What question did this study set out to answer?

This research aims to analyze protein-DNA interactions at the muscle creatine kinase gene enhancer during muscle cell differentiation.

November 10, 1989

In Vivo Footprinting of a Muscle Specific Enhancer by Ligation Mediated PCR

Key Points

This research aims to analyze protein-DNA interactions at the muscle creatine kinase gene enhancer during muscle cell differentiation.
Utilized a ligation mediated PCR footprinting technique to examine DNA interactions.
Focused on terminally differentiated muscle cells and compared them to myogenic and nonmuscle cells.
Identified footprints indicative of binding sites for the myogenic regulator MyoD1.
Footprints detected only in differentiated muscle cells with active MCK gene transcription.
Two footprints correspond to binding sites for MyoD1, indicating specific regulatory interactions.
Data suggest that additional mechanisms prevent MyoD1 binding in undifferentiated cells.

Abstract

In vivo protein-DNA interactions at the developmentally regulated enhancer of the mouse muscle creatine kinase (MCK) gene were examined by a newly developed polymerase chain reaction (PCR) footprinting procedure. This ligation mediated, single-sided PCR technique permits the exponential amplification of an entire sequence ladder. Several footprints were detected in terminally differentiated muscle cells where the MCK gene is actively transcribed. None were observed in myogenic cells prior to differentiation or in nonmuscle cells. Two footprints appear to correspond to sites that can bind the myogenic regulator MyoD1 in vitro, whereas two others represent muscle specific use of apparently general factors. Because MyoD1 is synthesized by undifferentiated myoblasts, these data imply that additional regulatory mechanisms must restrict the interaction between this protein and its target site prior to differentiation.

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