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1.
Bioessays ; 43(10): e2100038, 2021 10.
Article in English | MEDLINE | ID: mdl-34423467

ABSTRACT

Although the promyelocytic leukemia (PML) protein is renowned for regulating a wide range of cellular processes and as an essential component of PML nuclear bodies (PML-NBs), the mechanisms through which it exerts its broad physiological impact are far from fully elucidated. Here, we review recent studies supporting an emerging view that PML's pleiotropic effects derive, at least partially, from its role in regulating histone H3.3 chromatin assembly, a critical epigenetic mechanism. These studies suggest that PML maintains heterochromatin organization by restraining H3.3 incorporation. Examination of PML's contribution to H3.3 chromatin assembly in the context of the cell cycle and PML-NB assembly suggests that PML represses heterochromatic H3.3 deposition during S phase and that transcription and SUMOylation regulate PML's recruitment to heterochromatin. Elucidating PML' s contributions to H3.3-mediated epigenetic regulation will provide insight into PML's expansive influence on cellular physiology and open new avenues for studying oncogenesis linked to PML malfunction.


Subject(s)
Chromatin Assembly and Disassembly , Histones , Chromatin Assembly and Disassembly/genetics , Epigenesis, Genetic/genetics , Histones/genetics , Histones/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Promyelocytic Leukemia Protein/genetics , Promyelocytic Leukemia Protein/metabolism
2.
J Cell Sci ; 132(6)2019 03 26.
Article in English | MEDLINE | ID: mdl-30796101

ABSTRACT

The incorporation of the histone H3 variant, H3.3, into chromatin by the H3.3-specific chaperone DAXX and the ATP-dependent chromatin remodeling factor ATRX is a critical mechanism for silencing repetitive DNA. DAXX and ATRX are also components of promyelocytic nuclear bodies (PML-NBs), which have been identified as sites of H3.3 chromatin assembly. Here, we use a transgene array that can be visualized in single living cells to investigate the mechanisms that recruit PML-NB proteins (i.e. PML, DAXX, ATRX, and SUMO-1, SUMO-2 and SUMO-3) to heterochromatin and their functions in H3.3 chromatin assembly. We show that DAXX and PML are recruited to the array through distinct SUMOylation-dependent mechanisms. Additionally, PML is recruited during S phase and its depletion increases H3.3 deposition. Since this effect is abrogated when PML and DAXX are co-depleted, it is likely that PML represses DAXX-mediated H3.3 chromatin assembly. Taken together, these results suggest that, at heterochromatin, PML-NBs coordinate H3.3 chromatin assembly with DNA replication, which has important implications for understanding how transcriptional silencing is established and maintained.


Subject(s)
Co-Repressor Proteins/metabolism , Histones/metabolism , Molecular Chaperones/metabolism , Promyelocytic Leukemia Protein/metabolism , S Phase/physiology , Cell Cycle Proteins/metabolism , Cell Line , DNA Replication/physiology , Gene Silencing/physiology , HeLa Cells , Heterochromatin/metabolism , Histone Chaperones/metabolism , Humans , Nucleosomes/metabolism
3.
J Biol Chem ; 293(32): 12360-12377, 2018 08 10.
Article in English | MEDLINE | ID: mdl-29921582

ABSTRACT

The histone H3 variant H3.3 is a highly conserved and dynamic regulator of chromatin organization. Therefore, fully elucidating its nucleosome incorporation mechanisms is essential to understanding its functions in epigenetic inheritance. We previously identified the RNase P protein subunit, Rpp29, as a repressor of H3.3 chromatin assembly. Here, we use a biochemical assay to show that Rpp29 interacts with H3.3 through a sequence element in its own N terminus, and we identify a novel interaction with histone H2B at an adjacent site. The fact that archaeal Rpp29 does not include this N-terminal region suggests that it evolved to regulate eukaryote-specific functions. Oncogenic H3.3 mutations alter the H3.3-Rpp29 interaction, which suggests that they could dysregulate Rpp29 function in chromatin assembly. We also used KNS42 cells, an H3.3(G34V) pediatric high-grade glioma cell line, to show that Rpp29 1) represses H3.3 incorporation into transcriptionally active protein-coding, rRNA, and tRNA genes; 2) represses mRNA, protein expression, and antisense RNA; and 3) represses euchromatic post-translational modifications (PTMs) and promotes heterochromatic PTM deposition (i.e. histone H3 Lys-9 trimethylation (H3K9me3) and H3.1/2/3K27me3). Notably, we also found that K27me2 is increased and K36me1 decreased on H3.3(G34V), which suggests that Gly-34 mutations dysregulate Lys-27 and Lys-36 methylation in cis The fact that Rpp29 represses H3.3 chromatin assembly and sense and antisense RNA and promotes H3K9me3 and H3K27me3 suggests that Rpp29 regulates H3.3-mediated epigenetic mechanisms by processing a transcribed signal that recruits H3.3 to its incorporation sites.


