Molecular cloning of Drosophila HCF reveals proteolytic processing and self‐association of the encoded protein

Abstract

HCF-1 functions as a coactivator for herpes simplex virus VP16 and a number of mammalian transcription factors. Mature HCF-1 is composed of two subunits generated by proteolytic cleavage of a larger precursor at six centrally-located HCF PRO repeats. The resulting N-and Cterminal subunits remain tightly associated via two complementary pairs of self-association domains: termed SAS1N-SAS1C and SAS2N-SAS2C. Additional HCF proteins have been identified in mammals (HCF-2) and Caenorhabditis elegans (CeHCF). Both contain wellconserved SAS1 domains but do not undergo proteolytic processing. Thus, the significance of the cleavage and self-association of HCF-1 remains enigmatic. Here, we describe the isolation of the Drosophila HCF homologue (dHCF) using a genetic screen based on conservation of the SAS1 interaction. The N-terminal β-propeller domain of dHCF supports VP16-induced complex formation and is more similar to mammalian HCF-1 than other homologues. We show that fulllength dHCF expressed in Drosophila cells undergoes proteolytic cleavage giving rise to tightly associated N-and C-terminal subunits. As with HCF-1, the SAS1N and SAS1C elements of dHCF are separated by a large central region, however, this sequence lacks obvious homology to the HCF PRO repeats required for HCF-1 cleavage. The conservation of HCF processing in insect cells argues that formation of separate N-and C-terminal subunits is important for HCF function.HCF-1, also known as C1 factor, is a broadly-expressed nuclear protein involved in the regulation of cellular and viral gene expression. HCF-1 was first identified through its role in transcriptional activation by the herpes simplex virus (HSV) VP16 protein (Gerster and Roeder, 1988;Kristie et al., 1989). Association of HCF-1 with VP16 (also known as α-TIF or Vmw65) leads to the assembly of a multiprotein-DNA complex that includes the POU protein Oct-1 and the TAATGARAT element, a VP16-responsive DNA sequence present in all viral immediate-early (IE) gene promoters (reviewed in (Herr, 1998)). Nuclear localization of VP16 is dependent on association with HCF-1, which contributes a nuclear localization signal (NLS) located at its C-terminus (LaBoissière et al., 1999;Scarr et al., 2000;Wilson et al., 2000). In addition, HCF-1 contains its own activation domain and this is required for optimal transactivation by the VP16-induced complex ( The arrangement of functional domains in human HCF-1 is shown in Figure 1A. HCF-1 is translated as a 230-300-kDa precursor and processed into N-and C-terminal subunits through autocatalytic proteolytic cleavage at a series of six HCF PRO repeats located near the center of the precursor polypeptide (Wilson et al., 1993b;Kristie et al., 1995;Wilson et al., 1995b;Vogel and Kristie, 2000). The resulting subunits remain stably associated via two matched pairs of interaction domains: termed SAS1N-SAS1C and SAS2N-SAS2C . The SAS1C domain corresponds to a pair of fibronectin type 3 (Fn3) repeats, which interact with a 43-residue sequence (SAS1N) that is locate...

Host Cell Factor and an Uncharacterized SANT Domain Protein Are Stable Components of ATAC, a Novel dAda2A/dGcn5-Containing Histone Acetyltransferase Complex in Drosophila

“…Both rat and rabbit antibodies against dHCF detected a strong band of approximately 70 kDa, corresponding to the C-terminal dHCF fragment. This is consistent with published results, which showed that fulllength dHCF gets proteolytically cleaved to yield two polypeptides of approximately 120 and 70 kDa (24). However, we did not see a band of 160 kDa corresponding to the full-length protein, which suggests that the majority of dHCF gets processed after translation.…”

“…dHCF is a 1,500-amino-acid protein with a predicted molecular mass of 160 kDa (24). This protein is conserved among higher eukaryotes, and its human homologue HCF-1 has been studied in detail.…”

“…Another interesting feature of this 2,035-amino-acid protein is that it gets processed after translation to generate two polypeptides that remain noncovalently bound (43). Although dHCF lacks the conserved repeats required for the processing of mammalian HCF-1, it is still cleaved to generate two fragments of approximately 120 and 72 kDa that remain associated (24).…”

“…For expression in S2 cells, cDNAs were inserted into plasmids derived from pRmHa3 (6) or pMT (Invitrogen). pACXT-T7-dHCF-FLAG was a kind gift from Angus Wilson (24). For expression in Escherichia coli, cDNAs were inserted into pQE12 (QIAGEN).…”

Drosophila Myc Interacts with Host Cell Factor (dHCF) to Activate Transcription and Control Growth

“…HCF 4 -1 (an acronym for "host cell factor") was originally identified as a large cellular protein that is required for the transcription of viral genes in herpes simplex virus-infected cells, and it was later shown to be essential for cell cycle progression of normal, uninfected vertebrate cells (reviewed in 18). A single HCF homolog is also present in Drosophila (called dHCF), but no mutant phenotypes have been described so far (19,20). In mammalian cells, HCF-1 is cleaved after synthesis into two parts that remain physically associated; the C terminus is required for passage through mitosis, whereas the N terminus is important for entry into S-phase (21).…”