Dynein Light Chain 1 (DYNLT1) Interacts with Normal and Oncogenic Nucleoporins

The chimeric oncoprotein NUP98-HOXA9 results from the t(7;11)(p15;p15) chromosomal translocation and is associated with acute myeloid leukemia. It causes aberrant gene regulation and leukemic transformation through mechanisms that are not fully understood. NUP98-HOXA9 consists of an N-terminal portion of the nucleoporin NUP98 that contains many FG repeats fused to the DNA-binding homeodomain of HOXA9. We used a Cytotrap yeast two-hybrid assay to identify proteins that interact with NUP98-HOXA9. We identified Dynein Light Chain 1 (DYNLT1), an integral 14 KDa protein subunit of the large microtubule-based cytoplasmic dynein complex, as an interaction partner of NUP98-HOXA9. Binding was confirmed by in vitro pull down and co-immunoprecipitation assays and the FG repeat region of NUP98-HOXA9 was shown to be essential for the interaction. RNAi-mediated knockdown of DYNLT1 resulted in reduction of the ability of NUP98-HOXA9 to activate transcription and also inhibited the ability of NUP98-HOXA9 to induce proliferation of primary human hematopoietic CD34+ cells. DYNLT1 also showed a strong interaction with wild-type NUP98 and other nucleoporins containing FG repeats. Immunofluorescence analysis showed that DYNLT1 localizes primarily to the nuclear periphery, where it co-localizes with the nuclear pore complex, and to the cytoplasm. Deletion studies showed that the interactions of the nucleoporins with DYNLT1 are dependent predominantly on the C-terminal half of the DYNLT1. These data show for the first time that DYNLT1 interacts with nucleoporins and plays a role in the dysregulation of gene expression and induction of hematopoietic cell proliferation by the leukemogenic nucleoporin fusion, NUP98-HOXA9.

This study was aimed at identifying other NUP98-HOXA9interacting proteins and determining their role in the dysregulation of gene expression by NUP98-HOXA9. Traditional yeast twohybrid assays based on intranuclear transcriptional activation are likely to show a high number of false positives because of the transactivating properties of the FG repeat-rich N-terminus of NUP98-HOXA9 [13,25]. On the other hand, the Cytotrap yeast two-hybrid method is based on interactions occurring in the cytoplasm and is not affected by the transactivating properties of the bait protein. Using this technique, we identified DYNLT1, an integral protein subunit of the large microtubule-based cytoplasmic dynein complex [26,27,28], as a novel interaction partner of NUP98-HOXA9 that is important for the ability of NUP98-HOXA9 to dysregulate gene transcription.

Cytotrap Yeast Two Hybrid Analysis
Cytotrap yeast two-hybrid analysis was carried out following the manufacturer's instructions. Briefly, the pMYR vectors containing the K562 cDNA library and pSOS-HA-NUP98-HOXA9 were cotransformed into the temperature sensitive cdc25H yeast strain, plated into selective medium containing glucose as the carbon source and incubated at room temperature till colonies appeared. The colonies were replica-plated into positive selective medium containing galactose as the carbon source and incubated at 37uC till visible colonies appeared. Temperature resistant cdc25 yeast colonies growing at 37uC were isolated, patched into new plates containing selective media, grown, and replica plated again to confirm the positive growth and eliminate false positives. Cells from the patches were grown in liquid medium and pMYR vectors containing the positive clones were isolated. The isolated plasmids were transformed and amplified in E. coli and DNA was isolated. The purified DNA was sequenced using primers on both sides of the cDNA insert regions. The cDNA inserts were identified using the National Center for Biotechnology Information blast application (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Protein Binding Assays
Full length NUP98, NUP98-HOXA9 and its deletion mutants, as well as full length NUP62 and NUP153 and their deletion mutants, were produced using the TnT T7 quick coupled transcription/translation system (Promega) in the presence of Tran35S-label (MP Biomedicals). Protein binding assays were carried out as described previously [29]. Briefly, for a binding reaction, 10 ml of beads of immobilized recombinant protein (GST control, or GST-DYNLT1, or GST-DYNLT1 N or C terminus) were incubated for 1 h at 4uC with 34 ml of a mixture consisting of in vitro translated protein, transport buffer-Tween 20 (TB-T, which consists of 20 mm HEPES-KOH, pH 7.4, 110 mm potassium acetate, and 2 mm MgCl2 with 0.1% Tween 20) and 0.5 mg/ml Pefabloc (Roche). The unbound fraction was removed and the beads were washed three times in cold TB-T. One fourth of the unbound and all of the bound fractions were visualized by SDS-PAGE and autoradiography.
For binding assays using all recombinant proteins, purified recombinant DYNLT1 was incubated with immobilized GST, GST-NUP98-HOXA9, or GST-HOXA9 proteins in 34 ml mixtures in TB-T and processed as described above. Bands were visualized by staining with Coomassie blue.

DYNLT1 Antibody Preparation
Recombinant DYNLT1 protein was purified as described above and was submitted to Harlan Laboratories where rabbit immunizations and bleedings were done according to their standard protocols. Production bleeds with the highest concentration of DYNLT1 antibody as detected by immunoblotting on K562 cell lysate and purified DYNLT1 were selected for antibody purification. At each step of purification, serum and antibody fractions were tested by immunoblotting of K562 cell lysate (Fig. S1). Samples were subjected to SDS-PAGE and transferred to PVDF membranes. Membrane strips were prepared and incubated with dilutions of pre-bleed serum, test bleeds, production bleeds, samples from different stages of purification and collected fractions (Fig. S1). An HRP-conjugated anti-rabbit antibody was used as secondary antibody. Antiserum (Fig. S1, lane 1) was first precleared for 4 h at 4uC using 1 ml Affigel 15 beads (Bio-Rad) loaded with lysate containing 30 mg protein from E. coli BL21 bacteria expressing empty pGEX6P1 vector (Fig. S1, lane 2). The pre-cleared serum was added to a column containing 1 ml Affigel 15 beads loaded with 1 mg DYNLT1 protein and incubated overnight with rotation at 4uC. The flow-through fraction was depleted of DYNLT1 antibody as shown in Fig. S1, lane 3. The column was washed 6 times with 10 ml cold DPBS at 4uC. Bound antibody was eluted once with 4 ml 1 M NaCl in DPBS, once with 4 ml 0.1 M Glycine, pH 2.5 and once with 4 ml 0.1 M Glycine pH 2.5, 1 M NaCl, 1% CHAPS. For each elution, 0.5 ml fractions were collected, and glycine-containing fractions were neutralized with 25 ml of 1 M Tris-base. Antibody concentration in the various fractions was determined by Amido Black staining and immunoblotting of purified recombinant DYNLT1 or K562 lysate (Fig. S1, lanes 4-11). More than 90% of the antibody was detected in fractions 4, 5 and 6 of the 0.1 M Glycine pH 2.5 elution (Fig. S1, lanes 7-9); these fractions were stored at 280uC. Specificities of the antibodies were determined by immunobloting either purified recombinant proteins or K562 lysates. To further test the anti-DYNLT1 antibody, K562 cells were nucleofected with empty pRFP-C-RS vector, vector containing nonspecific shRNA, or vectors containing two different shRNAs against DYNLT1 (Origene) (see Table 1 for sequences). Lysates were subjected to SDS-PAGE and DYNLT1 was detected with the anti-DYNLT1 antibody.

Immunofluorescence Microscopy
For localization of DYNLT1, 25,000 HEPG2 (ATCC) cells were grown overnight on poly-L-lysine coated coverslips (Neuvitro), fixed with methanol/1mM EGTA for 10 min at 220uC, washed with PBS twice, and blocked with 2% normal goat serum in DPBS with 1% BSA, 0.1% Tween 20. Primary antibodies used were rabbit anti-DYNLT1 along with mouse MAb 414 (Abcam). The fluorescent secondary antibodies used were FITC-conjugated anti-rabbit IgG from goat (Santa Cruz Biotechnology) and rhodamine-conjugated anti-mouse IgG from goat (Millipore Corp.). The slides were mounted with Vectashield mounting medium with DAPI (Vector Laboratories). The images were captured using an Eclipse 80i fluorescent microscope (Nikon) and processed in Metamorph ver 6.3r2 software (Molecular Devices).

