ABSTRACT
The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.
ABSTRACT
Cohesin is a ring-shaped multiprotein complex that is crucial for 3D genome organization and transcriptional regulation during differentiation and development. It also confers sister chromatid cohesion and facilitates DNA damage repair. Besides its core subunits SMC3, SMC1A, and RAD21, cohesin in somatic cells contains one of two orthologous STAG subunits, STAG1 or STAG2. How these variable subunits affect the function of the cohesin complex is still unclear. STAG1- and STAG2-cohesin were initially proposed to organize cohesion at telomeres and centromeres, respectively. Here, we uncover redundant and specific roles of STAG1 and STAG2 in gene regulation and chromatin looping using HCT116 cells with an auxin-inducible degron (AID) tag fused to either STAG1 or STAG2. Following rapid depletion of either subunit, we perform high-resolution Hi-C, gene expression, and sequential ChIP studies to show that STAG1 and STAG2 do not co-occupy individual binding sites and have distinct ways by which they affect looping and gene expression. These findings are further supported by single-molecule localizations via direct stochastic optical reconstruction microscopy (dSTORM) super-resolution imaging. Since somatic and congenital mutations of the STAG subunits are associated with cancer (STAG2) and intellectual disability syndromes with congenital abnormalities (STAG1 and STAG2), we verified STAG1-/STAG2-dependencies using human neural stem cells, hence highlighting their importance in particular disease contexts.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatin/genetics , Chromosomal Proteins, Non-Histone/metabolism , Gene Expression Regulation , Nuclear Proteins/metabolism , Binding Sites , Cell Cycle Proteins/chemistry , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Diploidy , Humans , Nuclear Proteins/chemistry , Protein Binding , Protein Conformation , Proteolysis , Structure-Activity Relationship , CohesinsABSTRACT
In the nuclei of eukaryotic cells, the genetic information is organized at several levels. First, the DNA is wound around the histone proteins, to form a structure termed as chromatin fiber. This fiber is then arranged into chromatin loops that can cluster together and form higher order structures. This packaging of chromatin provides on one side compaction but also functional compartmentalization. The cohesin complex is a multifunctional ring-shaped multiprotein complex that organizes the chromatin fiber to establish functional domains important for transcriptional regulation, help with DNA damage repair, and ascertain stable inheritance of the genome during cell division. Our current model for cohesin function suggests that cohesin tethers chromatin strands by topologically entrapping them within its ring. To achieve this, cohesin's association with chromatin needs to be very precisely regulated in timing and position on the chromatin strand. Here we will review the current insight in when and where cohesin associates with chromatin and which factors regulate this. Further, we will discuss the latest insights into where and how the cohesin ring opens to embrace chromatin and also the current knowledge about the 'exit gates' when cohesin is released from chromatin.
