Skip to main content
PMC home page
Nucleus logoLink to Nucleus
. 2011 Nov 1;2(6):591–600. doi: 10.4161/nucl.2.6.18044

Chromatin maintenance by a molecular motor protein

Manjari Mazumdar 1,✉, Myong-Hee Sung 2, Tom Misteli 2
PMCID: PMC3324347  PMID: 22130187

Abstract

The kinesin motor protein KIF4 performs essential functions in mitosis. Like other mitotic kinesins, loss of KIF4 causes spindle defects, aneuploidy, genomic instability and ultimately tumor formation. However, KIF4 is unique among molecular motors in that it resides in the cell nucleus throughout interphase, suggesting a non-mitotic function as well. Here we identify a novel cellular function for a molecular motor protein by demonstrating that KIF4 acts as a modulator of large-scale chromatin architecture during interphase. KIF4 binds globally to chromatin and its absence leads to chromatin decondensation and loss of heterochromatin domains. KIF4-dependent chromatin decondensation has functional consequences by causing replication defects and global mis-regulation of gene expression programs. KIF4 exerts its function in chromatin architecture via regulation of ADP-ribosylation of core and linker histones and by physical interaction and recruitment of chromatin assembly proteins during S-phase. These observations document a novel function for a molecular motor protein in establishment and maintenance of higher order chromatin structure.

Molecular motor proteins have long been known to generate most cellular forces and to power many forms of intracellular movement.1,2 One of the major families of molecular motors are the kinesins.1 The vast majority of kinesins are plus-end directed microtubule motors which localize to the cytoplasm during interphase. As cells enter mitosis and the nuclear envelope breaks down, kinesins associate with microtubules and contribute to mitotic chromosome segregation including cytokinesis.3,4 The chromokinesins are a subfamily of kinesins, which are characterized by their ability to bind to chromatin.3,5-9 Chromokinesins act in various steps of mitosis, including chromosome condensation, metaphase alignment, chromosome segregation, cytokinesis and they help maintain genome stability.3,10-13 The chromokinesin KIF4 associates with mitoic chromosomes and the central spindle and contributes to faithful chromosome segregation.10 Loss of KIF4 leads to hypercondensation of mitotic chromosomes, various mitotic defects, chromosome mis-segregation, activation of DNA damage signaling and cells lacking KIF4 rapidly form tumors in nude mice.10,14 Partial or complete loss of KIF4 occurs in ~35% of a wide range of human cancer cells14 and KIF4 has been associated with metastatic potential.15,16

KIF4 is unique among kinesins in that it localizes to the nucleus during interphase rather than the cytoplasm10-12,17 and many of the observed phenotypes of KIF4 loss are difficult to attribute to its mitotic functions.3 For example, in cells lacking KIF4, chromosomes undergo premature condensation prior to breakdown of the nuclear envelope at the onset of mitosis.10 Furthermore, KIF4 physically interacts with several chromatin associated proteins including condensins,10 DNA methyltransferase complexes and poly-ADP-ribosyl polymerase 1 (PARP-1).18 Given its nuclear localization throughout most of the cell cycle and its co-purification with a chromatin complex19 we sought to probe the function of KIF4 in the interphase nucleus.

Results

The molecular motor KIF4 dynamically binds to chromatin in interphase

In mouse ES cells KIF4 localizes predominantly to the nucleus in a homogenously diffuse pattern excluding nucleoli, reminiscent of globally binding chromatin proteins such as the linker histones H1 and HMG proteins (Fig. 1A). KIF4 staining is resistant to serial extraction with detergent and 500 mM NaCl, but is lost from the interior of the nucleus upon DNase treatment (Fig. 1B and Fig. S1). A pool of DNase-resistant KIF4 remains at the nuclear lamina, likely as a consequence of its physical interaction with lamin A (Mazumdar M, unpublished observation). In serial salt extractions, KIF4 shows similar extraction properties as the linker histone H1 (Fig. 1C). To directly test the ability of KIF4 to bind DNA, electrophoretic mobility shift assays (EMSA) were performed using AT-rich heat shock element (HSE) probes, since KIF4 shows high sequence similarity to heat shock factor proteins HSF1 and HSF2 in its DNA-binding region. Endogenous KIF4, as well as the purified recombinant GST-tagged cysteine-rich C-terminal domain of KIF4, effectively bound to 55-mer and 33-mer AT-rich probes (Fig. 1E, lane 3). Binding was competed by an unlabelled 50x probe (Fig. 1E, lanes 6 and 13), whereas addition of anti-KIF4 antibody, but not an anti-GST control antibody, resulted in a supershift, confirming that KIF4 directly and specifically binds to DNA (Fig. 1E, lane 4, 8 and 12). To test chromatin-association properties of KIF4 in living cells, Fluorescence Recovery After Photobleaching (FRAP) was performed on KIF4GFP stably expressed at endogenous levels in previously characterized kif4-/y ES cells (KIF4KO; Fig. 1D; ref. 14). KIF4GFP showed similar recovery kinetics as the linker histone H1GFP, which has a residence time of 1–2 min on chromatin.20 Based on these in vitro and in vivo experiments, we conclude that KIF4 is a DNA-binding protein in the interphase nucleus.

graphic file with name nucl-2-591-g1.jpg

Open in a new tab

Figure 1. KIF4 is a nuclear DNA-binding protein. (A) Immunofluorescence localization in mouse ES cells using anti-KIF4 antibody (red) in the nucleoplasm of the mammalian nucleus; DNA (blue). Scale bar: 5 μm. (B) Sequential in situ extraction of HeLa cells with 1% Triton-X100, 500 mM NaCl in CSK buffer and 500 μg/ml DNase. KIF4 (green) is only partially extracted in detergent and high-salt, but lost upon DNase treatment, DNA (blue). (C) Serial salt extraction of HeLa nuclei. KIF4 shows similar salt extraction properties as histone H1. Input, 10% of total. (D) Fluorescence Recovery after Photobleaching of KIF4GFP and H1GFP in KIF4KO ES cells. Half the nucleus was photobleached. Values represent averages ± SD from at least five independent experiments. (E) Electrophoretic mobility shift assays of KIF4. KIF4 binds to AT-rich sequences via its C-terminal domain.

