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TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS.
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MedLine Citation:
PMID:  23353889     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with O-GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3-OGT co-localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3-OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step-wise model, involving TET-OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation.
Authors:
Rachel Deplus; Benjamin Delatte; Marie K Schwinn; Matthieu Defrance; Jacqui Méndez; Nancy Murphy; Mark A Dawson; Michael Volkmar; Pascale Putmans; Emilie Calonne; Alan H Shih; Ross L Levine; Olivier Bernard; Thomas Mercher; Eric Solary; Marjeta Urh; Danette L Daniels; François Fuks
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2013-01-25
Journal Detail:
Title:  The EMBO journal     Volume:  32     ISSN:  1460-2075     ISO Abbreviation:  EMBO J.     Publication Date:  2013 Mar 
Date Detail:
Created Date:  2013-03-06     Completed Date:  2013-04-23     Revised Date:  2014-03-14    
Medline Journal Info:
Nlm Unique ID:  8208664     Medline TA:  EMBO J     Country:  England    
Other Details:
Languages:  eng     Pagination:  645-55     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
5-Methylcytosine / metabolism
Amino Acid Sequence
Animals
Blotting, Western
Cell Proliferation
Cells, Cultured
Chromatin Immunoprecipitation
CpG Islands
Cytosine / analogs & derivatives,  metabolism
DNA Methylation*
DNA-Binding Proteins / metabolism*
Dioxygenases / metabolism*
Epigenesis, Genetic
Gene Expression Regulation*
Glycosylation
Histone-Lysine N-Methyltransferase / metabolism*
Histones / metabolism
Host Cell Factor C1 / metabolism
Humans
Immunoprecipitation
Mice
Mice, Knockout
Molecular Sequence Data
N-Acetylglucosaminyltransferases / metabolism*
Promoter Regions, Genetic / genetics
Proto-Oncogene Proteins / metabolism*
Transcription, Genetic*
Grant Support
ID/Acronym/Agency:
100140//Wellcome Trust
Chemical
Reg. No./Substance:
0/DNA-Binding Proteins; 0/HCFC1 protein, human; 0/Histones; 0/Host Cell Factor C1; 0/Proto-Oncogene Proteins; 0/TET2 protein, human; 1123-95-1/5-hydroxymethylcytosine; 6R795CQT4H/5-Methylcytosine; 8J337D1HZY/Cytosine; EC 1.-/TET3 protein, human; EC 1.13.11.-/Dioxygenases; EC 2.1.1.43/Histone-Lysine N-Methyltransferase; EC 2.1.1.43/Setd1A protein, human; EC 2.4.1.-/N-Acetylglucosaminyltransferases; EC 2.4.1.-/O-GlcNAc transferase
Comments/Corrections
Comment In:
Mol Cell. 2013 Feb 21;49(4):618-9   [PMID:  23438858 ]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): EMBO J
Journal ID (iso-abbrev): EMBO J
ISSN: 0261-4189
ISSN: 1460-2075
Publisher: Nature Publishing Group
Article Information
Copyright © 2013, European Molecular Biology Organization
open-access:
Received Day: 05 Month: 12 Year: 2012
Accepted Day: 21 Month: 12 Year: 2012
Print publication date: Day: 06 Month: 03 Year: 2013
Electronic publication date: Day: 25 Month: 01 Year: 2013
pmc-release publication date: Day: 25 Month: 01 Year: 2013
Volume: 32 Issue: 5
First Page: 645 Last Page: 655
PubMed Id: 23353889
ID: 3590984
Publisher Item Identifier: emboj2012357
DOI: 10.1038/emboj.2012.357

TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS Alternate Title:Regulation of OGT and SET1/COMPASS by TET2/3
Rachel Deplus1*
Benjamin Delatte1*
Marie K Schwinn2*
Matthieu Defrance1
Jacqui Méndez2
Nancy Murphy2
Mark A Dawson34
Michael Volkmar1
Pascale Putmans1
Emilie Calonne1
Alan H Shih5
Ross L Levine5
Olivier Bernard6
Thomas Mercher6
Eric Solary7
Marjeta Urh2
Danette L Danielsa2
François Fuksb1
1Laboratory of Cancer Epigenetics, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium
2Promega Corporation, Madison, WI, USA
3Department of Haematology, Cambridge Institute for Medical Research, Cambridge, UK
4Department of Pathology, Gurdon Institute, University of Cambridge, Cambridge, UK
5Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, USA
6INSERM UMR 985, Institut Gustave Roussy, Université Paris XI, Villejuif, France
7INSERM UMR 1009, Institut Gustave Roussy, Université Paris XI, Villejuif, France
aPromega Corporation, 2800 Woods Hollow Road, Madison, WI 53703, USA. Tel.:+1 608 274 4330; Fax:+1 608 277 2601; E-mail: danette.daniels@promega.com
bLaboratory of Cancer Epigenetics, Faculty of Medicine, Université Libre de Bruxelles, 808 Route de Lennik, Brussels 1070, Belgium. Tel.:+32 2 555 62 45; Fax:+32 2 555 62 57; E-mail: ffuks@ulb.ac.be
*These authors contributed equally to this work

Introduction

Epigenetic marking of the genome and regulation of chromatin are central to establishing tissue-specific gene expression programs, and hence to several biological processes (Bonasio et al, 2010). Until recently, the only known epigenetic mark on DNA was 5-methylcytosine (mC), established and propagated by DNA methyltransferases, and generally associated with gene repression (Suzuki and Bird, 2008; Cedar and Bergman, 2009; Ndlovu et al, 2011). The discovery of cystosine 5-hydroxymethylation (hmC), and of the Ten-Eleven Translocation family of enzymes (TET1, TET2, and TET3) that catalyse the conversion of mC to hmC has sparked great interest in uncovering the roles played by this mark and these proteins (Kriaucionis and Heintz, 2009; Tahiliani et al, 2009; Ito et al, 2010). Genome-wide profiling of the distribution of Tet1 and hmC in mouse ES cells has shown that both are important in regulation of pluripotency and cellular differentiation (Ficz et al, 2011; Pastor et al, 2011; Williams et al, 2011; Wu et al, 2011). Other data have linked TET1 to epigenetic repression complexes, notably SIN3A and PRC2 (Williams et al, 2011; Wu et al, 2011). Regarding TET2 and TET3, mouse Tet2 has been implicated in haematopoiesis and human TET2 mutations have been found in various leukaemias (Langemeijer et al, 2009), while studies in mouse germ cells have shown the importance of Tet3 in epigenetic reprogramming (Gu et al, 2011). However the mode of action, particularly of the TET2 and TET3 family members, is still poorly understood.


