scholarly journals Hsp70 gene association with nuclear speckles is Hsp70 promoter specific

2010 ◽  
Vol 191 (4) ◽  
pp. 711-719 ◽  
Author(s):  
Yan Hu ◽  
Matt Plutz ◽  
Andrew S. Belmont
Keyword(s):  
Heat Shock ◽  
Integration Site ◽  
Promoter Sequence ◽  
Artificial Chromosome ◽  
Nuclear Bodies ◽  
Nuclear Speckles ◽  
Transcript Accumulation ◽  
Nuclear Speckle ◽  
Hsp70 Promoter ◽  
Acid Processing

Many mammalian genes localize near nuclear speckles, nuclear bodies enriched in ribonucleic acid–processing factors. In this paper, we dissect cis-elements required for nuclear speckle association of the heat shock protein 70 (Hsp70) locus. We show that speckle association is a general property of Hsp70 bacterial artificial chromosome transgenes, independent of the chromosome integration site, and can be recapitulated using a 2.8-kilobase HSPA1A gene fragment. Association of Hsp70 transgenes and their transcripts with nuclear speckles is transcription dependent, independent of the transcribed sequence identity, but dependent on the Hsp70 promoter sequence. Transgene speckle association does not correlate with the amount of transcript accumulation, with large transgene arrays driven by different promoters showing no speckle association, but smaller Hsp70 transgene arrays with lower transcript accumulation showing high speckle association. Moreover, despite similar levels of transcript accumulation, Hsp70 transgene speckle association is observed after heat shock but not cadmium treatment. We suggest that certain promoters may direct specific chromatin and/or transcript ribonucleoprotein modifications, leading to nuclear speckle association.

scholarly journals Revealing RCOR2 as a regulatory component of nuclear speckles

2021 ◽  
Author(s):  
Carlos Rivera ◽  
Daniel Verbel ◽  
Duxan Arancibia ◽  
Anna Lappala ◽  
Marcela González ◽  
...  
Keyword(s):  
System Development ◽  
Cell Types ◽  
Nervous System Development ◽  
Nuclear Bodies ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Multiple Cell ◽  
Rna Maturation ◽  
Nuclear Processes ◽  
The Brain

Abstract Background Nuclear processes such as transcription and RNA maturation can be impacted by subnuclear compartmentalization in condensates and nuclear bodies. Here we characterize the nature of nuclear granules formed by REST corepressor 2 (RCOR2), a nuclear protein essential for pluripotency maintenance and central nervous system development. Results Using biochemical approaches and high-resolution microscopy, we reveal that RCOR2 is localized in nuclear speckles across multiple cell types, including neurons in the brain. RCOR2 forms complexes with nuclear speckle components such as SON, SRSF7, and SRRM2. When cells are exposed to chemical stress, RCOR2 behaves as a core component of the nuclear speckle and is stabilized by RNA. In turn, nuclear speckle morphology appears to depend on RCOR2. Specifically, RCOR2 knockdown results larger nuclear speckles, whereas overexpressing RCOR2 leads to smaller and rounder nuclear speckles. Conclusion Our study suggests that RCOR2 is a regulatory component of the nuclear speckle bodies, setting this co-repressor protein as a factor that controls nuclear speckles behavior.

scholarly journals Revealing RCOR2 as a regulatory component of nuclear speckles

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Carlos Rivera ◽  
Daniel Verbel-Vergara ◽  
Duxan Arancibia ◽  
Anna Lappala ◽  
Marcela González ◽  
...  
Keyword(s):  
System Development ◽  
Cell Types ◽  
Nervous System Development ◽  
Nuclear Bodies ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Multiple Cell ◽  
Rna Maturation ◽  
Nuclear Processes ◽  
The Brain