Luciferase Assay
For analysis for DYNLT1 deletion mutants, K562 cells were nucleofected with 5 mg pGL4.11 vector driven by the KBTBD10 promoter and 10 mg pcDNA-HA-NUP98-HOXA9 in combination with 10 mg of empty pcDNA3, pcDNA-DYNLT1, pcDNA-DYNLT1 N-terminus, or pcDNA-DYNLT1 C-terminus. For shRNA-mediated knockdown of DYNLT1, K562 cells were nucleofected with 5 mg pGL4.11 vector driven by the KBTBD10 promoter in combination with 10 mg of either empty pcDNA3, or pcDNA3 expressing HA-NUP98-HOXA9. In addition, either empty pRFP-C-RS vector (Origene-TR30014), vector containing non-specific shRNA (Origene-TR30015), or vectors containing two different shRNA against DYNLT1 (Origene-TF313329) were included ( Table 1). Knockdown of DYNLT1 was confirmed by immunoblotting with the anti-DYNLT1 antibody and the efficiency of knockdown was measured by densitometric analysis (Fig. S2). To control for efficiency of transfection, 0.5 mg of pRL-TK (Promega), which expresses Renilla luciferase was included. Five million cells were incubated with the DNA at room temperature for 10 min before electroporation and 10 min after electroporation, and were cultured in 10 ml IMDM media with 10% FBS, 2 mM L-glutamine, and 100 units/ml penicillin/ streptomycin. Luciferase activity was measured 48 h after electroporation using the Dual Luciferase Reporter Assay System (Promega) and the results were normalized to Renilla luciferase.

Retroviral Transduction and Primary Human CD34+ Cells Proliferation Assay
Frozen human CD34+ cells from healthy volunteers (Fred Hutchinson Cancer Research Center) were prestimulated and transduced with MSCV-IRES-YFP retrovirus expressing HA-NUP98-HOXA9 as previously described [17]. After 48 h, YFP positive cells were isolated using a MoFlo high-speed sorter (Dako) and expression of the NUP98-HOXA9 was confirmed by immunoblotting with anti-HOXA9 antibody. For DYNLT1 knockdown, NUP98-HOXA9 transfected CD34+ cells were grown for two weeks and nucleofected with either non-specific siRNA (Invitrogen-12935-300) or siRNA specific for DYNLT1 (Invitrogen-1299003) ( Table 1). An MTS cell proliferation assay was done using CellTiter 96H AQueous NonRadioactive Cell Proliferation Assay (Promega G5421) according to the manufacturer's instructions. Twenty five thousand transfected cells were seeded into a 96-well plate in triplicate and grown for 24 h, 48 h and 72 h in 100 ml of culture medium. Twenty ml of MTS/ phenazine methosulfate solution was added and the cells were incubated at 37uC for 2 h. The absorbance at 490 nm was measured using an ELISA plate reader. Knockdown of DYNLT1 was confirmed by immunoblotting using the anti-DYNLT1 antibody.

Results
Cytotrap Two-hybrid Analysis shows that NUP98-HOXA9 Interacts with Dynein Light Chain 1 (DYNLT1) We used a Cytotrap yeast two-hybrid assay to identify possible interaction partners of the oncoprotein NUP98-HOXA9. The traditional two-hybrid assay is based on intranuclear interactions and is likely to show many false positives when the bait contains a transactivation domain such as the N-terminus of NUP98-HOXA9 [13]. In the Cytotrap yeast two-hybrid assay, the cDNA library expressing the target genes is subcloned downstream of a myristylation signal that localizes the proteins to the plasma membrane and the bait protein is fused to hSOS which remains in the cytoplasm. A physical interaction between the bait protein and one of the cDNA targets, results in recruitment of the hSOS protein to the plasma membrane, which causes activation of the Ras signaling pathway. This allows the cdc25H strain to grow and form colonies at the restrictive temperature (37uC) in galactosecontaining medium. The bait protein was prepared by fusing NUP98-HOXA9 to hSOS and the target cDNA library was prepared from human K562 cells and subcloned into the pMYR vector. Expression of hSOS-NUP98-HOXA9 in the cdc25H yeast strain was confirmed by immunoblotting [16]. Controls for the assay were performed according to manufacturer's instructions as previously described [16]. The cdc25H heat-sensitive yeast strain was transformed with both the bait protein and the target cDNA library. Several hundred colonies appeared and were replicaplated onto galactose-containing medium to induce expression of the target library. Colonies growing on the galactose-containing medium were isolated and selection in stringent temperature and media was repeated for selection of true positive clones. The cDNA inserts from the positive clones were isolated and sequenced. Several of the positive clones encoded full length Dynein Light Chain 1 (DYNLT1).
