Regulator Of Chromosome Condensation Research Articles

Mutations in Microcephalin Cause Aberrant Regulation of Chromosome Condensation

Microcephalin (MCPH1) is a gene mutated in primary microcephaly, an autosomal recessive neurodevelopmental disorder in which there is a marked reduction in brain size. PCC syndrome is a recently described disorder of microcephaly, short stature, and misregulated chromosome condensation. Here, we report the finding that MCPH1 primary microcephaly and PCC syndrome are allelic disorders, both having mutations in the MCPH1 gene. The two conditions share a common cellular phenotype of premature chromosome condensation in the early G2 phase of the cell cycle, which, therefore, appears to be a useful diagnostic marker for individuals with MCPH1 gene mutations. We demonstrate that an siRNA-mediated depletion of MCPH1 is sufficient to reproduce this phenotype and also show that MCPH1-deficient cells exhibit delayed decondensation postmitosis. These findings implicate microcephalin as a novel regulator of chromosome condensation and link the apparently disparate fields of neurogenesis and chromosome biology. Further characterization of MCPH1 is thus likely to lead to fundamental insights into both the regulation of chromosome condensation and neurodevelopment.

The dynamic association of RCC1 with chromatin is modulated by Ran-dependent nuclear transport.

Regulator of chromosome condensation (RCC1) binding to chromatin is highly dynamic, as determined by fluorescence recovery after photobleaching analysis of GFP-RCC1 in stably transfected tsBN2 cells. Microinjection of wild-type or Q69L Ran markedly slowed the mobility of GFP-RCC1, whereas T24N Ran (defective in nucleotide loading) decreased it further still. We found significant alterations in the mobility of intranuclear GFP-RCC1 after treatment with agents that disrupt different Ran-dependent nuclear export pathways. Leptomycin B, which inhibits Crm1/RanGTP-dependent nuclear export, significantly increased the mobility of RCC1 as did high levels of actinomycin D (to inhibit RNA polymerases I, II, and III) or alpha-amanitin (to inhibit RNA polymerases II and III) as well as energy depletion. Inhibition of just mRNA transcription, however, had no affect on GFP-RCC1 mobility consistent with mRNA export being a Ran-independent process. In permeabilized cells, cytosol and GTP were required for the efficient release of GFP-RCC1 from chromatin. Recombinant Ran would not substitute for cytosol, and high levels of supplemental Ran inhibited the cytosol-stimulated release. Thus, RCC1 release from chromatin in vitro requires a factor(s) distinct from, or in addition to, Ran and seems linked in vivo to the availability of Ran-dependent transport cargo.

A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo.

The RanGTP gradient across the interphase nuclear envelope and on the condensed mitotic chromosomes is essential for many cellular processes, including nucleocytoplasmic transport and spindle assembly. Although the chromosome-associated enzyme RCC1 is responsible for RanGTP production, the mechanism of generating and maintaining the RanGTP gradient in vivo remains unknown. Here, we report that regulator of chromosome condensation (RCC1) rapidly associates and dissociates with both interphase and mitotic chromosomes in living cells, and that this mobility is regulated during the cell cycle. Our kinetic modeling suggests that RCC1 couples its catalytic activity to chromosome binding to generate a RanGTP gradient. Indeed, we have demonstrated experimentally that the interaction of RCC1 with the chromatin is coupled to the nucleotide exchange on Ran in vivo. The coupling is due to the stable binding of the binary complex of RCC1–Ran to chromatin. Successful nucleotide exchange dissociates the binary complex, permitting the release of RCC1 and RanGTP from the chromatin and the production of RanGTP on the chromatin surface.

13q Deletion in Chronic Lymphocytic Leukemia: Characterization of E4.5, a Novel Chromosome Condensation Regulator-like Guanine Nucleotide Exchange Factor

We report the characterization of a new gene (E4.5) that maps at chromosome band 13q14.3, a chromosomal area frequently deleted in chronic lymphocytic leukemia (CLL) and in other lymphoid malignancies. E4.5 gene encodes for a 4 kb mRNA expressed in various tissues and has an open reading frame of 531 amino acids. The predicted E4.5 protein shows strong homology with the human regulator of chromosome condensation (RCC1) protein, the principal GTP exchange factor for Ran protein. The E4.5 protein contains a BTB domain in its N-terminus, a protein-protein interaction motif. Therefore, we propose that E4.5 is a new member of the RCC1-related guanine nucleotide exchange factor (GEF) family with potent interaction with other proteins and unknown function. Until now, no tumor suppressor genes have been mapped in the 13q14.3 minimal deleted region (MDR) in patients with CLL. It has been proposed that loss of the 13q14.3 MDR may contribute to lymphoid neoplasia by altering the expression/function of genes located on 13q14.3 outside the MDR. The E4.5 is one of these genes with a potential role in the pathogenesis of CLL.

