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The PURB-HOTAIR complex regulates p53-dependent promoter-specific transcriptional activation

ABSTRACT: p53 executes its diverse functions via different transcriptional targets, but the precise mechanism of promoter-specific regulation by p53 remains largely unknown. Through biochemical purification, we identify PURB, a dual DNA/RNA-binding protein, which acts as a transcriptional co-repressor for p53 in a manner dependent on p53 acetylation status. PURB is overexpressed in human cancers while its knockdown induces p53-dependent activation of p21 but has no effect on other major promoters such as PUMA and Mdm2. In contrast to other p53 corepressors, PURB can recognize a unique DNA element at the p21 promoter, with the loss of this element not affecting p53-mediated transactivation but abrogating the ability of p53 to recruit PURB to the p21 promoter for repression. Mechanistically, PURB requires its sequence-specific binding with lncRNA HOTAIR to exert its repressive role. In turn, HOTAIR interacts directly with EZH2 and, bridged by the PURB/HOTAIR complex, p53 can recruit the EZH2 histone methyltransferase to target promoters for transcriptional repression. Further analysis of p53 targets indicates a number of promoters that may serve as a target for PURB-binding, suggesting that this mechanism of PURB-dependent promoter-specific regulation may not be limited to p21. These data establish a mode of LncRNA-mediated regulation of p53 transcription in a sequence-specific manner and reveal a previously unanticipated mechanism for acetylation-mediated promoter-specific regulation through a cis-regulatory element recognized by the PURB-HOTAIR complex.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Zhangchuan Xia

LAB HEAD: Dr. Wei Gu

PROVIDER: PXD042681 | Pride | 2025-04-24

REPOSITORIES: Pride

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			Action	DRS
	75360_92272.mzid.gz	Mzid
	75360_out.tgz	Other
	92272.mzXML	Mzxml

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Project description:Once thought to be transcriptional noise, large non-coding RNAs (lncRNAs) have recently been demonstrated to be functional molecules. Cell type-specific expression patterns of lncRNAs suggest that their transcription may be regulated epigenetically. Using a custom-designed microarray, here we examine the expression profile of lncRNAs in embryonic stem (ES) cells, lineage-restricted neuronal progenitor cells (NPC), and terminally differentiated fibroblasts. In addition, we also analyze the relationship between their expression and their promoter H3K4 and H3K27 methylation patterns. We find that numerous lncRNAs in these cell types undergo changes in the levels of expression and promoter H3K4me3 and H3K27me3. Interestingly, lncRNAs that are expressed at lower levels in ES cells exhibit higher levels of H3K27me3 at their promoters. Consistent with this result, knockdown of the H3K27me3 methyltransferase Ezh2 results in derepression of these lncRNAs in ES cells. Thus, our results establish a role for Ezh2-mediated H3K27 methylation in lncRNA silencing in ES cells and reveal that lncRNAs are subject to epigenetic regulation in a similar manner to that of protein-coding genes. lncRNA expression analysis was performed in 5 different cell types: (1) ES cells, (2) neuroprogenitors, (3) tail tip fibroblasts, (4) Ezh2 knockdown #1 ES cells, and (5) Ezh2 knockdown #2 ES cells. Total RNA was extracted and RNA quality was assessed via Agilent Bioanlayzer. mRNA was amplified using the Low RNA Linear Amplification Kit (Agilent Technologies) by standard manufacturer's protocol. Hybridization of experimental RNA labeled with Cy5 and Cy3-labeled Universal Reference RNA (Ambion) was carried out by standard protocol on a custom-designed array manufactured by Agilent Technologies. Briefly, three unique 60nt probes were designed for each entry in the Fantom3 database and were synthesized along with lineage marker control probes on a 2x105K format array. Upon array scanning, data was quality filtered, normalized by the Lowess method, and all probes corresponding to a given lncRNA were averaged using the UNC Microarray Database (UNCMD). Intraclass correlation analysis was carried out in the UNCMD.

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The PURB-HOTAIR complex regulates p53-dependent promoter-specific transcriptional activation

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