Global genome reorganization upon loss of KIF4

KIF4 is important for structural and functional integrity of mitotic chromosomes.3,10 In the absence of KIF4 mitotic chromosomes are hypercondensed at the earliest stages of mitosis even before the nuclear envelope breaks down.10 The ability of KIF4 to bind DNA and its participation in very early chromosome condensation led us to speculate that KIF4 might play a role in interphase chromosome structure. In line with such a function, electron-microscopy analysis revealed a dramatic loss of peripheral and internal heterochromatin domains in mouse KIF4KO ES cells (Fig. 2A, arrows and inset). Analysis of 40 randomly selected electron micrographs each for WT and KIF4KO ES cells demonstrated that all WT cells contained the typical blocks of condensed heterochromatin, whereas no morphologically discernible heterochromatin blocks could be found in any of the KIF4 KO ES cells. Defects in heterochromatin organization were confirmed by analysis of major satellite repeats by fluorescence in situ hybridization (FISH). Major satellite regions appeared decondensed in interphase nuclei of KIF4KO ES cells compared to wild-type E14 ES control cells (left panel) where they form distinct, highly compacted heterochromatin blocks (Fig. 2B). Furthermore, while chromosome painting probes in control E14 ES cells were unable to gain access to chromatin upon denaturation at low temperature (65°C), probes readily bound to chromatin in KIF4KO ES cells at that temperature, suggesting higher accessibility of chromatin in the absence of KIF4 (Fig. S2). Immunofluorescence analysis of KIF4KO ES cells with anti-HP1 and H3K9me3 antibodies revealed that about 30% of cells exhibit decondensed heterochromatin compared with only 6% of control ES cells (Fig. 2C and D). Consistent with chromatin decondensation, the absence of KIF4 resulted in activation of normally transcriptionally silent heterochromatic repeats in KIF4KO cells as judged by semi-quantitative RT-PCR (Fig. 2E), possibly as consequence of allowing access of the transcription machinery to normally repressed genome regions upon chromatin decondensation caused by loss of KIF4.21 These defects were primary consequences of KIF4 loss, since complementation of KIF4KO ES cells with full length KIF4GFP restored interphase nuclear and chromatin morphology (Fig. 2F). We conclude that loss of the molecular motor KIF4 from the interphase nucleus leads to global changes in higher order chromatin organization.

graphic file with name nucl-2-591-g2.jpg

Open in a new tab

Figure 2 (See opposite page). Global chromatin reorganization due to loss of KIF4. (A) Electron microscopy of wild type (WT) mouse E14 and KIF4KO ES cells. Loss of internal and peripheral heterochromatin is observed in KIF4KO cells. Inset, magnification of nuclear periphery. Arrows and arrowheads indicate the nuclear lamina and the associated heterochromatin. WT E14 cells show condensed heterchromatin in electron-dense regions that are lost in KIF4KO cells. Bar: 2 μm. Inset: 2 μm. (B) FISH for major satellite repeats. Pericentric heterochromatin domains of WT E14 and KIF4KO ES cells visualized by major satellite repeat DNA probes (red) and DAPI (blue). Arrows indicate chromocenters. KIF4KO ES cells exhibit a decondendensed heterochromatin domain phenotype. Bar: 5 μm. (C) Heterochromatin reorganization in interphase nuclei of KIF4KO ES cells stained with heterochromatin markers HP1α (green) and H3K9me3 (red). Arrows point to chromocenters. Bar: 5 μm. (D) Quantitation of the percentage of mouse WT E14 and KIF4KO ES cells with decondensed heterochromatin. Values represent averages ± SEM from at least three experiments. (E) Transcription activity of heterochromatic repeats in KIF4KO ES cells as compared with wild-type E14 control cells. Values represent averages ± SEM from at least three experiments. (F) Phenotypic recovery of chromatin defects. Stable expression of GFP-fused full-length (FL) and truncation mutants of KIF4 protein in KIF4KO ES cells . ΔMotor: aa373–1296, FL KIF4: aa 1–1296. C-terminal domain: aa 859–1296. DNA (blue), HP1 antibody (red).

To determine whether these effects involved the motor domain of KIF4 we generated a mutant lacking aa 1–372 which eliminates the KIF4 motor domain (ΔMotorKIF4) and a mutant containing only the C-terminal domain consisting of aa 859–1232. Stable KIF4KO ES cell lines expressing either full length KIF4, ΔMotorKIF4 or the C-terminal domain were established and analyzed for chromatin defects (Fig. 2E). Similar to full length KIF4, both the ΔMotorKIF4 and the KIF4 C-terminal domain restored normal heterochromatin morphology as measured by immunostaining with HP1α antibody. These observations demonstrate that the motor domain of KIF4 is not required for the observed chromatin defects and that the C-terminal domain that binds to DNA in vitro is sufficient for the chromatin maintenance function of KIF4 during interphase.