Subject(s)
Chromatin Assembly and Disassembly , Epigenesis, Genetic , Glioma/metabolism , Histones/metabolism , Nucleosomes/metabolism , Ribonucleases/metabolism , Ribonucleoproteins/metabolism , Transcription, Genetic , Glioma/genetics , Glioma/pathology , Histones/genetics , Humans , Methylation , Mutation , Nucleosomes/genetics , Ribonucleases/genetics , Ribonucleoproteins/genetics , Tumor Cells, Cultured
4.
Nat Commun ; 8: 14928, 2017 04 10.
Article in English | MEDLINE | ID: mdl-28393830

ABSTRACT

POT1 and TPP1 are part of the shelterin complex and are essential for telomere length regulation and maintenance. Naturally occurring mutations of the telomeric POT1-TPP1 complex are implicated in familial glioma, melanoma and chronic lymphocytic leukaemia. Here we report the atomic structure of the interacting portion of the human telomeric POT1-TPP1 complex and suggest how several of these mutations contribute to malignant cancer. The POT1 C-terminus (POT1C) forms a bilobal structure consisting of an OB-fold and a holiday junction resolvase domain. TPP1 consists of several loops and helices involved in extensive interactions with POT1C. Biochemical data shows that several of the cancer-associated mutations, partially disrupt the POT1-TPP1 complex, which affects its ability to bind telomeric DNA efficiently. A defective POT1-TPP1 complex leads to longer and fragile telomeres, which in turn promotes genomic instability and cancer.


Subject(s)
Shelterin Complex/chemistry , Shelterin Complex/metabolism , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/metabolism , Telomere/chemistry , Telomere/metabolism , Calorimetry , Crystallography, X-Ray , DNA/metabolism , HEK293 Cells , Humans , Mutant Proteins/metabolism , Mutation/genetics , Protein Binding , Structure-Activity Relationship , Telomerase/metabolism , Telomere-Binding Proteins/genetics
5.
Mol Biol Cell ; 27(7): 1154-69, 2016 Apr 01.
Article in English | MEDLINE | ID: mdl-26842893

ABSTRACT

In mammals, histone H3.3 is a critical regulator of transcription state change and heritability at both euchromatin and heterochromatin. The H3.3-specific chaperone, DAXX, together with the chromatin-remodeling factor, ATRX, regulates H3.3 deposition and transcriptional silencing at repetitive DNA, including pericentromeres and telomeres. However, the events that precede H3.3 nucleosome incorporation have not been fully elucidated. We previously showed that the DAXX-ATRX-H3.3 pathway regulates a multi-copy array of an inducible transgene that can be visualized in single living cells. When this pathway is impaired, the array can be robustly activated. H3.3 is strongly recruited to the site during activation where it accumulates in a complex with transcribed sense and antisense RNA, which is distinct from the DNA/chromatin. This suggests that transcriptional events regulate H3.3 recruited to its incorporation sites. Here we report that the nucleolar RNA proteins Rpp29, fibrillarin, and RPL23a are also components of this H3.3/RNA complex. Rpp29 is a protein subunit of RNase P. Of the other subunits, POP1 and Rpp21 are similarly recruited suggesting that a variant of RNase P regulates H3.3 chromatin assembly. Rpp29 knockdown increases H3.3 chromatin incorporation, which suggests that Rpp29 represses H3.3 nucleosome deposition, a finding with implications for epigenetic regulation.


