Phosphorylated Form Of RNA Polymerase Research Articles

G-Quadruplex Structures Colocalize with Transcription Factories and Nuclear Speckles Surrounded by Acetylated and Dimethylated Histones H3.

G-quadruplexes (G4s) are four-stranded helical structures that regulate several nuclear processes, including gene expression and telomere maintenance. We observed that G4s are located in GC-rich (euchromatin) regions and outside the fibrillarin-positive compartment of nucleoli. Genomic regions around G4s were preferentially H3K9 acetylated and H3K9 dimethylated, but H3K9me3 rarely decorated G4 structures. We additionally observed the variability in the number of G4s in selected human and mouse cell lines. We found the highest number of G4s in human embryonic stem cells. We observed the highest degree of colocalization between G4s and transcription factories, positive on the phosphorylated form of RNA polymerase II (RNAP II). Similarly, a high colocalization rate was between G4s and nuclear speckles, enriched in pre-mRNA splicing factor SC-35. PML bodies, the replication protein SMD1, and Cajal bodies colocalized with G4s to a lesser extent. Thus, G4 structures seem to appear mainly in nuclear compartments transcribed via RNAP II, and pre-mRNA is spliced via the SC-35 protein. However, α-amanitin, an inhibitor of RNAP II, did not affect colocalization between G4s and transcription factories as well as G4s and SC-35-positive domains. In addition, irradiation by γ-rays did not change a mutual link between G4s and DNA repair proteins (G4s/γH2AX, G4s/53BP1, and G4s/MDC1), accumulated into DNA damage foci. Described characteristics of G4s seem to be the manifestation of pronounced G4s stability that is likely maintained not only via a high-order organization of these structures but also by a specific histone signature, including H3K9me2, responsible for chromatin compaction.

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity.

Proteasome activity is required for diverse cellular processes, including transcriptional and epigenetic regulation. However, inhibiting proteasome activity can lead to an increase in transcriptional output that is correlated with enriched levels of trimethyl H3K4 and phosphorylated forms of RNA polymerase (Pol) II at the promoter and gene body. Here, we perform gene expression analysis and ChIP followed by sequencing (ChIP-seq) in MCF-7 breast cancer cells treated with the proteasome inhibitor MG132, and we further explore genome-wide effects of proteasome inhibition on the chromatin state and RNA Pol II transcription. Analysis of gene expression programs and chromatin architecture reveals that chemically inhibiting proteasome activity creates a distinct chromatin state, defined by spreading of the H3K4me3 mark into the gene bodies of differentially-expressed genes. The distinct H3K4me3 chromatin profile and hyperacetylated nucleosomes at transcription start sites establish a chromatin landscape that facilitates recruitment of Ser-5- and Ser-2-phosphorylated RNA Pol II. Subsequent transcriptional events result in diverse gene expression changes. Alterations of H3K36me3 levels in the gene body reflect productive RNA Pol II elongation of transcripts of genes that are induced, underscoring the requirement for proteasome activity at multiple phases of the transcriptional cycle. Finally, by integrating genomics data and pathway analysis, we find that the differential effects of proteasome inhibition on the chromatin state modulate genes that are fundamental for cancer cell survival. Together, our results uncover underappreciated downstream effects of proteasome inhibitors that may underlie targeting of distinct chromatin states and key steps of RNA Pol II-mediated transcription in cancer cells.

Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer.

Hormone therapy targeting estrogen receptor (ER) is the principal treatment for ER-positive breast cancers. However, many cancers develop resistance to hormone therapy while retaining ER expression. Identifying new druggable mediators of ER function can help to increase the efficacy of ER-targeting drugs. Cyclin-dependent kinase 8 (CDK8) is a Mediator complex-associated transcriptional regulator with oncogenic activities. Expression of CDK8, its paralog CDK19 and their binding partner Cyclin C are negative prognostic markers in breast cancer. Meta-analysis of transcriptome databases revealed an inverse correlation between CDK8 and ERα expression, suggesting that CDK8 could be functionally associated with ER. We have found that CDK8 inhibition by CDK8/19-selective small-molecule kinase inhibitors, by shRNA knockdown or by CRISPR/CAS9 knockout suppresses estrogen-induced transcription in ER-positive breast cancer cells; this effect was exerted downstream of ER. Estrogen addition stimulated the binding of CDK8 to the ER-responsive GREB1 gene promoter and CDK8/19 inhibition reduced estrogen-stimulated association of an elongation-competent phosphorylated form of RNA Polymerase II with GREB1. CDK8/19 inhibitors abrogated the mitogenic effect of estrogen on ER-positive cells and potentiated the growth-inhibitory effects of ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive breast cancer cells with CDK8/19 inhibitors strongly impeded the development of estrogen independence. In vivo treatment with a CDK8/19 inhibitor Senexin B suppressed tumor growth and augmented the effects of fulvestrant in ER-positive breast cancer xenografts. These results identify CDK8 as a novel downstream mediator of ER and suggest the utility of CDK8 inhibitors for ER-positive breast cancer therapy.