To confirm the interaction between NUP98-HOXA9 and DYNLT1, the plasmids encoding DYNLT1 along with either pSOS vector or vector expressing hSOS-NUP98-HOXA9 were reintroduced into the cdc25H yeast strain. Colonies were isolated and patched into positive selective medium and subjected to the restrictive temperature. Co-transfection of with the pSOS-NUP98-HOXA9 bait construct along with a plasmid expressing myristylated Lamin C into cdc25 strain served as the negative control. Cells co-transfected with the pSOS-NUP98-HOXA9 bait construct and a plasmid expressing myristylated SOS-binding protein (SB) served as the positive control. As shown in Fig. 1 growth was observed in all transformants incubated at 23uC. No growth was observed at 37uC in glucose containing medium because the cDNA library is cloned under a galactose-inducible promoter and was not expressed in the absence of galactose. When transferred to restrictive 37uC, no growth was seen in cells coexpressing pMYR-LaminC and pSOS-NUP98-HOXA9 indicating that NUP98-HOXA9 does not activate Ras signaling by targeting itself to the plasma membrane. Growth was present in cells co-expressing myristylated SB (SOS binding protein) and pSOS-NUP98-HOXA9 because the membrane-anchored SB protein recruits hSOS-NUP98-HOXA9 to the plasma membrane by binding to the hSOS portion. Cells co-transfected with hSOS-NUP98-HOXA9 and DYNLT1 showed a similar growth pattern suggesting a specific interaction between DYNLT1 and NUP98-HOXA9. The positive interaction observed with hSOS-NUP98-HOXA9 is not due to binding of DYNLT1 to the hSOS portion of the bait as there was no growth in cells transfected with the empty hSOS vector and DYNLT1 (Fig. 1).

DYNLT1 Interacts with the FG Repeat Region of NUP98-HOXA9
To establish a direct interaction between DYNLT1 and NUP98-HOXA9 an in-vitro binding assay was carried out using purified recombinant proteins. GST, GST-NUP98-HOXA9, or GST-HOXA9 were immobilized on beads and incubated with purified DYNLT1 protein. As shown in Fig. 2A, GST-NUP98-HOXA9 interacted with DYNLT1 whereas no interaction was observed for GST or GST-HOXA9. This suggests that DYNLT1 specifically interacts with the NUP98 portion of the NUP98-HOXA9 fusion protein.
To demonstrate that NUP98-HOXA9 interacts with DYNLT1 in vivo, co-immunoprecipitation was carried out using K562 cells transfected with HA-tagged NUP98-HOXA9. As the affinity and specificity of the commercially available DYNLT1 antibody were not optimal (data not shown), a rabbit polyclonal antibody was raised (Fig. S1) and used for immunoprecipitation. Whole cell lysates were subjected to immunoprecipitation with either rabbit Figure 6. C-terminus of DYNLT1 interacts with NUP98-HOXA9, NUP62 and NUP153. A) Full length DYNLT1 and deletion mutants. B) GST or GST-DYNLT1 or GST-DYNLT1 N-terminus or GST-DYNLT1 C-terminus with in vitro translated NUP98-HOXA9, NUP62 or NUP153. Bound and unbound fractions were resolved by SDS-PAGE and subjected to autoradiography. C) K562 cells were nucleofected with pGL4.11 vector driven by the KBTBD10 promoter and pcDNA-HA-NUP98-HOXA9 in combination with either empty pcDNA3, pcDNA-DYNLT1, pcDNA-DYNLT1 N-terminus, or pcDNA-DYNLT1 C-terminus. Firefly luciferase activity was measured 48 hours after transfection and normalized to a Renilla luciferase internal control. The numbers represent fold change over control (average of 3 independent experiments); error bars represent standard deviations. The P values indicated were obtained by a two-tailed t-test in comparison to NUP98-HOXA9 alone. doi:10.1371/journal.pone.0067032.g006 DYNLT1 Binds to Normal and Oncogenic Nucleoporins PLOS ONE | www.plosone.org IgG or anti-DYNLT1 antibody and probed for NUP98-HOXA9 with anti-HA antibody. As shown in Figs. 2B and 2C, NUP98-HOXA9 co-immunoprecipitated with endogenous DYNLT1, confirming their interaction within human myeloid cells.
