High impact information on HAP2

• Most surprisingly, the subunits of CP1 and HAP2/HAP3 are functionally interchangeable [1].
• DNA binding was assessed both in vitro and in vivo whereas subunit association and transcriptional activation were analyzed in vivo by using a bifunctional lexA-HAP2 fusion [2].
• Functional homologs of HAP2 and HAP3 have been conserved in HeLa cells where HAP2 activity corresponds to a chromatographic fraction designated CP1B [2].
• Here, we describe deletion and codon insertion mutagenesis of the Saccharomyces cerevisiae HAP2 subunit [2].
• The CYC1 gene of Saccharomyces cerevisiae is positively regulated by the HAP2 and HAP3 proteins, which form a heteromeric complex that binds to a CCAAT box in the upstream activation site, UAS2, and which activate transcription in a nonfermentable carbon source [3].

Biological context of HAP2

• Through 5' deletion analysis we have also found that the effects of heme and HAP2 on COX6 are mediated by sequences between 135 and 590 base pairs upstream of its initiation codon [4].
• The promoter of this gene contains a number of motifs for DNA-binding transcriptional activators, including three which show strong sequence homology to the core HAP2/HAP3/HAP4 binding motif [5].
• No other putative binding sites such as a Hap2/3/4/5p site and an Abf1p consensus site were functional with respect to glucose-regulated CAT8 expression [6].
• The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small essential core protein domain [7].
• Mutations in HAP2, HAP3 or HAP4 block derepression of a CIT1-lacZ gene fusion [8].

Associations of HAP2 with chemical compounds

• Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription [9].
• We show here that both HAP2 and HAP3 in yeast extracts bind to UAS2UP1 and give rise to a single protein-DNA complex, termed C, in nondenaturing polyacrylamide gels [10].
• PUT1 is inducible by proline, responds only slightly to carbon catabolite repression, and is not regulated by the cytochrome activator proteins HAP1 and HAP2 [11].
• The generality of the Sp1A results is supported by our finding that yeast glutamine-rich domains from HAP2 and MCM1 are also transcriptionally active in S. cerevisiae [12].
• The first peptide is based on the native sequence of a protein-binding domain within a heteromeric transcriptional activator, HAP2, identified from yeast Saccharomyces cerevisiae, with tyrosine (Y) present at the 1st, 8th and 15th amino acid positions, hence we denote this YYY15 [13].

Physical interactions of HAP2

• Within this region the binding sites for both the Hap2/3/4/5 complex and Hap1p were defined by gel retardation experiments and site-directed mutagenesis [14].
• Mutagenesis of the perfect consensus for HAP2/3/4 complex binding at position -542 resulted in considerable reduction of UBI4 promoter derepression with respiratory adaptation in HAP wild-type cells and abolished the reduced UBI4-LacZ derepression normally seen when aerobic cultures of the hap1 mutant are transferred from glucose to lactate [15].
• We describe a detailed genetic analysis of the DNA-binding regions in the HAP2/HAP3 CCAAT-binding heteromeric complex [16].
• We demonstrated that mutations of the HAP2/3/4 binding site and of the two STress-Responsive cis-Elements (STRE) did not abolish the early induction of GSY2, although the latter mutation led to a 20-fold drop in the transcriptional activity of the promoter, as determined from lacZ gene fusions [17].
• In this report we demonstrate that, in addition to these regulators, the Hap2/3/4/5 complex interacts specifically with a CAAT-box element in the JEN1 promoter, and acts to derepress JEN1 expression [18].

Regulatory relationships of HAP2

• These findings identify COX6 as the fourth respiratory protein gene that is known to be regulated positively by heme and HAP2 [4].
• This HAP3 mutation also suppresses mutations in a different region of HAP2 which promotes subunit assembly of the complex [16].
• KlAAC was not subject to control by KlHap2, in contrast to AAC2 which is regulated by the Hap2 complex in S. cerevisiae [19].

Other interactions of HAP2

• Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer [3].
• It has consensus binding sequences for ABF1 and HAP2 [9].
• However, we have found that HAP2 and HAP3 gene products are involved in the expression of HEM3 [20].
• Results are consistent with generalized control of mitochondrial replication directed by the HAP1-HAP2 system and heme-directed control of formation of all apocytochromes mediated by HAP1 [21].
• Replacement of this region with the activation domain of GAL4 restored activity, suggesting that it provides the principal activation domain to the bound HAP2/3/4 complex [3].

References