NF-κB and STAT1 control CXCL1 and CXCL2 gene transcription

Diabetes mellitus results from immune cell invasion into pancreatic islets of Langerhans, eventually leading to selective destruction of the insulin-producing β-cells. How this process is initiated is not well understood. In this study, we investigated the regulation of the CXCL1 and CXCL2 genes, which encode proteins that promote migration of CXCR2(+) cells, such as neutrophils, toward secreting tissue. Herein, we found that IL-1β markedly enhanced the expression of the CXCL1 and CXCL2 genes in rat islets and β-cell lines, which resulted in increased secretion of each of these proteins. CXCL1 and CXCL2 also stimulated the expression of specific integrin proteins on the surface of human neutrophils. Mutation of a consensus NF-κB genomic sequence present in both gene promoters reduced the ability of IL-1β to promote transcription. In addition, IL-1β induced binding of the p65 and p50 subunits of NF-κB to these consensus κB regulatory elements as well as to additional κB sites located near the core promoter regions of each gene. Additionally, serine-phosphorylated STAT1 bound to the promoters of the CXCL1 and CXCL2 genes. We further found that IL-1β induced specific posttranslational modifications to histone H3 in a time frame congruent with transcription factor binding and transcript accumulation. We conclude that IL-1β-mediated regulation of the CXCL1 and CXCL2 genes in pancreatic β-cells requires stimulus-induced changes in histone chemical modifications, recruitment of the NF-κB and STAT1 transcription factors to genomic regulatory sequences within the proximal gene promoters, and increases in phosphorylated forms of RNA polymerase II.

Nuclear ALG-2 Protein Interacts with Ca2+ Homeostasis Endoplasmic Reticulum Protein (CHERP) Ca2+-dependently and Participates in Regulation of Alternative Splicing of Inositol Trisphosphate Receptor Type 1 (IP3R1) Pre-mRNA

The intracellular Ca(2+) signaling pathway is important for the control of broad cellular processes from fertilization to cell death. ALG-2 is a Ca(2+)-binding protein that contains five serially repeated EF-hand motifs and interacts with various proteins in a Ca(2+)-dependent manner. Although ALG-2 is present both in the cytoplasm and in the nucleus, little is known about its nuclear function. Ca(2+) homeostasis endoplasmic reticulum protein (CHERP) was first identified as an endoplasmic reticulum protein that regulates intracellular Ca(2+) mobilization in human cells, but recent proteomics data suggest an association between CHERP and spliceosomes. Here, we report that CHERP, containing a Pro-rich region and a phosphorylated Ser/Arg-rich RS-like domain, is a novel Ca(2+)-dependent ALG-2-interactive target in the nucleus. Immunofluorescence microscopic analysis revealed localization of CHERP to the nucleoplasm with prominent accumulation at nuclear speckles, which are the sites of storage and modification for pre-mRNA splicing factors. Live cell time-lapse imaging showed that nuclear ALG-2 was recruited to the CHERP-localizing speckles upon Ca(2+) mobilization. Results of co-immunoprecipitation assays revealed binding of CHERP to a phosphorylated form of RNA polymerase II. Knockdown of CHERP or ALG-2 in HT1080 cells resulted in generation of alternatively spliced isoforms of the inositol 1,4,5-trisphosphate receptor 1 (IP3R1) pre-mRNA that included exons 41 and 42 in addition to the major isoform lacking exons 40-42. Furthermore, binding between CHERP and IP3R1 RNA was detected by an RNA immunoprecipitation assay using a polyclonal antibody against CHERP. These results indicate that CHERP and ALG-2 participate in regulation of alternative splicing of IP3R1 pre-mRNA and provide new insights into post-transcriptional regulation of splicing variants in Ca(2+) signaling pathways.

