Nucleolin as activator of TCF7L2 in human hematopoietic stem/progenitor cells

TO THE EDITOR: Nucleolin is a multifunctional factor of growing and cancer cells [1, 2]. It is a candidate molecular target for cancer therapy [2], aberrantly active in certain hematological malignancies [1, 3]. Biological processes involving nucleolin include, but are not limited to, gene transcription, chromatin remodeling, RNA metabolism, translation and cell-surface signaling [1–4]. Nucleolin is predominantly expressed in hematopoietic stem/progenitor cells (HSPCs) versus differentiated hematopoietic tissue, amplifies long-term culture-initiating cells and promotes execution of the HSC gene expression program [1, 3, 4]. It also counteracts GSK3β to promote Wnt signaling and facilitates Akt signaling and a cytokine-dependent long-term maintenance of HSPCs [3, 5]. Working out the role of nucleolin in stem cell-governing signaling will improve understanding of the molecular contexts of HSPCs. Wnt signaling in stem cell control can guide tissue renewal and regeneration and is hijacked in certain leukemia types [6–9]. Here we find that, in human CD34+ HSPCs, nucleolin is associated with the molecular signature regulation of Wnt signaling whose member transcription factor 7-like 2 (TCF7L2), implicated also in regeneration of hematopoietic lineages [7], is partially involved in the transcriptional upregulation of the signature genes. Furthermore, nucleolin is a TCF7L2 promoter-binding factor that activates TCF7L2. The study provides new insights into molecular network relevant to stem/progenitor cells in normal and malignant hematopoiesis and suggests that deregulated nucleolin may favor aberrant Wnt signaling in certain cancers. Figure 1A shows that gene signature regulation of Wnt signaling was enriched by nucleolin as was determined by gene set enrichment analysis (GSEA), and transcriptionally upregulated Wnt signaling regulators included TCF7L2 (Table S1A). GSEA used nucleolin-dependent expression profile from mobilized peripheral blood (MPB) HSPCs [4], that are exploited in hematological transplantology for hematopoietic reconstitution [10]. Involved in certain facets of hematopoiesis, TCF7L2 belongs to the TCF/LEF family of DNA-binding nuclear factors, and its association with N-terminally dephosphorylated (active) β-catenin leads to activation of TCF7L2-bound genes (Supplementary Information, page 4). TCF7L2 and N-terminally dephosphorylated β-catenin were significantly upregulated in HSPCs carrying nucleolin expression vector (HSPC-NCL) versus control HSPCs carrying expression vector of N-terminally truncated nucleolin, amino-acid (aa) residues 289-709 (HSPC-NCL-289-709), or with no cDNA (HSPCmock) (Fig. S1). In addition, nucleolin was upregulated ~4-fold and a signature of TCF7L2-bound genes, derived from CD34+MPB HPCs [7], was enriched in HSPC-NCL cells, indicating activation of TCF7L2-associated transcription (Fig. 1B, [4] and data not shown). Moreover, genes bound by TCF7L2 were overrepresented among the genes encoding Wnt signaling regulators upregulated in HSPC-NCL cells (Table S1) (48%, P < 0.016), and this included several reported as TCF7L2 downstream-regulated genes (Supplementary Information, page 4). Thus, in CD34+ HSPCs, nucleolin is associated with the molecular signature regulation of Wnt signaling and the transcriptional upregulation of the signature genes partially involves TCF7L2.

2 supplemented with 20% FCS and 20% conditioned medium from 5637 cells DSMZ). Cells were recently authenticated and tested for mycoplasma contamination.
Electrophoretic mobility shift assay (EMSA) and ChIP assay TCF7L2 gene promoter upstream of the translation start site was described in the previous study. S4 A search of this promoter (GenBank accession number AF522996) for potential nucleolin binding sites revealed the presence of two fragments that included the following sequences: nucleotides (nt) -1215 to -1208 (5'-TGAAATGA-3') and nt -427 to -420 (5'-TGAACTGA-3'). Double-stranded oligonucleotides from the TCF7L2 promoter used in EMSA contained nt -1232 to -1193 of the TCF7L2 promoter (TCF7L2-A) or nt -440 to -406 (TCF7L2-B). The respective derivatives of the oligonucleotides TCF7L2-A and TCF7L2-B were devoid of nt -1213 to -1209 (Mut TCF7L2-A), or nt -425 to -421 (Mut TCF7L2-B). Double-stranded oligonucleotide carrying two copies of the sequence 5'-CCCTTTGATCTTACC-3' that contains the optimal TCF binding motif S5 was used as control oligonucleotide. Nucleolinglutathione S-transferase (GST) fusion protein, comprising amino-acid residues 289-709 of nucleolin, and use thereof in EMSA was described previously. S2,S3 ChIP experiments with protein A/G MicroBeads (Miltenyi Biotec, Gladbach, Germany) followed a protocol provided by Miltenyi Biotec, as described. 3 Antibody specific for Nterminal peptide of nucleolin, S2,S3 affinity-purified on the peptide column, was used and controls included presaturation of this antibody with the blocking peptide. Immunoprecipitated DNA was quantified by real-time PCR. Primer sequences are summarized in Table S2.