Subject(s)
Chromatin Assembly and Disassembly , Histones , Nucleosomes/metabolism , Ribonucleases , Ribonucleoproteins , Chromosomal Proteins, Non-Histone , Epigenesis, Genetic , Humans
6.
Cell Rep ; 9(6): 2263-78, 2014 Dec 24.
Article in English | MEDLINE | ID: mdl-25497088

ABSTRACT

Telomeres protect the ends of cellular chromosomes. We show here that infection with herpes simplex virus 1 (HSV-1) results in chromosomal structural aberrations at telomeres and the accumulation of telomere dysfunction-induced DNA damage foci (TIFs). At the molecular level, HSV-1 induces transcription of telomere repeat-containing RNA (TERRA), followed by the proteolytic degradation of the telomere protein TPP1 and loss of the telomere repeat DNA signal. The HSV-1-encoded E3 ubiquitin ligase ICP0 is required for TERRA transcription and facilitates TPP1 degradation. Small hairpin RNA (shRNA) depletion of TPP1 increases viral replication, indicating that TPP1 inhibits viral replication. Viral replication protein ICP8 forms foci that coincide with telomeric proteins, and ICP8-null virus failed to degrade telomere DNA signal. These findings suggest that HSV-1 reorganizes telomeres to form ICP8-associated prereplication foci and to promote viral genomic replication.


Subject(s)
Herpesvirus 1, Human/physiology , Telomere/virology , Virus Replication , Cell Line , Chromosome Aberrations , DNA Damage , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Herpesvirus 1, Human/metabolism , Humans , Immediate-Early Proteins/genetics , Immediate-Early Proteins/metabolism , Proteolysis , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , Repetitive Sequences, Nucleic Acid , Serine Proteases/genetics , Serine Proteases/metabolism , Shelterin Complex/metabolism , Telomere/chemistry , Telomere/genetics , Telomere-Binding Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism
7.
Cancer Res ; 74(12): 3332-43, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24736545

ABSTRACT

BRD4 is implicated in the pathogenesis of a number of different cancers. It is also the target of translocation t(15;19) that accounts for the highly aggressive NUT midline carcinoma (NMC). We discovered that t(15;19) NMC cells display the ability to grow into stem cell-like spheres and express an exceptionally high level of the stem cell marker, SOX2. The BRD4-NUT fusion oncogene resulting from t(15;19) translocation is required for the abnormal activation of SOX2, which drives the stem cell-like proliferation and cellular transformation in NMC cells. SOX2 knockdown phenocopies the effects of BRD4-NUT inhibition, whereas ectopic SOX2 expression rescues the phenotype. The BRD4-NUT-induced abnormal SOX2 activation was observed in multiple NMC cell lines as well as in NMC primary tumors. We further demonstrate that BRD4-NUT oncoprotein recruits p300 to stimulate transcription activation and that inhibition of p300 represses SOX2 transcription in NMC cells. These studies identify this stem cell marker as a novel BRD4-NUT target that supports the highly aggressive transforming activity of t(15;19) carcinomas. Our study provides new mechanistic insights for understanding how alteration of BRD4 function by BRD4-NUT oncogene leads to the highly malignant NMC carcinoma. Because abnormal stem cell self-renewal is frequently observed during tumor formation and metastasis, the aberrant stem cell-like proliferation associated with BRD4 dysregulation observed in NMC carcinoma may have implications for studying the oncogenic mechanism of other BRD4-associated tumors.


