Pioneer activity distinguishes activating from non-activating SOX2 binding sites.

Genome-wide transcriptional activity involves the binding of many transcription factors (TFs) to thousands of sites in the genome. Pioneer TFs are a class of TFs that maintain open chromatin and allow non-pioneer TFs access to their target sites. Determining which TF binding sites directly drive transcription remains a challenge. Here, we use acute protein depletion of the pioneer TF SOX2 to establish its functionality in maintaining chromatin accessibility. We show that thousands of accessible sites are lost within an hour of protein depletion, indicating rapid turnover of these sites in the absence of the pioneer factor. To understand the relationship with transcription, we performed nascent transcription analysis and found that open chromatin sites that are maintained by SOX2 are highly predictive of gene expression, in contrast to all other SOX2 binding sites. We use CRISPR-Cas9 genome editing in the Klf2 locus to functionally validate a predicted regulatory element. We conclude that the regulatory activity of SOX2 is exerted mainly at sites where it maintains accessibility and that other binding sites are largely dispensable for gene regulation.

Acute Protein Depletion Strategies to Functionally Dissect the 3D Genome.

The organization of the genome inside the nucleus facilitates many nuclear processes. Because the nuclear genome is highly dynamic and often regulated by essential proteins, rapid depletion strategies are necessary to perform loss-of-function analyses. Fortunately, in recent years, various methods have been developed to manipulate the cellular levels of a protein directly and acutely. Here, we describe different methods that have been developed to rapidly deplete proteins from cells, with a focus on auxin inducible degron and dTAG methods, as these are most commonly used in 3D genome organization studies. We outline best practices for designing a knockin strategy, as well as generation and validation of knockin cell lines. Acute depletion strategies have been transformative for the study of the 3D genome and will be important tools for delineating the processes and factors that determine organization of the genome inside the nucleus.

WAPL maintains a cohesin loading cycle to preserve cell-type-specific distal gene regulation.

The cohesin complex has an essential role in maintaining genome organization. However, its role in gene regulation remains largely unresolved. Here we report that the cohesin release factor WAPL creates a pool of free cohesin, in a process known as cohesin turnover, which reloads it to cell-type-specific binding sites. Paradoxically, stabilization of cohesin binding, following WAPL ablation, results in depletion of cohesin from these cell-type-specific regions, loss of gene expression and differentiation. Chromosome conformation capture experiments show that cohesin turnover is important for maintaining promoter-enhancer loops. Binding of cohesin to cell-type-specific sites is dependent on the pioneer transcription factors OCT4 (POU5F1) and SOX2, but not NANOG. We show the importance of cohesin turnover in controlling transcription and propose that a cycle of cohesin loading and off-loading, instead of static cohesin binding, mediates promoter and enhancer interactions critical for gene regulation.