The binding of lamin B receptor to chromatin is regulated by phosphorylation in the RS region

106

Background: LBR is an inner nuclear membrane protein that participates in heterochromatin organization. Results: LBR recognizes specific histone modifications and induces chromatin compaction and transcriptional repression. Conclusion: LBR tethers epigenetically marked chromatin to the NE to repress transcription. Significance: This finding provides an implication of how transcriptional activities are repressed beneath the NE.

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Lamin B Receptor Recognizes Specific Modifications of Histone H4 in Heterochromatin Formation

Hirano

Hizume

Kimura

et al. 2012

106

“…The assembly and disassembly of the nuclear envelope in the cell cycle accompany association and dissociation of the inner nuclear membrane with and from chromatin, respectively. All major inner nuclear membrane proteins, such as the laminaassociated polypeptide 2 (LAP2) family, LBR, and emerin, are known to bind to chromatin in vitro (5,6) and to accumulate on the surface of chromatin during the anaphase-telophase transition in vivo (7). Therefore, the binding of these proteins to chromatin is thought to support the binding of the inner nuclear membrane to chromatin.…”

mentioning

confidence: 99%

Regulation of Binding of Lamin B Receptor to Chromatin by SR Protein Kinase and cdc2 Kinase in Xenopus Egg Extracts

Takano

Koyama

Ito

et al. 2004

Self Cite

Participation of multiple kinases in regulation of the binding of lamin B receptor (LBR) to chromatin was suggested previously (Takano, M., Takeuchi, M., Ito, H., Furukawa, K., Sugimoto, K., Omata, S., and Horigome, T. (2002) Eur. J. Biochem. 269, 943-953). To identify these kinases, regulation of the binding of the nucleoplasmic region (NK, amino acid residues 1-211) of LBR to sperm chromatin was studied using a cell cycle-dependent Xenopus egg extract in vitro. The binding was stimulated on specific phosphorylation of the NK fragment by an S-phase egg extract. Protein depletion with beads bearing SF2/ASF, which binds SR protein kinases, abolished this stimulation, suggesting that an SR protein kinase(s) is responsible for the activation of LBR. This was confirmed by direct phosphorylation and activation with recombinant SR protein-specific kinase 1. The binding of the NK fragment to chromatin pretreated with an S-phase extract was suppressed by incubation with an M-phase extract. Enzyme inhibitor experiments revealed that multiple kinases participate in the suppression. One of these kinases was shown to be cdc2 kinase using a specific inhibitor, roscovitine, and protein depletion with beads bearing p13, which specifically binds cdc2 kinase. Experiments involving a mutant NK fragment showed that the phosphorylation of serine 71 by cdc2 kinase is responsible for the suppression.The nuclear envelope separates the nucleoplasm from the cytoplasm and thereby organizes the nuclear architecture. The nuclear envelope consists of two lipid bilayers (inner and outer), nuclear pore complexes, and the nuclear lamina and undergoes repeated dynamic assembly and disassembly in every cell cycle in higher eukaryotes. Studies on the molecular mechanisms underlying nuclear envelope assembly and disassembly are important to understand the mechanisms underlying cell division and prenucleus formation. Studies on the organization of the nuclear architecture by the nuclear envelope during the cell cycle are also important to understand how mutants of nuclear envelope proteins, i.e. lamin A/C (1), emerin (2), and lamin B receptor (LBR) 1 (3), cause genetic diseases (4). The assembly and disassembly of the nuclear envelope in the cell cycle accompany association and dissociation of the inner nuclear membrane with and from chromatin, respectively. All major inner nuclear membrane proteins, such as the laminaassociated polypeptide 2 (LAP2) family, LBR, and emerin, are known to bind to chromatin in vitro (5, 6) and to accumulate on the surface of chromatin during the anaphase-telophase transition in vivo (7). Therefore, the binding of these proteins to chromatin is thought to support the binding of the inner nuclear membrane to chromatin. Indeed, LBR and LAP2 have been demonstrated to play functional roles in the interaction of the nuclear envelope and chromatin through in vitro experiments as follows. Depletion of LBR from nuclear envelope precursor vesicles suppresses nuclear assembly (8), neutralization of LBR by antibodies in a sea urc...

“…Preparation of Demembranated Sperm Chromatin-Demembranated sperm chromatin was prepared as described (33) and stored at Ϫ80°C at a concentration of 40,000/l.…”

Section: Methodsmentioning

confidence: 99%

Participation of a Fusogenic Protein, Glyceraldehyde-3-phosphate Dehydrogenase, in Nuclear Membrane Assembly

Nakagawa

Hirano

Inomata

et al. 2003

Self Cite

We found an autoimmune serum, K199, that strongly suppresses nuclear membrane assembly in a cell-free system involving a Xenopus egg extract. Four different antibodies that suppress nuclear assembly were affinity-purified from the serum using Xenopus egg cytosol proteins. Three proteins recognized by these antibodies were identified by partial amino acid sequencing to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1,6-bisphosphate aldolase, and the regulator of chromatin condensation 1. GAPDH is known to be a fusogenic protein. To verify the participation of GAPDH in nuclear membrane fusion, authentic antibodies against human and rat GAPDH were applied, and strong suppression of nuclear assembly at the nuclear membrane fusion step was observed. The nuclear assembly activity suppressed by antibodies was recovered on the addition of purified chicken GAPDH. A peptide with the sequence of amino acid residues 70 -94 of GAPDH, which inhibits GAPDH-induced phospholipid vesicle fusion, inhibited nuclear assembly at the nuclear membrane fusion step. We propose that GAPDH plays a crucial role in the membrane fusion step in nuclear assembly in a Xenopus egg extract cell-free system. The nuclear envelope (NE)1 of eukaryotes is composed of inner and outer nuclear membranes, nuclear pore complexes, and the nuclear lamina. NE breakdown during the mitotic prophase results in the dispersal of both nuclear membranes into the mitotic endoplasmic reticulum (ER) network. Inner nuclear membrane proteins such as lamin B receptor, laminaassociated polypeptide 2␤, and emerin reconcentrate on the surface of decondensing chromatin during the late anaphase, and then the NE is re-formed (1, 2). These intrinsic membrane proteins and lamins are believed to play a critical role in NE reassembly (3-5). NE assembly can be studied in vitro using extracts of meiotic or mitotic cells (6, 7). The assembly requires cytosolic factors and is inhibited by a non-hydrolyzable GTP analogue, N-ethylmaleimide, and a calcium chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid) (8). In vitro, NE assembly is initiated by the binding of membrane vesicles to decondensed chromatin in an energy-independent manner (9 -11). Once bound to chromatin, membrane vesicles fuse and flatten. The insertion of nuclear pore complexes and the expansion of the NE require both energy and cytosolic components (12). The fusion of vesicles on chromatin is inhibited by a non-hydrolyzable GTP analogue (8, 13) and requires Ran GTPase and a regulator of chromatin condensation 1, RCC1/Ran GTP exchange factor (14 -16). Remarkably, agarose beads coated with Ran allow the assembly of a nuclear pore complex-containing NE (17, 18). After the formation of a closed NE around chromatin, further NE growth requires nucleocytoplasmic transport and further membrane fusion (8,19). Analysis of membrane fractions of a Xenopus egg extract suggested that the population of vesicles required for NE assembly is not uniform and that different membranes might be required fo...