Analysis of the upstream regulatory region of a chicken skeletal myosin heavy chain gene.

The organization of the cis-acting regulatory elements of a chick myosin heavy chain gene has been investigated. The data show that a gene which is transcribed in vivo in the fast white embryonic musculature is also the major transcript expressed during myotube differentiation of primary myoblasts derived from 12-day embryonic chick leg muscles. The upstream region of this gene consists of 7500 base pairs, and we have tested the ability of these sequences to drive expression of the chloramphenicol acetyltransferase gene in developing primary muscle cultures. Deletion analyses of the upstream region show that negative regulatory elements are present within 2000 base pairs of the basal promoter elements, the CCAAT and TAATA boxes. Removal of these elements reveals the presence of a strong positive element located near the start site of transcription. Sequence analysis showed that the region also contains a sequence characteristic of an enhancer found in the immunoglobulin heavy chain gene, ATGCAAAT, the "octa" element. Gel band-shift assays show that this octa sequence binds a transacting factor present in muscle nuclear extracts, although footprint analysis indicates a limited interaction. Transient assays carried out with a fragment in which the octa sequence has been mutated, with the subsequent abolition of protein binding, shows that the particular interaction probably plays a role in negatively modulating the action of the strong positive promoter element.