Human RNA Methyltransferase BCDIN3D Regulates MicroRNA Processing

SummaryMicroRNAs (miRNAs) regulate key biological processes and their aberrant expression may lead to cancer. The primary transcript of canonical miRNAs is sequentially cleaved by the RNase III enzymes, Drosha and Dicer, which generate 5′ monophosphate ends that are important for subsequent miRNA functions. In particular, the recognition of the 5′ monophosphate of pre-miRNAs by Dicer is important for precise and effective biogenesis of miRNAs. Here, we identify a RNA-methyltransferase, BCDIN3D, that O-methylates this 5′ monophosphate and negatively regulates miRNA maturation. Specifically, we show that BCDIN3D phospho-dimethylates pre-miR-145 both in vitro and in vivo and that phospho-dimethylated pre-miR-145 displays reduced processing by Dicer in vitro. Consistently, BCDIN3D depletion leads to lower pre-miR-145 and concomitantly increased mature miR-145 levels in breast cancer cells, which suppresses their tumorigenic phenotypes. Together, our results uncover a miRNA methylation pathway potentially involved in cancer that antagonizes the Dicer-dependent processing of miR-145 as well as other miRNAs

Mechanisms of P/CAF auto-acetylation

8 pages, 7 figures.-- PMID: 12888487 [PubMed].-- PMCID: PMC169960.P/CAF is a histone acetyltransferase enzyme which was originally identified as a CBP/p300-binding protein. In this manuscript we report that human P/CAF is acetylated in vivo. We find that P/CAF is acetylated by itself and by p300 but not by CBP. P/CAF acetylation can be an intra- or intermolecular event. The intermolecular acetylation requires the N-terminal domain of P/CAF. The intramolecular acetylation targets five lysines (416–442) at the P/CAF C-terminus, which are in the nuclear localisation signal (NLS). Finally, we show that acetylation of P/CAF leads to an increment of its histone acetyltransferase (HAT) activity. These findings identify a new post-translation modification on P/CAF which may regulate its function.This study was funded by an EU grant to H.S.R., a Cancer Research UK grant to T.K. and by grants (PB-98-0468) and (SAF 2002-00741) from the Spanish Ministerio de Ciencia y Tecnología to M.M.B.Peer reviewe

Acetylation of importin-α nuclear import factors by CBP/p300.

Histone acetylases were originally identified because of their ability to acetylate histone substrates 1, 2 and 3. Acetylases can also target other proteins such as transcription factors 4, 5, 6 and 7. We asked whether the acetylase CREB-binding protein (CBP) could acetylate proteins not directly involved in transcription. A large panel of proteins, involved in a variety of cellular processes, were tested as substrates for recombinant CBP. This screen identified two proteins involved in nuclear import, Rch1 (human importin-α) and importin-α7, as targets for CBP. The acetylation site within Rch1 was mapped to a single residue, Lys22. By comparing the context of Lys22 with the sequences of other known substrates of CBP and the closely related acetylase p300, we identified G/SK (in the single-letter amino acid code) as a consensus acetylation motif. Mutagenesis of the glycine, as well as the lysine, severely impaired Rch1 acetylation, supporting the view that GK is part of a recognition motif for acetylation by CBP/p300. Using an antibody raised against an acetylated Rch1 peptide, we show that Rch1 was acetylated at Lys22 in vivo and that CBP or p300 could mediate this reaction. Lys22 lies within the binding site for a second nuclear import factor, importin-β. Acetylation of Lys22 promoted interaction with importin-β in vitro. Collectively, these results demonstrate that acetylation is not unique to proteins involved in transcription. Acetylation may regulate a variety of biological processes, including nuclear import

Histone H3 lysine 4 methylation is associated with the transcriptional reprogramming efficiency of somatic nuclei by oocytes.