Results
TET2 and TET3 associate with the O-GlcNAc transferase OGT

To aid in this understanding and further explore binding partners of these proteins, we performed an unbiased proteomic approach using HaloTag technology as previously described (Los et al, 2008; Daniels et al, 2012). To this end, we expressed the full-length TET1, TET2, and TET3 proteins as HaloTag (HT) fusions in HEK293T cells, covalently captured them on an HT affinity resin, eluted the interacting proteins, and purified these for mass spectrometry (LC/MS/MS) and spectral counting analysis (Materials and methods; Supplementary Figure 1). Figure 1A shows the silver stain gel for each complex isolation and the enrichment of numerous bands for each as compared to the HT alone control. Mass spectrometry of each revealed notably several transcriptional and epigenetic interacting proteins identified in biological replicates and enriched over the HT alone control, including the known TET1–SIN3A interaction and the absence of this with TET2 (Williams et al, 2011; Figure 1B). All TET proteins showed interaction with O-GlcNAc transferase, OGT, (Figure 1B), though TET2 and TET3 showed demonstrably higher enrichment. Given this, we focused all following experiments with the TET2 and TET3 family members. Two approaches were used to confirm the interaction between TET2 or TET3 and OGT: (i) western blotting with anti-OGT antibody, applied to samples of eluate from the above-mentioned HT-pulldown experiments (Figure 1C), and (ii) co-immunoprecipitations (Co-IPs) showing capture of both TET2 and TET3 from extracts of untransfected cells upon immunoprecipitation with an anti-OGT antibody, confirming the interaction of endogenous TET2 and TET3 with OGT (Figure 1D). Input loading controls of OGT are shown for each (Figures 1C and D). A variant of TET2 lacking the catalytic domain (TET2ΔCD) did not show interaction with OGT (Figures 1A and C; Supplementary Figure 2), indicating that the catalytic domain is important for the OGT interaction and demonstrating the specificity of the HT pulldown and mass spectrometry analysis. Supporting this, FLAG Co-IP experiments performed with FLAG-TET2 and -TET3 CD alone showed interaction with transfected or endogenous OGT (Supplementary Figure 3). Furthermore, to evaluate whether the interaction is direct, an in vitro protein isolation was performed using E. coli expressed and purified full-length OGT incubated with E. coli expressed HT-TET2CD or HT alone control covalently bound to resin. As shown in Supplementary Figure 3, OGT was specifically enriched on resin containing the TET2CD as compared to the control. Together, these data demonstrate that TET2 and TET3 interact directly with the OGT glycosyltransferase.

TET2 and TET3 promote OGT-mediated GlcNAcylation

OGT places O-GlcNAc modifications on numerous proteins, including transcription factors, epigenetic regulators, and also histones (Hart et al, 2007). GlcNAcylation can activate or inhibit protein activity and serve as a mark to recruit other proteins (Slawson and Hart, 2011). As several proteins that interact with OGT are also regulated by GlcNAcylation (Hart et al, 2007), we analysed TET2 and TET3 by mass spectrometry for the presence of O-GlcNAc modifications. Despite high levels of spectral counts (∼400) for both proteins, GlcNAcylation could not be detected for either protein (data not shown). Based upon these results, we predict if TET proteins are GlcNAcylated, they would be at low levels. As TET and OGT are both enzymes, we next sought to determine if their respective activities are altered by each other. To evaluate if the enzymatic activity of OGT might influence hydroxymethylation by the TET enzymes, we measured the overall abundance of the hmC mark in dot blot assay using a specific hmC antibody (Supplementary Figure 4) in the absence or presence of either Alloxan, an OGT inhibitor, or PUGNAc, which prevents removal of the O-GlcNAc modification by inhibiting the enzyme O-linked N-acetylglucosaminidase (OGA) (Capotosti et al, 2011; Fujiki et al, 2009). As shown in Figure 1E, no changes in overall hmC level were observed with these respective treatments (upper panel), while a western blot of lysates revealed the expected impact on global levels of O-GlcNAc for each (lower panel). Similarly, overexpression of HT-OGT did not increase levels of hmC, while overexpression of a TET2CD alone control did (Supplementary Figure 4). Hence, cytosine hydroxylation does not appear to require the GlcNAcylating activity of OGT. We then investigated if GlcNAcylation might depend on TET2 and/or TET3. To answer this question, we measured overall OGT activity in lysates of cells where RNAi was used to knock down TET2 or TET3 (cf. Supplementary Figure 5 for RNAi efficiency and controls). As shown in Figure 1F (left panel), we observed lower GlcNAcylation activity in TET2 or TET3 RNAi than in the control cells. Following with this, in a TET2 and TET3 double RNAi kd (referred to as TET2/3 kd) a discernable decrease in O-GlcNAc modified proteins is seen as revealed by western blot analysis using an anti-O-GlcNAc antibody (Figure 1F, right panel; Supplementary Figure 5). To demonstrate that decreased OGT activity was not due to change in levels of OGT, analysis of lysates from mock RNAi and TET2/3 kd cells revealed that protein levels of OGT were unchanged after depletion of TET2 and TET3 (Supplementary Figures 5 and 6). These combined data suggest that TET2/3 positively impacts the activity of OGT.

