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.
Collas
P
P
Department of Biochemistry, Norwegian College of Veterinary Medicine, Oslo, Norway. philippe.collas@basalmed.uio.no
Liang
M R
MR
Vincent
M
M
Aleström
P
P
D000107:Acetylation; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004004:Dichlororibofuranosylbenzimidazole / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D017930:Genes, Reporter; D006657:Histones / Q000378:metabolism*; D006877:Hydroxamic Acids / Q000378:metabolism; D015151:Immunoblotting; D017404:In Situ Hybridization, Fluorescence; D008156:Luciferases / Q000378:metabolism; D009687:Nuclear Proteins; D011233:Precipitin Tests; D012319:RNA Polymerase II / Q000378:metabolism*; D012326:RNA Splicing / Q000502:physiology*; D012261:Ribonucleoproteins; D019076:Transgenes; D015027:Zebrafish / Q000235:genetics*
D002843:Chromatin; D004791:Enzyme Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D009687:Nuclear Proteins; D012261:Ribonucleoproteins; C012589:trichostatin A; D004004:Dichlororibofuranosylbenzimidazole; D008156:Luciferases; D012319:RNA Polymerase II