Background

The activity and post-translational regulation of many chromatin-bound proteins are poorly studied due to technical difficulties in isolating them for biochemical analysis. This is, even more, the case with transcription factors, such as the basic Helix-loop-Helix (bHLH) transcription factors that often harbor scarce temporal and spatial pattern of expression in tissues or cellular models (Dennis et al., 2018). Protocol refinement helps to lift technical limitation when the amount of biological materials become a barrier to investigate molecular pathways (Gillotin and Guillemot, 2016). In our recent study, we endeavored to understand how proteolysis of the proneural bHLH transcription factor Ascl1 is regulated in cellular models of neuronal differentiation (Gillotin et al., 2018). For this, we adapted existing protocols for subcellular fractionation to obtain efficient separation of the cytoplasm, nuclear and chromatin fractions. To be able to perform biochemical analysis on the chromatin fraction, we either used sonication to shear nucleic acids or treated this fraction with benzonase to degrade nucleic acids. These steps were essential to perform Western blot analysis or protein pull-down assays (Figure 1). This approach successfully led to the analysis of Ascl1 ubiquitylation pattern in the cytoplasm and in the chromatin fractions (Gillotin et al., 2018) and can be extended to the study of other chromatin-bound proteins and to other post-translational modifications.


Figure 1. Workflow of the protocol. First, the collection of each subcellular fraction is performed by successive protein extractions with the buffer E1, then with the buffer E2 and with the buffer E3. Second, the chromatin fraction is either sonicated to shear the DNA or treated with benzonase to digest nucleic acids. Third, each fraction is biochemically analyzed for a protein of interest. The purity of each fraction should be controlled by Western blot for α-Tubulin, LaminB and Histone H3 depicting respectively the cytoplasmic fraction, the nuclear fraction and the chromatin fraction.