Cancer Biology

RNA polymerase II (RNAPII) transcribes DNA into mRNA and thereby plays a critical role in cellular protein production. In addition, RNAPII plays a central role in DNA damage responses. Measurements of RNAPII on chromatin may thus give insight into several essential processes in eukaryotic cells. During transcription, the C-terminal domain of RNAPII becomes post-translationally modified, and phosphorylation on serine 5 and serine 2 can be used as markers for the promoter proximal and productively elongating forms of RNAPII, respectively. Here, we provide a detailed protocol for the detection of chromatin-bound RNAPII and its serine 5– and serine 2–phosphorylated forms in individual human cells through the cell cycle. We have recently shown that this method can be used to study the effects of ultraviolet DNA damage on RNAPII chromatin binding and that it can even be used to reveal new knowledge about the transcription cycle itself. Other commonly used methods to study RNAPII chromatin binding include chromatin immunoprecipitation followed by sequencing or chromatin fractionation followed by western blotting. However, such methods are frequently based on lysates made from a large number of cells, which may mask population heterogeneity, e.g., due to cell cycle phase. With strengths such as single-cell analysis, speed of use, and accurate quantitative readouts, we envision that our flow cytometry method can be widely used as a complementary approach to sequencing-based methods to study effects of different stimuli and inhibitors on RNAPII-mediated transcription.

Graphical overview

Accurate chromosome segregation during mitosis requires the kinetochore, a large protein complex, which makes a linkage between chromosomes and spindle microtubes. An essential kinetochore component, CENP-C, is phosphorylated by Cyclin-B-Cyclin dependent kinase 1 (CDK1) that is a master kinase for mitotic progression, promoting proper kinetochore assembly during mitosis. Here, we describe an in vitro CDK1 kinase assay to detect CENP-C phosphorylation using Phos-tag SDS-PAGE without radiolabeled ATP. Our protocol has advantages in ease and safety over conventional phosphorylation assays using [γ-³²P]-ATP, which has potential hazards despite their better sensitivity. The protocol described here can be applicable to other kinases and be also useful for analysis of phospho-sites in substrates in vitro.

Cell synchronization is widely used in studying mechanisms involves in regulation of cell cycle progression. Through synchronization, cells at distinct cell cycle stage could be obtained. Thymidine is a DNA synthesis inhibitor that can arrest cell at G1/S boundary, prior to DNA replication. Here, we present the protocol to synchronize cells at G1/S boundary by using double thymidine block. After release into normal medium, cell population at distinct cell cycle phase could be collected at different time points.

Noncanonical Wnt signaling functions independently of the β-catenin pathway to control diverse developmental processes, and dysfunction of the pathway contributes to a number of human pathological conditions, including birth defects and metastatic cancer. Progress in the field, however, has been hampered by the scarcity of functional assays for measuring noncanonical Wnt signaling activity. We recently described the Wnt5a-Ror-Kif26b (WRK) reporter assay, which directly monitors a post-transcriptional regulatory event in noncanonical Wnt signaling. In this protocol, we describe the generation of the stable GFP-Kif26b reporter cell line and a quantitative reporter assay for detecting and measuring Wnt5a signaling activities in live cells via flow cytometry.

The flow cytometric quantitation of DNA content by DNA-binding fluorochrome, propidium iodide (PI) is the most widely used method for cell cycle analysis. However, the commonly used methods are time-consuming and labor-intensive and are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Here, we report an optimized simple protocol for rapid and simultaneous analysis of characteristic mitotic phosphorylated proteins and DNA content, permitting the quantification of cells in mitosis, G₁, S and G₂ stage in a single assay. The protocol detailed here employs detergent-based hypotonic solution to rapidly permeabilize cells and allows simultaneous staining of DNA with PI and mitotic marker, phospho-Histone H3, with specific antibody within 20 min. The protocol requires only inexpensive and commercial available reagents and also enables a rapid and complete analysis of cell cycle profile.