Profiling of promoter occupancy by the SND1 transcriptional coactivator identifies downstream glycerolipid metabolic genes involved in TNFα response in human hepatoma cells

Abstract

The NF-κB-inducible Staphylococcal nuclease and tudor domain-containing 1 gene (SND1) encodes a coactivator involved in inflammatory responses and tumorigenesis. While SND1 is known to interact with certain transcription factors and activate client gene expression, no comprehensive mapping of SND1 target genes has been reported. Here, we have approached this question by performing ChIP-chip assays on human hepatoma HepG2 cells and analyzing SND1 binding modulation by proinflammatory TNFα. We show that SND1 binds 645 gene promoters in control cells and 281 additional genes in TNFα-treated cells. Transcription factor binding site analysis of bound probes identified motifs for established partners and for novel transcription factors including HSF, ATF, STAT3, MEIS1/AHOXA9, E2F and p300/CREB. Major target genes were involved in gene expression and RNA metabolism regulation, as well as development and cellular metabolism. We confirmed SND1 binding to 21 previously unrecognized genes, including a set of glycerolipid genes. Knocking-down experiments revealed that SND1 deficiency compromises the glycerolipid gene reprogramming and lipid phenotypic responses to TNFα. Overall, our findings uncover an unexpected large set of potential SND1 target genes and partners and reveal SND1 to be a determinant downstream effector of TNFα that contributes to support glycerophospholipid homeostasis in human hepatocellular carcinoma during inflammation.

Figure 1.

Analysis of SND1 binding regions. HepG2 cells were treated with TNFα (50 ng/ml, 24 h) or left untreated (control) before performing ChIP-chip analysis for SND1 binding. (A) Bound probes and Venn diagrams of the corresponding genes. (B) Identification of the genomic location of SND1 binding regions using PinkThing. (C) Positional distribution of the identified SND1 binding regions relative to the 5′ end in control and TNFα-treated cells. Graphs (B and C) are representative of three experiments with similar results.

Figure 2.

Effect of TNFα treatment on the transcript expression of selected SND1 target genes in HepG2 cells. Transcript expression was determined by reverse transcription quantitative real time PCR in control and TNFα-treated (50 ng/ml, 8 h) HepG2 cells. Results are reported as means ± SD of 3–4 experiments and are expressed relative to the level in control cells, which is shown as a gray grid line. *P ≤ 0.05, **P ≤ 0.01 versus control cells.

Figure 3.

The TNFα-promoted increase in the SND1 mRNA level and the nuclear/cytoplasmic SND1 protein partitioning is not visible in SND1-silenced HepG2 cells. (A) The SND1 transcript level and (B) the SND1 protein content in nuclei and the cytoplasmic fraction were quantified in control (white bars) and TNFα-treated (50 ng/ml, 8 h) (dark bars) HepG2 cells, expressing basal (solid bars) or residual levels of SND1 after silencing endogenous SND1 (hatched bars). Aliquots of cells (3–5 × 10⁶ cells) were subjected to RNA isolation and individual PCR reactions. Other aliquots (3–5 × 10⁶ cells) were processed for the isolation of nucleus and cytoplasm and subjected to immunoblot analysis for SND1 and normalized with histone H3 and β-tubulin. Results are reported as means ± SD of three independent experiments and expressed relative to untreated cells expressing basal levels of SND1. **P ≤ 0.01, ***P ≤ 0.001 versus control cells; ≠≠≠P ≤ 0.001 versus cells expressing endogenous levels of SND1.

Figure 4.

SND1 silencing impedes the adaptation of cellular lipid levels to TNFα stimulation. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), free cholesterol (FC), triacylglycerol (TAG) and cholesteryl esters (CE) were quantified in control (white bars) and TNFα-treated (50 ng/ml, 8 h) (dark bars) HepG2 cells, expressing either basal (solid bars) or residual levels of SND1 after silencing endogenous SND1 (hatched bars). Lipids were extracted from HepG2 cell lysates, separated by thin-layer chromatography and quantified by optical densitometry as described in ‘Materials and Methods’ section. Results are reported as means ± SD of four independent experiments and expressed as nmol/mg protein. *P ≤ 0.05, ***P ≤ 0.001 versus control cells.

Figure 5.

SND1-mediated modulation of the TNFα effect on transcript levels of glycerophospholipid metabolism genes. The transcript levels of CHPT1 (A), LPGAT1 (B), LPIN1 (C) and PTDSS1 (D) and the transcriptional activity of the gene promoters (E) were determined in control (white bars) and TNFα-treated (50 ng/ml, 8 h for A–D and 24 h for E) (dark bars) HepG2 cells, expressing either basal level of SND1 (solid bars) or after silencing endogenous SND1 (hatched bars). Results are reported as means ± SD of four independent experiments and expressed relative to the values in untreated cells expressing basal levels of SND1. *P ≤ 0.05, **P ≤ 0.01 versus control cells; ≠P ≤ 0.05, ≠≠P ≤ 0.01, ≠≠≠P ≤ 0.001 versus SND1 non silenced cells.

Figure 6.

Implication of SND1 in the glycerolipid metabolic response of HepG2 cells to TNFα. SND1 is an NF-κB responsive gene. Proinflammatory cytokine TNFα activates several signaling pathways by binding to receptors TNFR1 and TNFR2, leading to increased SND1 transcriptional activity via binding of downstream transcription factor NF-κB (6). TNFα promotes the nuclear translocation of the transcriptional coactivator SND1, which binds to a set of target genes involved in glycerophospholipid homeostasis. When SND1 is silenced, SND1 protein does not accumulate into the nucleus and neither the TNFα-induced expression change of target genes nor the increase in cellular phosphatidylcholine levels is elicited. Other SND1 functions are displayed in green. A recent study demonstrated that SND1 initiates a molecular cascade that activates NF-κB, onco-miR-221 and angiogenic factor expression in human HCC (41), whereby the interplay between SND1 and NF-κB activation states might provide an amplification loop mediating the response of hepatocytes to tumorigenic and inflammatory stimuli.