Abstract Background Nuclear processes such as transcription and RNA maturation can be impacted by subnuclear compartmentalization in condensates and nuclear bodies. Here, we characterize the nature of nuclear granules formed by REST corepressor 2 (RCOR2), a nuclear protein essential for pluripotency maintenance and central nervous system development. Results Using biochemical approaches and high-resolution microscopy, we reveal that RCOR2 is localized in nuclear speckles across multiple cell types, including neurons in the brain. RCOR2 forms complexes with nuclear speckle components such as SON, SRSF7, and SRRM2. When cells are exposed to chemical stress, RCOR2 behaves as a core component of the nuclear speckle and is stabilized by RNA. In turn, nuclear speckle morphology appears to depend on RCOR2. Specifically, RCOR2 knockdown results larger nuclear speckles, whereas overexpressing RCOR2 leads to smaller and rounder nuclear speckles. Conclusion Our study suggests that RCOR2 is a regulatory component of the nuclear speckle bodies, setting this co-repressor protein as a factor that controls nuclear speckles behavior.

scholarly journals Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins

2020 ◽  
Vol 219 (9) ◽  
Author(s):  
Joseph Dopie ◽  
Michael J. Sweredoski ◽  
Annie Moradian ◽  
Andrew S. Belmont
Keyword(s):  
Mass Spectrometry ◽  
Signal Amplification ◽  
Protein Composition ◽  
Nuclear Bodies ◽  
Ratio Method ◽  
Tyramide Signal Amplification ◽  
Label Free ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Speckle Structure

We present a simple ratio method to infer protein composition within cellular structures using proximity labeling approaches but compensating for the diffusion of free radicals. We used tyramide signal amplification (TSA) and label-free mass spectrometry (MS) to compare proteins in nuclear speckles versus centromeres. Our “TSA-MS ratio” approach successfully identified known nuclear speckle proteins. For example, 96% and 67% of proteins in the top 30 and 100 sorted proteins, respectively, are known nuclear speckle proteins, including proteins that we validated here as enriched in nuclear speckles. We show that MFAP1, among the top 20 in our list, forms droplets under certain circumstances and that MFAP1 expression levels modulate the size, stability, and dynamics of nuclear speckles. Localization of MFAP1 and its binding partner, PRPF38A, in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase. Our results update older proteomic studies of nuclear speckles and should provide a useful reference dataset to guide future experimental dissection of nuclear speckle structure and function.

scholarly journals Transcription amplification by nuclear speckle association

2019 ◽  
Author(s):  
Jiah Kim ◽  
Nimish Khanna ◽  
Andrew S. Belmont
Keyword(s):  
Heat Shock ◽  
Liquid Droplet ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Nuclear Compartment ◽  
Endogenous Genes ◽  
Amplification Mechanism ◽  
Transcriptional Bursting ◽  
Transcriptional Amplification ◽  
Chromosome Regions

AbstractA significant fraction of active chromosome regions and genes reproducibly position near nuclear speckles, but the functional significance of this positioning is unknown. Here we show that Hsp70 BAC transgenes and endogenous genes turn on 2-4 mins after heat shock irrespective of their distance to nuclear speckles. However, we observe 12-56-fold and 3-7-fold higher transcription levels for speckle-associated Hsp70 transgenes and endogenous genes, respectively, after 1-2 hrs heat shock. Several fold higher transcription levels for several genes flanking the Hsp70 locus also correlate with speckle-association at 37 °C. Live-cell imaging reveals this modulation of Hsp70 transcription temporally correlates with speckle association/disassociation. Our results demonstrate stochastic gene expression dependent on positioning relative to a liquid-droplet nuclear compartment through a “transcriptional amplification” mechanism distinct from transcriptional bursting.

scholarly journals A novel nuclear speckle factor, USP42, promotes homologous recombination repair by resolving DNA double-strand break induced R-loop

2019 ◽  
Author(s):  
Misaki Matsui ◽  
Ryo Sakasai ◽  
Masako Abe ◽  
Yusuke Kimura ◽  
Shoki Kajita ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
Nuclear Bodies ◽  
Nuclear Speckles ◽  
Dsb Repair ◽  
Nuclear Speckle ◽  
Spatial Regulation ◽  
Intrinsically Disordered ◽  
Dna Double Strand Break

