scholarly journals RNA polymerase II interacts with the Hspa1b promoter in mouse epididymal spermatozoa

Reproduction ◽  
2009 ◽  
Vol 137 (6) ◽  
pp. 923-929 ◽  
Author(s):  
Donald C Wilkerson ◽  
Kevin D Sarge
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Stage ◽  
Western Blots ◽  
Pol Ii ◽  
Epididymal Spermatozoa ◽  
The One ◽  
Genome Activation ◽  
Embryonic Viability ◽  
Zygotic Genome

TheHspa1b(Hsp70.1) gene is one of the first genes expressed after fertilization, with expression occurring during the minor zygotic genome activation (ZGA) in the absence of stress. This expression can take place in the male pronucleus as early as the one-cell stage of embryogenesis. The importance of HSPA1B for embryonic viability during times of stress is supported by studies showing that depletion of this protein results in a significant reduction in embryos developing to the blastocyte stage. Recently, we have begun addressing the mechanism responsible for allowing expression ofHspa1bduring the minor ZGA and found that heat shock transcription factor (HSF) 1 and 2 bind theHspa1bpromoter during late spermatogenesis. In this report, we have extended those studies using western blots and chromatin immunoprecipitation assays and found that RNA polymerase II (Pol II) is present in epididymal spermatozoa and bound to theHspa1bpromoter. These present results, in addition to our previous results, support a model in which the binding of HSF1, HSF2, SP1, and Pol II to the promoter ofHspa1bwould allow the rapid formation of a transcription-competent state during the minor ZGA, thereby allowingHspa1bexpression.

61 REVERSIBLE INHIBITION OF BOVINE MINOR EMBRYONIC GENOME ACTIVATION IMPAIRS PRE-IMPLANTATION DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 138
Author(s):  
R. P. Nociti ◽  
R. V. Sampaio ◽  
V. F. M. H. de Lima ◽  
R. M. Schultz ◽  
P. J. Ross
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Reversible Inhibitor ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Embryonic Genome Activation ◽  
Transcriptional Inhibition ◽  
Embryonic Genome ◽  
Genome Activation

Bovine pre-implantation embryos can develop in the absence of gene expression up to the 8/16-cell stage, the time when the major embryonic genome activation (EGA) occurs. Some embryonic genes, however, are transcribed before EGA (minor EGA). This study used a reversible inhibitor of RNA Polymerase II (5,6 dichlorobenzimidazole 1-β-D-ribofuranoside; DRB) to assess the importance of minor EGA for development to the blastocyst stage. Oocytes were matured and inseminated in vitro, and the fertilized eggs were cultured in supplemented KSOMaa and allocated to different treatments 16 h post-insemination (hpi). Development was recorded at 44 and 72 hpi, and the incidence of blastocyst formation on Day 7 (IVF = Day 0) was recorded. Data were analysed by ANOVA followed by Duncan test. First, we tested different DRB concentrations [50 μM (D50), 75 μM (D75), 100 μM (D100), and dimethyl sulfoxide vehicle control (CTRL)] to block development to blastocyst when continuously present. Only embryos in CTRL produced blastocysts (45.0 ± 5.8%; 4 replicates with a total of 391 oocytes examined). No difference in development was observed at 44 h (57.9 ± 16.5, 53.3 ± 10.5, 60.5 ± 19.0, and 52.3 ± 5.8% for D50, D75, D100, and CTRL, respectively) and 72 h (78.9 ± 8.8, 66.1 ± 11.7, 71.5 ± 16.5, and 70.8 ± 5.6% for D50, D75, D100, and CTRL, respectively). Next, in 7 replicates (751 oocytes) we determined the effect of blocking transcription (50 μM DRB) spanning 2 embryo stages (periods of 28 h), initiated at 16 hpi (1&2C), 30 hpi (2&4C), and 44 hpi (4&8C). Controls included DRB treatment from 16 to 72 hpi (1–8C) and CTRL. There was no difference in development at 44 and 72 h. The incidence of blastocyst formation, however, was significantly decreased in all treatment groups compared with CTRL (27.7 ± 4.7; 15.1 ± 3.5; 23.3 ± 3.1; 20.5 ± 1.9; and 42.1 ± 3.2% for 1&2C, 2&4C, 4&8C, 1–8C, and CTRL, respectively). Finally, in 12 replicates (1499 oocytes), the effect of blocking transcription for 14-h periods, spanning mostly a unique cleavage stage, was evaluated. The DRB treatment (50 μM) started at 16 hpi (1C), 30 hpi (2C), 44 hpi (4C), and 58 hpi (8C). Furthermore, 1–16C and CTRL treatments were included. No difference in development at 44 and 72 h were observed. Development to the blastocyst was significantly lower from CTRL (46.0 ± 3.2%) in 2C, 4C, 8C, and 1–16C (28.9 ± 3.9, 26.1 ± 4.2, 30.1 ± 4.8, and 18.9 ± 3.2%, respectively) but not in 1C (34.7 ± 4.4%). In summary, continuous transcriptional inhibition using DRB resulted in a developmental block at the time of major EGA, similar to α-amanitin treatment (an irreversible RNA Polymerase II inhibitor). Transcriptional inhibition during single cleavage stages was sufficient to decrease the developmental potential of the embryo. We conclude that minor EGA has an important role for bovine development. This work was funded by NIH-NICHD R01HD070044 to P. J. Ross. R. P. Nociti was sponsored by CNPQ; R. V. Sampaio was sponsored by FAPESP.

