CXXC Domain Research Articles

A novel isoform of TET1 that lacks a CXXC domain is overexpressed in cancer.

TET1 oxidizes methylated cytosine into 5-hydroxymethylcytosine (5hmC), resulting in regulation of DNA methylation and gene expression. Full length TET1 (TET1FL) has a CXXC domain that binds to unmethylated CpG islands (CGIs). This CXXC domain allows TET1 to protect CGIs from aberrant methylation, but it also limits its ability to regulate genes outside of CGIs. Here, we report a novel isoform of TET1 (TET1ALT) that has a unique transcription start site from an alternate promoter in intron 2, yielding a protein with a unique translation start site. Importantly, TET1ALT lacks the CXXC domain but retains the catalytic domain. TET1ALT is repressed in embryonic stem cells (ESCs) but becomes activated in embryonic and adult tissues while TET1FL is expressed in ESCs, but repressed in adult tissues. Overexpression of TET1ALT shows production of 5hmC with distinct (and weaker) effects on DNA methylation or gene expression when compared to TET1FL. TET1ALT is aberrantly activated in multiple cancer types including breast, uterine and glioblastoma, and TET1 activation is associated with a worse overall survival in breast, uterine and ovarian cancers. Our data suggest that the predominantly activated isoform of TET1 in cancer cells does not protect from CGI methylation and likely mediates dynamic site-specific demethylation outside of CGIs.

Not All H3K4 Methylations Are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification

The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription factors/co-factors and epigenetic states at cis-regulatory elements. Here, we show that in addition to implementing H3K4me3 at promoters of bivalent genes, Mll2 (KMT2B)/COMPASS can also implement H3K4me3 at a subset of non-TSS regulatory elements, a subset of which shares epigenetic signatures of active enhancers. Our mechanistic studies reveal that association of Mll2's CXXC domain with CpG-rich regions plays aninstrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of loci, generation of catalytically mutant MLL2/COMPASS demonstrated that H3K4me3 implemented by this enzyme was essential for expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development. Our findings suggest that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent.

Isoform Switch of TET1 Regulates DNA Demethylation and Mouse Development

The methylcytosine oxidase TET proteins play important roles in DNA demethylation and development. However, it remains elusive how exactly they target substrates and execute oxidation. Interestingly, we found that, in mice, the full-length TET1 isoform (TET1e) is restricted to early embryos, embryonic stem cells (ESCs), and primordial germ cells (PGCs). By contrast, a short isoform (TET1s) is preferentially expressed in somatic cells, which lacks the N terminus including the CXXC domain, a DNA-binding module that often recognizes CpG islands (CGIs) where TET1 predominantly occupies. Unexpectedly, TET1s can still bind CGIs despite the fact that its global chromatin binding is significantly reduced. Interestingly, global chromatin binding, but not targeted binding at CGIs, is correlated with TET1-mediated demethylation. Finally, mice with exclusive expression of Tet1s failed to erase imprints in PGCs and displayed developmental defects in progeny. These data show that isoform switch of TET1 regulates epigenetic memory erasure and mouse development.

Estradiol-Estrogen Receptor α Mediates the Expression of the CXXC5 Gene through the Estrogen Response Element-Dependent Signaling Pathway.

17β-estradiol (E2), the primary circulating estrogen hormone, mediates physiological and pathophysiological functions of breast tissue mainly through estrogen receptor α (ERα). Upon binding to E2, ERα modulates the expression of target genes involved in the regulation of cellular proliferation primarily through interactions with specific DNA sequences, estrogen response elements (EREs). Our previous microarray results suggested that E2-ERα modulates CXXC5 expression. Because of the presence of a zinc-finger CXXC domain (ZF-CXXC), CXXC5 is considered to be a member of the ZF-CXXC family, which binds to non-methylated CpG dinucleotides. Although studies are limited, CXXC5 appears to participate as a transcription factor, co-regulator and/or epigenetic factor in the regulation of cellular events induced by various signaling pathways. However, how signaling pathways mediate the expression of CXXC5 is yet unclear. Due to the importance of E2-ERα signaling in breast tissue, changes in the CXXC5 transcription/synthesis could participate in E2-mediated cellular events as well. To address these issues, we initially examined the mechanism whereby E2-ERα regulates CXXC5 expression. We show here that CXXC5 is an E2-ERα responsive gene regulated by the interaction of E2-ERα with an ERE present at a region upstream of the initial translation codon of the gene.

