein and mRNA levels are commonly increased in CRC primary tissues compared to non-malignant specimens. Importantly, depletion of NAA40 inhibits cell proliferation and survival of CRC cell lines and increases their sensitivity to 5-Fluorouracil (5-FU) treatment. Moreover, the absence of NAA40 significantly delays the growth of human CRC xenograft tumors. Intriguingly, we found that NAA40 knockdown and loss of N-acH4 reduce the levels of symmetric dimethylation of histone H4 (H4R3me2s) through transcriptional downregulation of protein arginine methyltransferase 5 (PRMT5). NAA40 depletion and subsequent repression of PRMT5 results in altered expression of key oncogenes and tumor suppressor genes leading to inhibition of CRC cell growth. Consistent with this, NAA40 mRNA levels correlate with those of PRMT5 in CRC patient tissues. Taken together, our results establish the oncogenic function of the epigenetic enzyme NAA40 in colon cancer and support its potential as a therapeutic target.
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http://dx.doi.org/10.1038/s41419-019-1487-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411749PMC
March 2019

Calorie restriction breaks an epigenetic barrier to longevity.

Cell Cycle 2017 05 20;16(9):821-822. Epub 2017 Mar 20.

a Department of Biological Sciences , University of Cyprus , Nicosia , Cyprus.

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http://dx.doi.org/10.1080/15384101.2017.1304745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444356PMC
May 2017

Histone Acetyltransferases in Cancer: Guardians or Hazards?

Crit Rev Oncog 2017 ;22(3-4):195-218

University of Cyprus, Department of Biological Sciences, Nicosia, Cyprus.

Histone acetyltransferases (HATs) catalyzing N-epsilon-lysine or N-alpha-terminal acetylation on histone and non-histone substrates are important epigenetic regulators controlling gene expression and chromatin structure. Deregulation of these enzymes by genetic or epigenetic alterations accompanied by defects in gene transcription have been implicated in oncogenesis. Therefore, these enzymes are considered promising therapeutic targets, offering new horizons for epigenetic cancer therapy. However, recent observations suggest that these enzymes function as both oncogenes and tumor suppressors. In this review, we present the current evidence demonstrating that individual HATs can either prevent cancer cell proliferation or drive malignant transformation depending on the molecular context and cancer type. We therefore advocate that future therapeutic interventions targeted toward these enzymes should carefully consider the fact that HATs commonly have a two-sided role in carcinogenesis.
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http://dx.doi.org/10.1615/CritRevOncog.2017024506DOI Listing
April 2019

Functional characterisation of long intergenic non-coding RNAs through genetic interaction profiling in Saccharomyces cerevisiae.

BMC Biol 2016 12 7;14(1):106. Epub 2016 Dec 7.

Department of Biological Sciences, University of Cyprus, Nicosia, CY-1678, Cyprus.

Background: Transcriptome studies have revealed that many eukaryotic genomes are pervasively transcribed producing numerous long non-coding RNAs (lncRNAs). However, only a few lncRNAs have been ascribed a cellular role thus far, with most regulating the expression of adjacent genes. Even less lncRNAs have been annotated as essential hence implying that the majority may be functionally redundant. Therefore, the function of lncRNAs could be illuminated through systematic analysis of their synthetic genetic interactions (GIs).

Results: Here, we employ synthetic genetic array (SGA) in Saccharomyces cerevisiae to identify GIs between long intergenic non-coding RNAs (lincRNAs) and protein-coding genes. We first validate this approach by demonstrating that the telomerase RNA TLC1 displays a GI network that corresponds to its well-described function in telomere length maintenance. We subsequently performed SGA screens on a set of uncharacterised lincRNAs and uncover their connection to diverse cellular processes. One of these lincRNAs, SUT457, exhibits a GI profile associating it to telomere organisation and we consistently demonstrate that SUT457 is required for telomeric overhang homeostasis through an Exo1-dependent pathway. Furthermore, the GI profile of SUT457 is distinct from that of its neighbouring genes suggesting a function independent to its genomic location. Accordingly, we show that ectopic expression of this lincRNA suppresses telomeric overhang accumulation in sut457Δ cells assigning a trans-acting role for SUT457 in telomere biology.

