Cis-acting DNA elements can either up-regulate or down-regulate transcription by binding transcription factors which are either activators or repressors. Cis-acting elements are therefore functionally grouped as positive or negative-acting elements. One research aim o f this thesis was to identify potential promoter sequences o f MyHC genes as well as to test the effect o f an enhancer sequence on muscle specific up-regulation o f gene expression. While promoters are essential for accurate and efficient initiation o f transcription, enhancers will increase the rate o f transcription from promoters. These two cis-acting elements will be described in more detail in the following sections.
1 .2 .1 .1 .1 . Promoter elements
The function o f promoter elements is position and orientation dependent. They are located directly upstream to the transcription initiation site (Cap-site, see Fig. 1.2.1) and are typically about 100 bp in length (Dynan and Tijan, 1985). Most class II genes contain a 'TATA-box' about 25 to 30bp upstream o f the transcription initiation site with the consensus sequence TATA(A/T)A(A/T) (Breathnach and Chambon, 1981). There are genes which lack a TATA- box and have instead a GC-rich region centred around position -33; this GC-rich region seems to be a feature o f 'housekeeping' genes and is also found in some tissue-specific genes. The TATA-box plays an essential role in the accurate positioning o f the transcription start site (Breathnach and Chambon, 1981). There are several elements upstream to the TATA-box (or the GC-region) which will increase the transcription activity strongly and have been termed
'upstream promoter elements' (UPE) (Goodwin et al., 1990).
Such elements are the CCAAT-box and the SPl-box (consensus GGGCGG), both o f which are commonly found closely 5' to the TATA-box. Mutations or deletions o f these boxes can abolish gene transcription as found for a factor IX mutation o f the CCAAT-box
(Crossley and Brownlee, 1990), causing Haemophilia B. Both these regulatory elements bind so called 'constitutive' transcription factors which are found in all cell types, yet some tissue specificity for the CCAAT-box can occur. Other UPEs usually have a high response specificity, like the heat-shock element (HSE) or the glucocorticoid response element (GRE) or a high tissue-type specificity like the octamer motif ATGCAAAT (Mason et al. , 1985) for B-cells.
A number o f regulatory elements involved in muscle specific expression have been described for different genes. The CArG-box/CBAR (consensus CC(A-I-T rich)gGG) is essential for muscle specific expression o f embryonic and cardiac alkali MyLCs (Uetsuki et a l., 1990; Kurabayashi et al., 1990) and for sarcomeric actins (Bergsma et al., 1986; Miwa et al., 1987). The M-CAT (muscle-CAT) motif (consensus CATTCCT) was described for chicken cardiac Troponin T in skeletal muscle (Mar and Ordahl, 1988 and 1990). E-boxes (consensus CANNTG) are found in muscle specific promoters as well as in enhancers and are targets for the transcription factors belonging to the Myo-D family, which inhibit cell proliferation and activate the muscle differentiation program (see section 1.2 .1 .2 .)
Positive and negative 5' regulatory elements o f different MyHC genes have been described for the rabbit p-cardiac MyHC promoter (Cribbs et a l., 1989; Shimizu et a l., 1992a and 1992b), the human p-cardiac MyHC promoter (Vosberg et a l., 1992), and the rat embryonic MyHC promoter (Bouvagnet et al., 1987). The data will be discussed in Chapter 4 alongside the sequence of a newly identified promoter.
1.2. .1.2. Enhancer elements
The function o f enhancers is in general position and orientation independent, exceptions are the stereospecific enhancer complexes with binding sites for architectural proteins (reviewed by Tjian and Maniatis, 1994). Enhancers are found 5' or 3' to the gene or even within an exon or intron o f the gene. Enhancers increase the activity o f promoters without
having an activity on their own. They are made up o f several sequence units, each o f which contain a transcription factor binding site. These units can be variously arranged, leading to a different binding sequence o f transcription factors and thus to novel nucleoprotein complexes. It is the synergism o f the transcription factors bound to the enhancer and interacting with the promoter nucleoprotein complex that make an enhancer cell-type or development specific and upregulates transcription. The sequence motifs found in enhancers are often identical or very similar to the ones found in the upstream promoter elements (UPEs) o f the related gene, and therefore bind the same transcription factors. An example is the CACCC-box in the promoters and enhancer o f globin genes, reviewed in Millier et al. ,
1988.
Muscle specific enhancers have been described for the mouse muscle creatine kinase (MCK) gene (Jaynes et cd., 1986; Sternberg et al., 1988), the rat MyLC 1/3 gene locus (Donoghue et al. , 1988) and the human MyLC 1/3 gene locus (Rosenthal et al., 1990). The mouse MyLC enhancer revealed high homology for the DNA motifs CACCTGCTGC (noncoding strand) and CACATGCTCA (coding strand) with the MCK enhancer. The MyLC enhancer has a binding site for the transcription factors M APFl and MAPF2, which were originally found to bind to the skeletal a-actin promoter (Ernst et al., 1991). The enhancer contains further binding sites for the muscle specific transcription factors M yoD and MEF2 (see section 1.2 .1 .2 .)
1 .2 .1 .1 .3 . Negative regulatory elements
Negative regulatory elements (NREs) commonly bind repressors and can thus downregulate or even block transcription of genes according to cell type specific or developmental specific requirements. The negative counterpart to enhancers are so called 'silencers', which function in an orientation and position independent manner (for review see Renkawitz, 1990). Position dependent NREs are commonly found between promoters and
enhancers. The binding o f repressors to these NREs is thought to prevent the interaction between the enhancer nucleoprotein complex and the transcription initiation complex. A third NRE group are the transcription arrest sites which are located downstream to the transcription initiation site. Binding o f repressors to these sites will disrupt the elongation o f transcript.
1 .2 .1 .1 .4 . Chromosomal domain elements
The two main elements o f this group are the locus control elements and the boundary elements. Both have an effect on a large stretch o f DNA. The locus control elements (like the LCR upstream the (3-globin gene cluster) create an open chromatin domain where transcription factors can easily bind to their target sequences. The deletion o f such an element blocks the transcription o f all the genes contained in the affected locus domain. Boundary elements act as buffers by 'protecting' the flanked genes from the influence o f any positive or negative elements contained in the neighbouring regions (reviewed by Eissenberg and Elgin, 1991).