Allosteric control of mammalian DNA methyltransferases - a new regulatory paradigm.

In mammals, DNA methylation is introduced by the DNMT1, DNMT3A and DNMT3B methyltransferases, which are all large multi-domain proteins containing a catalytic C-terminal domain and an N-terminal part with regulatory functions. Recently, two novel regulatory principles of DNMTs were uncovered. It was shown that their catalytic activity is under allosteric control of N-terminal domains with autoinhibitory function, the RFT and CXXC domains in DNMT1 and the ADD domain in DNMT3. Moreover, targeting and activity of DNMTs were found to be regulated in a concerted manner by interactors and posttranslational modifications (PTMs). In this review, we describe the structures and domain composition of the DNMT1 and DNMT3 enzymes, their DNA binding, catalytic mechanism, multimerization and the processes controlling their stability in cells with a focus on their regulation and chromatin targeting by PTMs, interactors and chromatin modifications. We propose that the allosteric regulation of DNMTs by autoinhibitory domains acts as a general switch for the modulation of the function of DNMTs, providing numerous possibilities for interacting proteins, nucleic acids or PTMs to regulate DNMT activity and targeting. The combined regulation of DNMT targeting and catalytic activity contributes to the precise spatiotemporal control of DNMT function and genome methylation in cells.

Targeting MLL-CXXC Domain with Synthetic CpG Dinucleotides: Docking and Molecular Dynamics Simulation Based Approach

Mixed lineage leukemia (MLL) is an aggressive type of childhood leukemia characterized by the presence of MLL fusion proteins resultant of chromosomal translocations affecting the MLL gene. These resulting chimeric proteins act as transcriptional regulators that take control of MLL targets. MLL fuses with more than 60 different partner genes, these fusion proteins retain the CXXC domain which binds to nonmethylated CpG DNA. The Molecular docking of CXXC domain and CpG dinucleotides were performed, large scale and long time (10 ns each) molecular dynamics simulation on CXXC domain wild type and AF9-MLL Fusion Protein have been performed to study the mechanism at atomic level. The effects of point mutation on DNA bindings were also studied through Molecular Docking. The results confirm that this point mutation have efficacy of interrupt the DNA bindings. Moreover, in-silico studies suggest external synthetic CpG DNA efficiently binds to CXXC domain and may be potential lead molecule for further in-vitro studies.

Abstract 2659: A novel isoform of TET1 that lacks a CXXC domain is overexpressed in cancer

Abstract TET1 is a DNA demethylase that regulates DNA methylation, hydroxymethylation and gene expression patterns. It has a catalytic domain that oxidizes methylated cytosine into 5-hydroxymethylcytosine and can then be further oxidized or converted to un-methylated cytosine. TET1 has a CXXC domain that allows it to recognize and bind to long stretches of CpG dinucleotides, known as CpG islands (CGIs). Our previous work found TET1 to protect CGIs from aberrant methylation and that the CXXC domain limits its ability to regulate genes outside of CGIs. However, many of the methylation changes in cancer are outside CGIs. To determine if TET1 could possibly play a role in this hypomethylation, we searched for alternatively spliced forms of the protein. We identified a novel isoform of TET1 (TET1ALT) that lacks the CXXC domain but retains the demethylase domain. TET1ALT has a unique transcription start site from a strong alternate promoter upstream of exon 3, yielding a protein with a unique translation start site. We confirmed promoter activity using Luciferase constructs, and confirmed TET1ALT translation and function through overexpression experiments. Gene expression analyses by qPCR found that TET1ALT is silenced in mESCs but becomes activated in select embryonic and adult tissues while full length TET1 is favored in mESCs, but is repressed in adult tissues. Thus, there is an isoform switch during differentiation and TET1ALT is the major isoform expressed in adult cells. TET1ALT is over expressed in several cancer cell lines, including breast cancer. In conclusion, we have identified a novel isoform of TET1 that has the potential to regulate DNA methylation outside of CGIs and is the predominant isoform expressed in adult cells. We are investigating the hypothesis that TET1ALT may contribute to the methylation changes observed in cancer outside of CGIs, such as in gene bodies and in enhancers. Citation Format: Charly R. Good, Jozef Madzo, Shinji Maegawa, Nora Engel, Jaroslav Jelinek, Jean-Pierre Issa. A novel isoform of TET1 that lacks a CXXC domain is overexpressed in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2659.

FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function.

Forkhead box A1 (FOXA1) is an FKHD family protein that plays pioneering roles in lineage-specific enhancer activation and gene transcription. Through genome-wide location analyses, here we show that FOXA1 expression and occupancy are, in turn, required for the maintenance of these epigenetic signatures, namely DNA hypomethylation and histone 3 lysine 4 methylation. Mechanistically, this involves TET1, a 5-methylcytosine dioxygenase. We found that FOXA1 induces TET1 expression via direct binding to its cis-regulatory elements. Further, FOXA1 physically interacts with the TET1 protein through its CXXC domain. TET1 thus co-occupies FOXA1-dependent enhancers and mediates local DNA demethylation and concomitant histone 3 lysine 4 methylation, further potentiating FOXA1 recruitment. Consequently, FOXA1 binding events are markedly reduced following TET1 depletion. Together, our results suggest that FOXA1 is not only able to recognize but also remodel the epigenetic signatures at lineage-specific enhancers, which is mediated, at least in part, by a feed-forward regulatory loop between FOXA1 and TET1.

Abnormal X chromosome inactivation and sex-specific gene dysregulation after ablation of FBXL10.

Background Almost all CpG-rich promoters in the mammalian genome are bound by the multidomain FBXL10 protein (also known as KDM2B, JHDM1B, CXXC2, and NDY1). FBXL10 is expressed as two isoforms: FBXL10-1, a longer form that contains an N-terminal histone demethylase domain with C-terminal F-box, CXXC, PHD, RING, and leucine-rich repeat domains, and FBXL10-2, a shorter form that initiates at an alternative internal exon and which lacks the histone demethylase domain but retains all other annotated domains. Selective deletion of Fbxl10-1 had been reported to produce a low penetrance and variable phenotype; most of the mutant animals were essentially normal. We constructed mutant mouse strains that were either null for Fbxl10-2 but wild type for Fbxl10-1 or null for both Fbxl10-1 and Fbxl10-2.ResultsDeletion of Fbxl10-2 (in a manner that does not perturb expression of Fbxl10-1) produced a phenotype very different from the Fbxl10-1 mutant, with craniofacial abnormalities, neural tube defects, and increased lethality, especially in females. Mutants that lacked both FBXL10-1 and FBXL10-2 showed embryonic lethality and even more extreme sexual dimorphism, with more severe gene dysregulation in mutant female embryos. X-linked genes were most severely dysregulated, and there was marked overexpression of Xist in mutant females although genes that encode factors that bind to Xist RNA were globally downregulated in mutant female as compared to male embryos.ConclusionsFBXL10 is the first factor shown to be required both for the normal expression and function of the Xist gene and for normal expression of proteins that associate with Xist RNA; it is proposed that FBXL10 coordinates the expression of Xist RNA with proteins that associate with this RNA. The function of FBXL10 is largely independent of the histone demethylase activity of the long form of the protein.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0069-1) contains supplementary material, which is available to authorized users.

Tet3 Reads 5-Carboxylcytosine through Its CXXC Domain and Is a Potential Guardian against Neurodegeneration.

We report that the mammalian 5-methylcytosine (5mC) oxidase Tet3 exists as three major isoforms and characterized the full-length isoform containing an N-terminal CXXC domain (Tet3FL). This CXXC domain binds to unmethylated CpGs, but, unexpectedly, its highest affinity is toward 5-carboxylcytosine (5caC). We determined the crystal structure of the CXXC domain-5caC-DNA complex, revealing the structural basis of the binding specificity of this domain as a reader of CcaCG sequences. Mapping of Tet3FL in neuronal cells shows that Tet3FL is localized precisely at the transcription start sites (TSSs) of genes involved in lysosome function, mRNA processing, and key genes of the base excision repair pathway. Therefore, Tet3FL may function as a regulator of 5caC removal by base excision repair. Active removal of accumulating 5mC from the TSSs of genes coding for lysosomal proteins by Tet3FL in postmitotic neurons of the brain may be important for preventing neurodegenerative diseases.