Subject(s)
Carcinoma, Squamous Cell/metabolism , Cell Transformation, Neoplastic/metabolism , Nuclear Proteins/physiology , Oncogene Proteins, Fusion/physiology , SOXB1 Transcription Factors/metabolism , Antineoplastic Agents/pharmacology , Azepines/pharmacology , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic , Humans , Protein Binding , SOXB1 Transcription Factors/genetics , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Transcription, Genetic , Transcriptional Activation , Triazoles/pharmacology , p300-CBP Transcription Factors/metabolism
8.
J Cell Physiol ; 229(3): 259-65, 2014 Mar.
Article in English | MEDLINE | ID: mdl-23929405

ABSTRACT

For a gene to be expressed, the functions of multiple molecular machines must be coordinated at the site of transcription. To understand the role of nuclear organization in transcription, it is necessary to visualize the dynamic interactions of regulatory factors with chromatin and RNA. It is currently possible to localize individual transcription sites in single living mammalian cells by engineering reporter gene constructs to include sequence elements which permit the visualization of nucleic acids in vivo. Upon stable integration, these transgenes form chromatinized arrays, which can be imaged during activation to obtain high-resolution quantitative information about transcriptional dynamics. Modeling can suggest new hypotheses about gene regulation, which can be tested both in the single-cell imaging system and at endogenous genes. This gene-specific imaging strategy has the potential to reveal regulatory mechanisms, which would be difficult to imagine outside of single living cells.


Subject(s)
Cell Nucleus/metabolism , Cell Tracking/methods , Chromatin/metabolism , DNA/metabolism , Microscopy, Fluorescence , RNA/biosynthesis , Transcription, Genetic , Animals , Cells, Cultured , Chromatin Assembly and Disassembly , Genes, Reporter , Humans , Kinetics , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Transfection
9.
Epigenetics ; 8(10): 1101-13, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23949383

ABSTRACT

In eukaryotic organisms, histone posttranslational modifications (PTMs) are indispensable for their role in maintaining cellular physiology, often through their mediation of chromatin-related processes such as transcription. Targeted investigations of this ever expanding network of chemical moieties continue to reveal genetic, biochemical, and cellular nuances of this complex landscape. In this study, we present our findings on a novel class of histone PTMs: Serine, Threonine, and Tyrosine O-acetylation. We have combined highly sensitive nano-LC-MS/MS experiments and immunodetection assays to identify and validate these unique marks found only on histone H3. Mass spectrometry experiments have determined that several of these O-acetylation marks are conserved in many species, ranging from yeast to human. Additionally, our investigations reveal that histone H3 serine 10 acetylation (H3S10ac) is potentially linked to cell cycle progression and cellular pluripotency. Here, we provide a glimpse into the functional implications of this H3-specific histone mark, which may be of high value for further studies of chromatin.


Subject(s)
Chromatin/metabolism , Histones/metabolism , Serine/metabolism , Acetylation , Animals , Cell Cycle , Chromatography, Liquid , Drosophila/metabolism , Embryonic Stem Cells/metabolism , Female , HEK293 Cells , HeLa Cells , Humans , Male , Mice , Pluripotent Stem Cells/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae/metabolism , Species Specificity , Tandem Mass Spectrometry , Tetrahymena thermophila/metabolism
10.
J Biol Chem ; 288(27): 19882-99, 2013 Jul 05.
Article in English | MEDLINE | ID: mdl-23689370

ABSTRACT

Unlike the core histones, which are incorporated into nucleosomes concomitant with DNA replication, histone H3.3 is synthesized throughout the cell cycle and utilized for replication-independent (RI) chromatin assembly. The RI incorporation of H3.3 into nucleosomes is highly conserved and occurs at both euchromatin and heterochromatin. However, neither the mechanism of H3.3 recruitment nor its essential function is well understood. Several different chaperones regulate H3.3 assembly at distinct sites. The H3.3 chaperone, Daxx, and the chromatin-remodeling factor, ATRX, are required for H3.3 incorporation and heterochromatic silencing at telomeres, pericentromeres, and the cytomegalovirus (CMV) promoter. By evaluating H3.3 dynamics at a CMV promoter-regulated transcription site in a genetic background in which RI chromatin assembly is blocked, we have been able to decipher the regulatory events upstream of RI nucleosomal deposition. We find that at the activated transcription site, H3.3 accumulates with sense and antisense RNA, suggesting that it is recruited through an RNA-mediated mechanism. Sense and antisense transcription also increases after H3.3 knockdown, suggesting that the RNA signal is amplified when chromatin assembly is blocked and attenuated by nucleosomal deposition. Additionally, we find that H3.3 is still recruited after Daxx knockdown, supporting a chaperone-independent recruitment mechanism. Sequences in the H3.3 N-terminal tail and αN helix mediate both its recruitment to RNA at the activated transcription site and its interaction with double-stranded RNA in vitro. Interestingly, the H3.3 gain-of-function pediatric glioblastoma mutations, G34R and K27M, differentially affect H3.3 affinity in these assays, suggesting that disruption of an RNA-mediated regulatory event could drive malignant transformation.


