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Impact of NSD1 Alternative Transcripts in Actin Filament Formation and Cellular Division Pathways in Fibroblasts - PubMed

  • ️Mon Jan 01 2024

Impact of NSD1 Alternative Transcripts in Actin Filament Formation and Cellular Division Pathways in Fibroblasts

Giuseppina Conteduca et al. Genes (Basel). 2024.

Abstract

Germline variants in the NSD1 gene are responsible for Sotos syndrome, while somatic variants promote neoplastic cell transformation. Our previous studies revealed three alternative RNA isoforms of NSD1 present in fibroblast cell lines (FBs): the canonical full transcript and 2 alternative transcripts, termed AT2 (NSD1 Δ5Δ7) and AT3 (NSD1 Δ19-23 at the 5' end). The precise molecular pathways affected by each specific isoform of NSD1 are uncharacterized to date. To elucidate the role of these isoforms, their expression was suppressed by siRNA knockdown in FBs and protein expression and transcriptome data was explored. We demonstrate that one gene target of NSD1 isoform AT2 is ARP3 actin-related protein 3 homolog B (ACTR3B). We show that loss of both canonical NSD1 and AT2 isoforms impaired the ability of fibroblasts to regulate the actin cytoskeleton, and we observed that this caused selective loss of stress fibers. Our findings provide novel insights into NSD1 function by distinguishing isoform function and demonstrating an essential role of NSD1 in regulating the actin cytoskeleton and stress fiber formation in fibroblasts.

Keywords: NSD1; cell cycle; cytoskeleton; isoforms; neoplastic pathways.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1

Schematic representation of the study strategy. Significant modulation of target genes between the wild-type fibroblast group and siRNA treated cells was calculated by a GeneSpring differential expression analysis tool. A Gene Set Enrichment Analysis (GSEA) identified statistically significantly modulated gene sets.

Figure 2
Figure 2

Schematic representation of the NSD1 isoforms. (A) NSD1 gene (top) and NSD1 canonical protein (bottom) with all functional domains indicated. Open boxes denote coding exons and grey boxes denote the 5′ and 3′ untranslated regions. (B) Modular domain architecture of putative NSD1 protein isoforms. Predicted protein domains were detected using PFAM motif markseq server (

https://www.genome.jp/tools/motif/

, accessed on 26 July 2023) and trRosetta tool (

https://yanglab.nankai.edu.cn/trRosetta/

, accessed on 2 October 2023). Colored boxes highlight specific functional domains. PWWP: proline-tryptophan-tryptophan-proline domain; NLS: nuclear localization signal; PHD: plant homeodomain domain; AWS: associated with SET domains; SET: Su(var)3–9, Enhancer-of-zeste, Trithorax domain.

Figure 3
Figure 3

Expression analysis of NSD1 isoforms in fibroblasts by immunofluorescence. (A) Representative image of IF with rabbit polyclonal NSD1 antibody, HPA070333 for the exon 5 epitope. (B) Representative image of IF with mouse monoclonal NSD1 antibody, sc-130470, clone K47, binding to the N-terminal epitope. After 24 h of post anti-NSD1 siRNA treatment and untreated control cells, DAPI was used to stain the cell nucleus to locate cells and Alexa fluor 595-labelled NSD1 antibodies for NSD1 AT2, NSD1 AT3, and NSD1 AT1. (×100 magnification; blue = DAPI; red = NSD1. Scale bar = 50 µm).The arrows indicate NSD1 red signals.

Figure 4
Figure 4

Venn diagram comparing shared and unique DEGs following NSD1 siRNA-AT1, siRNA-AT2 and siRNA-AT3 treatment of fibroblasts. Common and uniquely upregulated genes (A) and downregulated genes (B) are indicated.

Figure 5
Figure 5

Protein–protein interaction network among differentially expressed genes between wild type fibroblasts group and treated with NSD1 siRNA-AT1 (A) or with NSD1 siRNA-AT2 (B) or with NSD1 siRNA-AT3 (C). Protein–protein interaction networks were generated using the STRING database to assess potential functional interactions among selected genes involved in actin cytoskeleton organization, cell differentiation and cell cycle regulation. Networks were expanded to assess likely indirect interactions. Nodes represent gene products, and edges represent protein–protein associations. Only the associations with an evidence score higher than 0.3 are shown, with colors indicating different kinds of evidence. The legend for evidence type is shown in the bottom right corner. Networks showed that the significantly modulated genes in the distinct experiments did not show direct interactions with NSD1, but they did show indirect interactions with a subset of other related gene products. Red circle showed NSD1 gene.

Figure 6
Figure 6

Lists of statistically significantly enriched gene sets from the Hallmark collection. Gene Set Enrichment Analysis (GSEA) was used to perform an unbiased biological reasoning of the expression data in different treatments. Gene sets with nominal p − values < 0.05 and FDR q − values < 0.05 were considered significantly enriched. Plots report the normalized enriched score of gene sets in decreasing order. The Y −axis reports the gene set names. (A) Gene sets enriched in the group treated with NSD1 siRNA-AT2. (B) Gene sets enriched in the group treated with NSD1 siRNA-AT3. It is evident that siRNA-AT2 treatment induces a downregulation of the mitotic spindle pathway, which is not observed after treatment with siRNA-AT3.

Figure 7
Figure 7

NSD1 loss in fibroblasts (FBs) impairs actin cytoskeleton organization and stress fiber structure. (A) Representative images of FBs 24 h post-transfection with anti NSD1 siRNA-AT2 showed a morphological transition into an amoeboid phenotype and (B) FBs not transfected with siRNA displayed a typical flat and elongated structure. (C) FBs transfected with anti-NSD1 siRNA-AT1 showed NSD1 isoform 1 knockdown and normal β-actin expression. (D) FBs transfected with anti Cy3 siRNA control. (E) FBs transfected with anti NSD1 siRNA-AT3 (×100 magnification; blue = DAPI; red = NSD1; green = β-actin. Scale bar = 50 µm). Immunofluorescence images with NSD1 antibody, sc-130470, clone K47, confirmed that AT2 isoform silencing changed stress fiber organization in fibroblasts, therefore we observed an ameboid phenotype induced by siRNA-AT2 treatment (A) with respect to the canonical flat, elongated morphology characteristic of fibroblasts. Since the cells in culture were heterogeneous, some differences of fluorescence intensity can be noted compared to the same treatment condition in Figure 3.

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