Removal of the C-terminal portion of the NUP98 moiety (D253-445) did not affect binding of NUP98-HOXA9 to DYNLT1. On the other hand, when the N-terminal of NUP98-HOXA9 was deleted (D1-253), the interaction was significantly reduced. The interaction of DYNLT1 with NUP98-HOXA9 was abolished by deletion of the entire NUP98 moiety (Fig. 3B). These data show that DYNLT1 interacts with the FG repeat-rich NUP98 moiety of NUP98-HOXA9 and that the first 253 amino acids of NUP98-HOXA9 are required for optimal interaction with DYNLT1. DYNLT1 Colocalizes with the Nuclear Pore Complex DYNLT1, also known as Tctex-1, is an integral 14 KDa protein subunit of the large microtubule-based cytoplasmic dynein complex [26,27,28]. The interaction of DYNLT1 with the FG repeat region of NUP98-HOXA9 suggests that DYNLT1 may interact with other FG repeat nucleoporins and localize to the nuclear pore complex. To determine the intracellular localization of DYNLT1 in relation to the nuclear pore complex, human HEPG2 cells were immunostained with rabbit anti-DYNLT1 and mouse MAb414 that highlights the nuclear pore complexes [30]. A FITC-conjugated anti-rabbit secondary antibody was used to detect DYNLT1 and a rhodamine-conjugated anti-mouse secondary antibody was used to highlight the nucleoporins. DYNLT1 was found throughout the cell and formed a ring around the nuclear periphery where it co-localized with the nuclear pore complexes (Fig. 4).

DYNLT1 Interacts with FG Repeat Nucleoporins
The immunolocalization of DYNLT1 to the nuclear rim and its binding to the FG repeat portion of NUP98 raised the possibility that DYNLT1 interacts with other FG repeat nucleoporins. To determine whether this is the case, GST-DYNLT1 immobilized on beads was incubated with in vitro translated NUP153, NUP62, or their deletion mutants (Fig. 5A). As shown in Fig. 5B, DYNLT1 interacts with both NUP153 and NUP62. The binding of DYNLT1 to NUP153 was mildly diminished by deletion of either the N-or C-terminus. On the other hand, removal of the FGrepeat containing N-terminus of NUP62 almost completely abolished the interaction with DYNLT1.
To confirm that DYNLT1 interacts with nucleoporins in vivo, K562 cell lysates were subjected to immunoprecipitation with either rabbit IgG or anti-DYNLT1 antibody and probed for NUP153 and NUP62 using the mouse MAb414 antibody and for NUP98 using the rabbit anti-NUP98 antibody. As shown in Fig. 5C, DYNLT1 coimmunoprecipitates with NUP153, NUP62 and NUP98.
To test whether the DYNLT1 C-terminus, that interacts with NUP98-HOXA9 plays a role in transcriptional dysregulation by NUP98-HOXA9, a luciferase reporter construct driven by the KBTBD10 promoter was introduced into K562 cells along with a construct expressing NUP98-HOXA9 in combination with full length DYNLT1 or DYNLT1 N-terminus or C-terminus. Overexpression of the full length DYNLT1 or the DYNLT1 Cterminus resulted in a significant increase in transcriptional activation of the reporter construct by NUP98-HOXA9, whereas In addition, the transfections included either empty vector, or vector expressing non-specific shRNA, or 2 vectors expressing different DYNLT1 shRNAs. Firefly luciferase activity was measured 48 hours after transfection and normalized to a Renilla luciferase internal control. The numbers represent fold change over control (average of 3 independent experiments); error bars represent standard deviations. The P value indicated was obtained by a two-tailed t-test. The knockdown of DYNLT1 were verified by immunoblotting with anti-HA or anti-DYNLT1 antibody. doi:10.1371/journal.pone.0067032.g007 overexpression of the DYNLT1 N-terminus did not result in increased transcriptional activity by NUP98-HOXA9 (Fig. 6C).

