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Expression profiling by high throughput sequencing of NDP52 II

ABSTRACT: NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear function for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We found that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene-expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover novel nuclear roles for NDP52 in gene expression and DNA structure regulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE188566 | GEO | 2021/11/17

REPOSITORIES: GEO

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Expression profiling by high throughput sequencing of NDP52 I

Project description:NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear function for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We found that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene-expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover novel nuclear roles for NDP52 in gene expression and DNA structure regulation.

2021-11-17 | GSE188563 | GEO

A constant level of Pol III is maintained in the nucleus by nuclear retention and degradation-supplementery

Project description:Pol III functions in the nucleus for transcription and cytoplasm as a viral-DNA sensor, but how cells maintain the cytoplasmic-to-nuclear partitioning of Pol III activity is unknown. Here, we found that Pol III forms transcription-dependent protein clusters in the nucleus and preferentially associates with assembly factors in the cytoplasm. Acute protein degradation of Pol III subunits followed by transient protein labeling showed that nuclear and cytoplasmic Pol III response differently after the loss of their subunits.

2023-05-29 | PXD042522 | iProX

In vivo live imaging of RNA polymerase II transcription factories in primary cells

Project description:Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as M-bM-^@M-^XM-bM-^@M-^Xtranscription factories,M-bM-^@M-^YM-bM-^@M-^Y but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9- mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments. Examination of genome occupancy of CDK9 and RNAPII that was performed by ChIP-seq in the MEL cell line as described (Soler et al. 2010, 2011) using CDK9 C20 antibody (Santa Cruz Biotechnology, C20, sc-484) and RNA Pol II antibody (Santa Cruz Biotechnology, N20, sc899),

2013-05-13 | E-GEOD-46849 | biostudies-arrayexpress

Plasmodium falciparum S-adenosylmethionine synthetase is essential for parasite survival through a complex interaction network with cytoplasmic and nuclear proteins

Project description:Life-cycle progression of the human malaria parasite Plasmodium falciparum in the human and mosquito hosts requires epigenetic mechanisms to regulate the stage-dependency of gene expression, including DNA and histone methylation. Methylation reactions require S-adenosylmethionine (SAM), a universal biological methyl donor whose synthesis is catalyzed by the enzyme S-adenosylmethionine synthetase (SAMS). In P. falciparum, PfSAMS is encoded by a single gene and has been suggested to be crucial for malaria pathogenesis and transmission; however, to date PfSAMS has not been fully characterized. To gain deeper insight into the function of PfSAMS, we generated a conditional gene knockdown (KD) using the pSLI-glmS system. We show that PfSAMS is expressed in the cytosol and nucleus of blood stage parasites. PfSAMS-KD results in reduced histone methylation marks and leads to impaired intraerythrocytic growth and gametocyte development. To further determine the interaction network of PfSAMS we performed BioID analyses. We identified 664 nuclear and 213 cytosolic interactors linked to biological processes like transcriptional and cell cycle regulation. Our interaction data further unveil PfSAMS-specific interaction clusters of proteins involved in DNA replication, transcription and translation as well as proteostasis. Our findings emphasize the key roles of PfSAMS during intraerythrocytic growth and sexual stage development.

2022-07-13 | PXD034111 | JPOST Repository

Ubiquitin ligase WWP2 targets RNA polymerase II in a DNA-PK-dependent transcription shutoff circuitry for DNA repair

Project description:DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII)-transcribed genes lead to inhibition of transcription. DNA protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remained unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII-transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of non-ubiquitylatable RPB1 abrogates the loading of Non-Homologous End-Joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shut-off circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.

2019-03-29 | PXD012606 | Pride

Control of chromatin structure by Spt6 has different consequences in coding and regulatory regions