TET2, TET3, and OGT show genome-wide co-localization, notably at CpG islands and at transcription start sites

We next undertook to map genome-wide binding of TET2, TET3, and OGT, looking for their possible co-occurrence at genomic targets. We performed crosslinking and chromatin isolation from HEK293T cells, expressing HT fusion proteins (Los et al, 2008; Hartzell et al, 2009) coupled with high-throughput sequencing (Supplementary Figure 7). Our results show that TET2, TET3, and OGT all localize primarily to CpG islands (CGI) and promoter regions (Figure 2A; Supplementary Figure 8), similar to previously observed localization for Tet1 in mouse ES cells (Ficz et al, 2011; Williams et al, 2011; Wu et al, 2011). Co-occurrence analysis of OGT binding sites revealed statistically significant overlaps with TET2 (42%) (Figure 2A) and TET3 (55%) (Supplementary Figure 8; Supplementary Table 2). The TET2–OGT and TET3–OGT targets are primarily contained within promoter regions, tightly clustered around transcription start sites (TSSs), with intermediate to high CpG content (ICP and HCP) (Figure 2A; Supplementary Figures 8 and 9; Supplementary Table 2).

TET2, TET3, and OGT genomic targets are enriched for GlcNAcylation, but not for 5hmC or 5mC

We then examined the DNA methylation, DNA hydroxymethylation, and protein GlcNAcylation status of selected TET2/3–OGT target sequences. As shown in Figure 2B (upper and middle panels), methylated DNA and hydroxymethylated DNA immunoprecipitations (MeDIP and hMeDIP) followed by quantitative PCR (qPCR) analysis revealed essentially no enrichment of 5mC or 5hmC, respectively, at these targets in either immunoprecipitate. Significant enrichment of O-GlcNAc was found for TET2/3–OGT targets using chromatin immunoprecipitation (ChIP)-qPCR against O-GlcNAc (Figure 2B, lower panel), indicating OGT activity at these chromatin sites. Together, these data show co-occurrence of TET2, TET3, and OGT, which in a subset analysed, lack DNA methylation and hydroxymethylation, but display protein GlcNAcylation.

TET2, TET3, and OGT co-localize with and influence H3K4 trimethylation at active promoters in human cells

The clustering of TET2/3–OGT primarily at TSSs and HCP promoters (cf. Figure 2A) led us next to examine the genomic overlap at promoters with H3K4 trimethylation (H3K4me3), an expected mark at these sites which is also correlated with transcriptional activity. As depicted in Figures 2C and D, ChIP-Seq with an antibody against H3K4me3 confirmed an expected high overlap (98%) of TET2/3–OGT targets with the H3K4me3 mark. We then wondered whether TET proteins might be involved in regulating H3K4me3, and therefore repeated ChIP-Seq experiment in cells containing a TET2 RNAi kd. We observed a significant reduction of H3K4me3 at over 78% of the TET2/3–OGT genomic targets (Figure 2E). A similar impact on global reduction of H3K4me3 is observed in western blot analysis of TET2/3 knockdown cellular lysates (Figure 2F). As reduction in H3K4me3 could occur through a variety of mechanisms, we then sought to determine if there was a connection to OGT activity. Supporting this hypothesis and similar to the above data with TET2/3 kd (Figure 2F), cells treated with the OGT inhibitor, Alloxan, show also a decrease in global H3K4me3 as compared to controls (Figure 2G). Thus, TET2/3–OGT targets are enriched for H3K4me3, which is regulated by both the TET proteins and OGT activity.

To correlate these data to gene activity, we found by ChIP-Seq a marked co-occupancy of TET2/3–OGT–H3K4me3 targets with RNA polymerase II (RNA Pol II) and by RNA Sequencing (RNA-Seq) analyses a significant and positive correlation with high expression levels (Supplementary Figure 10). To determine if there are effects on transcriptional levels after loss of TET and/or OGT, we then analysed by RT–qPCR a subset of TET2/3–OGT targets specifically decreased in H3K4me3 after TET kd, as identified in Figure 2E. As shown in Figure 2H, moderate, but reproducible and specific decreases in expression of targets after TET2/3 kd match patterns to those observed in OGT RNAi kd. Together, these data suggest that TET2/3–OGT–H3K4me3 targets are highly transcribed regions and for the subset studied, show concomitant transcriptional activation by both TET proteins and OGT.

