Abstract
FAcilitates Chromatin Transcription (FACT) is a complex of SSRP1 and SPT16 that is involved in chromatin remodeling during transcription, replication, and DNA repair. FACT has been mostly studied in cell-free or single cell model systems because general FACT knockout (KO) is embryonically lethal (E3.5). FACT levels are limited to the early stages of development and stem cell niches of adult tissues. FACT is upregulated in poorly differentiated aggressive tumors. Importantly, FACT inhibition (RNAi) is lethal for tumors but not normal cells, making FACT a lucrative target for anticancer therapy. To develop a better understanding of FACT function in the context of the mammalian organism under normal physiological conditions and in disease, we aimed to generate a conditional FACT KO mouse model. Because SPT16 stability is dependent on the SSRP1-SPT16 association and the presence of SSRP1 mRNA, we targeted the Ssrp1 gene using a CreERT2- LoxP approach to generate the FACT KO model. Here, we highlight the limitations of the CreERT2-LoxP (Rosa26) system that we encountered during the generation of this model. In vitro studies showed an inefficient excision rate of ectopically expressed CreERT2 (retroviral CreERT2) in fibroblasts with homozygous floxed Ssrp1. In vitro and in vivo studies showed that the excision efficiency could only be increased with germline expression of two alleles of Rosa26CreERT2. The expression of one germline Rosa26CreERT2 allele led to the incomplete excision of Ssrp1. The limited efficiency of the CreERT2-LoxP system may be sufficient for studies involving the deletion of genes that interfere with cell growth or viability due to the positive selection of the phenotype. However, it may not be sufficient for studies that involve the deletion of genes supporting growth, or those crucial for development. Although CreERT2-LoxP is broadly used, it has limitations that have not been widely discussed. This paper aims to encourage such discussions.
Highlights
Facilitates Chromatin Transcription (FACT), a heterodimeric complex of SSRP1 and SPT16, is a histone chaperone that is involved in chromatin remodeling through its binding to histone oligomers and modulation of nucleosome stability in the vicinity of ongoing RNA and DNA synthesis [1]
We showed that FACT stability is dependent on the dimerization of its two subunits (i.e., SPT16 and SSRP1) [8]
SPT16 is only stable when SSRP1 mRNA is present in the complex [8]
Summary
Facilitates Chromatin Transcription (FACT), a heterodimeric complex of SSRP1 and SPT16, is a histone chaperone that is involved in chromatin remodeling through its binding to histone oligomers and modulation of nucleosome stability in the vicinity of ongoing RNA and DNA synthesis [1]. Inhibition of FACT (RNAi-mediated) is tolerable for normal cells in vitro but causes growth arrest and death of tumor cells, including cancer stem cells [4,5,6,7]. To understand the role of FACT in postembryonic development, tumorigenesis, and tumor progression, we attempted to generate an inducible conditional FACT KO mouse model using the site-specific recombinase CreERT2 Since both SSRP1 and SPT16 are stable only when bound to each other and RNAi-mediated repression of SSRP1 expression effectively eliminates both subunits [4, 8], we targeted FACT by deleting part of the Ssrp gene
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