Conclusions: Overall, our work proposes that systematic application of this genetic approach could determine the functional significance of individual lncRNAs in yeast and other complex organisms.
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http://dx.doi.org/10.1186/s12915-016-0325-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5142380PMC
December 2016

Loss of Nat4 and its associated histone H4 N-terminal acetylation mediates calorie restriction-induced longevity.

EMBO Rep 2016 12 31;17(12):1829-1843. Epub 2016 Oct 31.

Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus

Changes in histone modifications are an attractive model through which environmental signals, such as diet, could be integrated in the cell for regulating its lifespan. However, evidence linking dietary interventions with specific alterations in histone modifications that subsequently affect lifespan remains elusive. We show here that deletion of histone N-alpha-terminal acetyltransferase Nat4 and loss of its associated H4 N-terminal acetylation (N-acH4) extend yeast replicative lifespan. Notably, nat4Δ-induced longevity is epistatic to the effects of calorie restriction (CR). Consistent with this, (i) Nat4 expression is downregulated and the levels of N-acH4 within chromatin are reduced upon CR, (ii) constitutive expression of Nat4 and maintenance of N-acH4 levels reduces the extension of lifespan mediated by CR, and (iii) transcriptome analysis indicates that nat4Δ largely mimics the effects of CR, especially in the induction of stress-response genes. We further show that nicotinamidase Pnc1, which is typically upregulated under CR, is required for nat4Δ-mediated longevity. Collectively, these findings establish histone N-acH4 as a regulator of cellular lifespan that links CR to increased stress resistance and longevity.
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http://dx.doi.org/10.15252/embr.201642540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167350PMC
December 2016

Depletion of histone N-terminal-acetyltransferase Naa40 induces p53-independent apoptosis in colorectal cancer cells via the mitochondrial pathway.

Apoptosis 2016 Mar;21(3):298-311

Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus.

Protein N-terminal acetylation is an abundant post-translational modification in eukaryotes implicated in various fundamental cellular and biochemical processes. This modification is catalysed by evolutionarily conserved N-terminal acetyltransferases (NATs) whose deregulation has been linked to cancer development and thus, are emerging as useful diagnostic and therapeutic targets. Naa40 is a highly selective NAT that acetylates the amino-termini of histones H4 and H2A and acts as a sensor of cell growth in yeast. In the present study, we examine the role of Naa40 in cancer cell survival. We demonstrate that depletion of Naa40 in HCT116 and HT-29 colorectal cancer cells decreases cell survival by enhancing apoptosis, whereas Naa40 reduction in non-cancerous mouse embryonic fibroblasts has no effect on cell viability. Specifically, Naa40 knockdown in colon cancer cells activates the mitochondrial caspase-9-mediated apoptotic cascade. Consistent with this, we show that caspase-9 activation is required for the induced apoptosis because treatment of cells with an irreversible caspase-9 inhibitor impedes apoptosis when Naa40 is depleted. Furthermore, the effect of Naa40-depletion on cell-death is mediated through a p53-independent mechanism since p53-null HCT116 cells still undergo apoptosis upon reduction of the acetyltransferase. Altogether, these findings reveal an anti-apoptotic role for Naa40 and exhibit its potential as a therapeutic target in colorectal cancers.
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http://dx.doi.org/10.1007/s10495-015-1207-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746217PMC
March 2016

N-alpha-terminal acetylation of histone H4 regulates arginine methylation and ribosomal DNA silencing.

PLoS Genet 2013 19;9(9):e1003805. Epub 2013 Sep 19.

Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus.

