# Determining the Mechanisms of RING1- and YY1-binding protein (RYBP) function and dysregulation in glioblastoma cells

> **NIH NIH R16** · WINTHROP UNIVERSITY · 2024 · $192,456

## Abstract

PROJECT SUMMARY / ABSTRACT
Glioblastoma multiforme (GBM) is the most common and most lethal tumor of the central nervous system, with
a median survival of less than 15 months (Stupp et al., 2009). GBM tumors display abnormal gene expression
profiles that contribute to their malignant features, and thus understanding the mechanisms of their aberrant
gene regulation is crucial to developing more effective, targeted therapies. An important class of proteins that
control gene expression patterns is the Polycomb (Pc) family. Specifically, the Pc protein RING1- and YY1-
binding protein (RYBP) exerts a tumor suppressive effect across cancer types, and RYBP is frequently
downregulated in GBM patients (Li et al., 2013; Hu et al., 2017). However, the specific role of RYBP in GBM and
the mechanisms leading to its reduced expression in GBM remain unclear. Therefore, we propose to (1)
determine the tumor suppressive effects of RYBP in glioblastoma cells. Our approach will be to stably transduce
GBM cells with lentivirus expressing either RYBP or a GFP control, and in parallel to stably transduce
immortalized astrocytes with lentivirus expressing shRNA against RYBP or a non-targeting control. We will
isolate single-cell clones and use them to (a) determine the in vitro phenotypic effects of RYBP ectopic
expression or knockdown on cell viability, migration, and invasion, and to (b) identify the transcriptional targets
of RYBP-mediated gene regulation using RNA-sequencing and chromatin immunoprecipitation-sequencing. We
also propose to (2) determine the mechanisms of RYBP silencing in glioblastoma. Specifically, we will (a)
elucidate the contribution of G-quadruplexes, which are secondary structures formed by G-rich regions of DNA
and RNA, to RYBP transcriptional regulation. To do this, we will treat cells with G4-stabilizing ligands or deplete
cells of G4-resolving enzymes via RNA interference, and measure the effects on RYBP expression. We will also
determine whether the putative G4-forming sequences in the RYPB promoter can form bona fide G4s in cells.
Additionally, we will (b) determine the role of microRNAs in inhibiting RYBP protein synthesis by using synthetic
inhibitors and luciferase assays. Collectively, we expect this project to reveal a novel tumor suppressive effect
for the transcription factor and chromatin regulator, RYBP, in GBM, and to identify the transcriptional targets
under its control. The impact of this project will be the placement of RYBP's function within a coordinated,
regulatory context that will expand our knowledge of potential molecular targets, thereby enlightening
development of GBM therapies.

## Key facts

- **NIH application ID:** 10936126
- **Project number:** 1R16NS141721-01
- **Recipient organization:** WINTHROP UNIVERSITY
- **Principal Investigator:** Daniel Brooks Stovall
- **Activity code:** R16 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $192,456
- **Award type:** 1
- **Project period:** 2024-09-17 → 2028-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10936126

## Citation

> US National Institutes of Health, RePORTER application 10936126, Determining the Mechanisms of RING1- and YY1-binding protein (RYBP) function and dysregulation in glioblastoma cells (1R16NS141721-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10936126. Licensed CC0.

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