Active transgenes in zebrafish are enriched in acetylated histone H4 and dynamically associate with RNA Pol II and splicing complexes

We have investigated the functional organization of active and silent integrated luciferase transgenes in zebrafish, with the aim of accounting for the variegation of transgene expression in this species. We demonstrate the enrichment of transcriptionally active transgenes in acetylated histone H4 and the dynamic association of the transgenes with splicing factor SC35 and RNA Pol II. Analysis of interphase nuclei and extended chromatin fibers by immunofluorescence and in situ hybridization reveals a co-localization of transgenes with acetylated H4 in luciferase-expressing animals only. Enrichment of expressed transgenes in acetylated H4 is further demonstrated by their co-precipitation from chromatin using anti-acetylated H4 antibodies. Little correlation exists, however, between the level of histone acetylation and the degree of transgene expression. In transgene-expressing zebrafish, most transgenes co-localize with Pol II and SC35, whereas no such association occurs in non-expressing individuals. Inhibition of Pol II abolishes transgene expression and disrupts association of transgenes with SC35, although inactivated transgenes remains enriched in acetylated histones. Exposure of embryos to the histone deacetylation inhibitor TSA induces expression of most silent transgenes. Chromatin containing activated transgenes becomes enriched in acetylated histones and the transgenes recruit SC35 and Pol II. The results demonstrate a correlation between H4 acetylation and transgene activity, and argue that active transgenes dynamically recruit splicing factors and Pol II. The data also suggest that dissociation of splicing factors from transgenes upon Pol II inhibition is not a consequence of changes in H4 acetylation.

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In situ dissection of domain boundaries affect genome topology and gene transcription in Drosophila

10.1101/776542 ◽

2019 ◽

Author(s):

Rodrigo G. Arzate-Mejía ◽

Angel Josué Cerecedo-Castillo ◽

Georgina Guerrero ◽

Mayra Furlan-Magaril ◽

Félix Recillas-Targa

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Regulation Of Gene Expression ◽

Topological Defects ◽

Rna Pol Ii ◽

Pol Ii ◽

Domain Boundaries ◽

Topologically Associated Domains ◽

Genetic Sequences

AbstractThe molecular mechanisms responsible for Topologically Associated Domains (TADs) formation are not yet fully understood. In Drosophila, it has been proposed that transcription is fundamental for TAD organization while the participation of genetic sequences bound by Architectural Proteins (APs) remains controversial. Here, we investigate the contribution of domain boundaries to TAD organization and the regulation of gene expression at the Notch gene locus in Drosophila. We find that deletion of domain boundaries results in TAD fusion and long-range topological defects that are accompanied by loss of APs and RNA Pol II chromatin binding as well as defects in transcription. Together, our results provide compelling evidence on the contribution of discrete genetic sequences bound by APs and RNA Pol II in the partition of the genome into TADs and in the regulation of gene expression in Drosophila.

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NET-prism enables RNA polymerase-dedicated transcriptional interrogation at nucleotide resolution

10.1101/246827 ◽

2018 ◽

Author(s):

Constantine Mylonas ◽

Peter Tessarz

Keyword(s):

Splicing Factors ◽

Elongation Factors ◽

Single Nucleotide ◽

Rna Pol Ii ◽

Pol Ii ◽

Genome Wide ◽

Regulatory Complexity ◽

Pol Ii Pausing ◽

Nucleotide Resolution ◽

The Impact

ABSTRACTThe advent of quantitative approaches that enable interrogation of transcription at single nucleotide resolution has allowed a novel understanding of transcriptional regulation previously undefined. However, little is known, at such high resolution, how transcription factors directly influence RNA Pol II pausing and directionality. To map the impact of transcription/elongation factors on transcription dynamics genome-wide at base pair resolution, we developed an adapted NET-seq protocol called NET-prism (Native Elongating Transcription by Polymerase-Regulated Immunoprecipitants in the Mammalian genome). Application of NET-prism on elongation factors (Spt6, Ssrp1), splicing factors (Sf1), and components of the pre-initiation complex (PIC) (TFIID, and Mediator) reveals their inherent command on transcription dynamics, with regards to directionality and pausing over promoters, splice sites, and enhancers/super-enhancers. NET-prism will be broadly applicable as it exposes transcription factor/Pol II dependent topographic specificity and thus, a new degree of regulatory complexity during gene expression.