TET2 and TET3 associate with HCF1, an OGT-modified target and component of the H3K4 methyltransferase complex SET1/COMPASS

We next wished to extend our observed TET2/3–OGT interaction and identify potential specific downstream protein targets that may connect to H3K4me3 regulation. With this in mind, we noticed in our initial proteomic analysis for TET2 and TET3 that besides OGT (cf. Figure 1B), Host Cell Factor 1 (HCF1) was also identified and similarly to OGT, was decreased in the TET2ΔCD isolation (Figure 3A). HCF1 was of interest as not only is it a known GlcNAcylation target of OGT (Capotosti et al, 2011), but also a component of the histone H3K4 methyltransferase complex, SET1/COMPASS (Wysocka et al, 2003; Lee et al, 2010). ChIP-Seq experiments for endogenous HCF1 showed overlap of HCF1 target genes with those of Halo-TET2, TET3, OGT, and H3K4me3 (Supplementary Figure 11A). We then desired to further the mechanistic understanding of the TET2- or TET3-OGT–HCF1 interactions with relationship to H3K4me3, and therefore examined the interactions of OGT using a similar HT proteomics approach including mass spectrometry analysis. Interacting partners of HT-OGT included the previously identified HCF1 protein (Capotosti et al, 2011), the TET2 and TET3 proteins (confirming the TET–OGT interaction), and interestingly, all components of SET1/COMPASS (Figure 3B). To determine if the interactions were dependent on OGT activity, we repeated the above experiments in the presence of the OGT inhibitor Alloxan. Such treatment resulted not only in the expected decrease in OGT–HCF1 interaction (Capotosti et al, 2011), but also in significant loss of capture of the SET1/COMPASS components, including the H3K4 methyltransferase SETD1A (Figure 3C). Normalized spectral abundance factors (NSAFs) showed the same trend (Supplementary Figure 11B), therefore validating the direct comparison of respective loss of spectral counts for each protein within the samples. Mass spectrometry analysis of HCF1 peptides from HT-OGT protein isolations with or without Alloxan treatment showed high levels of GlcNAcylation in untreated cells and reduction after Alloxan treatment (Supplementary Figure 12A), matching previously published results (Capotosti et al, 2011).

TET2/3 promotes GlcNAcylation of HCF1, and both TET and OGT activity favour the integrity of SET1/COMPASS and SETD1A binding to chromatin