KIF4 modulates global gene expression patterns

To ask whether loss of KIF4 has functional consequences on gene expression, we performed genome-wide transcription analysis of WT E14, or KIF4KO ES cells using Affymetrix GeneArray MOE430 2.0. In the absence of KIF4, 502 genes were differentially expressed in the KIF4KO ES cells (minimum fold change of 1.5, p < 0.05, two-sample Student T test; four replicates each). Of these, 275 had elevated expression and 227 had decreased levels in the KIF4KO ES cells compared with control ES cells. Mis-regulated genes were evenly distributed among all chromosomes (Fig. 3A). Gene-ontology analysis (Fig. 3B) indicates that the KIF4-dependent genes were involved in a wide range of cellular functions, with a slight preference for transcription or DNA-dependent processes. Some of the most deregulated genes were key components of lymphocyte function such as Runx1 (10-fold downregulated), CD44 (10-fold downregulated), CD84 antigen (4 fold upregulated), ILR17B (2-fold upregulated), TCF4 (2-fold upregulation) IL-1R (2 fold upregulation), IL-17RB (2-fold overexpression). These expression changes are of interest in the light of the fact that KIF4-null mice are highly susceptible to spontaneous lymphomas (M.M., unpublished observation). Interestingly, the KIF4 homolog BC12/GDD1 in rice, has recently been shown to modulate transcription globally.22

graphic file with name nucl-2-591-g3.jpg

Open in a new tab

Figure 3. Genome-wide misregulation of genes in KIF4KO ES cells. (A) The changes in KIF4KO compared with control ES cells is indicated in false color as log2 ratios, (red) overexpressed genes, (green) downregulated genes. (B) Gene ontology representation of differentially expressed genes in KIF4KO ES cells compared with all annotated genes. Four biological replicates of each WT E14 and KIF4KO ES cell were analyzed.

Altered histone ADP-ribosylation and replication defects in the absence of KIF4

While a role for KIF4 in mitosis is well established, our initial observation that loss of KIF4 leads to aberrant chromosome condensation prior to nuclear envelope breakdown suggested a role in interphase chromosome structure.3 With regards to a mechanistic basis for such a role, we focused on KIF4 interacting proteins. One prominent KIF4 interaction partner with a known role in chromatin structure is Poly-(ADP-ribose) Polymerase (PARP-1).23,24 The enzymatic activity of PARP-1 has been shown to be modulated by KIF4.18 PARP-1 catalyzes the covalent attachment of ADP-ribose from donor NAD+ molecules to a variety of target proteins resulting in linear or branched polymers of poly(ADP) ribose (PAR).25 Two prominent target proteins of poly(ADP)-ribosylation are the linker histone H1 and the core histone H2B and their poly-(ADP)-ribosylation affects higher order chromatin organization and nucleosome binding.26 To ask whether KIF4 is involved in poly(ADP)-ribosylation of chromatin components, we probed the ADP-ribosylation status of histones. ADP-ribosylation of core and linker histones was significantly increased in cells depleted of KIF4; no difference in PARP-1 levels was observed (Fig. 4A and B). We conclude that loss of KIF4 leads to hyper-poly-(ADP)-ribosylation of key architectural components of chromatin. This could potentially lead to “open”chromatin as seen in KIF4KO cells (Figs. 2andS2). In line with such a role, a recent study has demonstrated that KIF4 binds to SRE1 (serum responsive)–EBS (ETS binding site)-GRE (glucocorticoid responsive element) of the ApoD promoter under normal growth conditions and that PARP-1 binding to the same promoter under growth arrest condition is modulated by KIF4.27

graphic file with name nucl-2-591-g4.jpg

Open in a new tab

Figure 4. Altered Histone ADP-ribosylation and replication defects in the absence of KIF4. (A) Effect of KIF4 deletion on histone-ADP-ribosylation and PARP-1 examined in the context of chromatin structure. (B) Immunoblot of nuclear extract from WT and KIF4KO ES cells with anti-PARP-1, anti-histone H3 and Coomassie stain of the same protein gel blot shows little or no effect on PARP-1 protein levels in KIF4KO cells. (A,B) Purified nuclei from WT and KIF4KO ES cells were subjected to Microccocal Nuclease (MNase) digestion to produce monocleosomes. MNase-released soluble histones and other chromatin proteins were immunoblotted with Poly ADP-ribosyl (PAR) antibody to show ADP-ribosylation of histone proteins. Histone H1 is used as a loading control. (C and D) Slowed S-phase progression of KIF4 KO ES cells. FACS profile were obtained by 15 min pulse-labeling with BrdU followed by fixation and staining with fluorescently labeled anti-BrdU antibody to identify actively replicating cells and propidium iodide for DNA content. Higher DNA content and increased BrdU incorporation in KIF4KO cells compared with WT E14 ES cells indicate defects in S-phase progression. (E) The number of actively replicating cells at different stages of S phase in non-synchronous populations of WT and KIF4 KO ES cells were determined using the number of replicating foci formation that can be detected by fluorescence microscopy of individual cells. Cells were fixed after a 15 min pulse of BrdU and co-stained with anti-BrdU and anti-PCNA, a component of the replication machinery. Values represent averages obtained from 100 cells each ± S.E.M from at least three experiments. (F) Interaction of replication components with KIF4. Protein gel blot of immunoprecipitated chromatin assembly factors from non-synchronized (Non-sync) and S-phase synchronized HeLa cell nuclei. Chromatin fractions were immunoprecipitated with anti-KIF4 antibody and the protein gel blot was performed with the indicated antibodies. KIF4 specifically interacts with p150CAF, RbAp48 and histone chaperone Asf1 but not HIRA. (G) Effect of loss of KIF4 on replication factor association with S-phase chromatin. WT E14 and KIF4KO ES cells were synchronized in G1/S phase by overnight treatment with Aphidicolin (5 μg/ml) and released into fresh growth medium for 2.5 h to obtain S-phase cells. Purified nuclei from S-phase cells were subjected to extraction with the indicated concentration of NaCl and soluble proteins were immunoblotted with the indicated antibodies. A mixture of Asf1 antibodies was used to recognize both Asf1a and Asf1b isoforms on protein gel blots.

