Subnuclear Spatial Structuring of Chromatin and Polymerase II during Transcription Activation of the Zebrafish Zygotic Genome

Abstract

The relevance of spatial organization of chromatin to regulation of transcription is increasingly recognized [1]. Conversely, it was recently suggested that the level of general transcription activity affects subnuclear chromatin distribution [2]. Here, we ask how a general increase in transcription by polymerase II changes subnuclear chromatin distribution. Specifically, we used the transition of early zebrafish embryos from transcription quiescence to fully established transcription (Zygotic Genome Activation - ZGA) as a naturally occurring example of transcription level increase. General transcription activity occurs only following ZGA, i.e. 10 synchronous cell divisions after initial fertilization (three hours post fertilization). To make subnuclear distribution changes during ZGA accessible to super-resolution microscopy, we developed a dissociated cell culture protocol. Zebrafish embryos were dissociated at the 128-cell stage (three cell divisions before ZGA). Cultured cells developed transcription activity and morphological hallmarks of ZGA, confirming occurrence of native ZGA. We obtained preliminary results by wide-field fluorescence microscopy. Using fluorescently labeled histone proteins, which spontaneously bind DNA, we visualized a fine-grained chromatin structure in post-ZGA interphase nuclei in vivo. Utilizing antibody fragments (Fabs), we detected agglomerations of transcribing polymerase II in vivo. These agglomerations appeared as micron-sized, approximately spherical “transcription hot spots”. Few large transcription hot spots occurred at the 256-cell stage; in consecutive cell cycles more and smaller spots appeared. Any distinct signal of transcribing polymerase II disappeared during cell divisions, when no transcription occurs, thus confirming specificity of Fab-based detection of actively transcribing polymerase II. In the future, we will employ super-resolution microscopy to resolve the fine structure of chromatin and transcription hot spots in vivo throughout native zebrafish ZGA. [1] Gavrilov and Razin, Molecular Biology, 2015. [2] Popken et al., Nucleus, 2015.