As these data suggested that GlcNAcylation of HCF1 might be important for its interaction with SET1/COMPASS, we then performed an HT-SETD1A pulldown to study the SET1/COMPASS complex. Mass spectrometry analysis of the interacting proteins revealed all the known members of the complex, as well as OGT (Figure 3D). Additional analysis of the HCF1 peptides from this experiment revealed extensive O-GlcNAc modifications, indicating that HCF1 is heavily modified when associated with SETD1A (Figure 3E, left panel ‘RNAi Ctrl'). We next examined whether the TET proteins might influence SET1/COMPASS complex formation and/or specific GlcNAcylation of HCF1. In order to do this, we performed HT-SETD1A isolations in cells harbouring the TET2/3 double kd, which were shown to decrease global H3K4me3 (cf. Figure 2F). As shown in Figure 3D (and Supplementary Figure 11B as NSAF values with the same trends), we observed a significant loss of SET1/COMPASS components and OGT in the TET2/3 RNAi kd cells, coinciding with a significant decrease in GlcNAcylation of HCF1 as determined by mass spectrometry (Figure 3E; Supplementary Methods). To show further the direct effect of TET proteins specifically on SET1/COMPASS complex formation, proteomics experiments were additionally performed with HT-WDR82, the only other specific component of SET1/COMPASS besides SETD1A. These data revealed the same disruption of capture of SET1/COMPASS components in cells harbouring a TET2/3 knockdown as compared to mock (Supplementary Figure 12B). These results indicate that TET2/3 proteins, and OGT GlcNAcylation of HCF1 are important for the integrity of the SET1/COMPASS complex, suggesting a means by which H3K4me3 may be regulated. To further this understanding, bioluminescence resonance energy transfer (BRET) experiments were carried out in living cells to monitor the interaction of SETD1A with chromatin. In short, SETD1A was designed as an energy donor, expressed as a fusion to NanoLuc luciferase, and Histone H3.3 as an energy acceptor, expressed and fluorescently labelled as a fusion to HT (Figure 3F). As shown in Figure 3F, treatment of cells with an OGT inhibitor, Alloxan, (left panel) or analysis in TET2/3 RNAi knockdown cells (right panel) result in specific reduction of BRET signal, indicating decrease in interaction between SETD1A and Histone H3.3 after these respective treatments. Together, these data support that TET2/3 proteins and OGT activity are important not only for the integrity of the H3K4 methyltransferase SET1/COMPASS complex, but also binding of the methyltransferase component, SETD1A, to chromatin.

Tet2 knockout mouse tissue shows that Tet2 is needed for global GlcNAcylation and H3K4me3 at target promoters

We then endeavoured to extend our findings to mouse bone marrow tissue, a model where Tet2 has been implicated in haematopoiesis and Tet2 mutations have been identified and linked to myeloid neoplasms (Langemeijer et al, 2009; Moran-Crusio et al, 2011; Quivoron et al, 2011). For this, we started by performing ChIP-Seq for Tet2, H3K4me3, and O-GlcNAc. First, ChIP-Seq analysis with an anti-Tet2 antibody in bone marrow tissue revealed genome-wide binding profile of endogenous Tet2 mostly at CpG-rich regions and TSSs (Supplementary Figure 13). This binding profile is similar to the genomic distribution observed for HT-TET2 in HEK293T cell lines (see Supplementary Figures 9B and 14). Next, we examined the genome-wide overlap between O-GlcNAc and H3K4me3 with Tet2. As shown in Figure 4A and Supplementary Table 3, a significant degree of overlap was observed between Tet2 and both O-GlcNac and H3K4me3 modifications, again predominately at CpG-rich promoters. Further, ChIP-Seq for RNA Pol II as well as RNA-Seq in mouse bone marrow indicated that the Tet2/O-GlcNAc/H3K4me3 co-bound targets were enriched at many active genes (Figure 4B). To determine the specific dependency of GlcNAcylation and H3K4me3 upon the Tet proteins in mouse bone marrow, experiments were then performed in a Tet2 knockout mouse (Moran-Crusio et al, 2011). Supporting the data that loss of human TET2/3 results in decreased OGT activity and global GlcNAcylation (Figure 1F), we observe in the Tet2 knockout mouse, a correlating decrease in global levels of O-GlcNAc (Figure 4C) as determined by a western blot using an anti-O-GlcNAc antibody. ChIP-Seq for H3K4me3 in the Tet2 knockout mouse bone marrow tissue, reveals a significant reduction in H3K4me3 (Figure 4D) (mirroring also the decrease observed in TET2 knockdown HEK293T cells; Figure 2E). Strikingly, it is noteworthy that among targets which showed decrease in H3K4me3 in a Tet2 knockout mouse, we found several key regulators of haematopoiesis, including the Cebpa, Tal1, Runx1, and several Mll genes (Figure 4E). Taken together, in this biologically relevant Tet system, we find genomic co-localization of Tet2, O-GlcNAc, and H3K4me3 on transcriptional active genes and most importantly, show an essential implication of Tet2 for both OGT activity and H3K4me3, notably on genomic targets regulating haematopoiesis.