Subject(s)
Chromatin Assembly and Disassembly/physiology , Cytomegalovirus/metabolism , Histones/metabolism , Promoter Regions, Genetic/physiology , RNA, Viral/biosynthesis , Transcription, Genetic/physiology , Cell Line , Cytomegalovirus/genetics , Histones/genetics , Humans , Nucleosomes/genetics , Nucleosomes/metabolism , Protein Structure, Secondary , RNA, Viral/genetics
11.
Mol Biol Cell ; 24(9): 1454-68, 2013 May.
Article in English | MEDLINE | ID: mdl-23485562

ABSTRACT

Promyelocytic leukemia nuclear bodies (PML-NBs)/nuclear domain 10s (ND10s) are nuclear structures that contain many transcriptional and chromatin regulatory factors. One of these, Sp100, is expressed from a single-copy gene and spliced into four isoforms (A, B, C, and HMG), which differentially regulate transcription. Here we evaluate Sp100 function in single cells using an inducible cytomegalovirus-promoter-regulated transgene, visualized as a chromatinized transcription site. Sp100A is the isoform most strongly recruited to the transgene array, and it significantly increases chromatin decondensation. However, Sp100A cannot overcome Daxx- and α-thalassemia mental retardation, X-linked (ATRX)-mediated transcriptional repression, which indicates that PML-NB/ND10 factors function within a regulatory hierarchy. Sp100A increases and Sp100B, which contains a SAND domain, decreases acetyl-lysine regulatory factor levels at activated sites, suggesting that Sp100 isoforms differentially regulate transcription by modulating lysine acetylation. In contrast to Daxx, ATRX, and PML, Sp100 is recruited to activated arrays in cells expressing the herpes simplex virus type 1 E3 ubiquitin ligase, ICP0, which degrades all Sp100 isoforms except unsumoylated Sp100A. The recruitment Sp100A(K297R), which cannot be sumoylated, further suggests that sumoylation plays an important role in regulating Sp100 isoform levels at transcription sites. This study provides insight into the ways in which viruses may modulate Sp100 to promote their replication cycles.


Subject(s)
Antigens, Nuclear/metabolism , Autoantigens/metabolism , Chromatin Assembly and Disassembly , Cytomegalovirus/physiology , Promoter Regions, Genetic , Acetylation , Adaptor Proteins, Signal Transducing/metabolism , Co-Repressor Proteins , DNA Helicases/metabolism , Epigenesis, Genetic , HeLa Cells , Humans , Molecular Chaperones , Nuclear Proteins/metabolism , Promyelocytic Leukemia Protein , Protein Isoforms/metabolism , Protein Transport , Proteolysis , Sumoylation , Transcription Factors/metabolism , Transcription Initiation Site , Transcription, Genetic , Tumor Suppressor Proteins/metabolism , Virus Latency , X-linked Nuclear Protein
12.
Methods Mol Biol ; 977: 249-58, 2013.
Article in English | MEDLINE | ID: mdl-23436368

ABSTRACT

Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.


Subject(s)
Single-Cell Analysis/methods , Transcriptional Activation , Animals , Calcium Phosphates/chemistry , Cell Culture Techniques , Cell Line , Genes, Reporter , Humans , Transfection , Transgenes
13.
J Cell Sci ; 125(Pt 22): 5489-501, 2012 Nov 15.
Article in English | MEDLINE | ID: mdl-22976303