Post-translational modifications of histones play a key role in DNA-based processes, like transcription, by modulating chromatin structure. N-terminal acetylation is unique among the numerous histone modifications because it is deposited on the N-alpha amino group of the first residue instead of the side-chain of amino acids. The function of this modification and its interplay with other internal histone marks has not been previously addressed. Here, we identified N-terminal acetylation of H4 (N-acH4) as a novel regulator of arginine methylation and chromatin silencing in Saccharomyces cerevisiae. Lack of the H4 N-alpha acetyltransferase (Nat4) activity results specifically in increased deposition of asymmetric dimethylation of histone H4 arginine 3 (H4R3me2a) and in enhanced ribosomal-DNA silencing. Consistent with this, H4 N-terminal acetylation impairs the activity of the Hmt1 methyltransferase towards H4R3 in vitro. Furthermore, combinatorial loss of N-acH4 with internal histone acetylation at lysines 5, 8 and 12 has a synergistic induction of H4R3me2a deposition and rDNA silencing that leads to a severe growth defect. This defect is completely rescued by mutating arginine 3 to lysine (H4R3K), suggesting that abnormal deposition of a single histone modification, H4R3me2a, can impact on cell growth. Notably, the cross-talk between N-acH4 and H4R3me2a, which regulates rDNA silencing, is induced under calorie restriction conditions. Collectively, these findings unveil a molecular and biological function for H4 N-terminal acetylation, identify its interplay with internal histone modifications, and provide general mechanistic implications for N-alpha-terminal acetylation, one of the most common protein modifications in eukaryotes.
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http://dx.doi.org/10.1371/journal.pgen.1003805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778019PMC
March 2014

Beyond the histone tail: acetylation at the nucleosome dyad commands transcription.

Nucleus 2013 Sep-Oct;4(5):343-8. Epub 2013 Aug 9.

Department of Biological Sciences; University of Cyprus; Nicosia, Cyprus.

Post-translational modifications (PTMs) of histones have been implicated in cellular processes such as transcription, replication and DNA repair. These processes normally involve dynamic changes in chromatin structure and DNA accessibility. Most of the PTMs reported so far map on the histone tails and essentially affect chromatin structure indirectly by recruiting effector proteins. A recent study by Schneider and colleagues published in Cell (1) has uncovered the function of H3K122 acetylation found within the histone globular domain and specifically positioned on the DNA-bound surface of the nucleosome. Their findings demonstrate a direct effect of histone PTMs on chromatin dynamics, and propose that modifications located in different parts of the nucleosome employ distinct regulatory mechanisms.
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http://dx.doi.org/10.4161/nucl.26051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3899122PMC
August 2014

Cross-talk among epigenetic modifications: lessons from histone arginine methylation.

Biochem Soc Trans 2013 Jun;41(3):751-9

Department of Biological Sciences, University of Cyprus, 1678 Nicosia, Cyprus.

Epigenetic modifications, including those occurring on DNA and on histone proteins, control gene expression by establishing and maintaining different chromatin states. In recent years, it has become apparent that epigenetic modifications do not function alone, but work together in various combinations, and cross-regulate each other in a manner that diversifies their functional states. Arginine methylation is one of the numerous PTMs (post-translational modifications) occurring on histones, catalysed by a family of PRMTs (protein arginine methyltransferases). This modification is involved in the regulation of the epigenome largely by controlling the recruitment of effector molecules to chromatin. Histone arginine methylation associates with both active and repressed chromatin states depending on the residue involved and the configuration of the deposited methyl groups. The present review focuses on the increasing number of cross-talks between histone arginine methylation and other epigenetic modifications, and describe how these cross-talks influence factor binding to regulate transcription. Furthermore, we present models of general cross-talk mechanisms that emerge from the examples of histone arginine methylation and allude to various techniques that help decipher the interplay among epigenetic modifications.
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http://dx.doi.org/10.1042/BST20130003DOI Listing
June 2013

Distinct transcriptional outputs associated with mono- and dimethylated histone H3 arginine 2.

Nat Struct Mol Biol 2009 Apr 8;16(4):449-51. Epub 2009 Mar 8.

Gurdon Institute and Department of Pathology, Cambridge CB2 1QN, UK.

Dimethylation of histone H3 Arg2 (H3R2me2) maintains transcriptional silencing by inhibiting Set1 mediated trimethylation of H3K4. Here we demonstrate that Arg2 is also monomethylated (H3R2me1) in yeast but that its functional characteristics are distinct from H3R2me2: (i) H3R2me1 does not inhibit histone H3 Lys4 (H3K4) methylation; (ii) it is present throughout the coding region of genes; and (iii) it correlates with active transcription. Collectively, these results indicate that different H3R2 methylation states have defined roles in gene expression.
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http://dx.doi.org/10.1038/nsmb.1569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350867PMC
April 2009

Histone H3 tail clipping regulates gene expression.

Nat Struct Mol Biol 2009 Jan 14;16(1):17-22. Epub 2008 Dec 14.

