Julia M. Giger

Role of Antisense RNA in Coordinating Cardiac Myosin Heavy Chain Gene Switching

A novel mechanism of regulation of cardiac α and β myosin heavy chain gene by naturally occurring antisense transcription was elucidated via pre-mRNA analysis. Herein, we report the expression of an antisense β myosin heavy chain RNA in the normal rodent myocardium. The pattern of expression of the antisense βMHC RNA (β RNA) under altered thyroid state and in diabetes directly correlates with that of the α pre-mRNA/mRNA, whereas it negatively correlates with the β mRNA expression. Rapid amplification of the 5′ end shows that this antisense transcript originates 2 kb downstream of the β gene, and it is transcribed across the entire β gene from the opposite strand. Our results demonstrate that the β-α myosin heavy chain intergenic DNA possesses a bidirectional transcriptional activity, one direction transcribing the α gene, and the opposite direction transcribing the antisense β RNA. This process turns on the α expression, and it simultaneously turns off that of the β and thus coordinates α and β expression in an opposite fashion. Comparative analyses of the intergenic DNA sequence across five mammalian species revealed a conserved region that is proposed to be a common regulatory region for the α and antisense β promoter. This finding unravels the mechanism of cardiac α-β gene switching and implicates the role of cardiac myosin gene organization with their function.

Rapid muscle atrophy response to unloading: pretranslational processes involving MHC and actin.

Skeletal muscles, especially weight-bearing muscles, are very sensitive to changes in loading state. The aim of this paper was to characterize the dynamic changes in the unloaded soleus muscle in vivo following a short bout of hindlimb suspension (HS), testing the hypothesis that transcriptional events respond early to the atrophic stimulus. In fact, we observed that after only 1 day of HS, primary transcript levels of skeletal alpha-actin and type I myosin heavy chain (MHC) genes were significantly reduced by more than 50% compared with ground control levels. The degree of the decline for the mRNA expression of actin and type I MHC lagged behind that of the pre-mRNA levels after 1 day of HS, but by 2 and 7 days of HS, large decreases were observed. Although the faster MHC isoforms, IIx and IIb, began to be expressed in soleus after 1 day of HS, a relatively significant shift in mRNA expression from the slow MHC isoform type I toward these fast MHC isoforms did not emerge until 7 days of HS. One day of HS was sufficient to show significant decreases in mRNA levels of putative signaling factors serum response factor (SRF), suppressor of cytokine signaling-3 (SOCS3), and striated muscle activator of Rho signaling (STARS), although transcription factors yin-yang-1 (YY1) and transcriptional enhancing factor-1 (TEF-1) were not significantly affected by HS. The protein levels of actin and type I MHC were significantly decreased after 2 days of HS, and SRF protein was significantly decreased after 7 days HS. Our results show that after only 1 day of unloading, pre-mRNA and mRNA expression of muscle proteins and muscle-specific signaling factors are significantly reduced, suggesting that the downregulation of the synthesis side of the protein balance equation that occurs in atrophying muscle is initiated rapidly.

The CAAT-binding transcription factor 1/nuclear factor 1 binding site is important in β-myosin heavy chain antisense promoter regulation in rats

The rat heart expresses two myosin heavy chain (MHC) isoforms, β and α; these genes are arranged in tandem on the same chromosome. We have reported that an antisense (AS) β RNA starts in the intergenic (IG) region between β and α genes and extends to overlap the β gene. We propose that in adult rats, both the α sense and IG βAS RNA expression are activated by an IG bidirectional promoter and that the transcription of βAS RNA interferes with the sense β, resulting in low levels of β mRNA and high levels of α, a phenotype seen in a typical rat heart. A previous report examined the activity of the βAS promoter and showed that a 559 bp fragment of the βAS promoter (–2285 to –1726; relative to αMHC gene start site) injected into rat ventricle was activated in control heart, and decreased significantly in response to hypothyroidism (propylthiouracil induced) and diabetes (streptozotocin induced) and increased in hyperthyroid rats (T3 induced), similar in pattern to the endogenous βAS RNA. In the present paper, we demonstrate with electrophoretic mobility shift analyses that ventricular nuclear proteins are interacting with a nuclear factor 1/CAAT‐binding transcription factor 1 (NF1/CTF1) binding site, and a supershift assay indicates that the protein binding at this site is antigenetically related to the CTF1/NF1 factor. Moreover, a mutation of the CTF1/NF1 site within the 559 bp promoter region nearly abolished promoter activity in vivo in control, STZ‐ and PTU‐treated rats. Based on these findings, we conclude that the NF1 site is critical to βAS promoter regulation.