Discussion

Here, we present data showing an unrecognized link between DNA modifying enzymes, TETs, a master cellular sensor protein, OGT, and a histone modifying complex, SET1/COMPASS. These new findings combined with our initial goal to further understanding of TET2 and TET3 proteins, have allowed us to propose a cohesive and hierarchical model of interactions and to determine the cascade of their respective activities leading to H3K4me3 and transcriptional activation (Figure 5). The first sequence of events is the formation of TET2 or TET3–OGT interaction, which promotes GlcNAcylation by OGT on numerous proteins, including HCF1. HCF1 has been shown to be required for recruitment of SET1/COMPASS and H3K4me3 (Narayanan et al, 2007; Tyagi and Herr, 2009; Liu et al, 2010) and our data further elucidate its role as we show a GlcNAcylated HCF1 is important for the integrity of the SET1/COMPASS complex. Lastly, both TET proteins and OGT activity favour binding of SETD1A to chromatin, an event necessary for histone H3K4me3 and subsequent transcriptional activation.

As TET proteins have the potential to control DNA methylation fidelity by removing spurious or undesirable DNA methylation (Liu et al, 2010; Williams et al, 2011), the above model is likely not the only means these proteins utilize to influence H3K4me3. We hypothesize that TET proteins, in general, function as active guardians in promotion of transcriptional activation, which is supported by our data and previously published data of TET1 genomic localization primarily at regions high in H3K4me3 (Williams et al, 2011; Wu et al, 2011). In addition to their ability to reduce DNA methylation, the TET proteins might serve as scaffolding proteins beyond interaction with OGT as evidenced by the finding of several other interactors, and then recruit proteins required to establish a high-H3K4me3 chromatin environment. The TET–OGT interaction for example may be more broadly utilized to direct several epigenetic complexes to target sites or promote active transcription by GlcNAcylating not only HCF1, but also local histones or transcription factors at specific genomic localizations. Furthermore, similar to the dual function of TET1 in transcriptional regulation (Williams et al, 2011; Wu et al, 2011) our data suggest that TET3 might also provide a platform for repressive epigenetic complexes as it was shown also to interact with SIN3A. The TET proteins are thus more versatile and multifaceted than initially anticipated, performing important non-catalytic functions in addition to their known role of hydroxymethylation.

In addition to suggesting a broader role for the TET proteins, our study revealing a direct physical link between OGT and TET2/3 proteins provides new insight into the regulation and function of OGT in the cell. Although its connection with epigenetic regulatory events is by no means clear, new data are quickly emerging. In Drosophila, for example, OGT is reported to play a role in Polycomb function (Gambetta et al, 2009), and in human cells, O-GlcNAc modification on histone H2B is required for subsequent K120 monoubiquitination (Fujiki et al, 2011). The interaction evidenced here between TET2/3 and OGT may shed light on the link between the OGT glycosyltransferase and epigenetics and may add to the role of OGT observed in the regulation of MLL5 (Fujiki et al, 2009). Our study reveals an unforeseen genome-wide crosstalk between OGT and TET2/3 and a novel mode of regulation of OGT-directed O-GlcNAcylation. The precise mechanism of how TET impacts OGT activity is unclear and several possibilities exist (e.g., by other proteins associated with TET–OGT or by post-translational modifications). Our data allow however for a model wherein TET2 and TET3 might function as fine-tuning GlcNAcylation effectors, not only on epigenetic complexes such as SET1/COMPASS, but also other transcriptional regulation targets whose function is modulated by this modification.

In conclusion, our results reveal a relationship between TET2/3 and OGT with their direct interaction and influence on the H3K4 SET1/COMPASS complex shedding new light on the modes of action of TET proteins, impact upon chromatin biology, regulation of OGT activity, and the epigenetic mechanisms in which they participate.