Gurdon Institute and Department of Pathology, Tennis Court Road, Cambridge CB2 1QN, UK.

Induction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters. Here we identify a histone H3 endopeptidase activity in Saccharomyces cerevisiae that may regulate these events. The endopeptidase cleaves H3 after Ala21, generating a histone that lacks the first 21 residues and shows a preference for H3 tails carrying repressive modifications. In vivo, the H3 N terminus is clipped, specifically within the promoters of genes following the induction of transcription. H3 clipping precedes the process of histone eviction seen when genes become fully active. A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells. These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression.
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http://dx.doi.org/10.1038/nsmb.1534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350865PMC
January 2009

Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation.

Nature 2007 Oct 26;449(7164):928-32. Epub 2007 Sep 26.

Gurdon Institute and Department of Pathology, Tennis Court Road, Cambridge CB2 1QN, UK.

Modifications on histones control important biological processes through their effects on chromatin structure. Methylation at lysine 4 on histone H3 (H3K4) is found at the 5' end of active genes and contributes to transcriptional activation by recruiting chromatin-remodelling enzymes. An adjacent arginine residue (H3R2) is also known to be asymmetrically dimethylated (H3R2me2a) in mammalian cells, but its location within genes and its function in transcription are unknown. Here we show that H3R2 is also methylated in budding yeast (Saccharomyces cerevisiae), and by using an antibody specific for H3R2me2a in a chromatin immunoprecipitation-on-chip analysis we determine the distribution of this modification on the entire yeast genome. We find that H3R2me2a is enriched throughout all heterochromatic loci and inactive euchromatic genes and is present at the 3' end of moderately transcribed genes. In all cases the pattern of H3R2 methylation is mutually exclusive with the trimethyl form of H3K4 (H3K4me3). We show that methylation at H3R2 abrogates the trimethylation of H3K4 by the Set1 methyltransferase. The specific effect on H3K4me3 results from the occlusion of Spp1, a Set1 methyltransferase subunit necessary for trimethylation. Thus, the inability of Spp1 to recognize H3 methylated at R2 prevents Set1 from trimethylating H3K4. These results provide the first mechanistic insight into the function of arginine methylation on chromatin.
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http://dx.doi.org/10.1038/nature06160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350864PMC
October 2007

The polycomb group protein SUZ12 regulates histone H3 lysine 9 methylation and HP1 alpha distribution.

Chromosome Res 2007 10;15(3):299-314. Epub 2007 May 10.

Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA 94143, USA.

Regulation of histone methylation is critical for proper gene expression and chromosome function. Suppressor of Zeste 12 (SUZ12) is a requisite member of the EED/EZH2 histone methyltransferase complexes, and is required for full activity of these complexes in vitro. In mammals and flies, SUZ12/Su(z)12 is necessary for trimethylation of histone H3 on lysine 27 (H3K27me3) on facultative heterochromatin. However, Su(z)12 is unique among Polycomb Group Proteins in that Su(z)12 mutant flies exhibit gross defects in position effect variegation, suggesting a role for Su(z)12 in constitutive heterochromatin formation. We investigated the role of Suz12 in constitutive heterochromatin and discovered that Suz12 is required for histone H3 lysine 9 tri-methylation (H3K9me3) in differentiated but not undifferentiated mouse embryonic stem cells. Knockdown of SUZ12 in human cells caused a reduction in H3K27me3 and H3K9me3, and altered the distribution of HP1 alpha. In contrast, EZH2 knockdown caused loss of H3K27me3 but not H3K9me3, indicating that SUZ12 regulates H3-K9 methylation in an EZH2-independent fashion. This work uncovers a role for SUZ12 in H3-K9 methylation.
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http://dx.doi.org/10.1007/s10577-007-1126-1DOI Listing
August 2007

Composition and histone substrates of polycomb repressive group complexes change during cellular differentiation.

Proc Natl Acad Sci U S A 2005 Feb 31;102(6):1859-64. Epub 2005 Jan 31.

Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.