Solution structure of human MBD1 CXXC1.

Methylation of a CpG dinucleotide at cytosine C5 (mCpG)is a major modification in vertebrate genomes associatedwith epigenetic gene silencing. CpG methylation recruitsproteins which specifically recognise this motif and thesemethylated DNA binding proteins then recruit enzymeswhich chemically and physically alter chromatin, inducingtranscriptional repression. Although most CpG motifs aremethylated, short (500–2000 bp) CG-rich regions, knownas CpG islands (CGIs), found within *60 % of humangene promoters remain non-methylated (Bird 2002). Howthese CGIs contribute to epigentic regulation is an area ofcontinuing research.To investigate the possibility that proteins are targetedto these CGIs, Voo et al. (2000) used a ligand screen toidentify proteins that bind non-methylated CpGs. Theyidentified a protein, CFP1 (formerly CGBP) that contains acysteine rich zinc finger–CXXC (ZF-CXXC) domain.Since this discovery, several more CXXC containing pro-teins have been identified as reviewed recently by Longet al. (2013). CXXC domains can be differentiated intothree groups, type 1 which bind CpG and contain theKFGG motif, type 2 which do not bind CpG and lack theKFGG motif, and type 3 which lack the extended loop oftypes 1 and 2 but do bind to CpG (Fig. 3a). The conservedCXXCXXCX

Profile of Dinshaw J. Patel

Small-noncoding RNA molecules are the dark matter of molecular biology. From small interfering (si) RNAs that silence invading pathogens to microRNAs that fine-tune gene expression, noncoding RNAs carry out a vast number of functions within the cell. “It was generally believed in the early days of molecular biology that RNA was just a passive intermediate on the pathway from DNA to protein, but noncoding RNA architecture and its interactions with partners is much more intricate and interesting,” says structural biologist Dinshaw J. Patel. Elected to the National Academy of Sciences in 2009, Patel has spent his career deciphering the shapes of biomolecules and principles underlying molecular complex formation. In his Inaugural Article, Patel, the Abby Rockefeller Mauze Chair in Experimental Therapeutics at the Memorial Sloan Kettering Cancer Center, explores how the PAZ-binding domain of PIWI proteins forms a pocket capable of binding the 3′-ends of piRNAs, noncoding RNA molecules that help maintain the integrity of germ-line DNA (1). Dinshaw J. Patel. Image courtesy of Memorial Sloan Kettering Cancer Center. Crystal structures of Thermus thermophilus Argonaute (labeled and color-coded by domains) with bound 5′-phosphorylated guide DNA (in red) as a binary complex ( A ) and with target RNA (in blue) as a ternary complex in B . Reprinted with permission from Macmillan Publishers Ltd., ref. 5. Crystal structure of mammalian maintenance DNA methyltransferase DNMT1 (containing color-coded CXXC, BAH1, BAH2, and MTase domains) bound to a 19-mer CG-containing DNA in the presence of cofactor AdoHcy. Reproduced from ref. 14. Patel was born in Mumbai, India in 1942 and grew up in the Zoroastrian community. His father was a civil engineer, and in an education-rich setting, Patel immersed himself in the sciences. “Because India was a developing country, science was what we learned in textbooks, with minimal practical experience,” Patel explains. …

Sequence Analysis of a Subset of Plasma Membrane Raft Proteome Containing CXXC Metal Binding Motifs

In an attempt to identify the metal sensing proteins localized to mammalian plasma membrane, the authors screened a list of 300 raft associated proteins that are involved in cellular signaling mechanisms by searching the presence of metal thionin (CXXC) motifs. 50 proteins were found to possess CXXC motifs that could act as potential metal sensing proteins. The authors determined membrane topologies of the above CXXC motif containing proteins using TM-pred and analyzed the positions of their transmembrane (TM) domains using Bio-edit software. Based on the topology of CXXC domains, the authors classified all the raft-associated metal sensing proteins into six categories. They are (i) Exoplasmic tails with CXXC motif, (ii) Exoplasmic loops with CXXC motif, (iii) Cytosolic tails with CXXC motif, (iv) Cytosolic loop with CXXC motif, (v) TM domains with CXXC motifs, (vi) Proteins with multiple topologies of CXXC motif. The authors' study will lead to understanding of the raft-mediated mechanism of heavy metal sensing and signaling in mammalian cells.

Crystal Structure of Human DNA Methyltransferase 1

DNMT1 (DNA methyltransferase 1) is responsible for propagating the DNA methylation patterns during DNA replication. DNMT1 contains, in addition to a C-terminal methyltransferase domain, a large N-terminal regulatory region that is composed of an RFTS (replication foci targeting sequence) domain, a CXXC zinc finger domain and a pair of BAH (bromo adjacent homology) domains. The regulatory domains of DNMT1 mediate a network of protein–protein and protein–DNA interactions to control the recruitment and enzymatic activity of DNMT1. Here we report the crystal structure of human DNMT1 with all the structural domains (hDNMT1, residues 351–1600) in complex with S-adenosyl-l-homocysteine at 2.62Å resolution. The RFTS domain directly associates with the methyltransferase domain, thereby inhibiting the substrate binding of hDNMT1. Through structural analysis, mutational, biochemical and enzymatic studies, we further identify that a linker sequence between the CXXC and BAH1 domains, aside from its role in the CXXC domain-mediated DNMT1 autoinhibition, serves as an important regulatory element in the RFTS domain-mediated autoinhibition. In comparison with the previously determined structure of mouse DNMT1, this study also reveals a number of distinct structural features that may underlie subtle functional diversity observed for the two orthologues. In addition, this structure provides a framework for understanding the functional consequence of disease-related hDNMT1 mutations.

A CxxC domain that binds to unmethylated CpG is required for KDM2A to control rDNA transcription

Dysfunction of ribosome biogenesis is commonly found in cancers. Because the transcription of ribosomal RNA genes (rDNA) is a rate-limiting step in ribosome biogenesis and is elevated in many cancer cells, ribosomal RNA transcription can be a target for cancer therapy. In eukaryotes, ribosomal RNA is transcribed specifically in nucleoli by RNA polymerase I but not by RNA polymerase II. Therefore, ribosomal RNA transcription by RNA polymerase I would have a distinct nature compared to transcription by RNA polymerase II. Genomic DNA with proteins including histones constitutes chromatin. The structure of chromatin has plasticity and is regulated by chemical modifications of chromatin's components. We had reported that histone demethylase KDM2A reduced ribosomal RNA transcription in response to starvation. In this symposium, we reported our recent results showing the mechanism by which KDM2A was recruited to rDNA chromatin. We found that KDM2A bound to a rDNA promoter with unmethylated CpG dinucleotides via KDM2A CxxC-zinc finger motif. This binding was required for KDM2A to demethylate histone in the rDNA promoter and reduce rDNA transcription resulting from starvation. Further, this binding was detected before starvation, independent of the demethylase activity. We also found that the histone demethylation by KDM2A in response to starvation was detected only in the rDNA promoter, but not in a gene promoter transcribed by Pol II, the P2RX4 promoter. These results suggest that it is important to consider genome regions and cell conditions when developing epigenetic drugs.

RUNX1/CBFβ Dosage Is Critical for MLL Leukemias Development

Transcription factors RUNX1/CBFβ play critical roles in hematopoiesis. Both of them are frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. We have previously shown that MLL, RUNX1, and CBFβ interact and form a regulatory complex to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity remains unknown.To determine the impact of MLL fusion protein on RUNX1/CBFβ, we introduced either MLL, MLL-BP (longer N-terminal Flag-tagged MLL construct which contains CXXC domain; 1-1406), or MLL-fusions together with RUNX1, CBFβ, or both RUNX1 and CBFβ into 293T cells. MLL-BP and MLL fusions significantly decreased RUNX1 levels compared with controls (empty vector and MLL). CBFβ protein was mildly decreased by MLL-BP and MLL-fusions when expressed alone. However, when CBFβ was co-expressed with RUNX1, it was significantly decreased compared with controls. The expression levels of RUNX1 and CBFβ proteins in LSK cells from Mll-Af9 knock-in mice were significantly lower than those from wild-type (WT) mice. To confirm these findings in human acute myeloid leukemia (AML), we measured the expression of RUNX1 and CBFβ at both mRNA and protein levels in various leukemia cell lines. The expression levels of RUNX1 and CBFβ proteins were significantly decreased in AML cells with MLL fusion and MLL partial tandem duplication (MLL-PTD) compared with those in AML cells without MLL aberrations. MLL fusions still have CXXC domain. In MLL-PTD, the CXXC domain is duplicated. Our data showed that RUNX1 protein is not only down-regulated by MLL fusion proteins, but also by MLL-BP. Thus, to determine which region is involved in the down-regulation of RUNX1, we introduced a series of MLL deletion mutants into 293T cells and measured RUNX1 protein expression. MLL deletion mutants without CXXC domain had no effect on RUNX1 stability. The construct which contains point mutations in CXXC domain also lacked the ability to reduce RUNX1 expression. Furthermore, overexpression of only CXXC domain and flanking regions could down-regulate RUNX1 protein expression. These results suggest that MLL fusion proteins and the N-terminal MLL portion of MLL fusions down-regulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions.To understand the impact of RUNX1/CBFβ down-regulation on hematopoietic stem and progenitor cells (HSPCs), we generated RUNX1+/–/CBFβ+/– mice as a hypomorph model. The percentage of bone marrow (BM) LSK cells from RUNX1+/–/CBFβ+/– mice was significantly increased compared with that from WT mice. Using BM cells from these mice, we performed in vitro CFU assay and in vivo bone marrow transplantation (BMT) assay. BM cells from RUNX1+/–/CBFβ+/– mice provided more colonies in CFU assay compared with those from WT mice. To determine whether restoration of RUNX1 could repress the MLL mediated leukemogenesis, we retrovirally overexpressed WT RUNX1 in BM cells from Mll-Af9 knock-in mice. Using transduced BM cells, we performed in vitro CFU assay and in vivo BMT assay. RUNX1 overexpressed Mll-Af9 (Mll-Af9/RUNX1) cells underwent terminal differentiation after 2 times replating, while control vector transduced Mll-Af9 (Mll-Af9/Control) cells could still be replated more than 4 times. All the recipient mice transplanted with Mll-Af9/Control cells developed AML. In contrast, all the recipient mice transplanted with Mll-Af9/RUNX1 never develop AML. Furthermore, when we treated MLL leukemia cell lines with DOT1L inhibitor (EPZ-5676), RUNX1 protein levels in these MLL leukemia cell lines were significantly increased 48 hours after the treatment in comparing with controls treated with DMSO. However, there was no significant mRNA expression level change of RUNX1within 48 hours. Future studies are needed to fully understand the mechanism of whether this increasing RUNX1 protein level by DOT1L inhibitor is through blocking CXXC domain and flanking regions mediated degradation.In conclusion, MLL aberrations down-regulate RUNX1/CBFβ via their CXXC domain and flanking regions. Down-regulation of RUNX1/CBFβ plays critical role for MLL mediated leukemia development. Targeting RUNX1/CBFβ levels allows us to test novel therapies for MLL leukemias. DisclosuresMulloy:Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding.

TET1 regulates hypoxia-induced epithelial-mesenchymal transition by acting as a co-activator.

BackgroundHypoxia induces the epithelial-mesenchymal transition, EMT, to promote cancer metastasis. In addition to transcriptional regulation mediated by hypoxia-inducible factors, HIFs, other epigenetic mechanisms of gene regulation, such as histone modifications and DNA methylation, are utilized under hypoxia. However, whether DNA demethylation mediated by TET1, a DNA dioxygenase converting 5-methylcytosine, 5mC, into 5-hydroxymethylcytosine, 5hmC, plays a role in hypoxia-induced EMT is largely unknown.ResultsWe show that TET1 regulates hypoxia-responsive gene expression. Hypoxia/HIF-2α regulates the expression of TET1. Knockdown of TET1 mitigates hypoxia-induced EMT. RNA sequencing and 5hmC sequencing identified the set of TET1-regulated genes. Cholesterol metabolic process genes are among the genes that showed high prevalence and statistical significance. We characterize one of the genes, INSIG1 (insulin induced gene 1), to confirm its expression and the 5hmC levels in its promoter. Knockdown of INSIG1 also mitigates hypoxia-induced EMT. Finally, TET1 is shown to be a transcriptional co-activator that interacts with HIF-1α and HIF-2α to enhance their transactivation activity independent of its enzymatic activity. TET1 acts as a co-activator to further enhance the expression of INSIG1 together with HIF-2α. We define the domain in HIF-1α that interacts with TET1 and map the domain in TET1 that confers transactivation to a 200 amino acid region that contains a CXXC domain. The TET1 catalytically inactive mutant is capable of rescuing hypoxia-induced EMT in TET1 knockdown cells.ConclusionsThese findings demonstrate that TET1 serves as a transcription co-activator to regulate hypoxia-responsive gene expression and EMT, in addition to its role in demethylating 5mC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0513-0) contains supplementary material, which is available to authorized users.

CXXC4 mRNA levels are associated with clinicopathological parameters and survival of myelodysplastic syndrome patients

ObjectiveThe CXXC domain protein 4 (CXXC4) functions as a negative regulator of Wnt signaling and also regulates expression of the ten-eleven translocation 2 (TET2) protein for DNA methylation. This study detected levels of CXXC4 and TET2 mRNA to determine their association with survival of patients with myelodysplastic syndrome (MDS). MethodsLevels of TET2 and CXXC4 mRNA were analyzed in bone marrow samples from 154 MDS patients and 50 control subjects using qRT-PCR and subsequently associated these levels with clinicopathological characteristics and survival of MDS patients. ResultsLevels of TET2 and CXXC4 mRNA were significantly lower in MDS patients than that in controls (P=0.009 and P<0.001, respectively). Patients with advanced WHO subtypes (e.g., RAEB-1 and RAEB-2) exhibited a higher level of CXXC4 mRNA (P=0.020) compared to those with early stage subtypes (i.e., RA, RARS, RCMD, RCMD-RS, 5q-syndrome, and MDS-U). Moreover, levels of CXXC4 mRNA were associated with marrow blast levels (P=0.014) and neutrophil counts (P=0.039). Levels of CXXC4 mRNA and hemoglobin and IPSS cytopenias were associated with the overall survival (P=0.025) but not with the leukemia-free survival of MDS patients. The multivariate analysis demonstrated that the age of patients and levels of hemoglobin and marrow blast were independent risk factors for survival of MDS patients. ConclusionThis study demonstrated that the age of patients and levels of CXXC4 mRNA, hemoglobin, and marrow blast associated with survival of MDS patients.

TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells.

TET1 is a 5-methylcytosine dioxygenase and its DNA demethylating activity has been implicated in pluripotency and reprogramming. However, the precise role of TET1 in DNA methylation regulation outside of developmental reprogramming is still unclear. Here, we show that overexpression of the TET1 catalytic domain but not full length TET1 (TET1-FL) induces massive global DNA demethylation in differentiated cells. Genome-wide mapping reveals that 5-hydroxymethylcytosine production by TET1-FL is inhibited as DNA methylation increases, which can be explained by the preferential binding of TET1-FL to unmethylated CpG islands (CGIs) through its CXXC domain. TET1-FL specifically accumulates 5-hydroxymethylcytosine at the edges of hypomethylated CGIs, while knockdown of endogenous TET1 induces methylation spreading from methylated edges into hypomethylated CGIs. We also found that gene expression changes after TET1-FL overexpression are relatively small and independent of its dioxygenase function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically prevents aberrant methylation spreading into CGIs in differentiated cells.

Downregulation of RUNX1/CBFβ by MLL fusion proteins enhances hematopoietic stem cell self-renewal

RUNX1/CBFβ (core binding factor [CBF]) is a heterodimeric transcription factor complex that is frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. The MLL protein interacts with RUNX1 and prevents RUNX1 from ubiquitin-mediated degradation. RUNX1/CBFβ recruits MLL to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity has not been fully understood. In this report, we show that MLL fusion proteins and the N-terminal MLL portion of MLL fusions downregulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. We confirmed this finding in Mll-Af9 knock-in mice and human M4/M5 acute myeloid leukemia (AML) cell lines, with or without MLL translocations, showing that MLL translocations cause a hypomorph phenotype of RUNX1/CBFβ. Overexpression of RUNX1 inhibits the development of AML in Mll-Af9 knock-in mice; conversely, further reducing Runx1/Cbfβ levels accelerates MLL-AF9-mediated AML in bone marrow transplantation assays. These data reveal a newly defined negative regulation of RUNX1/CBFβ by MLL fusion proteins and suggest that targeting RUNX1/CBFβ levels may be a potential therapy for MLLs.