Ran-binding Protein 3 Links Crm1 to the Ran Guanine Nucleotide Exchange Factor

Ran-binding protein 3 (RanBP3) is an approximately 55-kDa protein that functions as a cofactor for Crm1-mediated nuclear export. RanBP3 stimulates export by enhancing the affinity of Crm1 for Ran.GTP and cargo. However, important additional functions for this cofactor may exist. We now report that RanBP3 associates with the Ran-specific guanine nucleotide exchange factor, regulator of chromosome condensation 1 (RCC1). This interaction was stimulated by the addition of Ran; moreover, Ran.GDP, Ran.GTP, and Ran without nucleotide could all stimulate complex formation between RanBP3 and RCC1 even though binding of Ran.GDP to RanBP3 alone was undetectable. RanBP3 could also promote binding of Crm1 to RCC1 in the presence of Ran. Binding of RanBP3 to RCC1 increased the catalytic activity of RCC1 toward Ran, and importantly, the ability of RanBP3 to stimulate RCC1 was not affected by the presence of Crm1. These data indicate that RanBP3 acts as a scaffold protein to promote the efficient assembly of export complexes. By tethering Crm1 to catalytically enhanced RCC1, RanBP3 may lower the entropic barrier for the loading of Ran.GTP onto Crm1. We propose that this provides an additional mechanism by which RanBP3 facilitates export.

Identification of casein kinase Iα interacting protein partners

Casein kinase Iα (CKIα) belongs to a family of serine/threonine protein kinases involved in membrane trafficking, RNA processing, mitotic spindle formation and cell cycle progression. In this report, we identified several CKIα interacting proteins including RCC1, high mobility group proteins 1 and 2 (HMG1, HMG2), Erf, centaurin-α 1, synaptotagmin IX and CPI-17 that were isolated from brain as CKIα co-purifying proteins. Actin, importin-α 1, importin-β, PP2Ac, centaurin-α 1, and HMG1 were identified by affinity chromatography using a peptide column comprising residues 214–233 of CKIα. We have previously shown that centaurin-α 1 represents a CKIα partner both in vitro and in vivo. The nuclear protein regulator of chromosome condensation 1 (RCC1) is a guanosine nucleotide exchange factor for Ran which is involved in nuclear transport and mitotic spindle formation. Here we show that CKIα and RCC1 interact in brain and in cultured cells. However, the interaction does not involve residues 217–233 of CKIα which are proposed from X-ray structures to represent an anchoring site for CKI partners. Formation of the RCC1/CKIα complex is consistent with the association of the kinase with mitotic spindles. In conclusion, we have identified a number of novel CKIα protein partners and their relations to CKI are discussed.

C-terminal domain of gyrase A is predicted to have a beta-propeller structure.

Two different type II topoisomerases are known in bacteria. DNA gyrase (Gyr) introduces negative supercoils into DNA. Topoisomerase IV (Par) relaxes DNA supercoils. GyrA and ParC subunits of bacterial type II topoisomerases are involved in breakage and reunion of DNA. The spatial structure of the C-terminal fragment in GyrA/ParC is not available. We infer homology between the C-terminal domain of GyrA/ParC and a regulator of chromosome condensation (RCC1), a eukaryotic protein that functions as a guanine-nucleotide-exchange factor for the nuclear G protein Ran. This homology, complemented by detection of 6 sequence repeats with 4 predicted beta-strands each in GyrA/ParC sequences, allows us to predict that the GyrA/ParC C-terminal domain folds into a 6-bladed beta-propeller. The prediction rationalizes available experimental data and sheds light on the spatial properties of the largest topoisomerase domain that lacks structural information.

PARF-1: an Arabidopsis thaliana FYVE-domain protein displaying a novel eukaryotic domain structure and phosphoinositide affinity.

