nfi-I affects behavior and life-span in C. elegans but is not essential for DNA replication or survival

Abstract

Background: The Nuclear Factor I (one) (NFI) family of transcription/replication factors plays essential roles in mammalian gene expression and development and in adenovirus DNA replication. Because of its role in viral DNA replication NFI has long been suspected to function in host DNA synthesis. Determining the requirement for NFI proteins in mammalian DNA replication is complicated by the presence of 4 NFI genes in mice and humans. Loss of individual NFI genes in mice cause defects in brain, lung and tooth development, but the presence of 4 homologous NFI genes raises the issue of redundant roles for NFI genes in DNA replication. No NFI genes are present in bacteria, fungi or plants. However single NFI genes are present in several simple animals including Drosophila and C. elegans, making it possible to test for a requirement for NFI in multicellular eukaryotic DNA replication and development. Here we assess the functions of the single nfi-1 gene in C. elegans.

Results: C. elegans NFI protein (CeNFI) binds specifically to the same NFI-binding site recognized by vertebrate NFIs. nfi-1 encodes alternatively-spliced, maternally-inherited transcripts that are expressed at the single cell stage, during embryogenesis, and in adult muscles, neurons and gut cells. Worms lacking nfi-1 survive but have defects in movement, pharyngeal pumping and egg-laying and have a reduced life-span. Expression of the muscle gene Ce titin is decreased in nfi-1 mutant worms.

Conclusion: NFI gene function is not needed for survival in C. elegans and thus NFI is likely not essential for DNA replication in multi-cellular eukaryotes. The multiple defects in motility, egg-laying, pharyngeal pumping, and reduced lifespan indicate that NFI is important for these processes. Reduction in Ce titin expression could affect muscle function in multiple tissues. The phenotype of nfi-1 null worms indicates that NFI functions in multiple developmental and behavioral systems in C. elegans, likely regulating genes that function in motility, egg-laying, pharyngeal pumping and lifespan maintenance.

Figure 1

A) Alternatively spliced products of the nfi-1 gene. The nfi-1 gene is shown as a line with exons as numbered solid boxes and alternatively spliced exons as gray boxes. Arrows show the direction of transcription. Below the line are cDNAs from the Kohara and TIGR libraries; the vertical bar indicates the 5' end of the cDNA. In yK213C10 the letters a and b above exon 2 denote that it is alternatively spliced generating 2a and 2b and the arrow on the right denote undetermined sequence in the cDNA. The asterisk (*) on yk42f10 denotes an alternative 3'splice acceptor site in exon 13 used in yk42f10 but not in CEESQ09. Below are depictions of cDNAs obtained by RT-PCR from total and polyA+ RNA of whole worms or isolated eggs using nfi-1 exon-specific primers (RT-PCR nfi-1 primers). Lastly we show cDNA obtained by PCR using SL1 primer and nfi-1 exon-specific primers (RT-PCR SL1-linked products). Some of the alternatively spliced cDNAs have been described previously [14]. B) Comparison of CeNFI-DBD with the consensus mouse NFI DBD.The sequence of the CeNFI-DBD (top line) is aligned to a consensus sequence from the 4 mouse NFI-DBDs (bottom line). Gaps in the mouse consensus indicate residues that are not identical between the 4 mouse NFI-DBDs. The dash in the 6^th aligned row indicates a single insertion in the CeNFI-DBD sequence needed to align it with the mouse consensus. Dark gray boxes show identical residues in CeNFI-DBD and mouse NFI-DBDs, light gray boxes show residues not identical but similar between CeNFI and mouse NFI-DBDs, unboxed residues with black letters show residues that are not similar between the CeNFI and mouse DBDs, and gray letters in the CeNFI-DBD above gaps in the mouse consensus indicate positions where the 4 mouse genes are not identical. 151 of 190 residues are identical in all 4 mouse NFI-DBDs while 60 of these 151 residues are different in the CeNFI-DBD and 27 of these 60 differences are non-conservative substitutions. The alignment was done in Macvector 6.5.3 using the ClustalW similarity matrix.

Figure 2

A) Specific NFI DNA-binding activity of recombinant CeNFI-H6. Partially purified recombinant H6-tagged CeNFI (containing parts of exons 2–8) and human NFI-C220 were incubated with a duplex oligonucleotide containing an NFI binding site (2.6, even lanes) or an oligo with a single point mutation that abolishes NFI binding (C2, odd lanes) and analyzed on a 6.5% non-denaturing polyacrylamide gel. Lanes 1 and 2, crude E. coli extract (neg. control); lanes 3 and 4, ~5 ng partially purified CeNFI-H6; lanes 5 and 6, ~40 ng partially purified CeNFI-H6; lanes 7 and 8, ~5 ng purified human NFI-C220H6. See bottom of panel for sequences of oligonucleotides. B) Specific NFI DNA-binding activity in worm extracts. Nuclear extracts of a mixed population of C. elegans were prepared and used in a gel mobility shift assay with an oligonucleotide that contains an NFI-binding site (lanes 1 & 2, 2.6) or the same oligo with a single point mutation that abolishes NFI binding (lane 3, C2). Lane 1, no extract; Lanes 2 and 3, C. elegans extract (~10 μg). See A for sequences of oligonucleotides.