DYNLT1 is Required for Transcriptional Activation by NUP98-HOXA9
NUP98-HOXA9 causes transcriptional dysregulation in human hematopoietic cells [12,13,17,19]. In the human myeloid K562 cell line, we have previously identified the KBTBD10 gene as a direct transcriptional target of NUP98-HOXA9 [19]. To test whether DYNLT1 plays a role in transcriptional dysregulation by NUP98-HOXA9, DYNLT1 was knocked down using two different anti-DYNLT1 shRNA constructs. A luciferase reporter construct driven by the KBTBD10 promoter was introduced into K562 cells along with a construct expressing NUP98-HOXA9, with or without non-specific shRNA or one of the two DYNLT1specific shRNA constructs. Knockdown of DYNLT1 was confirmed by immunoblotting (Figs. 7 and S2). Interestingly, knockdown of DYNLT1 by either of the shRNA constructs resulted in impairment of transcriptional activation of the KBTBD10 promoter by NUP98-HOXA9 (Fig. 7). No such impairment of transcriptional activation was observed with the non-specific shRNA construct. This indicates that DYNLT1 plays a role in transcriptional dysregulation by NUP98-HOXA9 and raises the possibility that DYNLT1 plays a role in the leukemogenic effects of NUP98-HOXA9.

Knockdown of DYNLT1 Inhibits NUP98-HOXA9 Mediated Proliferation of Primary Human CD34+ Cells
To demonstrate the role of DYNLT1 in NUP98-HOXA9mediated aberrant cell proliferation, human CD34+ primary cells were retrovirally transduced to express NUP98-HOXA9 from an MSCV-IRES-YFP vector. Transduced cells were isolated by sorting for YFP positivity, and expression of NUP98-HOXA9 was confirmed by immunoblotting (Fig. 8A). CD34+ cells expressing NUP98-HOXA9 were transfected with either nonspecific siRNA or DYNLT1 siRNA and were grown for 24 h, 48 h and 72 h and knockdown of DYNLT1 was confirmed by immunoblotting with anti-DYNLT1 antibody (Fig. 8B). An MTS cell proliferation assay was performed and showed that knockdown of DYNLT1 resulted in a significant inhibition of cell proliferation at 48 h and 72 h (Fig. 8C). These data suggest that DYNLT1 plays a role in the transformation of human hematopoietic cells by NUP98-HOXA9.

Discussion
DYNLT1 is an integral 14 KDa protein subunit of the large microtubule-based cytoplasmic dynein complex [26,27,28]  Cells positive for YFP were sorted and NUP98-HOXA9 was detected by immunoblotting with an anti-HOXA9 antibody; immunoblotting for actin was used as a loading control. B) Knockdown of DYNLT1 was confirmed by immunoblotting with the anti-DYNLT1 antibody; actin immunoblotting was used as a loading control.C) Sorted NUP98-HOXA9expressing human primary CD34+ cells were transfected by nucleofection with non-specific siRNA and siRNA specific for DYNLT1. Cells were seeded into a 96-well plate in triplicate and cell proliferation was measured 24 h, 48 h and 72 h later by an MTS assay using CellTiter 96H AQueous Nonradioactive Cell Proliferation Assay. The y axis represents cell proliferation as measured by the absorbance at 490 nm. The error bars represent standard deviation from three independent experiments; the p-values were calculated using a two-tailed t-test. doi:10.1371/journal.pone.0067032.g008 the LC8 family (DYNLL1, DYNLL2) [33]. The two DYNLT1 polypeptides within the cytoplasmic dynein complex form homodimers and also interact with the N-terminus of the intermediate chain DYNC1I1 [34,35,36]. The dynein complex is involved in a variety of cellular functions including transport of membranous organelles, mitotic spindle orientation, nuclear migration, and cell migration [37]. Dynactin is part of an actinrelated cytoplasmic protein complex that interacts with Dynein and is essential for its function [38,39,40].