scholarly journals The miR-430 locus with extreme promoter density is a transcription body organizer, which facilitates long range regulation in zygotic genome activation

2021 ◽  
Author(s):  
Yavor Hadzhiev ◽  
Lucy Wheatley ◽  
Ledean Cooper ◽  
Federico Ansaloni ◽  
Celina Whalley ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Gene Cluster ◽  
Core Promoter ◽  
Single Copy ◽  
Early Embryo ◽  
Zygotic Genome Activation ◽  
Pol Ii ◽  
A Minor ◽  
Genome Activation ◽  
Zygotic Genome

In anamniote embryos the major wave of zygotic genome activation (ZGA) starts during the mid-blastula transition. This major wave of ZGA is facilitated by several mechanisms, including dilution of repressive maternal factors and accumulation of activating transcription factors during the fast cell division cycles preceding the mid-blastula transition. However, a set of genes escape global genome repression and are activated substantially earlier, during what is called, the minor wave of genome activation. While the mechanisms underlying the major wave of genome activation have been studied extensively, the minor wave of genome activation is little understood. In zebrafish the earliest expressed RNA polymerase II (Pol II) transcribed genes are activated in a pair of large transcription bodies depleted of chromatin, abundant in elongating Pol II and nascent RNAs (Hadzhiev et al., 2019; Hilbert et al., 2021). This transcription body includes the miR-430 gene cluster required for maternal mRNA clearance. Here we explored the genomic, chromatin organisation and cis-regulatory mechanisms of the minor wave of genome activation occurring in the transcription body. By long read genome sequencing we identified a remarkable cluster of miR-430 genes with over 300 promoters and spanning 0.6 Mb, which represent the highest promoter density of the genome. We demonstrate that the miR-430 gene cluster is required for the formation of the transcription body and acts as a transcription organiser for minor wave activation of a set of zinc finger genes scattered on the same chromosome arm, which share promoter features with the miR-430 cluster. These promoter features are shared among minor wave genes overall and include the TATA-box and sharp transcription start site profile. Single copy miR-430 promoter transgene reporter experiments indicate the importance of promoter-autonomous mechanisms regulating escape from global repression of the early embryo. These results together suggest that formation of the transcription body in the early embryo is the result of high promoter density coupled to a minor wave-specific core promoter code for transcribing key minor wave ZGA genes, which are required for the overhaul of the transcriptome during early embryonic development.

scholarly journals Artificial Recruitment of TFIID, but Not RNA Polymerase II Holoenzyme, Activates Transcription in Mammalian Cells

2000 ◽  
Vol 20 (12) ◽  
pp. 4350-4358 ◽  
Author(s):  
David R. Dorris ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Pol Ii ◽  
Activation Domains ◽  
Synergistic Activation ◽  
The One ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT In yeast cells, transcriptional activation occurs when the RNA polymerase II (Pol II) machinery is artificially recruited to a promoter by fusing individual components of this machinery to a DNA-binding domain. Here, we show that artificial recruitment of components of the TFIID complex can activate transcription in mammalian cells. Surprisingly, artificial recruitment of TATA-binding protein (TBP) activates transiently transfected and chromosomally integrated promoters with equal efficiency, whereas artificial recruitment of TBP-associated factors activates only chromosomal reporters. In contrast, artificial recruitment of various components of the mammalian Pol II holoenzyme does not confer transcriptional activation, nor does it result in synergistic activation in combination with natural activation domains. In the one case examined in more detail, the Srb7 fusion failed to activate despite being associated with the Pol II holoenzyme and being directly recruited to the promoter. Interestingly, some acidic activation domains are less effective when the promoter is chromosomally integrated rather than transiently transfected, whereas the Sp1 glutamine-rich activation domain is more effective on integrated reporters. Thus, yeast and mammalian cells differ with respect to transcriptional activation by artificial recruitment of the Pol II holoenzyme.

scholarly journals Development, embryonic genome activity and mitochondrial characteristics of bovine–pig inter-family nuclear transfer embryos

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 273-285 ◽  
Author(s):  
Irina Lagutina ◽  
Helena Fulka ◽  
Tiziana A L Brevini ◽  
Stefania Antonini ◽  
Dario Brunetti ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Transfer ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Pol I ◽  
Embryonic Genome ◽  
Genome Activity

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine–pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency gene NANOG and alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos, NANOG was not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16–25-cell stage for pig–bovine iSCNT embryos and at the four-cell stage for bovine–pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus–cytoplasm compatibility between these animal species.

scholarly journals Chromatin accessibility established by Pou5f3, Sox19b and Nanog primes genes for activity during zebrafish genome activation

2019 ◽  
Author(s):  
Máté Pálfy ◽  
Gunnar Schulze ◽  
Eivind Valen ◽  
Nadine L. Vastenhouw
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Zebrafish Genome ◽  
Zygotic Genome Activation ◽  
Lineage Specification ◽  
Genome Activation ◽  
Zygotic Genome

ABSTRACTIn many organisms, early embryonic development is driven by maternally provided factors until the controlled onset of transcription during zygotic genome activation. The regulation of chromatin accessibility and its relationship to gene activity during this transition remains poorly understood. Here, we generated chromatin accessibility maps from genome activation until the onset of lineage specification. During this period, chromatin accessibility increases at regulatory elements. This increase is independent of RNA polymerase II-mediated transcription, with the exception of the hyper-transcribed miR-430 locus. Instead, accessibility often precedes the transcription of associated genes. Loss of the maternal transcription factors Pou5f3, Sox19b, and Nanog, which are known to be required for zebrafish genome activation, results in decreased accessibility at regulatory elements. Importantly, the accessibility of regulatory regions, especially when established by Pou5f3, Sox19b and Nanog, is predictive for future transcription. Our results show that the maternally provided transcription factors Pou5f3, Sox19b, and Nanog open up chromatin and prime genes for activity during zygotic genome activation in zebrafish.

scholarly journals Symmetrically dimethylated histone H3R2 promotes global transcription during minor zygotic genome activation in mouse pronuclei

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kohtaro Morita ◽  
Yuki Hatanaka ◽  
Shunya Ihashi ◽  
Masahide Asano ◽  
Kei Miyamoto ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Arginine Methylation ◽  
Dna Demethylation ◽  
Zygotic Genome Activation ◽  
H3k4 Methylation ◽  
Cell Stage ◽  
Paternal Genome ◽  
Genome Activation ◽  
Histone Arginine Methylation ◽  
Zygotic Genome

AbstractPaternal genome reprogramming, such as protamine–histone exchange and global DNA demethylation, is crucial for the development of fertilised embryos. Previously, our study showed that one of histone arginine methylation, asymmetrically dimethylated histone H3R17 (H3R17me2a), is necessary for epigenetic reprogramming in the mouse paternal genome. However, roles of histone arginine methylation in reprogramming after fertilisation are still poorly understood. Here, we report that H3R2me2s promotes global transcription at the 1-cell stage, referred to as minor zygotic genome activation (ZGA). The inhibition of H3R2me2s by expressing a histone H3.3 mutant H3.3R2A prevented embryonic development from the 2-cell to 4-cell stages and significantly reduced global RNA synthesis and RNA polymerase II (Pol II) activity. Consistent with this result, the expression levels of MuERV-L as minor ZGA transcripts were decreased by forced expression of H3.3R2A. Furthermore, treatment with an inhibitor and co-injection of siRNA to PRMT5 and PRMT7 also resulted in the attenuation of transcriptional activities with reduction of H3R2me2s in the pronuclei of zygotes. Interestingly, impairment of H3K4 methylation by expression of H3.3K4M resulted in a decrease of H3R2me2s in male pronuclei. Our findings suggest that H3R2me2s together with H3K4 methylation is involved in global transcription during minor ZGA in mice.

scholarly journals Structure of the p53/RNA polymerase II assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shu-Hao Liou ◽  
Sameer K. Singh ◽  
Robert H. Singer ◽  
Robert A. Coleman ◽  
Wei-Li Liu
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
P53 Protein ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Dna Binding Activity ◽  
Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

scholarly journals RecQL5 Promotes Genome Stabilization through Two Parallel Mechanisms—Interacting with RNA Polymerase II and Acting as a Helicase

2010 ◽  
Vol 30 (10) ◽  
pp. 2460-2472 ◽  
Author(s):  
M. Nurul Islam ◽  
David Fox ◽  
Rong Guo ◽  
Takemi Enomoto ◽  
Weidong Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genome Stability ◽  
Transcriptional Regulators ◽  
Helicase Activity ◽  
Parallel Mechanisms ◽  
Recq Helicase ◽  
Pol Ii ◽  
Kix Domain ◽  
Genome Stabilization

ABSTRACT The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

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