Table 1.

Current approaches to study mitotic gene bookmarking by transcription factors

ApproachApplicationLimitations
Tissue cultureFluorescence activated cell sortingPurified population of mitotic cells using antibodies against mitosis specific histone modifications (e.g., H3S10 and H3S28)Requires large number of cells depending upon downstream application (e.g., a transcription factor ChIP-seq requires larger cell number than an RNA-seq experiments). Can be cost-prohibitive.
Cell synchronizationEnrichment of mitotic population using cell-cycle inhibitors (e.g., nocodazole)Potential artifacts caused by treatment with chemicals. Imprecise enrichment of mitotic population (e.g., depending on the inhibitor used, cells may be synchronized at the boundary of G2–M phases, thus resulting in a heterogenous population of late G2–early M phase cells).
Cell biologicalFixed cell immunofluorescence microscopyVisualization of protein of interest (POI) localization to mitotic chromosomesAntibody specificity. Antibody accessibility to condensed mitotic chromosomes. Fixation artifacts.
Live cell microscopyDynamics of protein localization during mitosisMost approaches require fusion of POI with fluorescence proteins with possible issues associated with overexpression and/or interference of fluorescence proteins with physiologic activity of POI.
High-throughput Imaging (e.g., high-throughput imaging positioning mapping)Covisualization of multiple genes, transcripts and proteinsDeveloping reagents that work together is challenging. Specialized instrumentation and training is required for execution and interpretation of experiments.
BiochemicalTranscription factor (TF) chromatin immunoprecipitation (ChIP)Gene-specific (by qPCR) or genome-wide (by sequencing) protein-chromatin interactions using antibodies against POIAntibody specificity is a key variable and must be determined empirically using supporting approaches (e.g., IP and Western blot). Antibody accessibility to condensed mitotic chromosomes.
Histone posttranslational modification (PTM) ChIPOften done in combination with TF-ChIP to identify epigenetic characteristics of genes bookmarked by POIAlthough antibodies against most histone PTMs are well-characterized, multiple histone PTMs coexist on histone amino terminal tails. A limitation is the chromatin inaccessibility of an antibody against one histone PTM when another PTM is present, thus potentially missing a subset of genes that otherwise contain the PTM under experimental conditions.
FunctionalGlobal run-on sequencingWhole transcriptome analysis of nascently transcribed RNA in mitotically enriched cells in which the POI expression has been modified.Requires complex experimental design to ensure specific and selective detection of transcripts nascently transcribed during and immediately following mitosis in the presence or the absence of POI.
Inducible POI expression/downregulation using lentivirus. Degron systems for inducible degradation of POI at mitosis.Regulated expression of protein of interest at and during mitosis to determine functional relevance of mitotic gene bookmarkingFunctional link between mitotic gene bookmarking and activity of POI requires precise expression (or downregulation) of POI at mitosis, which can be challenging because of the short length of mitosis.