Heterochromatin domains are typically late replicating and are maintained by the interplay of structural chromatin proteins, chromatin assembly factors and histone modifiers.28-30 Since heterochromatin decondensation in the absence of KIF4 was only observed in about 30% of unsynchronized cells (Fig. 2D), we hypothesized that KIF4 plays a role in DNA replication and probed if any specific stage(s) of S phase was affected by the loss of KIF4. Fluorescence Activated Cell Sorting (FACS) analysis of unsynchronized populations of E14 control and KIF4KO ES cells after BrdU incorporation revealed an ~1.5-fold reduction in S-phase cells in the absence of KIF4 (Fig. 4C and D). To identify the stages of S-phase progression, we performed immunofluorescence microscopy after incorporation of BrdU and counterstaining with antibodies against the replication clamp PCNA. Single cell analysis showed a reduction of early S-phase cells and a concomitant increase in late S-phase cells upon loss of KIF4, suggesting a delay in S-phase entry and/or progression (Fig. 4E).

The accumulation of S-phase cells led us to hypothesize that KIF4 may exert its effect on chromatin organization via its role in recruiting chromatin factors during replication. DNase-extracted soluble chromatin fractions from non-synchronized and S-phase synchronized cells were immunoprecipitated with anti-KIF4 antibody to determine physical interactions of KIF4 with known replication-associated chromatin assembly factors. Multiple components of the S-phase chromatin assembly machinery including the replication clamp PCNA, the histone chaperone complex component pCAF150 and the NURD chromatin remodeling component RbAp48 and RbAp46 physically interact with KIF4 (Fig. 4F). Furthermore, Asf1, a histone H3.1 chaperone that acts in a DNA synthesis-dependent manner, physically associates with KIF4. In contrast HirA, a nucleosome assembly factor that acts as a chaperone of histone H3.3 in a DNA synthesis-independent manner31 does not interact with KIF4 (Fig. 4F). We conclude that several key replication-associated chromatin assembly and remodeling factors physically interact with KIF4 during S-phase.

These results prompted us to hypothesize that KIF4 may function during DNA replication by mediating the recruitment of chromatin assembly factors to chromatin. To probe whether KIF4 alters the association of key chromatin assembly factors with S-phase chromatin, the extraction properties of several factors involved in replication-coupled chromatin assembly were analyzed. Asf1 and the NURD chromatin-remodeling components RbAp48 and RbAp46 were more easily extracted from S-phase KIF4KO ES cells compared to control ES cells (Fig. 4G). In contrast, the replication-independent H3 chaperone HIRA and PCNA were not affected by loss of KIF4 (Fig. 4G). Based on these results, we speculate that KIF4 plays a role in the recruitment of Asf1 and NURD complex components to chromatin during replication either by altering the chromatin binding template or via protein-protein interactions.

Discussion

We have here identified a novel cellular function for a molecular motor protein. We demonstrate that the chromokinesin KIF4 functions as a structural chromatin protein in the interphase nucleus and contributes to faithful S-phase progression. KIF4 physically interacts with several key assembly proteins including the histone chaperone component p150 CAF-1 and NURD chromatin remodeling complex components. Immunoprecipitation of cross-linked isolated chromatin from HeLa cells with KIF4 antibody identifies association of PARP-1, RbAp48 and Asf1a and b with KIF4 chromatin (Fig. S3). Loss of KIF4 interferes with chromatin recruitment during S-phase of several architectural chromatin proteins including the histone chaperone Asf1, but not the replication-independent histone chaperone HIRA. As a consequence of reduced recruitment of these factors S-phase progression is slowed. A structural role for KIF4 explains the previously reported activation of DNA damage signaling pathways in KIF4-null ES cells14 likely brought about by replication-associated defects in chromatin assembly.32 In line with KIF4 mediated chromatin-associated S-phase defects, the observed chromatin phenotypes upon KIF4 loss are reminiscent of those seen in mutants of replication factors such as ORC2 or defective resolution of Holliday junctions in Bloom’s syndrome cells.33,34 The molecular interactions of chromokinesin KIF4 and the chromosome condensation and DNA replication machinery occur probably in the context of larger complexes since human KIF4A and condensin can be biochemically purified with condensin, the DNA methyltransferase DNMT3B, the chromatin remodelers SIN3A and hSNF2H and the histone deacetylase HDAC1.19 Interestingly, chromatin remodeling SWI/SNF complex component BAF170 directly associates with KIF4 as well as with lamins and replication origins.35 The association of KIF4 with ATP-dependent chromatin remodeling factors and histone modifiers such as HDACs, DNMT3B and PARP-1 in the normal interphase nucleus is intriguing as these complexes regulate chromatin accessibility for dynamic interactions both at the nucleosomal level and for maintaining higher-order chromatin structure. The changes in condensation /“opening” of chromatin observed here may imply that KIF4 functions as a nuclear scaffold either by DNA binding or via protein-protein interactions to regulate nuclear organization, gene expression and cell cycle events such as DNA replication. Because the large chromatin remodeling complexes such as SWI/SNF or NURD play non-redundant roles in various nuclear functions, it is highly likely that nuclear motor proteins such as KIF4 provide a dynamic platform to assemble these chromatin complexes. The interactions of KIF4 with epigenetic regulators may be necessary to ensure the formation of higher-order structure and its propagation through mitosis. This scenario is consistent with the global decondensation of chromatin in the interphase nucleus and hypercondensation of mitotic chromosomes in the absence of KIF4. KIF4 localizes along the length of condensed chromosome arms.10 It is intriguing to speculate that KIF4 interactions with centromeric heterochromatin assembly factors such as RbAp48 may serve to tether for kinetochore microtubules during mitosis, consistent with the observed spindle defects and chromosome mis-segregation.10

Our results point to a role of KIF4 in determining higher order chromatin structure. While we show that the effects on chromatin structure do not require the motor domain of KIF4, an open, intriguing question is whether the motor protein KIF4 functions in the nucleus in a more architectural role such as promoting the organization or movement of chromatin or nuclear bodies. If so, the motor protein would require a substrate to move along. While there is some evidence for tubulin and short, possibly, branched, networks of actin36,37 in the nucleus, it remains to be determined whether KIF4 interacts with these structures. Since KIF4 physically interacts with lamin A, an intriguing, yet untested, possibility is that KIF4 uses the lamins as a track.

