Identification and partial characterization of new cell density-dependent nucleocytoplasmic shuttling proteins and open chromatin

Abstract

The contact inhibition of proliferation (CIP) denotes the cell density-dependent inhibition of growth, and the loss of CIP represents a hallmark of cancer. However, the mechanism by which CIP regulates gene expression remains poorly understood. Chromatin is a highly complex structure consisting of DNA, histones, and trans-acting factors (TAFs). The binding of TAF proteins to specific chromosomal loci regulates gene expression. Therefore, profiling chromatin is crucial for gaining insight into the gene expression mechanism of CIP. In this study, using modified proteomics of TAFs bound to DNA, we identified a protein that shuttles between the nucleus and cytosol in a cell density-dependent manner. We identified TIPARP, PTGES3, CBFB, and SMAD4 as cell density-dependent nucleocytoplasmic shuttling proteins. In low-density cells, these proteins predominantly reside in the nucleus; however, upon reaching high density, they relocate to the cytosol. Given their established roles in gene regulation, our findings propose their involvement as CIP-dependent TAFs. We also identified and characterized potential open chromatin regions sensitive to changes in cell density. These findings provide insights into the modulation of chromatin structure by CIP.

The Role of Mechanotransduction in Contact Inhibition of Locomotion and Proliferation

“…In various biological processes, such as CI and other mechanotransductionmediated biological processes, gene regulation involves changes in chromatin structure due to epigenetic modifications. The DSP-MNase-proteogenomics method identifies TAFs and their binding loci [171]. The heatmaps represent the read coverage or signal intensities across genomic regions and DNA samples extracted from open chromatin using MNase (see Figure 6 for details).…”

“…This review will primarily focus on TAF proteins that directly or indirectly bind to DNA, such as transcription factors (TFs), co-factors, repressors, and RNA polymerase [172], all of which are influenced during CIP and downstream of mechanotransduction. While researchers have identified more than 40 TAFs that shuttle between the nucleus and cytosol in response to cell-cell contact and mechanical stimulation [171]. DNA-TAF complex is stabilized with a reversible cross-linker, dithiobis(succinimidyl propionate) (DSP).…”

“…Recently, a method to comprehensively determine TAFs and their binding loci on chromatin (DSP-MNase-proteogenomics) has been developed [171] (Figure 6).…”

“…Moreover, DSP-MNase-proteogenomics revealed that DNA fragments binding to TAFs are notably enriched upstream of the TSS (Figure 5B), as anticipated. This analysis enables the comparison of active genes across multiple samples and has shown that genes associated with the Hippo pathway are downregulated in high-density cells [171]. Another method known as DNase I hypersensitive sites (DHSs) proteomics, described in Figure 6 [87], also facilitates the quantitative comparison of multiple samples and the identification of TAFs that transiently interact with chromatin.…”

“…Methods to identify mechanosenstive TAFs. (A) DSP-MNase-proteogenomics[171]. DNA-TAF complex is stabilized with a reversible cross-linker, dithiobis(succinimidyl propionate) (DSP).…”