ABSTRACT

Histone H3.3 is a constitutively expressed H3 variant implicated in the epigenetic inheritance of chromatin structures. Recently, the PML-nuclear body (PML-NB)/Nuclear Domain 10 (ND10) proteins, Daxx and ATRX, were found to regulate replication-independent histone H3.3 chromatin assembly at telomeres and pericentric heterochromatin. As it is not completely understood how PML-NBs/ND10s regulate transcription and resistance to viral infection, we have used a CMV-promoter-regulated inducible transgene array, at which Daxx and ATRX are enriched, to delineate the mechanisms through which they regulate transcription. When integrated into HeLa cells, which express both Daxx and ATRX, the array is refractory to activation. However, transcription can be induced when ICP0, the HSV-1 E3 ubiquitin ligase required to reverse latency, is expressed. As ATRX and Daxx are depleted from the activated array in ICP0-expressing HeLa cells, this suggests that they are required to maintain a repressed chromatin environment. As histone H3.3 is strongly recruited to the ICP0-activated array but does not co-localize with the DNA, this also suggests that chromatin assembly is blocked during activation. The conclusion that the Daxx and ATRX pathway is required for transcriptional repression and chromatin assembly at this site is further supported by the finding that an array integrated into the ATRX-negative U2OS cell line can be robustly activated and that histone H3.3 is similarly recruited and unincorporated into the chromatin. Therefore, this study has important implications for understanding gene silencing, viral latency and PML-NB/ND10 function.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA Helicases/metabolism , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Single-Cell Analysis/methods , Transcription, Genetic , Cell Line, Tumor , Chromatin/metabolism , Co-Repressor Proteins , Cytomegalovirus/genetics , DNA Helicases/chemistry , HeLa Cells , Histones/metabolism , Humans , Molecular Chaperones , Nuclear Proteins/chemistry , Promoter Regions, Genetic/genetics , Protein Structure, Tertiary , Transcriptional Activation/genetics , Transgenes , X-linked Nuclear Protein
14.
Mol Cancer Res ; 10(3): 401-14, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22205726

ABSTRACT

The repair of DNA damage in highly compact, transcriptionally silent heterochromatin requires that repair and chromatin packaging machineries be tightly coupled and regulated. KAP1 is a heterochromatin protein and co-repressor that binds to HP1 during gene silencing but is also robustly phosphorylated by Ataxia telangiectasia mutated (ATM) at serine 824 in response to DNA damage. The interplay between HP1-KAP1 binding/ATM phosphorylation during DNA repair is not known. We show that HP1α and unmodified KAP1 are enriched in endogenous heterochromatic loci and at a silent transgene prior to damage. Following damage, γH2AX and pKAP1-s824 rapidly increase and persist at these loci. Cells that lack HP1 fail to form discreet pKAP1-s824 foci after damage but levels are higher and more persistent. KAP1 is phosphorylated at serine 473 in response to DNA damage and its levels are also modulated by HP1. Unlike pKAP1-s824, pKAP1-s473 does not accumulate at damage foci but is diffusely localized in the nucleus. While HP1 association tempers KAP1 phosphorylation, this interaction also slows the resolution of γH2AX foci. Thus, HP1-dependent regulation of KAP1 influences DNA repair in heterochromatin.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Heterochromatin/metabolism , Nuclear Proteins/metabolism , Phosphoserine/metabolism , Protein Serine-Threonine Kinases/metabolism , Repressor Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Animals , Ataxia Telangiectasia Mutated Proteins , Blotting, Western , Cell Fractionation , Chromobox Protein Homolog 5 , Gene Knockdown Techniques , Histones/metabolism , Humans , Immunohistochemistry , Mice , Models, Biological , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , NIH 3T3 Cells , Nuclear Proteins/chemistry , Phosphorylation , Repressor Proteins/chemistry , Substrate Specificity , Transgenes/genetics , Tripartite Motif-Containing Protein 28
15.
Mol Cell ; 40(6): 976-87, 2010 Dec 22.
Article in English | MEDLINE | ID: mdl-21172662