Loss of KIF4 has previously been shown to lead to mitotic chromatin defects, chromosome segregation defects, genomic instability, and ultimately, tumor formation.14 These effects are relevant since KIF4 is deleted or downregulated in up to 35% of human cancers.14 It has generally been assumed that these global defects are due to mitotic errors caused by loss of KIF4. Given its role in interphase chromosome structure, however, it is possible that the aberrant interphase chromosome structure and replication defects also contribute to premature chromosome condensation, genomic instability and tumor formation. Interestingly, KIF4, PARP1 and condensin have all been shown to interact with each other38 and interactions between condensin, KIF4 and PARP-110,18,38 may act in concert in chromosome structure maintenance and genome integrity.9 Importantly, PARP-1 also interacts with condensins38 and has recently been implicated in homologous recombination on damaged DNA.9,39 Furthermore, the interaction between condensin and PARP-1 is particularly strong during S phase. The heat shock protein HSF2 has been shown to recruit PP2A that inactivates condensin subunit CAP-G for transient “opening” of chromatin.40 PP2A has also been shown to be involved in recruiting KIF4 onto mitotic chromosome arms.9 Interestingly, PRC1, the protein regulator of cytokinesis and a KIF4 interacting protein, has been found to interact with HSF2 at the hsp70i promoter.41 The binding of KIF4 to heat shock elements (HSE) in vitro (Fig. 1) and the direct binding to both condensin10 and PARP-1 lead us to speculate that KIF4, PARP-1 and condensin may also collaborate in the regulation of higher order chromatin structure.

We demonstrate here that the KIF4 C-terminal domain is sufficient to bind DNA in vitro and for restoration of the chromatin defects caused by genetic deletion of KIF4 in ES cells (Fig. 2). The fact that PARP-1 binds to the C-terminal domain of KIF418 suggests the possibility that KIF4 modulates higher-order chromatin structure by regulating the action of PARP-1 by competitive binding to chromatin via the same domain.

In contrast to interphase chromatin which is highly decondensed in the absence of KIF4, loss of KIF4 has previously been shown to cause hypercondensation of chromosomes in early mitosis.10 These two apparently divergent effects may be the manifestation of a single cause. The S-phase related defects may alter chromatin structure such that when cells enter M phase, condensation cannot occur properly. This effect may occur via condensin complexes, which interact with KIF4 and are mis-localized on mitotic chromosomes upon loss of KIF4.10 Moreover, the defects observed in the progression through S-phase in the absence of KIF4 is consistent with the observation that the association of both KIF4 and several components of condensin complexes on chromatin peak during early stages of S phase.42 Thus KIF4 association with the chromosome condensation machinery may play a pivotal role in determining progression through the cell cycle. Together, these observations demonstrate that the microtubule-associated motor chromokinesin KIF4 not only functions in mitosis, but also has a key role in maintenance of interphase chromatin structure thus revealing a novel cellular role for a molecular motor protein.

Materials and Methods

DNA constructs and cell lines

Human KIF4 full-length clone in pEGFP-C1 plasmid was kindly provided by Jiang Wei, La Jolla, CA, USA.11 The stable ES cell clones were generated by introducing the expression vectors via transfection with Oligofectamine (Invitrogen), selection for resistance to G418, followed by identification of GFP-expressing clones by direct observation under epifluorescence. KIF4GFP expressing cells were selected from low expressing clones in the KIF4KO background to eliminate overexpressing clones. Histone H1GFP was previously described.20

Electrophoretic Mobility Shift (EMSA) assay

EMSA was performed with both HeLa cell nuclear extract and a homogeneously purified recombinant GST-tagged C-terminal KIF4.10 The AT-rich DNA probes (HSE) were selected by homology search of KIF4 sequence against all DNA-binding protein motifs.43,44 Probes were radio-labeled and gel shift assay with anti-KIF4 antibody was performed (see ref. 45). Nonspecific competitor DNA poly (dI•dC) or poly (dA•dT) were included in the binding reaction to minimize the binding of nonspecific proteins to the labeled target DNA. Unlabeled competitor sequence was also added to the binding reaction in 50-fold molar excess to out-compete any specific interactions.

Indirect immunofluorescence, in vivo microscopy and FRAP

The serial extraction of HeLa cell nuclei was performed with cytoskeletal buffer containing 1% Triton X-100 and/or 500 mM NaCl, and/or 500 μg/ml DNase (Boehringer). Indirect immunofluorescence microscopy was performed using fixation in 2% buffered paraformaldehyde in PBS for 15 min at room temperature followed by permeabilization with 0.1% TritonX-100 where necessary. Cells were mounted using DAPI-containing mounting medium (Vectashield; Vector Laboratories). Antibodies used were Histone H3 (tri methyl K9) (rabbit polyclonal, Abcam) and HP1α (mouse monoclonal, Chemicon); secondary antibodies were Alexa Fluor 488 or 568 goat anti- rabbit or anti-mouse (Invitrogen) as appropriate. Cells were observed on a confocal microscope (LSM 510 META; Carl Zeiss, Inc.) and images were acquired using a 63x 1.4 NA oil objective lens (Carl Zeiss, Inc.) using an optical step size of 0.2 or 0.5 µm. Images were generated and analyzed in Metamorph (Universal Imaging). For live-cell FRAP analysis, we grew cells to 50% confluence in Nalgene LabTek II chambers and photo-bleached half the nucleus in Zeiss LSM 510 confocal microscope using the 488-nm laser line of an argon laser at 37°C as described.20 Five single imaging scans were acquired, followed by a single bleach pulse of 5 sec. Single-section images were then collected at 1 sec intervals (120 images). For imaging, the laser power was attenuated to 1% of the bleach intensity. FRAP recovery curves were generated from background-subtracted images. The total nuclear fluorescence was determined for each image and compared with the initial total fluorescence to account for the amount of signal lost during the bleach pulse and during imaging as described20 and relative recoveries were determined as described.20 The standard Student’s t-test was used to determine the statistical significance of results. All quantitative values represent averages ± s.d. from at least 15 cells from three independent experiments.

