Dataset Information

Identification of functional cis-acting elements within the rat liver S14 promoter.

ABSTRACT: The structure of DNAase I hypersensitive site 1 (Hss-1), located adjacent to the 5' end of the rat liver S14 gene, is regulated by tissue-specific factors, and its formation correlates with the transcriptional activation of the S14 gene. We propose that tissue-specific trans-acting factors interacting with key cis-linked elements within this site function in the initiation of S14 gene transcription. To examine this hypothesis we used DNAase I footprint, gel shift and in vitro transcriptional analyses to identify cis-linked elements that function in the control of S14 gene transcription. Binding of rat liver nuclear proteins to the S14 promoter (from -8 to -464 bp) produced four DNAase I footprints (designated A-D). Gel shift studies showed that DNA-protein binding was tissue- and sequence-specific, differentially heat-sensitive, and abolished by proteinase K. The function of the four cis-acting elements was assessed by using an in vitro transcription initiation assay in which the S14 promoter was fused to a reporter gene (G-free cassette). Deletion studies showed that nuclear factors binding to regions A (-48 to -63 bp), B (-88 to -113 bp) and D (-286 to -310 bp) enhanced the rate of initiation of transcription, while proteins binding to region C (-227 to -244 bp) suppressed the rate of initiation of transcription. Based on oligonucleotide competition studies, we suggest that hepatic NF-1 (or a related protein) binding to the A region enhances the rate of initiation of S14 gene transcription. Since trans-acting factors interacting with regions B and D are found in liver but not in spleen or kidney, we suggest that the proteins interacting with these regions may be involved in the tissue-specific augmentation of S14 gene transcription.

SUBMITTER: MacDougald OA

PROVIDER: S-EPMC1130519 | biostudies-other | 1991 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:FBXL2 is an important ubiquitin E3 ligase component that modulates inflammatory signaling and cell cycle progression, but its molecular regulation is largely unknown. Here, we show that tumor necrosis factor alpha (TNF-α), a critical cytokine linked to the inflammatory response during skeletal muscle regeneration, suppressed Fbxl2 mRNA expression in C2C12 myoblasts and triggered significant alterations in cell cycle, metabolic, and protein translation processes. Gene silencing of Fbxl2 in skeletal myoblasts resulted in increased proliferative responses characterized by activation of mitogen-activated protein (MAP) kinases and nuclear factor kappa B and decreased myogenic differentiation, as reflected by reduced expression of myogenin and impaired myotube formation. TNF-α did not destabilize the Fbxl2 transcript (half-life [t1/2], ∼10 h) but inhibited SP1 transactivation of its core promoter, localized to bp -160 to +42 within the proximal 5' flanking region of the Fbxl2 gene. Chromatin immunoprecipitation and gel shift studies indicated that SP1 interacted with the Fbxl2 promoter during cellular differentiation, an effect that was less pronounced during proliferation or after TNF-α exposure. TNF-α, via activation of JNK, mediated phosphorylation of SP1 that impaired its binding to the Fbxl2 promoter, resulting in reduced transcriptional activity. The results suggest that SP1 transcriptional activation of Fbxl2 is required for skeletal muscle differentiation, a process that is interrupted by a key proinflammatory myopathic cytokine.IMPORTANCE Skeletal muscle regeneration and repair involve the recruitment and proliferation of resident satellite cells that exit the cell cycle during the process of myogenic differentiation to form myofibers. We demonstrate that the ubiquitin E3 ligase subunit FBXL2 is essential for skeletal myogenesis through its important effects on cell cycle progression and cell proliferative signaling. Further, we characterize a new mechanism whereby sustained stimulation by a major proinflammatory cytokine, TNF-α, regulates skeletal myogenesis by inhibiting the interaction of SP1 with the Fbxl2 core promoter in proliferating myoblasts. Our findings contribute to the understanding of skeletal muscle regeneration through the identification of Fbxl2 as both a critical regulator of myogenic proliferative processes and a susceptible gene target during inflammatory stimulation by TNF-α in skeletal muscle. Modulation of Fbxl2 activity may have relevance to disorders of muscle wasting associated with sustained proinflammatory signaling.

Dataset Information

Identification of functional cis-acting elements within the rat liver S14 promoter.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets