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Title: The demethylase NMAD-1 regulates DNA replication and repair in the Caenorhabditis elegans germline}
Article:
Abstract:
The biological roles of nucleic acid methylation, other than at the C5-position of cytosines in CpG dinucleotides, are still not well understood. Here, we report genetic evidence for a critical role for the putative DNA demethylase NMAD-1 in regulating meiosis in C. elegans. nmad-1 mutants have reduced fertility. They show defects in prophase I of meiosis, which leads to reduced embryo production and an increased incidence of males due to defective chromosomal segregation. In nmad-1 mutant worms, nuclear staging beginning at the leptotene and zygotene stages is disorganized, the cohesin complex is mislocalized at the diplotene and diakinesis stages, and chromosomes are improperly condensed, fused, or lost by the end of diakinesis. RNA sequencing of the nmad-1 germline revealed reduced induction of DNA replication and DNA damage response genes during meiosis, which was coupled with delayed DNA replication, impaired DNA repair and increased apoptosis of maturing oocytes. To begin to understand how NMAD-1 regulates DNA replication and repair, we used immunoprecipitation and mass spectrometry to identify NMAD-1 binding proteins. NMAD-1 binds to multiple proteins that regulate DNA repair and replication, including topoisomerase TOP-2 and co-localizes with TOP-2 on chromatin. Moreover, the majority of TOP-2 binding to chromatin depends on NMAD-1. These results suggest that NMAD-1 functions at DNA replication sites to regulate DNA replication and repair during meiosis.
Introduction:
Meiosis is the process by which a diploid cell divides to produce haploid gametes. Errors at any stage during the carefully orchestrated events of meiosis can result in improper chromosome segregation and subsequent aneuploidy( cells with an abnormal number of chromosomes). Aneuploidy is detected in at least 20% of human pregnancies and is the most frequent chromosomal abnormality[1]. Although most errors in meiosis and aneuploidy lead to miscarriage, aneuploidy in fetuses that survive is the most common cause of both developmental and intellectual disabilities[2]. Methylation of nucleic acid bases increases the repertoire of information carried in a DNA or RNA sequence. The extent and diversity of recognized DNA and RNA methylation changes has expanded in recent years with the development of new and more sensitive detection technologies[3–6]. DNA methylation has been proposed to regulate self-versus-nonself recognition, distinguishing DNA damaged strands in need of repair, genomic imprinting, X-chromosome inactivation, transposon suppression, and transcription[4, 7, 8]. Meanwhile RNA methylation has been implicated in regulating translation initiation, RNA stability, RNA localization, and function[3, 5, 6, 9]. Understanding the biological importance of these modifications has been facilitated by identifying the methylases and demethylases that modify nucleic acids. We and others previously identified methylation on the N6 position of adenines( 6mA) as a novel DNA modification in metazoans[10, 11]. The AlkB family of dealkylating enzymes demethylates a diverse repertoire of methylated DNA and RNA in a variety of species[12–14]. There are nine AlkB family members in humans( Alkbh1-8 and FTO) and five AlkB family members in C. elegans. We identified the AlkB family member NMAD-1( F09F7. 7) as a dealkylating enzyme in C. elegans which demethylates 6mA and the DNA damage modification 3mC on DNA[10]. Moreover, 6mA was increased in nmad-1 mutant worms as assessed by ultra-high performance liquid chromatography coupled with mass spectrometry( UHPLC-ms/ms). These results suggested that NMAD-1 is a 6mA DNA demethylase in vivo. Since our original publication multiple studies have identified 6mA DNA modifications in numerous eukaryotic species[11, 15–26] and that ALKBH4, the NMAD-1 homolog, demethylates 6mA in vitro[27]. In C. elegans, the level of 6mA on DNA has been shown to increase globally in response to mitochondrial stress and to mark genes important for stress response[26]. However, our recent work suggests that 6mA in DNA is difficult to measure and is less abundant than previously thought because of contamination with bacterial DNA, where 6mA is frequent, and because of the inability of current sequencing methods to accurately identify 6mA[28]. Because these DNA methylation events in eukaryotes are rare, it is still uncertain whether NMAD-1 functions in vivo as a 6mA and 3mC DNA demethylase. At this time, we cannot accurately map 6mA modifications in eukaryotes. In the absence of such a technique, we nonetheless wanted to investigate the role of NMAD-1 by characterizing nmad-1 mutant worms. We previously found that mutation of NMAD-1 reduced fertility in C. elegans by an unknown mechanism[10]. Our goal here was to examine how nmad-1 deficiency leads to infertility.
Discussion:
Here we show that the putative demethylase NMAD-1 plays a critical role in DNA replication and DNA damage repair during meiosis in C. elegans. NMAD-1 binds to the same sites on chromatin in the germline as TOP-2 and physically interacts directly with TOP-2 both in vitro and in vivo, suggesting that NMAD-1 acts on chromatin to regulate these processes in meiosis. Both the nmad-1 deletion and catalytic mutant strains have impaired fertility, an increased incidence of males, elevated RAD-51 foci, and reduced replication rates( Figs 1D, 3C and 5 and S1A Fig). These findings suggest that NMAD-1’s demethylation activity at DNA is central to its role in meiosis. We previously identified NMAD-1 as a 6mA DNA demethylase[10] and it has been proposed as a conserved 6mA DNA demetylase in the silkworm Bombyx mori[55] as well as in mice[27]. However, because of the low levels of 6mA DNA methylation in C. elegans( which appear to be even lower than initially reported[28]), we have been unable to determine whether 6mA DNA is the NMAD-1 substrate in C. elegans responsible for its profound effect on meiosis. Additional accurate sequencing technologies to map and quantify 6mA at single-base resolution in gDNA will need to be developed to reveal whether modifications to DNA are present and biologically significant for the role of NMAD-1 in regulating meiosis. Future studies will need to determine the NMAD-1 substrate that regulates DNA repair and replication in meiosis, which could be 6mA or 3mC DNA, or an unidentified other nucleic acid or even protein substrate. In prokaryotes, 6mA regulates DNA replication by marking the origin of replication and hemimethylation of the origin delays DNA replication[56, 57]. One possibility is that, in worms, 6mA marks origins of replication and needs to be removed by NMAD-1 for DNA replication to proceed. In any event, a defect in the NMAD-1 demethylase strongly disrupts replication, DSB repair, and chromosome segregation in meiosis, ultimately leading to embryo death and sterility. Deficiency of ALKBH4, the closest mammalian homolog to NMAD-1, has been reported to cause reproduction defects in mice and zebrafish[58, 59], suggesting that this dealkylating enzyme might play a conserved role in regulating DNA replication throughout eukaryotes. Together our findings reveal a new layer of regulation where this demethylating enzyme is required to physically interact with the DNA repair and DNA replication machinery on the chromatin to facilitate appropriate meiosis progression.
Errors in meiosis are the leading cause of miscarriages , as well as developmental and intellectual disabilities . We have identified that NMAD-1 , an enzyme which removes methyl moieties from nucleic acids , is essential for appropriate DNA damage response , DNA replication and meiosis in the nematode C . elegans . We have cytologically and genetically characterized the defects which occur due to deletion of NMAD-1 in the C . elegans germline . Additionally , we have begun to determine molecularly how NMAD-1 can regulate DNA replication , by demonstrating that NMAD-1 binds to components of the DNA replication machinery and is required for their appropriate localization to DNA . Characterizing how epigenetic modifications and the corresponding enzymes that add or remove epigenetic modifications can control the fundamental process of meiosis will have broad implications for understanding and eventually correcting errors in meiosis that disrupt normal development .