Electron microscopy

Transmission electron microscopy was performed at the National Cancer Institute's Frederick EM facility by Kunio Nageshima. Low passage parental E14 wild-type or KIF4KO ES cells were plated in 6-well plastic plates coated with gelatin at 60% cell density. Cells were fixed with 1% Gluteraldehyde in 0.1M cacodylate buffer. For both WT and KIF4KO ES cells over 50 different sections were imaged at a magnification of 8000X.

DNA and whole chromosome FISH

Mouse major satellite repeat FISH was performed in ES cells (as described in ref. 46) and chromosome territory FISH was performed (according to ref. 47). The karyotypically normal human fibroblast cell line MRC-5 was used for chromosome territory FISH. KIF4 was depleted from these cells by RNAi as described.10 Human X-chromosome specific whole chromosome paint probe was purchased from Cambio (Cat# 1153-X Cy3–01).

Salt extraction, micrococcal nuclease digestion, Immunoprecipitation and protein gel blotting

Salt extraction of various proteins from HeLa or ES cells and MNase digestion were performed after purification of nuclei from both cell types following refs.20,46 respectively. Immunoprecipitation and protein gel blotting using anti-KIF4A monoclonal antibody was performed as previously described.10 Anti-PARP-1 monoclonal antibody (clone C2–10) and anti-PAR monoclonal antibody (10HA) were from Trevigen. Polyclonal histone H1 antibody was a kind gift of M. Bustin, NCI, NIH, USA. Monoclonal antibodies against HIRA were kindly provided by P. Adams, Beatson Institute, Glasgow, UK. Rabbit polyclonal antibody against p150 CAF-1 and Asf1a and b were used at 1:2000 dilutions (kindly provided by G. Almouzni, Institute Pasteur, Paris). Polyclonal RbAp48/RbAp46 antibody was from Abcam (ab1765). Soluble chromatin-bound material from asynchronous and S-phase synchronized HeLa cells were immunoprecipitated following the method described in reference.48

Microarray and Statistical analysis of microarray data

Affymetrix whole mouse genome arrays MG 430.2 were used to profile transcript abundance. Four biological replicates each for KIF4KO and WT E14 ES cells were hybridized onto the microarrays. Differentially expressed genes were obtained by requiring a minimum fold change of 1.5 with p value < 0.05 based on a two-sample T test. Gene ontology analysis was performed using the biological process classification information. The GO categories for the 502 differentially expressed genes were tabulated. The same tabulation was applied also to all genes represented in the Affymetrix microarray to determine the background distribution. The GO categories enriched in the differentially expressed genes and their tabulation results were extracted. All computations and plotting were performed by custom-written programs in R.

Supplementary Material

Additional material

Supplementary PDF file supplied by authors.

nucl-2-591-s01.pdf (363.4KB, pdf)

Acknowledgments

We thank G. Almouzni, Institute Pasteur, Paris for providing the p150 CAF-1 and Asf1 antibodies, M. Bustin, NCI, NIH, USA for polyclonal histone H1 antibody, and P. Adams, Beatson Institute, Glasgow, UK, for monoclonal antibodies against HIRA. We thank C. Hassel, Indiana University Flow cytometry facility and S. Dasari for help with FACS analysis and K. Meaburn for help with FISH protocols. EM was performed by K. Nageshima at the NCI, EM facility. This research was in part supported by the Intramural Research Program of the National Institutes of Health (NIH), NCI, Center for Cancer Research and by start-up funds from Indiana University. M.M. conceived, performed and analyzed most experiments. M-H.S. analyzed the microarray data. M.M. and T.M. consulted on data interpretation, M.M. and T.M wrote the manuscript.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Footnotes

