Functional Analysis of Hif1 Histone Chaperone in Saccharomyces cerevisiae

Abstract

The Hif1 protein in the yeast Saccharomyces cerevisie is an evolutionarily conserved H3/H4-specific chaperone and a subunit of the nuclear Hat1 complex that catalyzes the acetylation of newly synthesized histone H4. Hif1, as well as its human homolog NASP, has been implicated in an array of chromatin-related processes including histone H3/H4 transport, chromatin assembly and DNA repair. In this study, we elucidate the functional aspects of Hif1. Initially we establish the wide distribution of Hif1 homologs with an evolutionarily conserved pattern of four tetratricopeptide repeats (TPR) motifs throughout the major fungal lineages and beyond. Subsequently, through targeted mutational analysis, we demonstrate that the acidic region that interrupts the TPR2 is essential for Hif1 physical interactions with the Hat1/Hat2-complex, Asf1, and with histones H3/H4. Furthermore, we provide evidence for the involvement of Hif1 in regulation of histone metabolism by showing that cells lacking HIF1 are both sensitive to histone H3 over expression, as well as synthetic lethal with a deletion of histone mRNA regulator LSM1. We also show that a basic patch present at the extreme C-terminus of Hif1 is essential for its proper nuclear localization. Finally, we describe a physical interaction with a transcriptional regulatory protein Spt2, possibly linking Hif1 and the Hat1 complex to transcription-associated chromatin reassembly. Taken together, our results provide novel mechanistic insights into Hif1 functions and establish it as an important protein in chromatin-associated processes.

The histone chaperoning pathway: from ribosome to nucleosome

“…Attempts have been made to separate out pre- and post-import machineries through biochemical fractionation of the cytosolic fraction. However, a number of factors identified to be cytosolic in these studies have been found to be nuclear when probed in intact cells; namely NASP [15,18], ASF1 [19–21], HAT1 [22,23] and RbAp46 [15,24,25]. The reason for this discrepancy could lie in the soluble nature of these proteins, rapidly leaking into the cytosolic fraction upon common sub-cellular fractionation methods, with insoluble proteins, such as lamins and tubulin, being poor controls for reporting on such mixings [15,26].…”

The histone chaperone Nrp1 is required for chromatin stability and nuclear division in Tetrahymena thermophila

“…Asf1 co-purifies with NASP in human cells. Similar to their human counterparts, yeast Asf1 interacts with Hif1, and the interaction is likely mediated by histones H3/H4 [ 13 ]. The AP-MS data of the co-purification with Asf1-FZZ from vegetatively growing cells and conjugation cells revealed the interaction of Asf1 and Nrp1 in T. thermophila [ 54 ].…”

“…In S. cerevisiae, Hif1 interacts with H3/H4, histone acetyltransferase complex, chromatin assembly proteins, DNA replication-associated proteins, DNA damage response proteins, transcription regulation-related proteins, and RNA polymerase II transcriptional pre-initiation complex assembly [ 13 ]. Hif1 buffers displaced histones during transcription and makes them available for immediate chromatin reassembly.…”

CAF-1 and Rtt101p function within the replication-coupled chromatin assembly network to promote H4 K16ac, preventing ectopic silencing

“…Strong evidence supports Asf1p functioning upstream of both CAF-1 and Rtt106p during DNA replication [ 4 , 19 – 25 , 28 – 30 ], and links between Asf1p and Hif1p during chromatin assembly have also been reported [ 40 , 41 , 42 , 61 ], but the distinct cellular functions of CAF-1, Rtt106p, and Hif1p have remained unclear. To evaluate their relationship and uncover distinct functions, we first examined silencing at the crippled HMR a e** locus ( Fig 1A ).…”