AbstractThe nucleus of mammalian cells is highly compartmentalized by nuclear bodies, including nuclear speckles. While nuclear bodies are known to function in regulating gene expression, their involvement in DNA repair has not been actively investigated. Here, our focused screen for nuclear speckle factors involved in homologous recombination (HR), which is a faithful DNA double-strand break (DSB) repair mechanism, revealed that nuclear speckle factors regulating transcription are potentially involved in the regulation of HR. Among the top hits, we provide evidence showing that USP42, which is a deubiquitylating enzyme and a hitherto unidentified nuclear speckles factor, promotes HR by facilitating BRCA1 recruitment to DSB sites and DNA-end resection. We further showed that USP42 localizes to nuclear speckles via an intrinsically disordered region, which is required for efficient HR. Furthermore, we established that USP42 interacts with DHX9, which possesses DNA-RNA helicase activity, and is required for efficient resolution of DSB-induced R-loop. Mechanistically, USP42 antagonizes mono-ubiquitylation of DHX9 that is evoked after DSB induction. In conclusion, our data propose a model in which a novel nuclear speckle factor, USP42, facilitates DSB-induced R-loop resolution, BRCA1 loading to DSB sites and preferential DSB repair by HR, indicating the importance of spatial regulation of DSB repair choice mediated by nuclear bodies.Significant statementDefects in the repair of DNA double-strand break (DSB), which is one of the most harmful DNA insults, cause human diseases including cancers. It has been suggested that DSBs generated in the coding region tend to be repaired by homologous recombination (HR) that is error-free DSB repair pathway. To reveal the spatial regulation of HR, in this study, we investigated the potential contribution of nuclear bodies, especially nuclear speckles, to HR, identifying a deubiquitylating enzyme USP42 as a HR promoting factor. We found that USP42 deubiquitylates DHX9, facilitates resolution of DNA-RNA hybrid structure and enhances HR through BRCA1 loading to DSB sites.ClassificationBiological Sciences, Cell Biology

scholarly journals RNAs as proximity labeling media for identifying nuclear speckle positions relative to the genome

2018 ◽  
Author(s):  
Weizhong Chen ◽  
Zhangming Yan ◽  
Simin Li ◽  
Norman Huang ◽  
Xuerui Huang ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Genomic Sequence ◽  
Single Cells ◽  
Regulation Of Gene Expression ◽  
Nuclear Genome ◽  
Fold Increase ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Interaction Sites ◽  
Genomic Regions

AbstractNuclear speckles are interchromatin structures enriched in RNA splicing factors. Determining their relative positions with respect to the folded nuclear genome could provide critical information on co-and post-transcriptional regulation of gene expression. However, it remains challenging to identify which parts of the nuclear genome are in proximity to nuclear speckles, due to physical separation between nuclear speckle cores and chromatin. We hypothesized that noncoding RNAs including small nuclear RNAs, 7SK and Malat1, which accumulate at the periphery of nuclear speckles (nsaRNA,nuclearspeckleassociated RNA), may extend to sufficient proximity to the nuclear genome. Leveraging a transcriptome-genome interaction assay (MARGI), we identified nsaRNA-interacting genomic sequences, which exhibited clustering patterns (nsaPeaks) in the genome, suggesting existence of relatively stable interaction sites for nsaRNAs in nuclear genome. Posttranscriptional pre-mRNAs, which are known to be clustered to nuclear speckles, exhibited proximity to nsaPeaks but rarely to other genomic regions. Furthermore, CDK9 proteins that localize to the vicinity of nuclear speckles produced ChIP-seq peaks that overlapped with nsaPeaks. Our combined DNA FISH and immunofluorescence analysis in 182 single cells revealed a 3-fold increase in odds for nuclear speckles to localize near an nsaPeak than its neighboring genomic sequence. These data suggest a model that nsaRNAs locate in sufficient proximity to nuclear genome and leave identifiable genomic footprints, thus revealing the parts of genome proximal to nuclear speckles.

scholarly journals Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells

1994 ◽  
Vol 14 (8) ◽  
pp. 5309-5317
Author(s):  
S P Murphy ◽  
J J Gorzowski ◽  
K D Sarge ◽  
B Phillips
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Embryonal Carcinoma ◽  
Embryonic Stem ◽  
Binding Activity ◽  
Hsp70 Promoter ◽  
Ec Cells