References

  • 1.Hirokawa N, Noda Y, Tanaka Y, Niwa S. Kinesin superfamily motor proteins and intracellular transport. Nat Rev Mol Cell Biol. 2009;10:682–96. doi: 10.1038/nrm2774. [DOI] [PubMed] [Google Scholar]
  • 2.Bisbal M, Wojnacki J, Peretti D, Ropolo A, Sesma J, Jausoro I, et al. KIF4 mediates anterograde translocation and positioning of ribosomal constituents to axons. J Biol Chem. 2009;284:9489–97. doi: 10.1074/jbc.M808586200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Mazumdar M, Misteli T. Chromokinesins: multitalented players in mitosis. Trends Cell Biol. 2005;15:349–55. doi: 10.1016/j.tcb.2005.05.006. [DOI] [PubMed] [Google Scholar]
  • 4.Walczak CE, Cai S, Khodjakov A. Mechanisms of chromosome behaviour during mitosis. Nat Rev Mol Cell Biol. 2010;11:91–102. doi: 10.1038/nrm2832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Wu G, Chen PL. Structural requirements of chromokinesin Kif4A for its proper function in mitosis. Biochem Biophys Res Commun. 2008;372:454–8. doi: 10.1016/j.bbrc.2008.05.065. [DOI] [PubMed] [Google Scholar]
  • 6.Wu G, Zhou L, Khidr L, Guo XE, Kim W, Lee YM, et al. A novel role of the chromokinesin Kif4A in DNA damage response. Cell Cycle. 2008;7:2013–20. doi: 10.4161/cc.7.13.6130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Wang SZ, Adler R. Chromokinesin: a DNA-binding, kinesin-like nuclear protein. J Cell Biol. 1995;128:761–8. doi: 10.1083/jcb.128.5.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Tahara K, Takagi M, Ohsugi M, Sone T, Nishiumi F, Maeshima K, et al. Importin-beta and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid. J Cell Biol. 2008;180:493–506. doi: 10.1083/jcb.200708003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Takemoto A, Maeshima K, Ikehara T, Yamaguchi K, Murayama A, Imamura S, et al. The chromosomal association of condensin II is regulated by a noncatalytic function of PP2A. Nat Struct Mol Biol. 2009;16:1302–8. doi: 10.1038/nsmb.1708. [DOI] [PubMed] [Google Scholar]
  • 10.Mazumdar M, Sundareshan S, Misteli T. Human chromokinesin KIF4A functions in chromosome condensation and segregation. J Cell Biol. 2004;166:613–20. doi: 10.1083/jcb.200401142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Zhu C, Jiang W. Cell cycle-dependent translocation of PRC1 on the spindle by Kif4 is essential for midzone formation and cytokinesis. Proc Natl Acad Sci USA. 2005;102:343–8. doi: 10.1073/pnas.0408438102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kurasawa Y, Earnshaw WC, Mochizuki Y, Dohmae N, Todokoro K. Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation. EMBO J. 2004;23:3237–48. doi: 10.1038/sj.emboj.7600347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Ohsugi M, Adachi K, Horai R, Kakuta S, Sudo K, Kotaki H, et al. Kid-mediated chromosome compaction ensures proper nuclear envelope formation. Cell. 2008;132:771–82. doi: 10.1016/j.cell.2008.01.029. [DOI] [PubMed] [Google Scholar]
  • 14.Mazumdar M, Lee JH, Sengupta K, Ried T, Rane S, Misteli T. Tumor formation via loss of a molecular motor protein. Curr Biol. 2006;16:1559–64. doi: 10.1016/j.cub.2006.06.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Steeg PS. New insights into the tumor metastatic process revealed by gene expression profiling. Am J Pathol. 2005;166:1291–4. doi: 10.1016/S0002-9440(10)62348-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Anjomshoaa A, Lin YH, Black MA, McCall JL, Humar B, Song S, et al. Reduced expression of a gene proliferation signature is associated with enhanced malignancy in colon cancer. Br J Cancer. 2008;99:966–73. doi: 10.1038/sj.bjc.6604560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Lee YM, Lee S, Lee E, Shin H, Hahn H, Choi W, et al. Human kinesin superfamily member 4 is dominantly localized in the nuclear matrix and is associated with chromosomes during mitosis. Biochem J. 2001;360:549–56. doi: 10.1042/0264-6021:3600549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Midorikawa R, Takei Y, Hirokawa N. KIF4 motor regulates activity-dependent neuronal survival by suppressing PARP-1 enzymatic activity. Cell. 2006;125:371–83. doi: 10.1016/j.cell.2006.02.039. [DOI] [PubMed] [Google Scholar]
  • 19.Geiman TM, Sankpal UT, Robertson AK, Chen Y, Mazumdar M, Heale JT, et al. Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery. Nucleic Acids Res. 2004;32:2716–29. doi: 10.1093/nar/gkh589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Misteli T, Gunjan A, Hock R, Bustin M, Brown DT. Dynamic binding of histone H1 to chromatin in living cells. Nature. 2000;408:877–81. doi: 10.1038/35048610. [DOI] [PubMed] [Google Scholar]
  • 21.Chen ES, Zhang K, Nicolas E, Cam HP, Zofall M, Grewal SI. Cell cycle control of centromeric repeat transcription and heterochromatin assembly. Nature. 2008;451:734–7. doi: 10.1038/nature06561. [DOI] [PubMed] [Google Scholar]
  • 22.Li J, Jiang J, Qian Q, Xu Y, Zhang C, Xiao J, et al. Mutation of rice BC12/GDD1, which encodes a kinesin-like protein that binds to a GA biosynthesis gene promoter, leads to dwarfism with impaired cell elongation. Plant Cell. 2011;23:628–40. doi: 10.1105/tpc.110.081901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Krishnakumar R, Gamble MJ, Frizzell KM, Berrocal JG, Kininis M, Kraus WL. Reciprocal binding of PARP-1 and histone H1 at promoters specifies transcriptional outcomes. Science. 2008;319:819–21. doi: 10.1126/science.1149250. [DOI] [PubMed] [Google Scholar]