A full-length cDNA encoding a novel protein named PARF-1 was isolated from Arabidopsis thaliana. PARF-1 is the first eukaryotic protein to be identified that displays a domain structure which includes a FYVE-finger domain, a Pleckstrin Homology (PH) domain, as well as multiple Regulator of Chromosome Condensation-1 (RCC1) repeats. Northern blot analysis revealed that PARF-1 mRNA is present at high levels in flowers, but only at very low levels in other tissues. Recombinant PARF-1 fusion proteins expressed in E. coli were found to display unique binding specificities for monophosphorylated phosphoinositide lipids. The unusual domain structure of PARF-1 combined with its phosphoinositide specificity suggests that it may fulfil unexpected functions in higher plants.

FYVE zinc-finger proteins in the plant model Arabidopsis thaliana: identification of PtdIns3P-binding residues by comparison of classic and variant FYVE domains

Classic FYVE zinc-finger domains recognize the phosphoinositide signal PtdIns3P and share the basic (R/K)1(R/K)HHCR6 (single-letter amino acid codes) consensus sequence. This domain is present in predicted PtdIns3P 5-kinases and lipases from Arabidopsis thaliana. Other Arabidopsis proteins, named PRAF, consist of a pleckstrin homology (PH) domain, a regulator of chromosome condensation (RCC1) guanine nucleotide exchange factor repeat domain, and a variant FYVE domain containing an Asn residue and a Tyr residue at positions corresponding to the PtdIns3P-interacting His4 and Arg6 of the basic motif. Dot-blot and liposome-binding assays were used in vitro to examine the phospholipid-binding ability of isolated PRAF domains. Whereas the PH domain preferentially bound PtdIns(4,5)P2, the variant FYVE domain showed a weaker charge-dependent binding of phosphoinositides. In contrast, specificity for PtdIns3P was obtained by mutagenic conversion of the variant into a classic FYVE domain (Asn4,Tyr6 → His4,Arg6). Separate substitutions of the variant residues were not sufficient to impose preferential binding of PtdIns3P, suggesting a co-operative effect of these residues in binding. A biochemical function for PRAF was indicated by its ability to catalyse guanine nucleotide exchange on some of the small GTPases of the Rab family, permitting a discussion of the biological roles of plant FYVE proteins and their regulation by phosphoinositides.

Chromatin docking and exchange activity enhancement of RCC1 by histones H2A and H2B.

The Ran guanosine triphosphatase (GTPase) controls nucleocytoplasmic transport, mitotic spindle formation, and nuclear envelope assembly. These functions rely on the association of the Ran-specific exchange factor, RCC1 (regulator of chromosome condensation 1), with chromatin. We find that RCC1 binds directly to mononucleosomes and to histones H2A and H2B. RCC1 utilizes these histones to bind Xenopus sperm chromatin, and the binding of RCC1 to nucleosomes or histones stimulates the catalytic activity of RCC1. We propose that the docking of RCC1 to H2A/H2B establishes the polarity of the Ran-GTP gradient that drives nuclear envelope assembly, nuclear transport, and other nuclear events.

Structural Basis for Guanine Nucleotide Exchange on Ran by the Regulator of Chromosome Condensation (RCC1)

RCC1 (regulator of chromosome condensation), a β propeller chromatin-bound protein, is the guanine nucleotide exchange factor (GEF) for the nuclear GTP binding protein Ran. We report here the 1.8 Å crystal structure of a Ran•RCC1 complex in the absence of nucleotide, an intermediate in the multistep GEF reaction. In contrast to previous structures, the phosphate binding region of the nucleotide binding site is perturbed only marginally, possibly due to the presence of a polyvalent anion in the P loop. Biochemical experiments show that a sulfate ion stabilizes the Ran•RCC1 complex and inhibits dissociation by guanine nucleotides. Based on the available structural and biochemical evidence, we present a unified scenario for the GEF mechanism where interaction of the P loop lysine with an acidic residue is a crucial element for the overall reaction.

Ran is associated with chromosomes during starfish oocyte meiosis and embryonic mitoses

Ran is an abundant small G protein that has a central role in nuclear transport during interphase. Ran function depends on distinct pools of RanGTP in the nucleus and RanGDP in the cytoplasm, which are maintained by compartmentalisation of the nucleotide exchange and GTPase activating proteins for Ran. RCC1 (Regulator of Chromosome Condensation) is the only known guanine nucleotide exchange factor for Ran and is associated with chromatin. RanGAPl together with RanBPl are the primary GTPase activator proteins and are located in the cytoplasm.Ran function in nuclear transport is clearly inactivated after the nuclear envelope has disassembled during mitosis. There is now evidence that Ran plays a role in organising the spindle. Experiments using well-characterised cell-free extracts from meiotic metaphase II arrestedXenopuseggs suggest that Ran regulates microtubules involved in spindle assembly (Carazo-Salaset al., 1999; Kalabet al., 1999; Ohbaet al., 1999; Wilde & Zheng, 1999). These experiments demonstrate that high levels of RanGTP or GTP-locked forms of Ran result in aster formation and can cause formation of bipolar spindles in the absence of centrosomes or chromosomes. In contrast, reduction of RanGTP blocks centriole- and chromatin-dependent aster formation.

