ZNF597 is a maternally expressed imprinted gene in the Holstein breed.

Genomic imprinting is an epigenetic phenomenon that leads to the preferential expression of genes from either the paternal or maternal allele. Imprinted genes play important roles in mammalian growth and development and a central role in placental function. ZNF597 and NAA60 are two paternally imprinted genes in the human ZNF597-NAA60 imprinted locus, both of which show biallelic expression in the mouse, but their imprinting status in cattle is still unknown. In this study, we examined the allelic expression of ZNF597 and NAA60 in adult bovine placental and somatic tissues. By comparing the mRNA-based genotypes with the genomic DNA-based genotypes, we identified monoallelic expression of ZNF597 in the placenta and in seven other tissues, including the cerebrum, heart, liver, spleen, lung, kidney, and muscle. Nevertheless, analysis revealed biallelic expression of the NAA60 gene in these tissues. Moreover, we tested the imprinting status of ZNF597 and confirmed that the maternal allele is expressed in the bovine placenta. To determine the role of DNA methylation in regulating monoallelic/imprinted expression of bovine ZNF597, the methylation status of two CpG-enriched regions in the bovine ZNF597-NAA60 locus was analyzed using the bisulfite sequencing method. Differentially methylated regions were detected on ten CpG loci in the bovine ZNF597 promoter region. In summary, the bovine ZNF597 gene is a maternally expressed gene, and its expression is regulated by DNA methylation, whereas the NAA60 gene is not imprinted in cattle.

Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase.

N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine N(ε)-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the ß7-ß8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved ß3-ß4 long loop participates in the regulation of hNaa60 activity.

An organellar nα-acetyltransferase, naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity.

N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or Nα-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi's structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.