Abstract
Skeletal muscle atrophy is a highly-prevalent and debilitating condition that remains poorly understood at the molecular level. Previous work found that aging, fasting, and immobilization promote skeletal muscle atrophy via expression of activating transcription factor 4 (ATF4) in skeletal muscle fibers. However, the direct biochemical mechanism by which ATF4 promotes muscle atrophy is unknown. ATF4 is a member of the basic leucine zipper transcription factor (bZIP) superfamily. Because bZIP transcription factors are obligate dimers, and because ATF4 is unable to form highly-stable homodimers, we hypothesized that ATF4 may promote muscle atrophy by forming a heterodimer with another bZIP family member. To test this hypothesis, we biochemically isolated skeletal muscle proteins that associate with the dimerization- and DNA-binding domain of ATF4 (the bZIP domain) in mouse skeletal muscle fibers in vivo. Interestingly, we found that ATF4 forms at least five distinct heterodimeric bZIP transcription factors in skeletal muscle fibers. Furthermore, one of these heterodimers, composed of ATF4 and CCAAT enhancer-binding protein β (C/EBPβ), mediates muscle atrophy. Within skeletal muscle fibers, the ATF4–C/EBPβ heterodimer interacts with a previously unrecognized and evolutionarily conserved ATF–C/EBP composite site in exon 4 of the Gadd45a gene. This three-way interaction between ATF4, C/EBPβ, and the ATF–C/EBP composite site activates the Gadd45a gene, which encodes a critical mediator of muscle atrophy. Together, these results identify a biochemical mechanism by which ATF4 induces skeletal muscle atrophy, providing molecular-level insights into the etiology of skeletal muscle atrophy.
Highlights
Skeletal muscle atrophy is a highly-prevalent and debilitating condition that remains poorly understood at the molecular level
This suggested that full-length activating transcription factor 4 (ATF4) may be too unstable in skeletal muscle fibers to be suitable for tandem affinity purification (TAP), which would be consistent with previous findings that full-length ATF4 is highly unstable due to rapid degradation [31,32,33,34,35,36]
ATF4 forms at least five distinct heterodimeric basic leucine zipper transcription factor (bZIP) transcription factors
Summary
ATF4 is unable to form stable homodimers [19], and a heterodimerization partner of ATF4 in skeletal muscle has never been found. In vitro analyses of interactions between human bZIP family members found that, compared with its affinity for another ATF4 bZIP domain, an individual ATF4 bZIP domain has a higher affinity for the bZIP domains of at least 30 other bZIP family members [20, 21] Consistent with this finding, ATF4 heterodimers are known to play important roles in nonmuscle cells [22,23,24,25,26,27,28], and several ATF4 target genes contain non-palindromic ATF4 regulatory elements, indicating regulation by an ATF4 heterodimer (29 –31). We conducted an unbiased search for ATF4 heterodimerization partners in mouse skeletal muscle fibers
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