BACKGROUND: When the nuclei of mammalian somatic cells are transplanted to amphibian oocytes in the first meiotic prophase, they are rapidly induced to begin transcribing several pluripotency genes, including Sox2 and Oct4. The more differentiated the donor cells of the nuclei, the longer it takes for the pluripotency genes to be activated after the nuclear transfer to oocytes. We have used this effect in order to investigate the role of histone modifications in this example of nuclear reprogramming. RESULTS: Reverse transcription polymerase chain reaction analysis shows that the transcriptional reprogramming of pluripotency genes, such as Sox2 and Oct4, takes place in transplanted nuclei from C3H10T1/2 cells and from newly differentiated mouse embryonic stem cells. We find that the reprogramming of 10T1/2 nuclei is accompanied by an increased phosphorylation, an increased methylation and a rapidly reduced acetylation of several amino acids in H3 and other histones. These results are obtained by the immunofluorescent staining of transplanted nuclei and by Western blot analysis. We have also used chromatin immunoprecipitation analysis to define histone modifications associated with the regulatory or coding regions of pluripotency genes in transplanted nuclei. Histone phosphorylation is increased and histone acetylation is decreased in several regulatory and gene coding regions. An increase of histone H3 lysine 4 dimethylation (H3K4 me2) is seen in the regulatory regions and gene coding region of pluripotency genes in reprogrammed nuclei. Furthermore, histone H3 lysine 4 trimethylation (H3K4 me3) is observed more strongly in the regulatory regions of pluripotency genes in transplanted nuclei that are rapidly reprogrammed than in nuclei that are reprogrammed slowly and are not seen in beta-globin, a gene that is not reprogrammed. When 10T1/2 nuclei are incubated in Xenopus oocyte extracts, histone H3 serine 10 (H3S10) is strongly phosphorylated within a few hours. Immunodepletion of Aurora B prevents this phosphorylation. CONCLUSION: We conclude that H3K4 me2 and me3 are likely to be important for the efficient reprogramming of pluripotency genes in somatic nuclei by amphibian oocytes and that Aurora B kinase is required for H3S10 phosphorylation which is induced in transplanted somatic cell nuclei.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

DDX3X RNA helicase affects breast cancer cell cycle progression by regulating expression of KLF4.

DDX3X is a multifunctional RNA helicase with documented roles in different cancer types. Here, we demonstrate that DDX3X plays an oncogenic role in breast cancer cells by modulating the cell cycle. Depletion of DDX3X in MCF7 cells slows cell proliferation by inducing a G1 phase arrest. Notably, DDX3X inhibits expression of Kruppel-like factor 4 (KLF4), a transcription factor and cell cycle repressor. Moreover, DDX3X directly interacts with KLF4 mRNA and regulates its splicing. We show that DDX3X-mediated repression of KLF4 promotes expression of S-phase inducing genes in MCF7 breast cancer cells. These findings provide evidence for a novel function of DDX3X in regulating expression and downstream functions of KLF4, a master negative regulator of the cell cycle

Nucleosome-Interacting Proteins Regulated by DNA and Histone Methylation

SummaryModifications on histones or on DNA recruit proteins that regulate chromatin function. Here, we use nucleosomes methylated on DNA and on histone H3 in an affinity assay, in conjunction with a SILAC-based proteomic analysis, to identify “crosstalk” between these two distinct classes of modification. Our analysis reveals proteins whose binding to nucleosomes is regulated by methylation of CpGs, H3K4, H3K9, and H3K27 or a combination thereof. We identify the origin recognition complex (ORC), including LRWD1 as a subunit, to be a methylation-sensitive nucleosome interactor that is recruited cooperatively by DNA and histone methylation. Other interactors, such as the lysine demethylase Fbxl11/KDM2A, recognize nucleosomes methylated on histones, but their recruitment is disrupted by DNA methylation. These data establish SILAC nucleosome affinity purifications (SNAP) as a tool for studying the dynamics between different chromatin modifications and provide a modification binding “profile” for proteins regulated by DNA and histone methylation

Dynamic distribution of the replacement histone variant H3.3 in the mouse oocyte and preimplantation embryos

Upon fertilization, the gametes undergo a drastic reprogramming that includes changes in DNA methylation and histone modifications. Currently, it is not known whether replacement of the major histones by histone variants is also involved in these processes. Here we have examined the expression and localization of the histone variant H3.3 in early mouse embryogenesis. We show that H3.3 is present in the oocyte as a maternal factor. It is then incorporated preferentially into the male pronucleus before genome activation, pointing towards an asymmetry in histone composition between the two pronuclei. This is in line with the male pronucleus bearing transcriptional activation first. The same distribution was observed when we followed the localisation of a tagged version of H3.3. We detected H3.3 in the nuclei of mouse embryos in all of the stages analysed, from the zygote to the blastocyst stage, suggesting that the epigenetic mechanisms in the early embryo not only involve changes in histone modifications but may also include histone replacement

Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway.