In yeast, dynein light chain (Dyn2) localizes to the nuclear pore complex through its interaction with the FG repeat-containing nucleoporin Nup159, and it has been suggested that Dyn2 may play a role in organizing the natively unfolded FG repeats within the nuclear pore complex and promoting oligomerization of nucleoporin subcomplexes [41,42]. There is evidence that ubiquitylation of NUP159 is necessary for recruitment of Dyn2 to the nuclear pore complex and this interaction facilitates the migration of nuclei to daughter cells during mitosis [43]. Homology search using the Saccharomyces Genome Database (SGD) http://www.yeastgenome.org/showed that yeast Dyn2 protein shares homology with both human DYNLL1 (50%) and DYNLL2 (47%) but not with DYNLT1.
In higher eukaryotes, several reports have shown evidence for an association between dynein and the nuclear envelope and/or the nuclear pore complex. These associations mostly occur during mitosis and are generally indirect. For example, in mammalian cells dynein intermediate chains have been localized to the nuclear envelope during prophase [44,45]. Similarly, in Drosophila, dynein mediates the attachment of centrosomes to the nuclear envelope during mitosis [46]. Payne et al. showed that several nucleoporins coimmunoprecipitate with dynactin in the mammalian zygote; they suggested that the interaction between nucleoporins and dynactin is indirect and is mediated by vimentin [47]. It has also been shown in mammalian cells that dynein is recruited to the nuclear pore complex during the G2 phase through an interaction between the adaptor molecule, BICD2 and the nucleoporin RANBP2 (NUP358) [48]. Dynein light chain DYNLL1 and dynactin have also been shown to co-immunoprecipitate with the nucleoporin TPR in HeLa cells during mitosis [49]. The interaction with dynein was confirmed by pulldown assays using TPR prepared in a reticulocyte lysate system; however since the latter contains cellular proteins, it remains unclear whether the interaction is direct. A recent study has shown that during prophase the nucleoporin NUP133 anchors the dynein/ dynactin complex to the nuclear envelope indirectly through interactions with CENP-F and NudE/EL [50]. Thus it appears that dynein interacts transiently and indirectly with components of the nuclear pore complex to facilitate cell division.
In contrast, the data presented here demonstrate, for the first time to our knowledge, that a cytoplasmic dynein component directly interacts with nucleoporins in mammalian cells and that the interaction occurs in interphase. We have shown that DYNLT1 co-localizes with the nuclear pore complex and binds to the nucleoporins NUP98, NUP62 and NUP153 as well as to the oncogenic nucleoporin fusion NUP98-HOXA9. All of these DYNLT1-interacting nucleoporins contain the highly conserved FG repeat regions [1]. In the case of NUP62 and NUP98-HOXA9 these FG repeat regions are required for binding to DYNLT1 (Figs. 3 and 5).
The function of DYNLT1 at the nuclear pore complex remains to be determined. One possibility is that it may have a function in nucleocytoplasmic transport. The nuclear localization of some viral and cellular proteins is facilitated by the cytoskeleton, particularly microtubules, by mediating their perinuclear accu-mulation [51]. For example, Roth et al. found evidence for the involvement of microtubules in the nuclear import of some proteins, such as the cancer-related proteins p53, pRB, and PTHrP, but not others [52,53]. Similarly, microtubules play a role in the nuclear import of STAT1 and STAT5B [54,55]. There is substantial evidence that the role of microtubules in nucleocytoplasmic trafficking of cellular proteins is mediated by dynein; examples include the nuclear import of the mineralocorticoid receptor, STAT5B, and NFkB [55,56,57,58]. The role of the cytoskeleton and dynein in the nucleocytoplasmic transport of viral components is also very well documented, and different viral proteins interact with different dynein subunits, including DYNLT1 [59,60,61,62].
