Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry.

Chromatin-bound proteins have been conventionally measured through subcellular fractionation followed by immunoblotting or by immunofluorescence microscopy. Here, we present Chromoflow, a protocol for the quantitative analyses of protein levels on chromatin in single cells and throughout the cell cycle using flow cytometry. We describe steps for harvesting cells and for nuclear extraction, and a barcoding strategy to multiplex samples from different conditions that reduces antibody staining variability and eliminates the need for normalization.1,2 We then detail procedures for data acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Alonso-Gil et al. (2023).3.

NIPBL and cohesin: new take on a classic tale.

Cohesin folds the genome in dynamic chromatin loops and holds the sister chromatids together. NIPBLScc2 is currently considered the cohesin loader, a role that may need reevaluation. NIPBL activates the cohesin ATPase, which is required for topological entrapment of sister DNAs and to fuel DNA loop extrusion, but is not required for chromatin association. Mechanistic dissection of these processes suggests that both NIPBL and the cohesin STAG subunit bind DNA. NIPBL also regulates conformational switches of the complex. Interactions of NIPBL with chromatin factors, including remodelers, replication proteins, and the transcriptional machinery, affect cohesin loading and distribution. Here, we discuss recent research addressing how NIPBL modulates cohesin activities and how its mutation causes a developmental disorder, Cornelia de Lange Syndrome (CdLS).

Different NIPBL requirements of cohesin-STAG1 and cohesin-STAG2.

Cohesin organizes the genome through the formation of chromatin loops. NIPBL activates cohesin's ATPase and is essential for loop extrusion, but its requirement for cohesin loading is unclear. Here we have examined the effect of reducing NIPBL levels on the behavior of the two cohesin variants carrying STAG1 or STAG2 by combining a flow cytometry assay to measure chromatin-bound cohesin with analyses of its genome-wide distribution and genome contacts. We show that NIPBL depletion results in increased cohesin-STAG1 on chromatin that further accumulates at CTCF positions while cohesin-STAG2 diminishes genome-wide. Our data are consistent with a model in which NIPBL may not be required for chromatin association of cohesin but it is for loop extrusion, which in turn facilitates stabilization of cohesin-STAG2 at CTCF positions after being loaded elsewhere. In contrast, cohesin-STAG1 binds chromatin and becomes stabilized at CTCF sites even under low NIPBL levels, but genome folding is severely impaired.

The CDK Pef1 and protein phosphatase 4 oppose each other for regulating cohesin binding to fission yeast chromosomes.

Cohesin has essential roles in chromosome structure, segregation and repair. Cohesin binding to chromosomes is catalyzed by the cohesin loader, Mis4 in fission yeast. How cells fine tune cohesin deposition is largely unknown. Here, we provide evidence that Mis4 activity is regulated by phosphorylation of its cohesin substrate. A genetic screen for negative regulators of Mis4 yielded a CDK called Pef1, whose closest human homologue is CDK5. Inhibition of Pef1 kinase activity rescued cohesin loader deficiencies. In an otherwise wild-type background, Pef1 ablation stimulated cohesin binding to its regular sites along chromosomes while ablating Protein Phosphatase 4 had the opposite effect. Pef1 and PP4 control the phosphorylation state of the cohesin kleisin Rad21. The CDK phosphorylates Rad21 on Threonine 262. Pef1 ablation, non-phosphorylatable Rad21-T262 or mutations within a Rad21 binding domain of Mis4 alleviated the effect of PP4 deficiency. Such a CDK/PP4-based regulation of cohesin loader activity could provide an efficient mechanism for translating cellular cues into a fast and accurate cohesin response.