Nup98 recruits the Wdr82-Set1A/COMPASS complex to promoters to regulate H3K4 trimethylation in hematopoietic progenitor cells

Abstract

Recent studies have shown that a subset of nucleoporins (Nups) can detach from the nuclear pore complex and move into the nuclear interior to regulate transcription. One such dynamic Nup, called Nup98, has been implicated in gene activation in healthy cells and has been shown to drive leukemogenesis when mutated in patients with acute myeloid leukemia (AML). Here we show that in hematopoietic cells, Nup98 binds predominantly to transcription start sites to recruit the Wdr82-Set1A/COMPASS (complex of proteins associated with Set1) complex, which is required for deposition of the histone 3 Lys4 trimethyl (H3K4me3)-activating mark. Depletion of Nup98 or Wdr82 abolishes Set1A recruitment to chromatin and subsequently ablates H3K4me3 at adjacent promoters. Furthermore, expression of a Nup98 fusion protein implicated in aggressive AML causes mislocalization of H3K4me3 at abnormal regions and up-regulation of associated genes. Our findings establish a function of Nup98 in hematopoietic gene activation and provide mechanistic insight into which Nup98 leukemic fusion proteins promote AML.

Keywords: Nup98; Set1A; Wdr82; acute myeloid leukemia; histone 3 Lys4 trimethylation; transcription.

Figure 1.

Nup98 binds to transcription start sites adjacent to regions of H3K4me3 in mouse HPCs. (A) ChIP-seq trace showing a characteristic peak for Nup98 binding (top) and H3K4me3 binding (bottom) to an example gene, Sec23a. (B) Correlation between H3K4me3 binding and Nup98 binding near transcription start sites. H3K4me3 data were derived from previously published data (Bernt et al. 2011). (C) Pie chart showing the percentage of peaks found in a particular region of the genome for Nup98, Runx1 (Wu et al. 2012), and HOXB4 (Fan et al. 2012) ChIP-seq data sets. (D) GO analysis results for genes whose promoters were bound by Nup98. Gene clusters and P-values were identified using DAVID open access software. Gene clusters that were found to be among the most highly expressed in HPCs are highlighted in red.

Figure 2.

A Nup98 protein variant that localizes only in the nucleoplasm interacts with Wdr82. (A) Localization of human GFP-Nup98 (panel 1), a mutant of Nup98 that does not bind the NPC (GFP-Nup98ΔCTD) (panel 2), and a control protein (GFP) (panel 3). GFP-Nup98ΔCTD localizes in GLFG bodies, as has been reported previously (Griffis et al. 2002). Bars, 2 µm. (Panels 4–6) A merge of GFP and Hoechst staining is shown for each condition. (B) Silver-stained sodium dodecyl sulfate (SDS)-PAGE showing proteins that copurified with GFP and GFP-Nup98ΔCTD. Bands corresponding to proteins of interest are indicated at the right. (C) Table showing most enriched proteins in GFP-Nup98ΔCTD co-IP lysates. Proteins of interest are highlighted in red. The numbers of peptides identified for each protein in control GFP immunoprecipitation and GFP-Nup98ΔCTD immunoprecipitation are indicated. (D) Western blots showing input protein and purified protein from GFP-Nup98ΔCTD (lanes 2,4, respectively) and a control protein (GFP) (lanes 1,3, respectively). Input lanes were loaded with 0.625% of starting lysate. (E) Immunofluorescence (IF) assays showing localization of Wdr82 (panels 1,5), GFP-Nup98ΔCTD (panels 2,6), Hoechst (panels 3,7), and a merge of Wdr82 and GFP-Nup98ΔCTD localization (panels 4,8). Zoomed images of the dotted box that appears in panels 1–4 are shown in panels 5–8. The average fold enrichment and standard deviation of Wdr82 focus staining relative to nuclear background are shown in the bottom right corner of panel 5. Bars: panels 1–4, 10 µm; panels 5–8, 2 µm.

Figure 3.

Wdr82 is required for Set1A recruitment to chromatin and H3K4me3. (A) Pie chart showing the percentage of Set1A peaks found in different regions of the genome. (B) GO analysis of genes whose promoters are bound by Set1A. Groups corresponding to one of the most highly expressed gene clusters in HPCs (Supplemental Fig. S1B) are highlighted in red. (C) ChIP-seq traces showing Set1A or H3K4me3 binding to chromatin in control knockdown or Wdr82 knockdown cells. Red arrows indicate regions of interest. (D) ChIP-seq traces showing H3K4me3 binding to genomic regions in control knockdown or Wdr82 knockdown cells. (E) Genome-wide histogram showing the binding of H3K4me3 in relation to promoter regions for the experimental conditions indicated.

Figure 4.

Nup98 is required for Set1A recruitment to chromatin and H3K4me3. (A) Model showing the potential role of Nup98–Wdr82 interaction in HPCs. Perhaps Nup98 is required to recruit the WSC complex to chromatin. (B) Overlap of Set1A promoter peaks with Nup98 promoter peaks from experiments conducted in mouse HPCs. (C) Genome-wide histogram showing the binding of Nup98, Set1A, or a control ChIP (IgG) in relation to promoter regions in wild-type HPCs. (D) ChIP-seq trace showing Nup98 binding, Set1A binding in control or Nup98 knockdown conditions, or H3K4me3 binding in control, Nup98 knockdown cells, or Wdr82 knockdown cells in relation to the Hjurp gene promoter. Note that Nup98 knockdown was carried out for 24 h, while Wdr82 knockdown was carried out for 48 h except where indicated in D. (E) Genome-wide histogram showing the binding of Set1A in relation to promoter regions in control knockdown (blue) or Nup98 knockdown (red) cells. (F) Graph showing the number of promoters bound by Set1A in control knockdown or Nup98 knockdown cells. (G) Genome-wide histogram showing the binding of H3K4me3 in relation to promoter regions in control knockdown (blue), Nup98 knockdown (red), or Wdr82 knockdown (yellow) or for a control IgG ChIP (brown). Dotted traces indicate data that are the same as shown in Figure 3E.

Figure 5.

Nup98-Nsd1 expression disrupts H3K4me3 through direct and indirect mechanisms. (A) Model showing a possible mechanism by which Nup98-Nsd1 triggers misregulation of genes that it binds. Perhaps Nup98-Nsd1 recruits Wdr82–Set1A to unnatural binding sites to trigger H3K4me3 and gene activation. (B,C) ChIP-seq traces for Nup98-Nsd1 (top trace) or H3K4me3 in Nup98-Nsd1-expressing (second trace) or wild-type (third trace) cells. RNA-seq tracks for Nup98-Nsd1 or wild-type cells are shown below the ChIP-seq tracks. Red arrows indicate regions of interest to compare between wild-type and Nup98-Nsd1 cells. The asterisk indicates exogenous HOXA9 overexpression, which was used to immortalize our wild-type HPC cell line (Calvo et al. 2000).

Figure 6.

Model for Nup98 function in wild-type and leukemic cells. (A) In wild-type cells, Nup98 is required for recruitment of the WSC complex to transcription start sites and thereby promotes H3K4me3 and gene activation. Nup98 is not recruited to promoters of developmental genes, such as those of the HOX locus and Meis1. (Right side of diagram) Gene expression at these loci is silent, and cells are poised for differentiation. (B) In Nup98-Nsd1-expressing cells, the Nup98 portion of the translocation recruits Set1A activity to the wrong binding sites, which promotes unusual H3K4me3 patterns and constitutive activation of genes that regulate HPC differentiation (shown in the right side of diagram).