Phosphorylation of the HP1β hinge region sequesters KAP1 in heterochromatin and promotes the exit from naïve pluripotency

Weihua Qin,Ignasi Forné,Christopher B Mulholland,M Cristina Cardoso,Irina Solovei,Enes Ugur,Miha Modic,Michael Wierer,Axel Imhof,Heinrich Leonhardt,Martha Smets,Sebastian Bultmann

doi:10.1093/nar/gkab548

Abstract

Heterochromatin binding protein HP1β plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1β−/− embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1β is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1β−/− and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1−/− cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.

Highlights

Heterochromatin binding protein HP1 is a non-histone chromosomal protein and has a function in the establishment and maintenance of higher-order chromatin structures and gene silencing [1,2]
DAPI staining of DNA showed alteration in chromocenter number and size in the HP1β−/− compared to the wt mouse embryonic stem cells (mESCs) (Figure 1A–C)
We found that the dysregulated genes in HP1β−/− and HP1␤ S89A cells overlapped with the pluripotency cell fate (PCF) genes identified previously [50] (Supplementary Figure S5B)

Summary

Introduction

Heterochromatin binding protein HP1 is a non-histone chromosomal protein and has a function in the establishment and maintenance of higher-order chromatin structures and gene silencing [1,2]. HP1 homologues contain two conserved functional domains, an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD), linked by a hinge region. Recent studies testing the capacity of HP1 to induce phase separation revealed that only HP1␣ formed phaseseparated droplets [8,9]. This phase separation correlates with the formation of heterochromatin compartments (chromocenters) in the nucleus. We found that the charge of the hinge IDR (IDR-H) is a distinctive feature of HP1 homologues and plays a decisive role in liquid-liquid phase separation (LLPS) [10,11] and that HP1␤ undergoes phase separation in a histone H3K9me dependent manner [11]. HP1␣/␤ together with other chromatin binding proteins, such as SUV39H1 and KAP1, coalesce around the solid chromatin scaffold [12,13,14,15]

Methods

Results

Conclusion