Model of the Ran-RCC1 Interaction using Biochemical and Docking Experiments

RCC1, the regulator of chromosome condensation, is the guanine nucleotide exchange factor (GEF) for the nuclear Ras-like GTP-binding protein Ran. Its structure was solved by X-ray crystallography and revealed a seven-bladed β-propeller, one side of which was proposed to be the interaction site with Ran. To gain more insight into this interaction, alanine mutagenesis studies were performed on conserved residues on the surface of the structure. Purified mutant proteins were analysed by steady-state kinetic analysis of their GEF activities towards Ran. A number of residues were identified whose mutation affected either the KMor kcatof the overall reaction, or had no effect. Mutants were further analysed by plasmon surface resonance in order to get more information on individual steps of the complex reaction pathway. Ran-GDP was coupled to the sensor chip and reacted with RCC1 mutants to categorise them into different groups, demonstrating the usefulness of plasmon surface resonance in the study of complex multi-step kinetic processes. A docking solution of Ran-RCC1 structures in combination with sequence analysis allows prediction of the site of interaction between RCC1 and Ran and proposes a model for the Ran-RCC1 structure which corresponds to and extends the biochemical data. Three invariant residues which most severely affect the kcatof the reaction, D128, D182 and H304, are located in the centre of the Ran-RCC1 interface and interfere with switch II and the phosphate binding area. The structural model suggests that different guanine nucleotide exchange factors use a similar interaction site on their respective GTP-binding proteins, but that the molecular mechanisms for the release of nucleotides are likely to be different.

Crystallization and preliminary X-ray analysis of human RCC1, the regulator of chromosome condensation.

RCC1, the regulator of chromosome condensation, is the guanine nucleotide-exchange factor (GEF) of the GTP-binding protein Ran. Its GEF activity on Ran makes it a key element in nucleo-cytoplasmic transport and cell-cycle regulation. Crystals of human RCC1 suitable for X-ray analysis have been obtained using the seeding technique in hanging drops with sodium citrate as a precipitant. The crystals diffract to 1.7 A at 100 K and belong to the space group P1, with unit-cell parameters a = 49.5, b = 84.3, c = 84.9 A, alpha = 113.0, beta = 103.9,gamma = 103.3 degrees. The Matthews parameter (Vm) and the self-rotation function are consistent with three molecules in the unit cell, which is confirmed by the averaged single isomorphous replacement (SIR) electron-density map.

Identification of a large Myc-binding protein that contains RCC1-like repeats.

The protooncogene MYC plays an important role in the regulation of cellular proliferation, differentiation, and apoptosis and has been implicated in a variety of human tumors. MYC and the closely related MYCN encode highly conserved nuclear phosphoproteins (Myc and NMyc) that apparently function as transcription factors in the cell. We have identified a large and highly conserved nuclear protein that interacts directly with the transcriptional activating domain of Myc (designated "protein associated with Myc" or Pam). Pam contains an extended amino acid sequence with similarities to a protein known as regulator of chromosome condensation (RCC1), which may play a role in the function of chromatin. The gene encoding Pam (PAM) is expressed in all of the human tissue examined, but expression is exceptionally abundant in brain and thymus. Pam binds specifically to Myc, but not NMyc. The region in Myc required for binding to Pam includes a domain that is essential for the function of Myc and that is frequently mutated in Burkitt's lymphomas. PAM is located within a 300-kb region on chromosome 13q22.

Molecular screening for P-element insertions in a large genomic region of Drosophila melanogaster using polymerase chain reaction mediated by the vectorette.

As an alternative to existing methods for the detection of new insertions during a transposon mutagenesis, we adapted the method of vectorette ligation to genomic restriction fragments followed by PCR to obtain genomic sequences flanking the transposon. By combining flies containing a defined genomic transposon with an excess of flies containing unrelated insertion sites, we demonstrate the specificity and sensitivity of the procedure in the detection of integration events. This method was applied in a transposon-tagging screen for BJ1, the Drosophila homolog of the vertebrate gene Regulator of Chromosome Condensation (RCCI). Genetic mobilization of a single genomic P element was used to generate preferentially new local insertions from which integrations into a genomic region surrounding the BJ1 gene were screened. Flies harboring new insertions were phenotypically selected on the basis of the zeste1-dependent transvection of white. We detected a single transposition to a 13-kb region close to the BJ1 gene among 6650 progeny that were analyzed. Southern analysis of the homozygous line confirmed the integration 3 kb downstream of BJ1.

Nucleocytoplasmic recycling of the nuclear localization signal receptor alpha subunit in vivo is dependent on a nuclear export signal, energy, and RCC1.

Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The alpha subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the beta subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.

Replication of herpes simplex virus type 1 DNA is inhibited in a temperature-sensitive mutant of BHK-21 cells lacking RCC1 (regulator of chromosome condensation) and virus DNA remains linear.

tsBN2, a temperature-sensitive (ts) growth mutant of the hamster cell line BHK-21, has a point mutation in the RCC1 (regulator of chromosome condensation) gene, and prematurely enters mitosis at 39.5 degrees C, a nonpermissive temperature. In this mutant at 39.5 degrees C infectious progeny of herpes simplex virus type 1 (HSV-1) was not produced and replication of HSV-1 DNA was inhibited. HSV-1 DNA from virus particles is normally circularized upon infection, and circularized HSV-1 DNA molecules can serve as template for DNA replication. In tsBN2 at 39.5 degrees C, HSV-1 DNA appeared to remain linear after infection, suggesting the obstruction of HSV-1 DNA circularization, which could account for failure of HSV-1 DNA replication. In transient replication assays performed in tsBN2 at 39.5 degrees C, through superinfection with HSV-1 helper virus, there was no evidence of replication of circular DNA of the hybrid plasmid containing the HSV-1 replication origin. Production of mRNAs of HSV-1 early genes required for HSV-1 DNA replication was decreased in tsBN2 at 39.5 degrees C. Therefore, RCC1 was assumed to be involved in the formation of an HSV-1 DNA configuration suitable for replication (that is circularization) and the supply of proteins required for replication of the circularized HSV-1 DNAs.

The thermolability of nuclear protein import in tsBN2 cells is suppressed by microinjected Ran-GTP or Ran-GDP, but not by RanQ69L or RanT24N.

The nuclear protein regulator of chromosome condensation 1 (RCC1) stimulates guanine nucleotide exchange on a protein, Ran, that is required for nuclear protein import. In the present report, we confirm that RCC1 is also required for nuclear protein import in tsBN2 hamster cells in vivo. The thermolability of nuclear protein import in tsBN2 cells was suppressed by microinjection of purified Ran-GTP into the cytoplasm, but Ran-GDP also relieved the import deficiency, suggesting either that both forms of Ran are active in import in vivo or that tsBN2 cells at restrictive temperature retain a mechanism to convert Ran-GDP to Ran-GTP. To distinguish between these possibilities, nuclear protein import in tsBN2 cells was tested in the presence of Ran mutants, one deficient in GTP hydrolysis (RanQ69L), and one with weak binding to GDP and little or no binding to GTP (RanT24N). Microinjection of the mutant RanQ69L inhibited import in vivo in either the GTP- or GDP-bound form at both the permissive and nonpermissive temperatures. RanT24N-GDP inhibited import in vivo at the permissive temperature and failed to stimulate nuclear protein import at the nonpermissive temperature. The implications of these results for the roles of RCC1 and Ran in nuclear protein import in vivo are discussed.

A gene (RPGR) with homology to the RCC1 guanine nucleotide exchange factor is mutated in X–linked retinitis pigmentosa (RP3)

X-linked retinitis pigmentosa (xlRP) is a severe progressive retinal degeneration which affects about 1 in 25,000 of the population. The most common form of xlRP, RP3, has been localised to the interval between CYBB and OTC in Xp21.1 by linkage analysis and deletion mapping. Identification of microdeletions within this region has now led to the positional cloning of a gene, RPGR, that spans 60 kg of genomic DNA and is ubiquitously expressed. The predicted 90 kD protein contains in its N-terminal half a tandem repeat structure highly similar to RCC1 (regulator of chromosome condensation), suggesting an interaction with a small GTPase. The C-terminal half contains a domain, rich in acidic residues, and ends in a potential isoprenylation anchorage site. The two intragenic deletions, two nonsense and three missense mutations within conserved domains provide evidence that RPGR (retinitis pigmentosa GTPase regulator) is the RP3 gene.