Project description:Spt6 is a highly conserved factor that carries out important functions in transcription and chromatin structure. To gain new insights into Spt6, we measured nucleosome occupancy along Saccharomyces cerevisiae chromosome III in an spt6 mutant and found that the level of nucleosomes is greatly reduced accross some but not all coding regions. In addition, genome-wide location analyses of RNA polymerase II showed that the nucleosome loss in the spt6 mutant occurs over highly-transcribed genes. Unexpectedly, the effects of the spt6 mutation on nucleosome levels did not correlate with its effects on mRNA levels, suggesting that Spt6 plays distinct roles in controlling chromatin structure across coding regions and in transcriptional regulation. We studied one case of transcriptional regulation by Stp6, at the CHA1 gene, and showed that regulation likely occurs by Spt6 controlling the position of the +1 nucleosome. These results, along with previous studies, suggest that Spt6 regulates transcription by controlling chromatin structure over regulatory regions, and its effects on nucleosome levels over coding regions likely serve and independent function. In order to examine the genome-wide localization of RNAPII and Spt6 in Saccharomyces cerevisiae, RNAPII and Spt6 along with associated DNA sequences were immunoprecipitated using anti-8WG16 and anti-HA antibodies, respectively. The RNAPII and Spt6 chromatin immunoprecipitation was performed in duplicate from WT cells as described below. The extracted DNA was hybridized to a DNA microarray containing an average of 4 probes per kilobase across the whole yeast genome. The combined datasets are available in the supplemental files of the related publication.

2010-12-01 | E-GEOD-21787 | biostudies-arrayexpress

In vivo live imaging of RNA polymerase II transcription factories in primary cells

Project description:Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as ‘‘transcription factories,’’ but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9- mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments.

2013-05-13 | GSE46849 | GEO

Chromatin-wide profiling of the protein kinase DYRK1A reveals a role as a transcriptional activator of cell growth related genes

Project description:DYRK1A is a dosage-sensitive protein kinase that fulfills key roles during development and in tissue homeostasis, and its dysregulation results in human pathologies. DYRK1A is present in both the nucleus and cytoplasm of mammalian cells, although its nuclear function remains unclear. Genome-wide analysis of DYRK1A-associated loci reveals that the kinase is recruited preferentially to promoters of genes actively transcribed by RNA polymerase II (RNAPII), which are functionally associated with translation, RNA processing and cell cycle. DYRK1A-bound promoter sequences are highly enriched in a conserved palindromic motif, which is necessary to drive DYRK1A-dependent transcriptional activation. DYRK1A phosphorylates the carboxy-terminal domain (CTD) of RNAPII at Ser2 and Ser5. Depletion of DYRK1A results in reduced association of RNAPII at the target promoters as well as hypophosphorylation of the CTD of RNAPII along the target gene bodies. Accordingly, we propose that DYRK1A acts as a transcriptional regulator by acting as a novel CTD kinase. Occupancy of the kinase DYRK1A in two different cell lines and in two different growing conditions.

2015-03-11 | E-GEOD-63712 | biostudies-arrayexpress

Single molecule nanoscopy reveals that nuclear myosin VI regulates the spatial organization of mammalian transcription initiation

Project description:During transcription, RNA Polymerase II (RNAPII) is spatially organised into clusters that correlate with transcription activity. However, the assembly, organisation and stability of these transcription units remains unknown. Here, we have combined single molecule imaging with genomics approaches in order to explore the role of nuclear myosin VI in the nanoscale organisation of RNAPII. We can reveal that myosin VI acts as the molecular anchor that holds RNAPII into these transcription factories. Perturbation of myosin VI leads to the disruption of RNAPII localisation, changes in chromatin organisation and subsequently a decrease in gene expression. This role of Myosin VI is particularly important for transcription events following stimulation, where multiple genes are simultaneously activated.

2020-05-31 | GSE149448 | GEO

Global changes in mRNA abundance drive differential shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

Project description:Alterations in global mRNA decay can broadly impact multiple upstream and downstream stages of gene expression. For example, accelerated cytoplasmic mRNA degradation can trigger a reduction in mammalian RNA polymerase II (RNAPII) transcription, although signals that connect these seemingly distal processes remain largely unknown. Here, we used tandem mass tag labeling with mass spectrometry to chart how changes in Xrn1-dependent mRNA degradation impact nuclear-cytoplasmic protein distribution in human cells. Notably, accelerating mRNA decay through expression of a gammaherpesviral endonuclease known to coordinate with Xrn1 drove nuclear relocalization of many RNA binding proteins. Particularly enriched in the relocalized subset were factors linked to the poly(A) tail. Conversely, cells lacking Xrn1 exhibited changes in the localization and/or abundance of numerous factors linked to mRNA turnover. Based on these data, we uncovered a new role for cytoplasmic poly(A) binding protein in repressing RNAPII transcription upon its mRNA decay-induced translocation to the nucleus.

2018-10-24 | PXD009487 | Pride

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