Studying Chromatin Epigenetics with Fluorescence Microscopy.

Epigenetic modifications of histones (methylation, acetylation, phosphorylation, etc.) are of great importance in determining the functional state of chromatin. Changes in epigenome underlay all basic biological processes, such as cell division, differentiation, aging, and cancerous transformation. Post-translational histone modifications are mainly studied by immunoprecipitation with high-throughput sequencing (ChIP-Seq). It enables an accurate profiling of target modifications along the genome, but suffers from the high cost of analysis and the inability to work with living cells. Fluorescence microscopy represents an attractive complementary approach to characterize epigenetics. It can be applied to both live and fixed cells, easily compatible with high-throughput screening, and provide access to rich spatial information down to the single cell level. In this review, we discuss various fluorescent probes for histone modification detection. Various types of live-cell imaging epigenetic sensors suitable for conventional as well as super-resolution fluorescence microscopy are described. We also focus on problems and future perspectives in the development of fluorescent probes for epigenetics.

Fluorescence imaging of epigenetic genome modifications.

Epigenome contains a lot of information about cell state. Epigenetic analysis includes primarily sequence-based methods, which provide detailed data on distribution of modifications along the genome, but are poorly applicable for screenings. Specific fluorescence labeling and imaging of epigenetic modifications is an attractive complementary approach. It is currently based mainly on histone modifications study. We expect that inclusion of DNA modifications into imaging-based study would empower the method. In this review we discuss methods for fluorescence imaging of DNA modifications (mainly 5-methylcytosine). It opens an easy way to single cell analysis and high-throughput screening. Moreover, tracking epigenome changes in live cells becomes possible with genetically encoded probes.

Role of RNA Biogenesis Factors in the Processing and Transport of Human Telomerase RNA.

Telomerase RNA has long been considered to be a noncoding component of telomerase. However, the expression of the telomerase RNA gene is not always associated with telomerase activity. The existence of distinct TERC gene expression products possessing different functions were demonstrated recently. During biogenesis, hTR is processed by distinct pathways and localized in different cell compartments, depending on whether it functions as a telomerase complex component or facilitates antistress activities as a noncoding RNA, in which case it is either processed in the mitochondria or translated. In order to identify the factors responsible for the appearance and localization of the exact isoform of hTR, we investigated the roles of the factors regulating transcription DSIF (Spt5) and NELF-E; exosome-attracting factors ZCCHC7, ZCCHC8, and ZFC3H1; ARS2, which attracts processing and transport factors; and transport factor PHAX during the biogenesis of hTR. The data obtained revealed that ZFC3H1 participates in hTR biogenesis via pathways related to the polyadenylated RNA degradation mechanism. The data revealed essential differences that are important for understanding hTR biogenesis and that are interesting for further investigations of new, therapeutically significant targets.