Coordination of di-acetylated histone ligands by the ATAD2 bromodomain

Open access article. Creative Commons Attribution 4.0 International license (CC BY 4.0) appliesThe ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four -helices. ATAD2 functions as a coactivator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up-regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the “RVF” shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.Ye

Coordination of kinase and phosphatase activities by Lem4 enables nuclear envelope reassembly during mitosis

et al.Mitosis in metazoa requires nuclear envelope (NE) disassembly and reassembly. NE disassembly is driven by multiple phosphorylation events. Mitotic phosphorylation of the protein BAF reduces its affinity for chromatin and the LEM family of inner nuclear membrane proteins; loss of this BAF-mediated chromatin-NE link contributes to NE disassembly. BAF must reassociate with chromatin and LEM proteins at mitotic exit to reform the NE; however, how its dephosphorylation is regulated is unknown. Here, we show that the C. elegans protein LEM-4L and its human ortholog Lem4 (also called ANKLE2) are both required for BAF dephosphorylation. They act in part by inhibiting BAF's mitotic kinase, VRK-1, in vivo and in vitro. In addition, Lem4/LEM-4L interacts with PP2A and is required for it to dephosphorylate BAF during mitotic exit. By coordinating VRK-1- and PP2A-mediated signaling on BAF, Lem4/LEM-4L controls postmitotic NE formation in a function conserved from worms to humans.The NIH-funded Caenorhabditis Genetic Center provided some C. elegans strains. I.F.D. was an EMBO Long-Term Fellow, C.A. a Spanish Ministry of Science and Innovation Fellow, and M.G. a Human Frontier Science Program Organization Fellow.Peer reviewe

Coordination of kinase and phosphatase activities by Lem4 enables nuclear envelope reassembly during mitosis

Resumen del trabajo presentado al 4th Spanish Worm Meeting, celebrado en Carmona (Sevilla) del 14 al 15 de marzo de 2013.-- et al.The nuclear envelope (NE) comprises inner and outer nuclear membranes that fuse at nuclear pore complexes. In metazoa the NE is broken down upon entry into mitosis and reformed upon mitotic exit; how this happens is not fully understood. The LEM domain protein family shares a ~40 amino acid domain, first identified in the proteins Lap2, Emerin and Man1. Most LEM proteins contain transmembrane domains, reside in the INM and interact with the nuclear lamina. One described function of the LEM domain is to interact with the essential and highly conserved chromatin-binding protein Barrier-to-autointegration factor (BAF). These BAF-LEM interactions serve as an important link between chromatin and the NE, both through the maintenance of nuclear architecture and during post-mitotic NE reassembly. Numerous studies have shown that NE breakdown (NEBD) and reformation are controlled by protein phosphorylation. Members of the Vaccinia-Related Kinase (VRK) family of mitotic kinases phosphorylate BAF in mitosis and meiosis; this modification strongly reduces the affinity of BAF for chromatin and slightly weakens its affinity for LEM proteins. Phosphorylation of BAF by VRK-1 is therefore proposed to be essential to break the link between chromatin, BAF and LEM proteins upon entry into mitosis. However the mechanism that permits BAF re-association with chromatin upon mitotic exit is unclear. Protein phosphatases regulate numerous processes during mitotic progression but their roles in mitotic exit are largely uncharacterized. A protein phosphatase 2A complex comprising PP2A-CA, PP2A-R1A and PP2A-B55α has been shown to regulate mitotic exit in human cells, although the target(s) of this complex are unknown. Here we show that a PP2A complex regulates BAF chromatin recruitment during mitotic exit and is required to enable BAFʼs essential function in NE assembly. In addition, we show that the uncharacterized LEM protein Lem4 is essential for BAF recruitment to chromatin upon mitotic exit in C. elegans and human cells. Lem4 depletion or mutation causes NE morphology defects and BAF hyperphosphorylation. In vivo and in vitro data show that BAF phosphorylation is dependent on VRK-1 and is counteracted by protein phosphatase 2A (PP2A). We propose an evolutionarily conserved model whereby Lem4 coordinates the control of BAF dephosphorylation by interacting with and inhibiting VRK-1 and recruiting a PP2A complex to BAF. This results in BAF dephosphorylation and chromatin recruitment, thereby facilitating NE assembly during mitotic exit.Peer reviewe

The Caenorhabditis elegans SUN protein UNC-84 interacts with lamin to transfer forces from the cytoplasm to the nucleoskeleton during nuclear migration.

Nuclear migration is a critical component of many cellular and developmental processes. The nuclear envelope forms a barrier between the cytoplasm, where mechanical forces are generated, and the nucleoskeleton. The LINC complex consists of KASH proteins in the outer nuclear membrane and SUN proteins in the inner nuclear membrane that bridge the nuclear envelope. How forces are transferred from the LINC complex to the nucleoskeleton is poorly understood. The Caenorhabditis elegans lamin, LMN-1, is required for nuclear migration and interacts with the nucleoplasmic domain of the SUN protein UNC-84. This interaction is weakened by the unc-84(P91S) missense mutation. These mutant nuclei have an intermediate nuclear migration defect-live imaging of nuclei or LMN-1::GFP shows that many nuclei migrate normally, others initiate migration before subsequently failing, and others fail to begin migration. At least one other component of the nucleoskeleton, the NET5/Samp1/Ima1 homologue SAMP-1, plays a role in nuclear migration. We propose a nut-and-bolt model to explain how forces are dissipated across the nuclear envelope during nuclear migration. In this model, SUN/KASH bridges serve as bolts through the nuclear envelope, and nucleoskeleton components LMN-1 and SAMP-1 act as both nuts and washers on the inside of the nucleus

ATAD2 is an epigenetic reader of newly synthesized histone marks during DNA replication

ATAD2 (ATPase family AAA domain-containing protein 2) is a chromatin regulator harboring an AAA+ ATPase domain and a bromodomain, previously proposed to function as an oncogenic transcription co-factor. Here we suggest that ATAD2 is also required for DNA replication. ATAD2 is co-expressed with genes involved in DNA replication in various cancer types and predominantly expressed in S phase cells where it localized on nascent chromatin (replication sites). Our extensive biochemical and cellular analyses revealed that ATAD2 is recruited to replication sites through a direct interaction with di-acetylated histone H4 at K5 and K12, indicative of newly synthesized histones during replication-coupled chromatin reassembly. Similar to ATAD2-depletion, ectopic expression of ATAD2 mutants that are deficient in binding to these di-acetylation marks resulted in reduced DNA replication and impaired loading of PCNA onto chromatin, suggesting relevance of ATAD2 in DNA replication. Taken together, our data show a novel function of ATAD2 in cancer and for the first time identify a reader of newly synthesized histone di-acetylation-marks during replication