Hutchinson-Gilford progeria syndrome (HGPS) is an incurable premature aging disease. Identifying deregulated biological processes in HGPS might thus help define novel therapeutic strategies. Fibroblasts from HGPS patients display defects in nucleocytoplasmic shuttling of the GTP-bound form of the small GTPase Ran (RanGTP), which leads to abnormal transport of proteins into the nucleus. We report that microtubule stabilization in HGPS cells sequestered the nonclassical nuclear import protein Transportin-1 (TNPO1) in the cytoplasm, thus affecting the nuclear localization of its cargo, including the nuclear pore protein NUP153. Consequently, nuclear Ran, nuclear anchorage of the nucleoporin TPR, and chromatin organization were disrupted, deregulating gene expression and inducing senescence. Inhibiting N-acetyltransferase 10 (NAT10) ameliorated HGPS phenotypes by rebalancing the nuclear to cytoplasmic ratio of TNPO1. This restored nuclear pore complex integrity and nuclear Ran localization, thereby correcting HGPS cellular phenotypes. We observed a similar mechanism in cells from healthy aged individuals. This study identifies a nuclear import pathway affected in aging and underscores the potential for NAT10 inhibition as a possible therapeutic strategy for HGPS and perhaps also for pathologies associated with normal aging

The non-coding snRNA 7SK controls transcriptional termination, poising, and bidirectionality in embryonic stem cells

BACKGROUND: Pluripotency is characterized by a unique transcriptional state, in which lineage-specification genes are poised for transcription upon exposure to appropriate stimuli, via a bivalency mechanism involving the simultaneous presence of activating and repressive methylation marks at promoter-associated histones. Recent evidence suggests that other mechanisms, such as RNA polymerase II pausing, might be operational in this process, but their regulation remains poorly understood. RESULTS: Here we identify the non-coding snRNA 7SK as a multifaceted regulator of transcription in embryonic stem cells. We find that 7SK represses a specific cohort of transcriptionally poised genes with bivalent or activating chromatin marks in these cells, suggesting a novel poising mechanism independent of Polycomb activity. Genome-wide analysis shows that 7SK also prevents transcription downstream of polyadenylation sites at several active genes, indicating that 7SK is required for normal transcriptional termination or control of 3′-UTR length. In addition, 7SK suppresses divergent upstream antisense transcription at more than 2,600 loci, including many that encode divergent long non-coding RNAs, a finding that implicates the 7SK snRNA in the control of transcriptional bidirectionality. CONCLUSIONS: Our study indicates that a single non-coding RNA, the snRNA 7SK, is a gatekeeper of transcriptional termination and bidirectional transcription in embryonic stem cells and mediates transcriptional poising through a mechanism independent of chromatin bivalency.GCB was funded by an EMBO Long-Term Post-Doctoral Fellowship and a Marie Curie Intra-European Fellowship for Career Development. PA was supported by a Royal Society Newton International Fellowship and a Corpus Christi College research fellowship. This work was supported by Cancer Research UK, European Research Council (Advanced Grant, TK), EMBL (PB) and Swedish Research Council (GCB)

The Snf2 Homolog Fun30 acts as a homodimeric ATP-dependent chromatin-remodeling enzyme

The Saccharomyces cerevisiae Fun30 (Function unknown now 30) protein shares homology with an extended family of Snf2-related ATPases. Here we report the purification of Fun30 principally as a homodimer with a molecular mass of about 250 kDa. Biochemical characterization of this complex reveals that it has ATPase activity stimulated by both DNA and chromatin. Consistent with this, it also binds to both DNA and chromatin. The Fun30 complex also exhibits activity in ATP-dependent chromatin remodeling assays. Interestingly, its activity in histone dimer exchange is high relative to the ability to reposition nucleosomes. Fun30 also possesses a weakly conserved CUE motif suggesting that it may interact specifically with ubiquitinylated proteins. However, in vitro Fun30 was found to have no specificity in its interaction with ubiquitinylated histones