Different E-box regulatory sequences are functionally distinct when placed within the context of the troponin I enhancer.

Basic helix-loop-helix (bHLH) regulatory proteins are known to bind to a single DNA consensus sequence referred to as an E-box. The E-box is present in the regulatory elements of many developmentally controlled genes, including most muscle-specific genes such as troponin I (TnI). Although the E-box consensus is minimally defined as CANNTG, the adjacent nucleotides of functional E-boxes are variable for genes regulated by the bHLH proteins. In order to examine how E-box regulatory regions containing different internal and flanking nucleotides function when placed within the context of a single regulatory element, the E-box region (14 bp) present within the TnI enhancer was substituted with the corresponding E-box sequences derived from the muscle-specific M-creatine kinase (MCK) and cardiac alpha-actin regulatory elements as well as from the immunoglobulin kappa (Ig kappa) enhancer. Within the TnI enhancer, the E-box sequence derived from cardiac alpha-actin was inactive whereas the corresponding sequence from the MCK right E-box efficiently restored wild-type enhancer activity in muscle cells. Intermediate levels of gene activity were observed for TnI enhancers containing E-boxes derived from the MCK left E-box site or from the Ig kappa E2 E-box. DNA binding studies of MyoD:E12 protein complexes with each substituted TnI enhancer confirmed that DNA binding activity in vitro mimics the relative strength of the enhancers in vivo. These studies demonstrate that the specific nucleotide composition of individual E-boxes, which are contained within the regulatory elements of most if not all muscle-specific genes, contributes to the complex regulatory mechanisms governing bHLH-mediated gene expression.     

In vivo functional analysis of in vitro protein binding sites in the immunoglobulin heavy chain enhancer.

We have systematically investigated the functional role of protein binding sites within the mouse immunoglobulin heavy chain enhancer which we previously identified by in vitro binding studies (1,2). Each binding site was deleted, mutant enhancers were cloned 3' of the chloramphenicol acetyl transferase gene in the vector pA10CAT2 and transfected into plasmacytoma cells. We demonstrate that the newly identified site E, located at 324-338 bp, is important for enhancer function; previously identified sites B(uE1), Cl(uE2), C2(uE3) and C3 were also shown to be important for enhancer activity. Sites A and D are not required for IgH enhancer function, as assayed by our methods. Thus, including the octamer site, six protein binding sites which bind at least six different proteins are important for enhancer function in vivo.     

Purification of a nuclear trans-acting factor involved in the regulated transcription of a human immunoglobulin heavy chain gene

The expression of the rearranged human immunoglobulin gamma 1 heavy chain gene (HIG1) was shown to be induced through its enhancer by the positive regulatory trans-acting factor(s) that was contained only in cells of B lineage. The trans-acting factors were purified from mouse myeloma NS1 cells, and HIG1-inducing activity was found mainly in fractions of molecular weight 53-127 kd and in a fraction eluted from a heparin-Sepharose column with 0.5 M KCI. This semipurified fraction contained proteins binding to the conserved octamer sequence, ATGCAAAT, in the promoter region, as well as to sequences in the enhancer region. The 0.5 M KCI eluates from a heparin-Sepharose column were applied to a DNA affinity column of synthetic oligonucleotides of the octamer sequence and the sequence TATTTTAGGAAGCAAA in the HpaII-BgIII region of the HIG1 gene enhancer. The protein eluted from the enhancer sequence-specific DNA affinity column showed a strong inducing activity for the HIG1 gene, and the molecular weight of a predominant protein was 96 kd.