DYNLT1 is also implicated in functions independent of the cytoplasmic dynein complex [63]. DYNLT1 was shown to mediate neuritogenesis during hippocampal neuronal development in a dynein-independent manner [64]. It was also identified as an activator of G-protein signaling in association with several receptor proteins like the Trk neurotrophin receptor, bone morphogenetic receptor type II and the parathyroid hormone receptors [65,66,67,68]. DYNLT1 also interacts with Lfc, a guanine nucleotide exchange factor in a phosphorylation dependent manner to regulate neurogenesis [69,70]. RP3, a dynein light chain subunit, localizes to the nucleus and is involved in transcriptional regulation [71]. Localization studies showed a sub-population of DYNLT1 in the nucleus [63]. We have also observed a population of nuclear DYNLT1 (Fig. 4) and have shown that DYNLT1 plays a role in transcriptional dysregulation by NUP98-HOXA9 (Fig. 7).
Structural analysis showed that each DYNLT1 core contains two a-helices followed by four b-strands [35]. We have separated the a-helix-containing regions form the b-strand-containing regions and found that functional interactions with NUP98-HOXA9 or nucleoporins occurred through the b-strand region (Fig. 6). The interactions between DYNLT1 and other components of the dynein complex also occur through this b-stand containing region, suggesting that the interactions of DYNLT1 with nucleoporins and with components of the dynein complex may be mutually exclusive [36]. Thus it is possible that DYNLT1 functions within the nuclear pore complex independent of the dynein complex. In yeast dynein light chains are integrated into the nuclear pore complex and provide structural support to the nuclear pore complex components [41,42]. DYNLT1 may play a similar role in the mammalian nuclear pore complex.
Our data suggest a role for DYNLT1 in leukemogenesis. NUP98-HOXA9 is a leukemogenic oncoprotein that acts as an aberrant transcription factor [12,13,17,19]. The N-terminal FG repeat region of NUP98-HOXA9 has been implicated in the ability of NUP98-HOXA9 to dysregulate transcription and to cause leukemic transformation [12,13,14,19]. Here we have shown that DYNLT1 interacts with this FG repeat region and that depletion of DYNLT1 impairs the ability of NUP98-HOXA9 to dysregulate transcription and to induce proliferation of primary human CD34+ cells (Figs. 7 and 8). These data suggest that DYNLT1 cooperates with NUP98-HOXA9 in leukemogenesis. Indeed, a study in a leukemia mouse model showed that the axonemal dynein light chain 4 (Dnalc4) and dynactin 3 (Dctn3) cooperate with NUP98-HOXA9 in mediating leukemogenesis [72]. Dynein components have also been implicated in the pathogenesis of other tumors. For example, upregulation of DYNLL1 was shown to promote cell proliferation and tumorigenesis by stimulating cyclin-dependent kinase 2 activity in hormone-responsive cancers [73,74]. Similarly, dynein light chain DYNLRB1 is upregulated in patients with hepatocellular carcinoma [75].
To summarize, we have identified DYNLT1 as a component of the nuclear pore complex and a novel interacting partner for the oncoprotein NUP98-HOXA9 that cooperates with it in transcriptional deregulation and in the induction of proliferation of primary human CD34+ hematopoietic precursors. These data establish a direct link between dynein and the nuclear pore complex and suggest a role for DYNLT1 in the pathogenesis of AML caused by NUP98 fusions. Further studies are needed to determine the function of DYNLT1 at the nuclear pore complex, its role in nucleocytoplasmic transport, and the mechanisms by which it contributes to transcriptional dysregulation by oncogenic NUP98 fusions. (TIF) Figure S2 Densitometric quantification of shRNA-mediated DYNLT1 knockdown. K562 cells were transfected with a firefly luciferase construct driven by the KBTBD10 promoter with either empty vector or vector expressing NUP98-HOXA9. In addition, the transfections included either empty vector, or vector expressing non-specific shRNA, or vectors expressing 2 different DYNLT1 shRNAs. The intensities of the DYNLT1 bands in Fig. 7 were quantified using Chemidoc Quantity One software (BioRad). The DYNLT1 band intensities are shown as % of the empty vector control in lane 1.