ACSM Fellow Ken Baldwin and Colleagues Discover a New Antisense Retrograde Mechanism of Gene Regulation (2006)

INTRODUCTION Muscle and exercise physiologists have been easy to characterize. They measure breathing by blowing up Douglas bags; they measure cardiac output by injecting green dye into veins and tapping arteries for blood sampling. Even if they only take small blood samples, they are known to stay up for days taking samples every few minutes. As well, they collect all manner of body fluids from men and women in deserts, on mountaintops, and in Earth orbit. They take and give muscle biopsies that they subsequently slice or grind up to explore. Clearly, exercise and muscle physiologists are the real current day "blood and guts" of contemporary scientists. As with any scientific discipline, muscle and exercise physiology have both undergone a breathtaking evolution during the past four decades, and this is elegantly illustrated in the work of Prof. Kenneth M. Baldwin, Ph.D., FACSM. Truthfully, this former collegiate wrestler can beat a mean air guitar, get down, and sweat with the best of them, but his recent work is clearly brilliant in nature. Dr. Baldwin's work is featured in this News Brief because it represents a milestone regarding fundamental mechanisms regulating gene transcription in skeletal muscle. A PHYSIOLOGIST'S SENSE: PATTERN RECOGNITION The Baldwin Laboratory's discovery relates to the area of the regulation and counterregulation of slow (β) and fast (α) myosin isoform expressions. Beyond the very sophisticated technical analysis, success relied on recognition of some rather elemental facts. First, when the expression (amount) of α myosin heavy chain (MHC) isoform is increased in cardiac or skeletal muscle, expression of the divergent slow (β) isoform is decreased. In his illustrious career, Dr. Baldwin has studied myosin isoform shifts in response to several stresses including thyroid toxicity, malnutrition, loading, and unloading, but the patterns on myosin isoform transition (i.e., increase in one class of isoform and suppression of the other) were common. Second, Dr. Baldwin appreciated the significance of the close location of genes encoding fast and slow myosin isoforms on the same chromosome. Third, however, to make their discovery, the Baldwin Laboratory's success required one more bold element, that of thinking outside the box. BREAKING THE RULE: UPSTREAM GENE SUPPRESSION BY ANTISENSE RNA The current field may seem like alphabet soup for those of us who went to school before the discovery of the genetic code or the double helix; hence, Figure 1 derived from the article of Haddad et al. (2) in the Baldwin Laboratory should be helpful in understanding both the biology and genius of the findings of Haddad and Baldwin. Consult any textbook and most any contemporary research paper, reading of the genetic code (i.e., the transcription of genes to the corresponding messenger RNA [mRNA]) is said to occur in a downstream direction. This is undeniably true. Consulting the same references will reveal that adjacent genes on the same chromosome can be separated by long sequences on noncoding DNA sequences. Relating to Figure 1, the nucleic acid sequence in genomic DNA is "read" (transcribed) from left to right. Typically, the beginning (upstream or untranslated end) of genes contains promotor areas to which specific molecules (transcription factors) bind and trigger the downstream transcription. With publication of the human, rat, mouse, and other genomes, many scientists are now focused on the factors that regulate gene expression by either promoting or blocking transcriptional regulation. In other words, given that the genes are known, massive attention is now directed on understanding the mechanisms regulating promoter binding and activation of downstream gene transcription. Consulting the promotor region of the slow myosin β isoform, a contemporary approach to understanding the regulation of slow myosin expression might beto identify what binds to the upstream (left side, 5′-untranslated region) promotor region and how that transcription factor stimulates downstream (left to right) transcription. Alternatively, another approach might be to figure out what factor or factors interfere with transcription factor formation or its binding to the βMHC gene promotor region. Well, in the past, the Baldwin Laboratory was no exception to common practice, that of investigating the role of transcriptional regulation in myosin isoform expression. But, as already described, the counterregulatory nature of myosin isoform expression and the downstream position of the dominant α isoform gene in determining MHC isoform switches in vivo led Baldwin and colleagues to focus on the DNA sequence between the genes, the so-called intergenic DNA. It looked to be a noncoding DNA sequence, but was it? This is where the real novelty of the work of discovery happened.Figure 1: Model of coordinated striated muscle fast-slow isoform switching. Antisense βRNA starts in the intergenic region between genes coding for β- and α-myosin isoforms, and it extends all the way to the first intron of the βMHC gene. Cardiac MHC genes are located on the same chromosome. Each gene is approximately 25 kilobase (kb) in length, and they are separated by a short, intergenic 4-kb segment. βP is the basal promoter of the βMHC gene transcribing the sense (left-toright direction as diagrammed) to yield βMHC pre-mRNA. The αP is the basal promoter of the αMHC gene transcribing the sense αMHC premRNA. The antisense βP is an intergenic promoter transcribing the antisense βRNA, which inhibits the processing of the sense β pre-mRNA into mature β mRNA. Both sense α and antisense β transcriptions are regulated by a common promoter region (CPR). Note that MHC genes are not to Scale because emphasis is on showing the intergenic region. Figure courtesy of K.M. Baldwin.Dr. Baldwin and his investigative team focused on the intergenic DNA sequence between genes encoding for fast and slow myosin isoform. Through ingenious and laborious work, these investigators found that transcription factor binding to the common promoter region (CPR) on the intergenic DNA between slow and fast genes sends both downstream and upstream (retrograde) signals. The upstream signaling molecule is an antisense RNA that binds to the βMHC gene, blocking its transcription. Simultaneously, binding to the CPR causes αMHC gene transcription in the typical downstream (left to right) fashion. Hence, both sense α and antisense βMHC transcriptions are reciprocally regulated by the CPR. SUMMARY Physiologists know about control theory and the presence of negative and positive feedback and feedforward regulation. Exercise physiologists know that feedforward regulation of glycemia occurs during hard exercise (1), but all physiologists know that negative feedback regulation is the most common means of controlling physiological systems including blood glucose concentration, which is the prototypical example of a parameter regulated by negative feedback. Why then is anyone surprised that a dual control, simultaneous negative feedback and feedforward mechanisms could reciprocally coordinate the expression of two genes? Perhaps, in retrospect of the Baldwin Laboratory discoveries, some might say something such as "sure, retrograde negative feedback of gene regulation is to be expected based on basic biological principles." Nonetheless, Drs. Baldwin and Haddad and others in the Department of Physiology and Biophysics, University of California-Irvine are to be complimented for their pioneering achievements. Certainly, they have contributed to our understanding of the reciprocal regulation of myosin isoform expression in cardiac and skeletal muscles. More than that, they have set a standard for other physiologists to emulate in this postgenomic era. They have shown how knowledge of muscle and organ systems physiology can be a key for moving physiology dramatically forward. And, in so doing, they have discovered a previously unrecognized mechanism of gene-to-gene cross talk by means of an antisense RNA. Importantly, the Baldwin Laboratory has provided a seminal observation that is at the heart of coordinated sarcomeric gene regulation. Bravo Baldwin and colleagues. Acknowledgment The author thanks K.M. Baldwin for the figure and V.J. Caiozzo for constructive comments.