The new nucleoporin: Regulator of transcriptional repression and beyond

Transcriptional regulation is a complex process that requires the integrated action of many multi-protein complexes. The way in which a living cell coordinates the action of these complexes in time and space is still poorly understood. Recent work has shown that nuclear pores, well known for their role in 3′ processing and export of transcripts, also participate in the control of transcriptional initiation. We have recently begun to explore how nuclear pores interface with the well-described machinery that regulates initiation. This work led to the discovery that specific nucleoporins are required for binding of the repressor protein Mig1 to its site in target promoters. Nuclear pores are therefore involved in repressing, as well as activating, transcription. Here we discuss in detail the main models explaining our result and consider what each implies about the roles that nuclear pores play in the regulation of gene expression

Dynein Light Chain 1 (DYNLT1) interacts with normal and oncogenic nucleoporins

<div><p>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 <i>in vitro</i> 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.</p></div

The Nuclear Pore Complex Mediates Binding of the Mig1 Repressor to Target Promoters

All eukaryotic cells alter their transcriptional program in response to the sugar glucose. In Saccharomyces cerevisiae, the best-studied downstream effector of this response is the glucose-regulated repressor Mig1. We show here that nuclear pore complexes also contribute to glucose-regulated gene expression. NPCs participate in glucose-responsive repression by physically interacting with Mig1 and mediating its function independently of nucleocytoplasmic transport. Surprisingly, despite its abundant presence in the nucleus of glucose-grown nup120Δ or nup133Δ cells, Mig1 has lost its ability to interact with target promoters. The glucose repression defect in the absence of these nuclear pore components therefore appears to result from the failure of Mig1 to access its consensus recognition sites in genomic DNA. We propose that the NPC contributes to both repression and activation at the level of transcription

Dissection of the Transformation of Primary Human Hematopoietic Cells by the Oncogene NUP98-HOXA9

NUP98-HOXA9 is the prototype of a group of oncoproteins associated with acute myeloid leukemia. It consists of an N-terminal portion of NUP98 fused to the homeodomain of HOXA9 and is believed to act as an aberrant transcription factor that binds DNA through the homeodomain. Here we show that NUP98-HOXA9 can regulate transcription without binding to DNA. In order to determine the relative contributions of the NUP98 and HOXA9 portions to the transforming ability of NUP98-HOXA9, the effects of NUP98-HOXA9 on primary human CD34+ cells were dissected and compared to those of wild-type HOXA9. In contrast to previous findings in mouse cells, HOXA9 had only mild effects on the differentiation and proliferation of primary human hematopoietic cells. The ability of NUP98-HOXA9 to disrupt the differentiation of primary human CD34+ cells was found to depend primarily on the NUP98 portion, whereas induction of long-term proliferation required both the NUP98 moiety and an intact homeodomain. Using oligonucleotide microarrays in primary human CD34+ cells, a group of genes was identified whose dysregulation by NUP98-HOXA9 is attributable primarily to the NUP98 portion. These include RAP1A, HEY1, and PTGS2 (COX-2). Their functions may reflect the contribution of the NUP98 moiety of NUP98-HOXA9 to leukemic transformation. Taken together, these results suggest that the effects of NUP98-HOXA9 on gene transcription and cell transformation are mediated by at least two distinct mechanisms: one that involves promoter binding through the homeodomain with direct transcriptional activation, and another that depends predominantly on the NUP98 moiety and does not involve direct DNA binding

Non-specific and DYNLT1-specific shRNA/siRNA sequences.

<p>Non-specific and DYNLT1-specific shRNA/siRNA sequences.</p

DYNLT1 interacts with the FG repeat region of NUP98-HOXA9.

<p><i>A)</i> Full length NUP98, NUP98-HOXA9 and NUP98-HOXA9 deletion mutants. Vertical lines represent FG repeats. DBD = DNA-binding domain. <i>B)</i> GST or GST-DYNLT1 were immobilized on beads and incubated with <i>in vitro</i> translated full length NUP98, NUP98-HOXA9, or deletion mutants of NUP98-HOXA9. Bound and unbound fractions were resolved by SDS-PAGE and subjected to autoradiography.</p

C-terminus of DYNLT1 interacts with NUP98-HOXA9, NUP62 and NUP153.

<p><i>A)</i> Full length DYNLT1 and deletion mutants. <i>B</i>) GST or GST-DYNLT1 or GST-DYNLT1 N-terminus or GST-DYNLT1 C-terminus with <i>in vitro</i> translated NUP98-HOXA9, NUP62 or NUP153. Bound and unbound fractions were resolved by SDS-PAGE and subjected to autoradiography. <i>C)</i> K562 cells were nucleofected with pGL4.11 vector driven by the <i>KBTBD10</i> 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.</p

Cytoplasmic yeast two-hybrid analysis identifies DYNLT1 as an interacting partner for NUP98-HOXA9.

<p>K562 cDNA library in pMyr vector was co-transformed with pSOS-NUP98-HOXA9 into cdc25H yeast strain. Isolated colonies were patched on galactose-containing medium and transferred to 37°C. Patches growing at 37°C were re-spotted on plates containing selective medium supplemented with either glucose (repressing) or galactose (de-repressing) as sole carbon source and incubated either at 23°C (permissive) or 37°C (restrictive) for 3–5 days. Plasmids were isolated from yeast cells growing at 37°C in galactose containing medium and retransformed along with pSOS-NUP98-HOXA9 to confirm the interactions. Several positive cDNA clones were sequenced and identified as DYNLT1. Cells containing pSOS-NUP98-HOXA9 with pMYR-LaminC or empty pSOS vector with pMyr-DYNLT1 were spotted as negative controls. Cells containing pSOS-NUP98-HOXA9 with pMYR-SB (SOS Binding protein) were spotted as a positive control. Growth at 37°C in galactose containing medium indicates a positive interaction.</p

Knockdown of DYNLT1 inhibits proliferation of cells expressing NUP98-HOXA9.

<p>A) Primary human CD34+ cells were retrovirally transduced with either control MSCV-IRES-YFP vector or vector expressing NUP98-HOXA9. 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-HOXA9-expressing 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 96® 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.</p