Inhibition of cdk9 during Herpes Simplex Virus 1 Infection Impedes Viral Transcription

During herpes simplex virus 1 (HSV-1) infection there is a loss of the serine-2 phosphorylated form of RNA polymerase II (RNAP II) found in elongation complexes. This occurs in part because RNAP II undergoes ubiquitination and proteasomal degradation during times of highly active viral transcription, which may result from stalled elongating complexes. In addition, a viral protein, ICP22, was reported to trigger a loss of serine-2 RNAP II. These findings have led to some speculation that the serine-2 phosphorylated form of RNAP II may not be required for HSV-1 transcription, although this form is required for cellular transcription elongation and RNA processing. Cellular kinase cdk9 phosphorylates serine-2 in the C-terminal domain (CTD) of RNAP II. To determine if serine-2 phosphorylated RNAP II is required for HSV-1 transcription, we inhibited cdk9 during HSV-1 infection and measured viral gene expression. Inhibition was achieved by adding cdk9 inhibitors 5,6-dichlorobenzimidazone-1-β-D-ribofuranoside (DRB) or flavopiridol (FVP) or by expression of a dominant–negative cdk9 or HEXIM1, which in conjunction with 7SK snRNA inhibits cdk9 in complex with cyclin 1. Here we report that inhibition of cdk9 resulted in decreased viral yields and levels of late proteins, poor formation of viral transcription-replication compartments, reduced levels of poly(A)+ mRNA and decreased RNA synthesis as measured by uptake of 5-bromouridine into nascent RNA. Importantly, a global reduction in viral mRNAs was seen as determined by microarray analysis. We conclude that serine-2 phosphorylation of the CTD of RNAP II is required for HSV-1 transcription.

Molecular mechanism of WW‐domain binding protein‐2 coactivation function in estrogen receptor signaling

The link between breast cancer and estrogen receptor (ER) is well established. The ER is a hormone-inducible transcription factor that, upon binding to its ligand, regulates the expression of a variety of genes mainly involved in cell proliferation and differentiation. Coactivators are proteins recruited by the hormone-activated receptor, which allow or enhance the ER transactivation functions by acting as chromatin remodeling enzymes or adaptors between ER and the transcriptional machinery. Our laboratory has previously identified the WW-domain binding protein-2 (WBP-2) as a bona fide coactivator of ER. However, the molecular mechanism underlying WBP-2 coactivation function was not clear yet. In this study, we explore and identify the mechanism by which WBP-2 acts as coactivator of ER. Our data show that WBP-2 is involved in the regulation of ER target genes, and its expression is required for the proper expression of some ER target genes. To clarify the molecular mechanism by which WBP-2 regulates ER function, we performed chromatin immunoprecipitation assays. We demonstrate here that WBP-2 binds to the ER target gene promoter pS2 promoter and is required for the binding of the phosphorylated form of RNA polymerase II (associated with active transcription/elongation) to the same promoter. Furthermore, we also show that WBP-2 is essential for the recruitment of the histone acetyl transferase p300, an important chromatin modifier enzyme and for histone acetylation at the same target region. Collectively, our data indicate that WBP-2 enhances ER transactivation function at certain genes by facilitating the recruitment and/or the stabilization of a histone modifier enzyme that favors a relaxed chromatin structure, permissive of transcription.

Superresolution and Single Molecule Imaging of Transcription by Reflected Light Sheet Microscopy

Understanding gene expression in mammalian cells requires techniques that allow to probe abundance, dynamics and distribution of the key molecular players such as transcription factors, RNA Polymerase and RNA inside the nucleus. To circumvent ensemble averaging of biomolecular dynamics, single molecule sensitivity inside the cell is essential, while superresolution imaging beyond the diffraction limit is needed to resolve the structural architecture of macromolecular assemblies involved in transcription.We achieve both high temporal and spatial resolution in single cells using a new variant of light sheet microscopy. This new technique, Reflected Light Sheet Microscopy, employs a small mirror near the cell to project a light sheet into the nucleus. This optical sectioning reduces out-of-focus background and allows video-rate detection of individual fluorescent proteins and superresolved STORM imaging in mammalian nuclei.In live cells, tracking of single transcription factors labeled with a fluorescent protein reveals the kinetics of free diffusion as well as non specific and specific binding to DNA. Furthermore, STORM imaging in fixed cells using fluorescently labeled antibodies against different phosphorylated forms of RNA polymerase II reveals a homogenous distribution of transcription throughout the nucleus. This superresolution view of mammalian transcription provides no evidence for transcription factories.

Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing

An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. We have identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). Our results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. Our data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control.

Adult Stem Cells Exhibit Global Suppression of RNA Polymerase II Serine-2 Phosphorylation

Adult stem cells, which are characterized by their capacity for self-renewal and differentiation, participate in tissue homeostasis and response to injury. They are thought to enter a state of relative quiescence, known as reversible cell cycle arrest, but the underlying molecular mechanisms remain poorly characterized. Previous data from our laboratory has shown that housekeeping gene expression is downregulated in melanocyte stem cells (MelSCs), suggesting a global suppression of mRNA transcription. We now show, using antibodies against specific phosphorylated forms of RNA polymerase II (RNApII), that adult MelSCs do not undergo productive mRNA transcription elongation, while RNApII is activated and initialized, ready to synthesize mRNA upon stimulation, and that the RNApII kinase CDK9 is absent in adult MelSCs. Interestingly, other adult stem cells also, including keratinocyte, muscle, spermatogonia, and hematopoietic stem cells, showed a similar absence of RNApII phosphorylation. Although it is difficult to show the functional significance of this observation in vivo, CDK9 inhibition resulted in enhanced survival of cells that are deprived from survival factors. We conclude that the absence of productive mRNA transcription is an early, specific, and conserved characteristic of adult stem cells. Downregulation of mRNA transcription may lead to decreased rates of metabolism, and protection from cellular and genetic damage. Screening heterogeneous tissues, including tumors, for transcriptionally quiescent cells may result in the identification of cells with stem cell-like phenotypes.

Genotoxic stress causes the accumulation of the splicing regulator Sam68 in nuclear foci of transcriptionally active chromatin

DNA-damaging agents cause a multifaceted cellular stress response. Cells set in motion either repair mechanisms or programmed cell death pathways, depending on the extent of the damage and on their ability to withstand it. The RNA-binding protein (RBP) Sam68, which is up-regulated in prostate carcinoma, promotes prostate cancer cell survival to genotoxic stress. Herein, we have investigated the function of Sam68 in this cellular response. Mitoxantrone (MTX), a topoisomerase II inhibitor, induced relocalization of Sam68 from the nucleoplasm to nuclear granules, together with several other RBPs involved in alternative splicing, such as TIA-1, hnRNP A1 and the SR proteins SC35 and ASF/SF2. Sam68 accumulation in nuclear stress granules was independent of signal transduction pathways activated by DNA damage. Using BrU labelling and immunofluorescence, we demonstrate that MTX-induced nuclear stress granules are transcriptionally active foci where Sam68 and the phosphorylated form of RNA polymerase II accumulate. Finally, we show that MTX-induced relocalization of Sam68 correlates with changes in alternative splicing of its mRNA target CD44, and that MTX-induced CD44 splicing depends on Sam68 expression. These results strongly suggest that Sam68 is part of a RNA-mediated stress response of the cell that modulates alternative splicing in response to DNA damage.

Phosphorylation of the Pol II CTD by KIN28 Enhances BUR1/BUR2 Recruitment and Ser2 CTD Phosphorylation Near Promoters

Cyclin-dependent kinase BUR1/BUR2 appears to be the yeast ortholog of P-TEFb, which phosphorylates Ser2 of the RNA Pol II CTD, but the importance of BUR1/BUR2 in CTD phosphorylation is unclear. We show that BUR1/BUR2 is cotranscriptionally recruited to the 5' end of ARG1 in a manner stimulated by interaction of the BUR1 C terminus with CTD repeats phosphorylated on Ser5 by KIN28. Impairing BUR1/BUR2 function, or removing the CTD-interaction domain in BUR1, reduces Ser2 phosphorylation in bulk Pol II and eliminates the residual Ser2P in cells lacking the major Ser2 CTD kinase, CTK1. Impairing BUR1/BUR2 or CTK1 evokes a similar reduction of Ser2P in Pol II phosphorylated on Ser5 and in elongating Pol II near the ARG1 promoter. By contrast, CTK1 is responsible for the bulk of Ser2P in total Pol II and at promoter-distal sites. In addition to phosphorylating Ser2 near promoters, BUR1/BUR2 also stimulates Ser2P formation by CTK1 during transcription elongation.