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Sequential Entry of Components of Gene Expression Machinery into Daughter Nuclei

Molecular Biology of the Cell ◽

10.1091/mbc.e02-10-0669 ◽

2003 ◽

Vol 14 (3) ◽

pp. 1043-1057 ◽

Cited By ~ 90

Author(s):

Kannanganattu V. Prasanth ◽

Paula A. Sacco-Bubulya ◽

Supriya G. Prasanth ◽

David L. Spector

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Mrna Processing ◽

Mrna Splicing ◽

Splicing Factors ◽

Rna Pol Ii ◽

Pol Ii ◽

General Transcription Factors ◽

Sequential Entry ◽

Processing Factors

In eukaryotic cells, RNA polymerase II (RNA pol II) transcription and pre-mRNA processing are coordinated events. We have addressed how individual components of the transcription and pre-mRNA processing machinery are organized during mitosis and subsequently recruited into the newly formed daughter nuclei. Interestingly, localization studies of numerous RNA pol II transcription and pre-mRNA processing factors revealed a nonrandom and sequential entry of these factors into daughter nuclei after nuclear envelope/lamina formation. The initiation competent form of RNA pol II and general transcription factors appeared in the daughter nuclei simultaneously, but prior to pre-mRNA processing factors, whereas the elongation competent form of RNA pol II was detected even later. The differential entry of these factors rules out the possibility that they are transported as a unitary complex. Telophase nuclei were competent for transcription and pre-mRNA splicing concomitant with the initial entry of the respective factors. In addition, our results revealed a low turnover rate of transcription and pre-mRNA splicing factors during mitosis. We provide evidence to support a model in which the entry of the RNA pol II gene expression machinery into newly forming daughter nuclei is a staged and ordered process.

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The functional organization of RNAs in the mammalian cell nucleus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140129 ◽

1995 ◽

Vol 53 ◽

pp. 750-751

Author(s):

S. Huang ◽

D.L. Spector

Keyword(s):

Functional Organization ◽

Mrna Splicing ◽

Splicing Factors ◽

Specific Antibodies ◽

Rna Transcripts ◽

Eukaryotic Nucleus ◽

Hiv Tat ◽

Relationship Of ◽

The Relationship

Several models have been proposed for the functional organization of RNA within the eukaryotic nucleus and for the relationship of this organization to the distribution of pre-mRNA splicing factors. One model suggests that RNAs which must be spliced are capable of recruiting splicing factors to the sites of transcription from storage and/or reassembly sites. In order to further evaluate this model we have transiently transfected HeLa cells with constructs which express RNA transcripts containing introns, lacking introns, or containing an intron with a deletion at the 3' splice site. The expression of RNAs was detected by in situ hybridization and their association with splicing factors was evaluated by immunostaining using specific antibodies (Y12, SC35) in the same cells. We have found that the majority of the RNA transcripts produced from constructs which express intron-containing genes such as β-globin, tropomyosin, and HIV tat are associated with splicing factors. In contrast, RNAs lacking introns, such as βgalactosidase, and adenovirus VAI, are not associated with splicing factors in the nucleus.

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Organization of transcription and pre-mRNA splicing within the mammalian cell nucleus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014021x ◽

1995 ◽

Vol 53 ◽

pp. 768-769

Author(s):

D.L. Spector ◽

S. Huang ◽

S. Kaurin

Keyword(s):

Rna Polymerase Ii ◽

Cell Nucleus ◽

Functional Organization ◽

Mrna Splicing ◽

Splicing Factors ◽

Interchromatin Granule Clusters ◽

Interchromatin Granule ◽

Nuclear Regions ◽

Relationship Of ◽

Perichromatin Fibrils

We have been interested in the organization of RNA polymerase II transcription and pre-mRNA splicing within the cell nucleus. Several models have been proposed for the functional organization of RNA within the eukaryotic nucleus and for the relationship of this organization to the distribution of pre-mRNA splicing factors. One model suggests that RNAs which must be spliced are capable of recruiting splicing factors to the sites of transcription from storage and/or reassembly sites. When one examines the organization of splicing factors in the nucleus in comparison to the sites of chromatin it is clear that splicing factors are not localized in coincidence with heterochromatin (Fig. 1). Instead, they are distributed in a speckled pattern which is composed of both perichromatin fibrils and interchromatin granule clusters. The perichromatin fibrils are distributed on the periphery of heterochromatin and on the periphery of interchromatin granule clusters as well as being diffusely distributed throughout the nucleoplasm. These nuclear regions have been previously shown to represent initial sites of incorporation of 3H-uridine.

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