ABSTRACT

DNA double-strand breaks (DSBs) are repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR), but cellular repair processes remain elusive. We show here that the ATP-dependent chromatin-remodeling factors, ACF1 and SNF2H, accumulate rapidly at DSBs and are required for DSB repair in human cells. If the expression of ACF1 or SNF2H is suppressed, cells become extremely sensitive to X-rays and chemical treatments producing DSBs, and DSBs remain unrepaired. ACF1 interacts directly with KU70 and is required for the accumulation of KU proteins at DSBs. The KU70/80 complex becomes physically more associated with the chromatin-remodeling factors of the CHRAC complex, which includes ACF1, SNF2H, CHRAC15, and CHRAC17, after treatments producing DSBs. Furthermore, the frequency of NHEJ as well as HR induced by DSBs in chromosomal DNA is significantly decreased in cells depleted of either of these factors. Thus, ACF1 and its complexes play important roles in DSBs repair.


Subject(s)
Adenosine Triphosphatases/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Breaks, Double-Stranded , DNA Repair , Transcription Factors/metabolism , Antigens, Nuclear/metabolism , Cells, Cultured , DNA Polymerase III/metabolism , DNA-Binding Proteins/metabolism , Green Fluorescent Proteins/metabolism , Humans , Kinetics , Ku Autoantigen , Nucleoproteins/metabolism , Ultraviolet Rays
16.
Cell ; 141(6): 970-81, 2010 Jun 11.
Article in English | MEDLINE | ID: mdl-20550933

ABSTRACT

DNA double-strand breaks (DSBs) initiate extensive local and global alterations in chromatin structure, many of which depend on the ATM kinase. Histone H2A ubiquitylation (uH2A) on chromatin surrounding DSBs is one example, thought to be important for recruitment of repair proteins. uH2A is also implicated in transcriptional repression; an intriguing yet untested hypothesis is that this function is conserved in the context of DSBs. Using a novel reporter that allows for visualization of repair protein recruitment and local transcription in single cells, we describe an ATM-dependent transcriptional silencing program in cis to DSBs. ATM prevents RNA polymerase II elongation-dependent chromatin decondensation at regions distal to DSBs. Silencing is partially dependent on E3 ubiquitin ligases RNF8 and RNF168, whereas reversal of silencing relies on the uH2A deubiquitylating enzyme USP16. These findings give insight into the role of posttranslational modifications in mediating crosstalk between diverse processes occurring on chromatin.


Subject(s)
Cell Cycle Proteins/metabolism , Chromatin/metabolism , DNA Breaks, Double-Stranded , DNA-Binding Proteins/metabolism , Gene Silencing , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins , Cell Line, Tumor , DNA Damage , Histones/metabolism , Humans , Transcription, Genetic , Ubiquitin Thiolesterase/metabolism , Ubiquitination
17.
PLoS One ; 5(4): e10272, 2010 Apr 21.
Article in English | MEDLINE | ID: mdl-20422051

ABSTRACT

BACKGROUND: Gene activation is thought to occur through a series of temporally defined regulatory steps. However, this process has not been completely evaluated in single living mammalian cells. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the timing and coordination of gene activation events, we tracked the recruitment of GCN5 (histone acetyltransferase), RNA polymerase II, Brd2 and Brd4 (acetyl-lysine binding proteins), in relation to a VP16-transcriptional activator, to a transcription site that can be visualized in single living cells. All accumulated rapidly with the VP16 activator as did the transcribed RNA. RNA was also detected at significantly more transcription sites in cells expressing the VP16-activator compared to a p53-activator. After alpha-amanitin pre-treatment, the VP16-activator, GCN5, and Brd2 are still recruited to the transcription site but the chromatin does not decondense. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that a strong activator can rapidly overcome the condensed chromatin structure of an inactive transcription site and supercede the expected requirement for regulatory events to proceed in a temporally defined order. Additionally, activator strength determines the number of cells in which transcription is induced as well as the extent of chromatin decondensation. As chromatin decondensation is significantly reduced after alpha-amanitin pre-treatment, despite the recruitment of transcriptional activation factors, this provides further evidence that transcription drives large-scale chromatin decondensation.