Global Transcriptional Repression in C. elegans Germline Precursors by Regulated Sequestration of TAF-4

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Genomic Distribution of Transcripts and DNA Associated Proteins in One Percent of the Genome of Erythroid and Myeloid Cells.

The NIHGRI has sponsored a multi-group enterprise to use a variety of approaches to map DNA elements in one per cent of the human genome-the ENCODE Project. As part of this effort we have mapped sites of active transcription (transcriptionally active regions or TARS) for myeloid cells including the undifferentiated and retinoic acid treated promyelocytic NB4 cell line and normal neutrophils, as well as the erythroid cell line K562. The results show a large number of intergenic and intronic transcripts, particularly in neutrophils. One feature of note is the amount of cell type specific intergenic transcription in the HoxA cluster. In addition we have been developing simple methods to map the sites of polyadenylation of RNAs and applied this to several cell types. This method also indicates the presence of polyadenylated transcripts derived from the multiple introns of known genes as well as intergenic regions.In parallel with the transcript mapping, we have been performing microarray based chromatin immunoprecipitation studies (Chip-chip) on several factors, analyzing them on oligonucleotide arrays tiling the ENCODE selected genomic regions. These include various phosphorylated forms of RNA polymerase, various methylated forms of histone H3, certain DNA sequence specific transcription factors, and some other factors either broadly involved in chromatin remodeling or specifically found to be associated with certain sequences in the beta globin cluster. Among other things, our results as well as those of other members of the ENCODE consortium, provide a signature motif commonly associated with promoters.

TFIID and Spt-Ada-Gcn5-Acetyltransferase Functions Probed by Genome-wide Synthetic Genetic Array Analysis Using a Saccharomyces cerevisiae taf9-ts Allele

TAF9 is a TATA-binding protein associated factor (TAF) conserved from yeast to humans and shared by two transcription coactivator complexes, TFIID and SAGA. The essentiality of the TAFs has made it difficult to ascertain their roles in TFIID and SAGA function. Here we performed a genomic synthetic genetic array analysis using a temperature-sensitive allele of TAF9 as a query. Results from this experiment showed that TAF9 interacts genetically with: (1) genes for multiple transcription factor complexes predominantly involving Mediator, chromatin modification/remodeling complexes, and regulators of transcription elongation; (2) virtually all nonessential genes encoding subunits of the SWR-C chromatin-remodeling complex and both TAF9 and SWR-C required for expressing the essential housekeeping gene RPS5; and (3) key genes for cell cycle control at the G1/S transition, as well as genes involved in cell polarity, cell integrity, and protein synthesis, suggesting a link between TAF9 function and cell growth control. We also showed that disruption of SAGA by deletion of SPT20 alters histone-DNA contacts and phosphorylated forms of RNA polymerase II at coding sequences. Our results raise the possibility of an unappreciated role for TAF9 in transcription elongation, perhaps in the context of SAGA, and provide further support for TAF9 involvement in cell cycle progression and growth control.

Distribution of different phosphorylated forms of RNA polymerase II in relation to Cajal and PML bodies in human cells: an ultrastructural study

The mammalian nucleus is a highly organised organelle that contains many subcompartments with roles in DNA replication and repair, gene expression and RNA processing. Cajal and promyelocytic leukaemia (PML) bodies are discrete nuclear structures with specific molecular signatures. RNA polymerase II and many transcription factors have been identified within these compartments by immunofluorescence microscopy, suggesting a role in polymerase II assembly or transcriptional activity. Here, we have examined the presence of different phosphorylated forms of polymerase II and newly made RNA in Cajal and PML bodies using high-resolution imaging of ultrathin cryosections (approximately 120 nm thick) with fluorescence and electron microscopes. We show that Cajal bodies contain polymerase II phosphorylated on Ser5, and not the Ser2-phosphorylated (active) form or newly made RNA. The presence of polymerase II in the absence of transcriptional activity suggests that Cajal bodies have roles in polymerase assembly or transport, but not in gene transcription. PML bodies contain no detectable polymerase II or nascent RNA in HeLa cells, at the resolution achieved by electron microscopy, but are often surrounded by these markers at distances>25 nm. These results support the view that although PML bodies are present in transcriptionally active areas of the nucleus, they are not generally sites of polymerase II assembly, transport or activity.

