Human EFO1p exhibits acetyltransferase activity and is a unique combination of linker histone and Ctf7p/Eco1p chromatid cohesion establishment domains

Abstract

Proper segregation of chromosomes during mitosis requires that the products of chromosome replication are paired together-termed sister chromatid cohesion. In budding yeast, Ctf7p/Eco1p is an essential protein that establishes cohesion between sister chromatids during S phase. In fission yeast, Eso1p also functions in cohesion establishment, but is comprised of a Ctf7p/Eco1p domain fused to a Rad30p domain (a DNA polymerase) both of which are independently expressed in budding yeast. In this report, we identify and characterize the first candidate human ortholog of Ctf7p/Eco1p, which we term hEFO1p (human Establishment Factor Ortholog). As in fission yeast Eso1p, the hEFO1p open reading frame extends well upstream of the C-terminal Ctf7p/Eco1p domain. However, this N-terminal extension in hEFO1p is unlike Rad30p, but instead exhibits significant homology to linker histone proteins. Thus, hEFO1p is a unique fusion of linker histone and cohesion establishment domains. hEFO1p is widely expressed among the tissues tested. Consistent with a role in chromosome segregation, hEFO1p localizes exclusively to the nucleus when expressed in HeLa tissue culture cells. Moreover, biochemical analyses reveal that hEFO1p exhibits acetyltransferase activity. These findings document the first characterization of a novel human acetyltransferase, hEFO1p, that is comprised of both linker histone and Ctf7p/Eco1p domains.

Figure 1

Identification of Ctf7p-like proteins in higher eukaryotes. (A) Schematic illustrating Eso1p and conceptual translations of cDNAs that encode Ctf7p-like proteins. E values (a measurement of similarity) for Ctf7p domains are shown in black. The N-terminal region of Eso1p that exhibits homology to Rad30p is shown in dark gray. Regions that extend upstream of the Ctf7p region in human and Drosophila are shown in light gray. (B) Amino acid alignments reveal the high degree of conservation of the Ctf7p domain for a selection of coding sequences. Residues are numbered according to conceptual translations of cDNA sequences, except for contig sequences which were assigned a starting residue number of 1. Accession numbers are as follows: Schizosaccharomyces pombe Eso1p (BAA95122), Saccharomyces cerevisiae Ctf7p/Eco1p (NP 116683), Drosophila melanogaster (AAF50579.1), Mus musculus ORF (MGC:30637), Homo sapiens KIAA1191 (AB067498) and Homo sapiens contig (comprised of N94144, BG288675 and BE247544). Other mouse (AAH08220, BAB26905 and AA881675) and human orthologs were also identified.

Figure 1

Identification of Ctf7p-like proteins in higher eukaryotes. (A) Schematic illustrating Eso1p and conceptual translations of cDNAs that encode Ctf7p-like proteins. E values (a measurement of similarity) for Ctf7p domains are shown in black. The N-terminal region of Eso1p that exhibits homology to Rad30p is shown in dark gray. Regions that extend upstream of the Ctf7p region in human and Drosophila are shown in light gray. (B) Amino acid alignments reveal the high degree of conservation of the Ctf7p domain for a selection of coding sequences. Residues are numbered according to conceptual translations of cDNA sequences, except for contig sequences which were assigned a starting residue number of 1. Accession numbers are as follows: Schizosaccharomyces pombe Eso1p (BAA95122), Saccharomyces cerevisiae Ctf7p/Eco1p (NP 116683), Drosophila melanogaster (AAF50579.1), Mus musculus ORF (MGC:30637), Homo sapiens KIAA1191 (AB067498) and Homo sapiens contig (comprised of N94144, BG288675 and BE247544). Other mouse (AAH08220, BAB26905 and AA881675) and human orthologs were also identified.

Figure 2

Amino acid alignments for conceptual translations of candidate human Ctf7p family members. See text for accession numbers and details.

Figure 3

Expression and conceptual translation of hEFO1p. (A) Nucleotide and corresponding amino acid sequence for hEFO1p. (B) Northern blot analysis reveals that hEFO1p is expressed in most of the human tissues tested.

Figure 3

Expression and conceptual translation of hEFO1p. (A) Nucleotide and corresponding amino acid sequence for hEFO1p. (B) Northern blot analysis reveals that hEFO1p is expressed in most of the human tissues tested.

Figure 4

hEFO1p and Tetrahymena linker histone alignments. Top: the schematic depicts the region of conservation between Tetrahymena linker histone proteins and hEFO1p. The MLH polyprotein cleavage products α, δ, γ and β linker histones are shaded (AAC18874). Bottom: amino acid alignment reveals the high degree of conservation (E of 1 × 10 ^–11) between hEFO1p and Tetrahymena γ and β linker histone proteins. Identical residues are boxed in black; conserved residues are boxed in gray.

Figure 5

Localization of hEFO1p in mammalian cells. GFP-tagged hEFO1p versus GFP vector alone constructs were transfected into HeLa cells and localization assessed 24 h later. Cell images obtained using the GFP channel reveal that hEFO1p specifically localizes to the nucleus while GFP is uniformly distributed throughout the cell. Cell images obtained by differential interference contrast microscopy (DIC) are also shown (* denotes nuclei in transfected cells). GFP expression was not observed in untransfected cells.

Figure 6

Expression and acetyltransferase activity of human EFO1p and budding yeast Ctf7p. (A) GST–EFO1p and GST–Ctf7p expressed in bacterial cells and visualized using an antibody directed against GST (α GST). Acetyltransferase activity of human EFO1p and budding yeast Ctf7p using an antibody directed against acetylated lysines (α Ac-K) previously described to detect Ctf7p acetylation reactions (15).