Western blotting
Immunoblot analysis followed standard procedures, and normalized band intensity is shown as a percentage of cells nucleofected with empty expression vector (Figures 2B and S2) or of the HSPC-mock sample ( Figure S1). Antibodies, validated previously, were as follows: antibody specific for N-terminal peptide of nucleolin, S2,S3 purified on the peptide column, and commercially available antibodies described in Table S3. 3
Gene set enrichment analysis (GSEA) GSEA was performed using GSEA v2.2 software (Broad Institute, Cambridge, MA, USA), S6 and statistical difference was determined by 1000 gene set permutations, timestamp seed for permutation. Minimum gene set size was set to 15; max_probe was used to collapse multiple probe sets / gene. The nucleolin-dependent gene expression profile, comprising data obtained with MPB HSPCs from 3 patients (HSPC-NCL cells versus HSPC-mock control cells), was described and the necessary controls, including the validation of differential gene expression by qRT-PCR, were reported. 4 Nucleolin protein levels were ~4-fold higher in HSPC-NCL versus HSPC-mock cells. 4 Other gene sets used for GSEAs were as follows: in Figure 1A, gene ontology gene set regulation of Wnt signaling pathway and, in Figure 1B, TCF7L2-bound gene set, derived from human CD34+ MPB HPCs. 7

Supplementary Discussion
Wnt signals are relevant to regulation of HSPCs and the strength of Wnt signaling regulates hematopoiesis. 8,9,S7 On the other hand, deregulated Wnt signaling, linked to worse clinical outcome for a subset of human cancers, S8 is causatively associated with leukemogenesis and is of importance to the properties of LSCs. 8,9,S9,S10,S11 The effect of nucleolin on Wnt signaling regulators including TCF7L2 suggests its relevance to regulation of Wnt signaling.
TCF7L2 (also known as TCF4) is upregulated in HSCs/HSPCs S12 and is capable of enhancing GATA2-mediated transcriptional activation. 7 Furthermore, TCF7L2 is involved in hematopoietic regeneration and differentiation and cooperates with lineage master regulators to affect expression of key hematopoietic genes. 7 On the other hand, its expression is elevated in CD34+ hematopoietic cells from patients in chronic myeloid leukemia (CML) blast crisis and TCF7L2 has a role in transcriptional changes in CML. S13 In addition, overexpression of TCF7L2 in mantle cell lymphoma-initiating cells is associated with aberrant Wnt activity critical for their maintenance and survival. S14 Additionally, installation of a cancerpromoting Wnt/SIX1 signaling axis by the MLL-AF9 oncoprotein in AML LSCs involves TCF7L2. S15 TCF7L2 participates as well in aberrant Wnt activity in certain non-hematological malignancies. S5,S16-S18 TCF7L2 is a member of the TCF/LEF family of DNA-binding nuclear factors that are effectors of the Wnt signaling pathway, and interaction of β-catenin with TCF7L2 leads to activation of TCF7L2-bound genes. S5,S17,S19 In addition, this interaction also serves as a target for cancer therapy. S14,S17,S18,S20 The herein-reported finding that nucleolin, as a TCF7L2 promoter-binding factor, upregulates TCF7L2 is novel and, in line with our published data, 3,5 nucleolin as well elevates the level of N-terminally dephosphorylated β-catenin that, as was demonstrated by Staal and colleagues, S21 transduces Wnt signals. Thereby, the signature of TCF7L2-bound genes is enriched by nucleolin ( Figure 1B). Furthermore, genes bound by TCF7L2 are overrepresented among the upregulated genes encoding regulators of Wnt signaling. This includes BAMBI, LRP4, MDFIC and SOX4, that were shown to be TCF7L2occupied S22,S23 and modulatable via TCF7L2 S18,S24-S27 and β-catenin S18,S24,S28-S30 in non-HSPCs. Thus, transcriptional upregulation of Wnt signaling regulators by nucleolin partially involves TCF7L2.