Changes in the substrate specificities of factors that irreversibly modify the histone components of chromatin are expected to have a profound effect on gene expression through epigenetics. Ezh2 is a histone-lysine methyltransferase with activity dependent on its association with other components of the Polycomb Repressive Complexes 2 and 3 (PRC2/3). Ezh2 levels are increasingly elevated during prostate cancer progression. Other PRC2/3 components also are elevated in cancer cells. Overexpression of Ezh2 in tissue culture promotes formation of a previously undescribed PRC complex, PRC4, that contains the NAD+-dependent histone deacetylase SirT1 and isoform 2 of the PRC component Eed. Eed2 is expressed in cancer and undifferentiated embryonic stem (ES) cells but is undetectable in normal and differentiated ES cells. The distinct PRCs exhibit differential histone substrate specificities. These findings suggest that formation of a transformation-specific PRC complex may have a major role in resetting patterns of gene expression by regulating chromatin structure.
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http://dx.doi.org/10.1073/pnas.0409875102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC548563PMC
February 2005

Genomic approaches that aid in the identification of transcription factor target genes.

Exp Biol Med (Maywood) 2004 Sep;229(8):705-21

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 533706, USA.

It is well-established that deregulation of the transcriptional activity of many different genes has been causatively linked to human diseases. In cancer, altered patterns of gene expression are often the result of the inappropriate expression of a specific transcriptional activator or repressor. Functional studies of cancer-specific transcription factors have relied upon the study of candidate target genes. More recently, gene expression profiling using DNA microarrays that contain tens of thousands of cDNAs corresponding to human mRNAs has allowed for a large-scale identification of genes that respond to increased or decreased levels of a particular transcription factor. However, such experiments do not distinguish direct versus indirect target genes. Coupling chromatin immunoprecipitation to micro-arrays that contain genomic regions (ChIP-chip) has provided investigators with the ability to identify, in a high-throughput manner, promoters directly bound by specific transcription factors. Clearly, knowledge gained from both types of arrays provides complementary information, allowing greater confidence that a transcription factor regulates a particular gene. In this review, we focus on Polycomb group (PcG) complexes as an example of transcriptional regulators that are implicated in various cellular processes but about which very little is known concerning their target gene specificity. We provide examples of how both expression arrays and ChIP-chip microarray-based assays can be used to identify target genes of a particular PcG complex and suggest improvements in the application of array technology for faster and more comprehensive identification of directly regulated target genes.
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http://dx.doi.org/10.1177/153537020422900803DOI Listing
September 2004

Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27.

Genes Dev 2004 Jul;18(13):1592-605

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.

Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27.
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http://dx.doi.org/10.1101/gad.1200204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC443521PMC
July 2004

Identification of the polycomb group protein SU(Z)12 as a potential molecular target for human cancer therapy.

Mol Cancer Ther 2003 Jan;2(1):113-21

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.

We have previously identified SU(Z)12 as an E2F target gene. Because many E2F target genes encode proteins that are critical for the control of cell proliferation, we have further characterized the regulation and expression of SU(Z)12. To understand the molecular mechanisms responsible for expression of SU(Z)12 mRNA, we have analyzed the promoter region. We found that the SU(Z)12 gene is controlled by dual promoters, one of which functions bidirectionally. In addition to the E2F binding site, we have identified two binding sites for T cell factor (TCF)/beta-catenin complexes. Using gel mobility shift assays, we demonstrated that both TCF sites can be bound by TCF4. TCF/beta-catenin complexes have been shown to be a critical regulator of gene expression in tumors of the colon, breast, and liver. Accordingly, we have used chromatin immunoprecipitation assays to confirm that TCF4/beta-catenin complexes are bound to the SU(Z)12 promoter in colon cancer cells but not in HeLa cells. We next adapted the chromatin immunoprecipitation assay for use with primary colon tumor samples, and, using matched pairs of normal and tumor tissue obtained from several different colon cancer patients, we demonstrate that levels of beta-catenin bound to the SU(Z)12 promoter are increased in colon tumors. Finally, we show that the SU(Z)12 mRNA is up-regulated in a number of different human tumors, including tumors of the colon, breast, and liver. Recent studies have found that SU(Z)12 is a component of the Drosophila ESC-E(Z) and the human EED-EZH2 Polycomb chromatin remodeling complexes. Therefore, we suggest that SU(Z)12, which may modulate the tumor phenotype by changing gene expression profiles, may be a logical target for the design of a new antitumor agent
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January 2003