Materials and methods
HT mammalian pulldown assay

HEK293T cells (12 × 106 cells) were plated in a 150-mm dish and grown to 70–80% confluency (∼18 h). The cells were then transfected with 30 μg of plasmid DNA using FuGENE HD Transfection Reagent (Promega) for 24 h, according to manufacturer's protocol. Cells expressing HT-fusion proteins or HT-Ctrl were incubated in mammalian lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, and 0.1% sodium deoxycholate) supplemented with Protease Inhibitor cocktail (Promega) and RQ1 RNase-Free DNase (Promega) for 10 min on ice. Lysate was then homogenized with a syringe and centrifuged at 14 000 g for 5 min to pellet cellular debris. Clarified lysate was incubated with HaloLink Resin (Promega) that had been pre-equilibrated in resin wash buffer (TBS and 0.05% IGEPAL CA-640; Sigma) for 15 min at 22°C with rotation. Resin was then washed five times with wash buffer after the initial binding of complexes to the resin and this, to remove non-specific interactions, and protein interactors were eluted with SDS elution buffer (50 mM Tris–HCl, pH 7.5, and 1% SDS). This number of washes reveals minimal non-specific interactions retained in control samples as determined by silver staining (Figure 1A). Such approach show quantitatively significantly lower levels of background as compared to standard Flag purifications using this affinity purification and washing procedure (Daniels et al, 2012). Affinity purified complexes were then analysed by nano LC/MS/MS (MSBioworks) and western blotting.

Chromatin immunoprecipitation

HEK293T or bone marrow cells were crosslinked 10 min at room temperature with 1% formaldehyde, then the reaction was quenched by addition of 0.125 M glycine and washed twice with 1 × cold PBS. ChIP experiments were performed according to the Transcription ChIP kit (Diagenode) protocol. Sonication was performed with bioruptor (Diagenode) in cold water+ice with the following settings: 2 cycles of 10 min, sonication strength set on high, with intervals of 30 s ON/OFF. 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O-linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C. After extensive washing steps, ChIPed DNA was eluted and de-crosslinked overnight at 65°C, then purified using QIAquick PCR purification kit (Qiagen). 3 μl of enriched fragmented DNA or 3 μl of input, supplemented with 0.5 μM of primers and SYBR Green master mix was subjected to 40 cycles of PCR using LightCycler 480 II (Roche). Percentage of input recovered after immunoprecipitation was calculated using the ΔCt formula: (2−(Ct IP—Ct Input)) × 100. Primer sequences are described in Supplementary Table 1.



Notes

Promega Corporation is the commercial owner by assignment of patents of the HaloTag technology and its applications.

RD, MD and MV were supported by the Belgian FNRS. FF is an FNRS ‘Senior Research Associate'. BD is an ‘FNRS Aspirant'. FF's laboratory was funded by grants from the FNRS and Télévie, the ‘Interuniversity Attraction Poles' (IAP Phase VII no P7/03) and by the ‘Action de Recherche Concertée' (AUWB-2010-2015 ULB-No 7). MKS, JM, NM, MU, and DLD are supported by Promega Corporation. We wish to thank Kazusa DNA Research Institute for providing vectors, MSBioworks for all mass spectrometry analysis, Dr Martin Rosenberg for thoughtful advice and continued support, and Prof. David Jeruzalmi for critical reading of the manuscript.

Author contributions: RD, BD, and MKS designed experiments, performed research, and interpreted data. Protein partner purifications were done and analysed by MKS, JM, NM, MU, and DLD. Confirmations of interaction were done by RD and MKS. BD and MV performed the Dot Blot experiments. Cloning, RNAi experiments, and expression studies were done by RD, PP and EC. RD performed OGT enzymatic assays. ChIP was performed by BD and MAD. MeDIP and hMeDIP were performed by BD. ChIP-Seq experiments were performed by BD and bioinformatic analyses were conducted by MD. AHS, RLL, ES, TM, and OB provided mouse bone marrow tissues. BRET experiments were done by MKS. DLD and FF designed experiments, interpreted data, and directed the study. RD, BD, MD, and MKS prepared the figures. DLD and FF wrote the manuscript.


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Keywords: chromatin, epigenetics, TET proteins.

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