Novel Splice Variants of ING4 and Their Possible Roles in the Regulation of Cell Growth and Motility

Abstract

The ING4 (inhibitor of growth family member 4) was identified in our laboratory as a candidate tumor suppressor gene (1) that is down-regulated in glioblastoma cells (2) and head and neck squamous cell carcinoma (3). ING4 suppresses cell growth (1, 4, 5), suppresses the loss of contact inhibition (6), inhibits angiogenesis (2), and down-regulates the stability of hypoxiainducible factor (HIF) 3 -␣ (7) through a physical interaction with HIF prolyl hydroxylase (HPH)-2C (8), resulting in repressed HIF activation in a chromatin-dependent manner. More recently, it was reported that ING4 acetylates histone H4 lysine 5, 8, and 12 with a histone acetyltransferase, HBO1/ MYST2 (MYST2 is MOZ, YBF2/SAS3, SAS2 and TIP60 histone acetyltransferase 2) (9), and interacts with methylated histone H3 (10). Thus, ING4 seems to play several major roles in cells like other ING family proteins (11) that are conserved from yeasts to vertebrates (12).In this study, we describe three novel splice variants of ING4, ING4_v2 (a 3-bp skip form), ING4_v3 (a 9-bp skip form), and ING4_v4 (a 12-bp skip form), besides ING4_v1 (the originally enrolled ING4, the longest form). These variants are produced from the alternative use of two splice donor sites at the end of exon 4 and two splice acceptor sites at the start of exon 5 of the ING4 gene, although one of the three variants, ING4_v4, was previously attributed to a common deletion mutation (6). The alternative RNA splicing of the ING4 pre-mRNA causes a partial loss of an NLS and affects nuclear localization of the proteins. We show that the small deletion in the ING4 protein leads to functional differences between the ING4 variants. Growth suppressive effects of the variants that have the partially missing NLS were attenuated by weaker activity on p21 WAF1 induction. In addition, ING4_v4 showed attenuated suppressive effects on cell spreading and migration compared with the original ING4 (ING4_v1). On the other hand, ING4_v2 only lost the suppressive effect on cell spreading, suggesting an important role for ING4_v2 on the regulation of cell migration. ING4_v4 played dominant-negative roles on these ING4_v1 effects. In addition, we found that ING4_v1 and ING4_v2, but not ING4_v4, have a binding affinity to Liprin ␣1 that may play a role in the regulation of focal adhesion disassembly (13,14). In addition, only ING_v1 has a binding affinity to G3BP2a, which is involved in Ras signaling, NF-B signaling, and the ubiquitin proteasome system (15-17). These differences may affect the function of ING4 splice variants. 3 The abbreviations used are: HIF, hypoxia-inducible factor; NLS, nuclear localization signal; Liprin ␣1, LAR-interacting protein ␣1; G3BP2, RasGTPase activating protein SH3 domain-binding protein 2; PHD, plant homeodomain; NF-B, nuclear factor of B; HPH-2, HIF prolyl hydroxylase 2; ESE, exonic splicing enhancer.

Detection of novel mRNA splice variants of human ING4 tumor suppressor gene

“…In addition, multiple variants of ING1 and ING2 have been identified in Xenopus laevis . While this paper was under revision, new ING4 variants produced by alternative splicing at the exon 4-5 boundary have been described (Tsai and Lin, 2006;Unoki et al, 2006). These reports, together with our data, strongly support the notion that ING4 functions may be differentially affected by alternative splicing.…”

“…Interestingly, all the wobble variants were detected in normal tissues. Using a different approach, Unoki et al (2006) documented the expression of the same variants in normal and tumor tissues; moreover, they showed functional differences among ING4 variants.…”

“…This region is retained by all the variants, including the ING4-D4AA, which carries a deletion of amino acids 127-130 (KGKK) within the NLS1 domain. A partially overlapping deletion (129-132, KKKG) abrogates the nuclear localization (Unoki et al, 2006), suggesting that KG residues 130-132, rather than KK 129-130, are required for ING4 proper cellular localization. This is in keeping with a previous report showing that mutations of residues 129-130 do not affect nuclear localization of ING4 (Zhang et al, 2005).…”

“…ING4 has been shown previously to display nuclear localization driven by its bipartite NLS1, a region also important for interaction with p53 (Zhang et al, 2005). A second nuclear localization signal (NLS2), located at the C terminus, has been recently shown not required for ING4 nuclear localization (Unoki et al, 2006). To investigate the cellular localization of the novel ING4 variants immunofluorescence and cell fractionation experiments in ING4-myc transfected cells were performed.…”

“…Similarly to the other ING family members, ING4 was described to associate with p53 and to upregulate p53-inducible genes ; this is mediated by the bipartite NLS1, also essential for nuclear localization (Zhang et al, 2005). A second, nonfunctional, NLS2 is located at the C-terminal (Unoki et al, 2006). A recent study demonstrated a role of ING4 in the regulation of growth of transplanted glioblastoma through a physical association with the p65 (RelA) subunit of NF-kB and subsequent downregulation of NF-kB responsive genes involved in angiogenesis.…”