Do nuclear bodies in oocytes of Tenebrio molitor (Coleoptera: Polyphaga, Tenebrionidae) contain two forms of RNA polymerase II?

Late vitellogenic oocytes of the mealworm beetle, Tenebrio molitor, which are transcriptionally inert, contain numerous fibrogranular nuclear bodies (NBs). Previously, we have shown that these NBs contain both unphosphorylated and phosphorylated forms of RNA polymerase II (pol II) [Tissue Cell 33 (2001) 549]. The conclusion on the presence of phosphorylated pol II was based on our immunoelectron experiments with monoclonal antibody (mAb) H5 against the phosphorylated serine-2 of the carboxy-terminal domain (CTD) of pol II. Because the specificity of mAb H5 was recently questioned by demonstration of its cross-reaction with SR-proteins [J. Struct. Biol. 140 (2002) 154], we re-examined here the occurence of pol II in T. molitor oocyte NBs using other appropriate antibodies. We confirm the presence of phosphorylated pol II in NBs using the affinity-purified polyclonal antibody against the phosphorylated CTD. Using double immunogold labeling with this antibody plus mAb 8WG16 against the unphosphorylated CTD, we confirm the presence of two forms of pol II in NBs. Additionally, the presence of pol II in NBs was verified here using mAb ARNA3 against the epitope outside CTD. We suggest that at the transcriptionally inactive stage, T. molitor oocyte NBs represent storage domains for pol II disengaged from the transcription.

Platinum-Induced Autoantibodies Target Nucleoplasmic Antigens Related to Active Transcription

Research on autoimmune diseases has revealed that autoimmunity can be induced by heavy metals such as mercury and gold. Following the introduction of platinum-containing catalytic converters in automobiles, the emission of platinum compounds constitutes an abundant environmental pollutant, however, potential immunological hazards resulting from platinum-containing emissions were not yet examined. In our previous studies on molecular mechanisms of heavy metal-induced autoimmunity, we showed a platinum-dependent subcellular redistribution of the autoantigen fibrillarin from the nucleolus to the nucleoplasm. Since H-2s mice constitute a valuable model to study the role of heavy metals in the development of systemic autoimmunity, we treated susceptible B10.S mice with hexachloroplatinate (Na2PtCl6, Pt4+) to examine whether platinum induces the production of autoantibodies. The present study shows for the first time that chronic administration of Pt4+ generated an autoimmune response in mice which targets distinct nucleoplasmic antigens. Dual-labeling revealed substantial colocalization of these nucleoplasmic autoantigens with (i) nascent RNA, (ii) the active, phosphorylated form of RNA polymerase II, and partial overlap with (iii) acetylated histone 4 protein, and (iv) 20S proteasomes in dendritic cells isolated from platinum-treated mice. The results suggest that platinum elicits antibodies against antigens associated with active sites of transcription which may be subject to proteasomal processing during heavy metal-induced autoimmunity.

The Cdk9 and cyclin T subunits of TAK/P-TEFb localize to splicing factor-rich nuclear speckle regions.

TAK/P-TEFb is an elongation factor for RNA polymerase II-directed transcription that is thought to function by phosphorylating the C-terminal domain of the largest subunit of RNA polymerase II. TAK/P-TEFb is composed of Cdk9 and cyclin T and serves as the cellular cofactor for the human immunodeficiency virus transactivator Tat protein. In this study, we examined the subcellular distribution of Cdk9 and cyclin T1 using high resolution immunofluorescence microscopy and found that Cdk9 and cyclin T1 localized throughout the non-nucleolar nucleoplasm, with increased signal present at numerous foci. Both Cdk9 and cyclin T1 showed only limited colocalization with different phosphorylated forms of RNA polymerase II. However, significant colocalization with antibodies to several splicing factors that identify nuclear 'speckles' was observed for Cdk9 and especially for cyclin T1. The pattern of Cdk9 and cyclin T1 distribution was altered in cells treated with transcription inhibitors. Transient expression of cyclin T1 deletion mutants indicated that a region in the central portion of cyclin T1 is important for accumulation at speckles. Furthermore, cyclin T1 proteins that accumulated at speckles were capable of recruiting Cdk9 and the HIV Tat protein to this compartment in overexpression experiments. These results suggest that cyclin T1 functions to recruit its binding partners to nuclear speckles and raises the possibility that nuclear speckles are a site of TAK/P-TEFb function.