Figure 3

Expression of endogenous nfi-1 mRNA in embryo and adults. N2 worms were fixed and hybridized with digoxigenin (DIG)-labeled antisense nfi-1 probe (from plasmid yk42f10) and bound probe was detected using alkaline phosphatase-conjugated anti-DIG antibodies. Panels: a, 2 cell embryo; b, 4 cell embryo; c, 24 cell embryo; d, beginning gastrulation; e, mid-gastrulation; f, late gastrulation; g, comma stage; h, 1.5-fold stage and i, 2-fold stage. Embryos in a-d and f-i are positive for staining. We are currently investigating the apparent loss of signal at mid-gastrulation (panel e). Panels j-m, antisense probe, adults with exposed internal organs: mature oocytes; intestine; gonadal germ cells; n-o, control sense probe, no specific staining is seen. Right panels k, m, o are two-fold magnifications of those on the left.

Figure 4

A) Deletion in C. elegans nfi-1 gene. The relative locations of confirmed exons (boxes) are shown. Square brackets indicate the location of the NFI-1 DNA-binding domain and the region deleted in nfi-1 mutant. The arrows indicate the position of the Tc1 insertion in an intron of the nfi-1 gene in strain NL747 pk240 and locations of PCR primers used in the screening for the deletion. B) Single-worm PCR reactions on N2 worm and nfi-1 homozygous mutant isolated by sib-selection. The arrows indicate a 1007 bp and 2969 bp PCR products corresponding to the nfi-1 mutant (qa524) and wild type alleles respectively. Lane 2 is 1 kb DNA ladder. C) RT-PCR on N2 worms and nfi-1 homozygous mutants. The arrows indicate a 480 bp and 320 bp RT-PCR products amplified using total RNA obtained from N2 worms (lane 1). nfi-1 mutants show loss of nfi-1 transcripts (lane 2). Lane (3) is 100 bp DNA ladder. D) Loss of NFI DNA-binding activity in extracts of nfi-1 mutants. Nuclear extracts of a mixed population of N2 worms and nfi-1 mutants were prepared and used in a gel mobility shift assay with an oligonucleotide (2.6) that contains an NFI-binding site (lanes 3, 5) or the same oligo with a single point mutation that abolishes NFI binding (C2) (lane 4, 6). See Fig. 2A for sequences of oligonucleotides. Extract of nfi-1 mutants show loss of NFI DNA-binding activity (lanes 5, 6). Lanes 1 & 2, no extract.

Figure 5

Locomotion in nfi-1 mutants. Single young adults were spotted in the center of fresh plates and left for 10 min. (A, D) Photographs of N2 worms and nfi-1 mutants; (B, E) Track patterns of N2 worms and nfi-1 mutants; (C, F) Track patterns of N2 worms and nfi-1 mutants with higher magnification. Note less regular tracks in nfi-1 mutant vs. N2 worms.

Figure 6

A) Egg-laying defect in nfi-1 mutants and transgenic rescue. Bagging was measured in wild-type N2, nfi-1, N2 worms carrying the transgenic array qaEx507(N2 qaEx507) and nfi-1 worms carrying this array (nfi-1 qaEx507). Bars represent % of bagging as the mean of 3–4 independent experiments and error bars show the standard deviation. 30–75 worms of each genotype were scored in each independent experiment. N2 and N2 qaEx507showed <5% bagging. The nfi-1 mutant worms showed ~30% bagging while the rescued nfi-1 qaEx507showed <5% bagging. B) Shortened life span in nfi-1 mutants and transgenic rescue. Survival curves for the strains described above N2 (n = 57), nfi-1 (n = 58), N2 qaEx507 (n = 52) and nfi-1 qaEx507 (n = 31) are shown. Kaplan-Meier analysis (SPSS11 software) was use to determine median, percentile and p values (log rank test) and Excel was used to construct survival curves. The array generated ~50% rescue of the life-span. The experiment was repeated twice with similar results.

Figure 7

Haploinsufficiency of nfi-1 locus. Egg-laying defect in progeny of nfi-1 heterozygous mutant animals are shown. Bagging was scored in all progeny of two nfi-1(qa524/+) heterozygous worms (n = 146) derived from eggs laid over 7 hours. All worms were genotyped by single-worm PCR. Bars represent % of bagging in wild type (+/+), nfi-1 heterozygous (qa524/+) and nfi-1 homozygous worms (qa524/qa524). The number of worms scored of each genotype are shown above the bars.

Figure 8

Down regulation of Ce titin expression in nfi-1 mutants assessed by QPCR. Bars represent fold changes in Ce titin transcript level in wild type N2 vs. nfi-1 mutant worms. RNA samples were obtained from 3 independent synchronized adult worm populations for each genotype.

Figure 9

Expression pattern of the nfi-1::GFP reporter transgenes. The nfi-1 locus and structure of the nfi-1-GFP fusion constructs are shown (A). Nfi-1 coding regions are shown in black, gray boxes indicate alternatively spliced exons, untranslated regions are in white. GFP is shown as a hatched box. Expression of nfi-1-GFP reporter constructs was observed in embryos (B), intestinal cells (C), body wall muscles (D, E), pharynx (F), egg-laying muscles (G), several head (H) and tail neurons (I). Expression was assessed using a FXA Nikon microscope (B-D, F-I) and a Bio-Rad confocal microscope (E).