Abstract
ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33(ING1b), but not of p47(ING1a), induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47(ING1a) inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33(ING1b) affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT.ING1.PCNA protein complexes.
Highlights
The ING1 candidate tumor suppressor gene expresses a family of alternatively spliced mRNAs encoding proteins that localize to the nucleus and that are growth inhibitory [1,2,3,4]
We were able to find a weak physical association between ING1 proteins and PCAF, which is a member of the hGNC5 family of histone acetyltransferase (HAT) [24], we were unable to find any interaction between ING1 proteins and hGCN5 (Fig. 1C)
In this study we demonstrated that similar to yeast ING1 proteins [23], human ING1 proteins associate with HATs (Figs. 1–3), co-precipitate HAT activity (Fig. 4), and regulate the acetylation of histones in vitro and in vivo (Figs. 4 – 6)
Summary
Cell Culture—Primary normal human diploid fibroblasts (Hs68; ATCC CRL#1635) and established human glioblastoma cells (SNB19; ATCC CRL#2219) were used in these studies, since Hs68 cells are phenotypically normal, and SNB19 cells have relatively high levels of endogenous ING1 proteins. For Western blots (Fig. 6), cells were co-transfected with ING1 and GFP expression constructs at an ING1:GFP plasmid ratio of 4:1. 48 h later, green cells were sorted by FACS and harvested for Western blot assays. FACS Analysis and Cell Sorting—For analysis of apoptosis, cells electroporated with ING1 expression constructs were harvested at 48 h, fixed in 70% ethanol/PBS, on ice for 1 h after which they were subjected to analysis or were kept at Ϫ20 °C for no more than 1 week. For preparation of lysates from ING1/GFP transfectants (Fig. 6), electroporated cells were harvested at 48 h, rinsed in PBS, and kept alive in media containing 1% serum on ice until being sorted by FACS. Green cells (positive transfectants) were collected, harvested, and their lysates were used for Western blot experiments
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