Th e HAP Activator Protei ns

The products of the MAPI, HAP2 and HAP3 regulatory loci in yeast activate transcription of two cytochrome c genes, CYC1 and CYC7 (Pfeifer eta!., 1987a and 1987b). There are two upstream activation sites adjacent to the CYC1 gene; UAS1 which is activated by HAP1, in a reaction dependent on haem, by HAP1, and UAS2 which is activated by the combined action of HAP2 and HAP3 (Guarente etal., 1984; Pinkham and Guarente, 1985).

Within UAS1 there are two distinct subsites, A and B, both of which are required for activity. Region B forms part of a recognition site that binds to a yeast factor, RC2, In vitro (Arcangioli and Lescure, 1985). When cells are grown under haem-defflcient conditions, extracts do not demonstrate RC2 binding activity ~ neither is activity restored by addition of haem to the extracts.

Pfeifer and coworkers (1987a) investigated the activity of the HAP1 gene product In vitro and found that the protein bound in region B of UAS1 to the same DNA sequences as RC2, but that binding of these factors was mutually exclusive. Moreover, haem was shown to stimulate the binding of MAPI to UAS1 but not RC2, even though haem was required for the In vivo synthesis of RC2. This would suggest that haem is directly involved In the activation of UAS1. Haem may function as a ligand that binds to HAP1 or, alternatively, the effect of haem could be indirect.

occurring via a second protein that modifies HAP1 in some way.

The role of RC2 in activation is not known. However, the regulation of the activity of RC2 and HAP1 suggests that RC2 Is not a repressor that is inactivated by the inducer haem. It Is possible that RC2 is a negative regulator that modulates UAS1 activity by competing with HAP1 for binding. Another possibility is that RC2 could be a positive regulatory factor involved in the activation of the adjacent UAS2. An additional factor, RAF (Region A Factor), distinct from HAP1, has been identified, and it has been suggested that it Is the complex of RAF and HAP1 that is required for transcriptional activation.

The CYC7 gene which encodes the minor form of cytochrome c In yeast is regulated coordinately with CYC1 (Pfeifer etal., 1987b). In addition, loss of function mutations in the HAP1 gene abolish the activity of the CYC7 UAS as well as UAS1. HAP1 binds to a site in the CYC7 UAS that bears no obvious sequence similarity to the HAP1 binding site in UAS1 of the CYC1 gene. Furthermore, the binding site of HAP1 to the CYC7 site, like its binding to UAS1, is stimulated by haem (Pfeifer et a!., 1987a and b).

In general, genes coordinated by the same regulator are marked by a consensus sequence in their S'-flanking DNA. The case of CYC1 and CYC7 is an obvious exception to this rule with the best alignment of the binding site sequences yielding only a match of seven nucleotides that are scattered across the 23 bp bound (Pfeifer et a!., 1987b). How then does HAP1 recognise two different DNA sequences?

HAP1 could contain physically distinct DNA binding regions, one of which recognises UAS1 and the other CYC7 UAS. However, dramatically increasing the dosage of HAP1 results only in a two-fold increase in CYC7 expression (K. Pfeifer, unpublished data), suggesting that the supply of HAP1 does not limit CYC7 expression to any great extent. Another possibility is that a single DNA binding domain of HAP1 could recognise both UAS1 and CYC7 UAS. This idea is consistent with the findings of Pfeifer etal. (1987b), namely that truncated HAP1 proteins do not separate the binding domains for the two sites. Secondly, the specific contacts made between HAP1 and the two DNA sites are similar, and finally, UAS1 and CYC7 sequences compete with each other, with comparable affinity, for binding to HAP1. How this single DNA binding domain recognises these two divergent sequences remains to be elucidated but the answer may in fact lie in the DNA itself in

that the DNA sequences of the UAS1 and CYC7 sites could create some common structure that contributes to recognition by MAPI (Pfeifer etal., 1987b; Rhodes and Klug. 1986).

Activation of UAS2 of the CYC1 gene is highly regulated by catabolite repression and is repressed about 30-fold by a shift of cells from lactose to glucose containing medium. The activity of UAS2 depends on the products of both the HAP2 and HAP3 genes (Guarente et ah, 1984). Mutations in either HAP2 or HAP3 result in the inability to activate UAS2 and a global defect in the expression of genes involved in respiratory metabolism.

Analysis of the UASs of the HEM1 gene (Keng and Guarente, 1987), C0X4 (Schneider and Guarente, unpublished data) and UAS2 reveals that all these HAP2-HAP3 dependent UASs contain the sequence TN(A/G)TTGGT. Wild type UAS2 contains the sequence TGGTTGGT but a G to A transition in UAS2 (termed UAS2UP1) alters this to TGATTGGT, thus Increasing the homology to the HEM1 and C0X4 UASs and resulting in a 10-fold increase in UAS2 activity In vivo (Guarente etal., 1984). Linker scanning analysis of UAS2 has confirmed the critical nature of the consensus element for activity of the site (Forsburg and Guarente, 1988). In addition, this element is homologous to the CCAAT box of higher eukaryotes. Moreover, major groove contacts made by HAP2-HAP3 in UAS2UP1, as determined by méthylation interference experiments, and the CCAAT box binding factor are identical (Olesen etal., 1987).

Olesen and coworkers (1987) have shown that both HAP2 and HAP3 in yeast extracts bind to UAS2UP1 and give rise to a single DNA-protein complex in native polyacrylamide gels. The binding of either HAP2 or HAP3 is abolished if either of these two proteins is synthesised In a strain mutant in the complementary HAP gene, implying that the binding of HAP2 and HAP3 is interdependent. It may be that the two proteins bind in very close proximity to one another and stabilise each other's binding cooperatively, or they exist as a heterodimer.

A series of experiments were carried out by Hahn and Guarente (1988) in an attempt to distinguish between the aforementioned possibilities. HAP2 and HAP3 were tagged by gene fusion to E. coll LexA and lac Z genes, respectively, and the products purified through four chromatographic steps. The co-purification of LexA-HAP2, HAP3-B-gaIactosidase, and UAS2 binding activity demonstrated that HAP2 and HAP3 associate in the absence of DNA to form a multisubunit activation complex - a complex which must be capable of at least three functions. First,

the complex must bind specifically to UAS2 and related sequences In the yeast genome. It must activate transcription once bound at UAS2, and finally it must be able to respond to signals generated under inducing conditions in media containing a non-fermentable carbon source. To date, it would appear that these functions are not distributed evenly between the two proteins. Although both HAP2 and HAP3 are required for binding to UAS2UP1, either HAP2 and HAP3 both make contacts with the DNA, or one protein makes all the contacts and is held in the proper conformation by the other. In addition, activation of transcription by the bound complex appears to require both proteins (Olesen and Guarente, unpublished data).

Recently Chodosh and coworkers (1988) demonstrated that the subunits of the human CCAAT-binding protein, CBP1, and yeast HAP2/HAP3 heterodimer are functionally interchangeable in that the yeast/human hybrid complexes which are formed retain the ability to specifically recognise eukaryotic CCAAT-containing transcription elements. A CCAAT motif has also been found in the UASs of several yeast genes regulated by HAP2 and HAP3 (Keng and Guarente, 1987), suggesting that the HAP2/HAP3 complex is a global activator of respiratory genes, functioning specifically by recognising CCAAT elements located in the UASs of this family.