  • 24.Tulin A, Stewart D, Spradling AC. The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development. Genes Dev. 2002;16:2108–19. doi: 10.1101/gad.1003902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.D'Amours D, Desnoyers S, D'Silva I, Poirier GG. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions. Biochem J. 1999;342:249–68. doi: 10.1042/0264-6021:3420249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kim MY, Zhang T, Kraus WL. Poly(ADP-ribosyl)ation by PARP-1: ‘PAR-laying’ NAD+ into a nuclear signal. Genes Dev. 2005;19:1951–67. doi: 10.1101/gad.1331805. [DOI] [PubMed] [Google Scholar]
  • 27.Levros LC, Jr., Do Carmo S, Edouard E, Legault P, Charfi C, Rassart E. Characterization of nuclear factors modulating the apolipoprotein D promoter during growth arrest: implication of PARP-1, APEX-1 and ERK1/2 catalytic activities. Biochim Biophys Acta. 2010;1803:1062–71. doi: 10.1016/j.bbamcr.2010.04.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Lu J, Gilbert DM. Proliferation-dependent and cell cycle regulated transcription of mouse pericentric heterochromatin. J Cell Biol. 2007;179:411–21. doi: 10.1083/jcb.200706176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Quivy JP, Gerard A, Cook AJ, Roche D, Almouzni G. The HP1-p150/CAF-1 interaction is required for pericentric heterochromatin replication and S-phase progression in mouse cells. Nat Struct Mol Biol. 2008;15:972–9. doi: 10.1038/nsmb.1470. [DOI] [PubMed] [Google Scholar]
  • 30.Taddei A, Roche D, Sibarita JB, Turner BM, Almouzni G. Duplication and maintenance of heterochromatin domains. J Cell Biol. 1999;147:1153–66. doi: 10.1083/jcb.147.6.1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Tagami H, Ray-Gallet D, Almouzni G, Nakatani Y. Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell. 2004;116:51–61. doi: 10.1016/S0092-8674(03)01064-X. [DOI] [PubMed] [Google Scholar]
  • 32.Sanchez-Molina S, Mortusewicz O, Bieber B, Auer S, Eckey M, Leonhardt H, Friedl AA, Becker PB. Role for hACF1 in the G2/M damage checkpoint. Nucleic Acids Res 2011; 39: 8445-56. [DOI] [PMC free article] [PubMed]
  • 33.Pflumm MF, Botchan MR. Orc mutants arrest in metaphase with abnormally condensed chromosomes. Development. 2001;128:1697–707. doi: 10.1242/dev.128.9.1697. [DOI] [PubMed] [Google Scholar]
  • 34.Wechsler T, Newman S, West SC. Aberrant chromosome morphology in human cells defective for Holliday junction resolution. Nature 2011; 471-642-6. [DOI] [PMC free article] [PubMed]
  • 35.Euskirchen GM, Auerbach RK, Davidov E, Gianoulis TA, Zhong G, Rozowsky J, et al. Diverse roles and interactions of the SWI/SNF chromatin remodeling complex revealed using global approaches. PLoS Genet. 2011;7:e1002008. doi: 10.1371/journal.pgen.1002008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Xu K, Luduena RF. Characterization of nuclear betaII-tubulin in tumor cells: a possible novel target for taxol. Cell Motil Cytoskeleton. 2002;53:39–52. doi: 10.1002/cm.10060. [DOI] [PubMed] [Google Scholar]
  • 37.Andrin C, Hendzel MJ. F-actin-dependent insolubility of chromatin-modifying components. J Biol Chem. 2004;279:25017–23. doi: 10.1074/jbc.M401805200. [DOI] [PubMed] [Google Scholar]
  • 38.Heale JT, Ball AR, Jr., Schmiesing JA, Kim JS, Kong X, Zhou S, et al. Condensin I interacts with the PARP-1-XRCC1 complex and functions in DNA single-strand break repair. Mol Cell. 2006;21:837–48. doi: 10.1016/j.molcel.2006.01.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Sugimura K, Takebayashi S, Taguchi H, Takeda S, Okumura K. PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA. J Cell Biol. 2008;183:1203–12. doi: 10.1083/jcb.200806068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Xing H, Wilkerson DC, Mayhew CN, Lubert EJ, Skaggs HS, Goodson ML, et al. Mechanism of hsp70i gene bookmarking. Science. 2005;307:421–3. doi: 10.1126/science.1106478. [DOI] [PubMed] [Google Scholar]
  • 41.Murphy LA, Wilkerson DC, Hong Y, Sarge KD. PRC1 associates with the hsp70i promoter and interacts with HSF2 during mitosis. Exp Cell Res. 2008;314:2224–30. doi: 10.1016/j.yexcr.2008.04.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Khoudoli GA, Gillespie PJ, Stewart G, Andersen JS, Swedlow JR, Blow JJ. Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition. Curr Biol. 2008;18:838–43. doi: 10.1016/j.cub.2008.04.075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Rabindran SK, Giorgi G, Clos J, Wu C. Molecular cloning and expression of a human heat shock factor, HSF1. Proc Natl Acad Sci USA. 1991;88:6906–10. doi: 10.1073/pnas.88.16.6906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Schuetz TJ, Gallo GJ, Sheldon L, Tempst P, Kingston RE. Isolation of a cDNA for HSF2: evidence for two heat shock factor genes in humans. Proc Natl Acad Sci USA. 1991;88:6911–5. doi: 10.1073/pnas.88.16.6911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Mosser DD, Theodorakis NG, Morimoto RI. Coordinate changes in heat shock element-binding activity and HSP70 gene transcription rates in human cells. Mol Cell Biol. 1988;8:4736–44. doi: 10.1128/mcb.8.11.4736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Meshorer E, Misteli T. Chromatin in pluripotent embryonic stem cells and differentiation. Nat Rev Mol Cell Biol. 2006;7:540–6. doi: 10.1038/nrm1938. [DOI] [PubMed] [Google Scholar]
  • 47.Parada LA, McQueen PG, Munson PJ, Misteli T. Conservation of relative chromosome positioning in normal and cancer cells. Curr Biol. 2002;12:1692–7. doi: 10.1016/S0960-9822(02)01166-1. [DOI] [PubMed] [Google Scholar]
  • 48.Groth A, Corpet A, Cook AJ, Roche D, Bartek J, Lukas J, et al. Regulation of replication fork progression through histone supply and demand. Science. 2007;318:1928–31. doi: 10.1126/science.1148992. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional material

Supplementary PDF file supplied by authors.

nucl-2-591-s01.pdf (363.4KB, pdf)

Articles from Nucleus are provided here courtesy of Taylor & Francis

RESOURCES