Subject(s)
Chromatin Assembly and Disassembly , Cytological Techniques , Transcriptional Activation/genetics , Alpha-Amanitin/pharmacology , Binding Sites , Cell Cycle Proteins , Cell Line, Tumor , Etoposide/metabolism , Humans , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Transport , RNA Polymerase II/metabolism , Time Factors , Transcription Factors/metabolism , p300-CBP Transcription Factors/metabolism
18.
J Cell Sci ; 120(Pt 14): 2301-7, 2007 Jul 15.
Article in English | MEDLINE | ID: mdl-17606985

ABSTRACT

The development of non-invasive methods of visualizing proteins and nucleic acids in living cells has provided profound insight into how they move and interact with each other in vivo. It is possible to evaluate basic mechanisms of gene expression, and to define their temporal and spatial parameters by using this methodology to label endogenous genes and make reporter constructs that allow specific DNA and RNA regulatory elements to be localized. This Commentary highlights recent reports that have used these techniques to study nuclear organization, transcription factor dynamics and the kinetics of RNA synthesis. These studies show how imaging gene expression in single living cells can reveal new regulatory mechanisms. They also expand our understanding of the role of chromatin and RNA dynamics in modulating cellular responses to developmental and environmental signals.


Subject(s)
Gene Expression Profiling/methods , Gene Expression , Genetic Techniques , Animals , Chromatin/physiology , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fluorescence , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins , Luminescent Proteins , Nuclear Proteins/metabolism , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Red Fluorescent Protein
19.
Science ; 304(5678): 1797-800, 2004 Jun 18.
Article in English | MEDLINE | ID: mdl-15205532

ABSTRACT

Understanding gene expression requires the ability to follow the fate of individual molecules. Here we use a cellular system for monitoring messenger RNA (mRNA)expression to characterize the movement in real time of single mRNA-protein complexes (mRNPs) in the nucleus of living mammalian cells. This mobility was not directed but was governed by simple diffusion. Some mRNPs were partially corralled throughout the nonhomogenous nuclear environment, but no accumulation at subnuclear domains was observed. Following energy deprivation, energy-independent motion of mRNPs was observed in a highly ATP-dependent nuclear environment; movements were constrained to chromatin-poor domains and excluded by newly formed chromatin barriers. This observation resolves a controversy, showing that the energetic requirements of nuclear mRNP trafficking are consistent with a diffusional model.


Subject(s)
Cell Nucleus/metabolism , Proteins/metabolism , RNA, Messenger/metabolism , Active Transport, Cell Nucleus , Adenosine Triphosphate/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Line, Tumor , Chromatin/metabolism , Cytoplasm/metabolism , Diffusion , Energy Metabolism , Fluorescence Recovery After Photobleaching , Globins/genetics , Globins/metabolism , Green Fluorescent Proteins , Humans , In Situ Hybridization, Fluorescence , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Peroxisomes/metabolism , Protein Biosynthesis , Proteins/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Transcription, Genetic , Transfection
20.
Cell ; 116(5): 683-98, 2004 Mar 05.
Article in English | MEDLINE | ID: mdl-15006351

ABSTRACT

We have developed an inducible system to visualize gene expression at the levels of DNA, RNA and protein in living cells. The system is composed of a 200 copy transgene array integrated into a euchromatic region of chromosome 1 in human U2OS cells. The condensed array is heterochromatic as it is associated with HP1, histone H3 methylated at lysine 9, and several histone methyltransferases. Upon transcriptional induction, HP1alpha is depleted from the locus and the histone variant H3.3 is deposited suggesting that histone exchange is a mechanism through which heterochromatin is transformed into a transcriptionally active state. RNA levels at the transcription site increase immediately after the induction of transcription and the rate of synthesis slows over time. Using this system, we are able to correlate changes in chromatin structure with the progression of transcriptional activation allowing us to obtain a real-time integrative view of gene expression.


Subject(s)
Chromosomal Proteins, Non-Histone , Gene Expression Regulation , Gene Silencing , Immunohistochemistry/methods , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Chromatin/chemistry , Chromatin/metabolism , Chromobox Protein Homolog 5 , DNA/metabolism , Heterochromatin/genetics , Heterochromatin/metabolism , Histones/metabolism , Humans , In Situ Hybridization, Fluorescence , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription, Genetic , Transgenes
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