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

scholarly journals Analysis of Influenza B Virus NS1 Protein Trafficking Reveals a Novel Interaction with Nuclear Speckle Domains

2008 ◽  
Vol 83 (2) ◽  
pp. 701-711 ◽  
Author(s):  
Jana Schneider ◽  
Bianca Dauber ◽  
Krister Melén ◽  
Ilkka Julkunen ◽  
Thorsten Wolff
Keyword(s):  
Gene Expression ◽  
Protein Domain ◽  
Replication Capacity ◽  
Influenza B Virus ◽  
Viral Gene ◽  
Influenza B ◽  
Ns1 Protein ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
B Virus

ABSTRACT Many proteins that function in the transcription, maturation, and export of metazoan mRNAs are concentrated in nuclear speckle domains, indicating that the compartment is important for gene expression. Here, we show that the NS1 protein of influenza B virus (B/NS1) accumulates in nuclear speckles and causes rounding and morphological changes of the domains, indicating a disturbance in their normal functions. This property was located within the N-terminal 90 amino acids of the B/NS1 protein and was shown to be independent of any other viral gene product. Within this protein domain, we identified a monopartite importin α binding nuclear localization signal. Reverse-genetic analysis of this motif indicated that nuclear import and speckle association of the B/NS1 protein are required for the full replication capacity of the virus. In the late phase of virus infection, the B/NS1 protein relocated to the cytoplasm, which occurred in a CRM1-independent manner. The interaction of the B/NS1 protein with nuclear speckles may reflect a recruitment function to promote viral-gene expression. To our knowledge, this is the first functional description of a speckle-associated protein that is encoded by a negative-strand RNA virus.

Organization of the Drosophila melanogaster hsp70 heat shock regulation unit

1987 ◽  
Vol 7 (3) ◽  
pp. 1055-1062
Author(s):  
J Amin ◽  
R Mestril ◽  
P Schiller ◽  
M Dreano ◽  
R Voellmy
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Synergistic Effect ◽  
Consensus Sequence ◽  
Relative Orientation ◽  
Hsp70 Promoter ◽  
Promoter Sequences ◽  
Highly Active ◽  
Sequence Elements ◽  
Five Elements

Expression from the Drosophila melanogaster hsp70 promoter was controlled by a regulatory unit that was composed of two sequence elements that resembled the heat shock consensus sequence. The unit functioned in both orientations and at different distances from downstream promoter sequences. Each element of the unit alone was essentially inactive. Association of two elements resulted in a dramatic increase of transcription from the hsp70 promoter. This synergistic effect was independent of the relative orientation of the elements and, to a large extent, of the distance between them. Duplication of a region containing only one element also yielded a highly active, heat-regulated promoter. Genes with three to five elements were three to four times more active than those with a single regulatory unit.

Modifying expression of the engrailed gene of Drosophila melanogaster

Development ◽  
1988 ◽  
Vol 104 (Supplement) ◽  
pp. 85-93 ◽  
Author(s):  
Stephen J. Poole ◽  
Thomas B. Kornberg
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Heat Shock ◽  
P Element ◽  
Drosophila Embryo ◽  
Hsp70 Promoter ◽  
Normal Period ◽  
In Cells

The engrailed gene is required for segmentation of the Drosophila embryo and is expressed in cells constituting the posterior developmental compartments. In mutant embryos lacking engrailed function, portions of the cuticular pattern in each segment are deleted, resulting in fusion of adjacent denticle bands. Using P-element-mediated transposition, we generated flies that express the engrailed gene under the control of an hsp70 promoter, and found that ectopic, heat-shock-induced, engrailed expression caused pattern defects similar to those in embryos lacking engrailed function. Sensitivity to heat shock was only during the cellular blastoderm and early gastrulation periods. This window of sensitivity corresponds to the time when wildtype engrailed protein localizes into segmentally reiterated stripes and represents only a small portion of the normal period of engrailed gene expression.

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