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Characterization of the Regulation and Function of Zinc Finger Protein 593 (Zfp593) in Skeletal Muscle
Author(s) -
McLeod Marc,
Waddell David
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.856.18
Subject(s) - myogenesis , zinc finger , reporter gene , biology , skeletal muscle , microbiology and biotechnology , zinc finger transcription factor , denervation , muscle atrophy , transcription factor , myocyte , gene expression , gene , genetics , endocrinology
Skeletal muscle atrophy is a debilitating medical condition that can arise due to aging, cancer, corticosteroid use, and denervation. In order to better characterize the molecular genetic events of neurogenic atrophy, mouse gastrocnemius muscle was isolated 3 and 14 days after sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Zinc Finger Protein 593 (Zfp593) is expressed in skeletal muscle and is significantly induced in response to denervation. Quantitative PCR (qPCR) analysis showed that Zfp593 is more highly expressed in proliferating myoblasts compared to differentiated myotubes, while Western blot analysis confirmed that Zfp593 is expressed at the protein level in proliferating and differentiated mouse muscle cells. In order to characterize the transcriptional regulation of Zfp593, fragments of the proximal promoter region located immediately upstream from the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C 2 C 12 mouse muscle cells alone or in combination with myogenic regulatory factor expression plasmids, resulting in a differential effect on reporter activity. Furthermore, analysis of the promoter region of Zfp593 revealed four conserved E‐box elements in the proximal regulatory region, which are known myogenic regulatory factor (MRF) binding sites. Therefore, in order to determine how these elements may regulate the transcription of Zfp593, the E‐Boxes where either mutated or deleted and the resulting mutant reporter constructs were then transfected into C 2 C 12 mouse myoblasts in combination with ectopic expression of MRFs. Additionally, it is predicted that the Zfp593 protein contains a zinc finger domain and is believed to function as a transcription factor. Therefore, to assess if Zfp593 is localized to the nucleus, we fused Zfp593 with green fluorescent protein (GFP), transfected the Zfp593‐GFP expression plasmid into C 2 C 12 mouse myoblasts cells, visualized by confocal microscopy, and confirmed that Zfp593 does localize to the nucleus. Finally, the Zfp593 transcript possess a particularly long 3′‐untranslated region (UTR) region, so we cloned several fragments of the UTR, fused them with a reporter gene, and found that the UTR region has a negative regulatory effect on reportor gene expression in muscle cells. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that Zfp593, a potential modulator of gene expression itself, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting. Support or Funding Information The work was support by University of North Florida Transformational Learning Opportunity grants to D.W. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .