Meiotic division is a specific process of gametogenesis, where the DNA from the maternal cell replicates and consequently divides in two turns of cell division, into new haploid germ cells. In Caenorhabditis elegans
, this includes the pairing of homologous chromosomes, which occurs during the leptotene and zygotene stages of prophase I (transition zone of the germ line). During this highly dynamic process, the two homologous copies of each chromosome find each other within the nucleus through an active search process that enables the chromosomes to distinguish the “self versus non-self” and to assume a side-by-side alignment [1
]. The process of pairing is coupled with a synaptonemal complex assembly between the formation of homologs and crossovers (COs), which provides sufficient tension to align the chromosomes on the spindle. As the chromosome pairing, which is a prerequisite for CO formation, is completed by the exit from the transition zone of the gonad, the failure of the COs formation results in randomly segregated chromosomes during the first meiotic division, and subsequent, aneuploidy [2
The processes connected with meiotic progression are complex and involve several regulatory steps that are influenced by the chromatin state. Modifications of DNA are modulating the accessibility of the nucleosomal DNA, which are critical for transcription, replication, recombination, and DNA damage repair [4
]. DNA transcription in germ cells is tightly regulated and represents ~21% of the transcription of all known C. elegans
], mostly expressing only the proteins involved in or regulating the meiotic progression.
Additionally, changes in the nuclear structure and in both the DNA and RNA polymerases activities can also be caused by nuclear phospholipids [6
]. Previous studies showed that in in vitro models, the addition of positively charged lipids lead to chromosome condensation, while negatively charged lipids caused decondensation [12
]. Phosphatidylinositol 4,5-bisphosphate (PIP2) is one of the most studied phosphoinositides. PIP2 localization to plasma membrane, nuclear speckles, small foci in the nucleoplasm, and to the nucleolus were described in mammalian cells [13
]. It is known that nuclear PIP2 acts as a transcription activator or repressor interacting with various protein complexes of RNA polymerases I and II, or with the histone demethylase [15
]. PIP2 regulates the RNA polymerase II transcription apparently by the direct interaction with histone H1 and H3, shielding the positive charges of the histones and thus competing with their ability to bind DNA [19
]. Additionally, PIP2 can play a role in RNA polymerase II transcription repression, through binding to the myristoylated transcriptional co-repressor brain acid-soluble protein 1 (BASP1). Toska et al. showed that BASP1 requires PIP2 binding for the recruitment of histone deacetylase 1 (HDAC1) to chromatin, which led to histone deacetylation and thus decreased the promoter accessibility for transcriptional machinery [20
Phosphoinositides metabolism is very complex thus, to overcome this, a simple organism, Caenorhabditis elegans
, has been used to investigate the roles of phosphoinositides and its kinases or phosphatases [21
]. In C. elegans
, some enzymes have a unique role, such as Type I PIP kinase (PPK-1 kinase, homolog of PI5PK), thus, we can manipulate specific phosphoinositide species. PPK-1 is a specific kinase that is so far known to be responsible for PIP2 synthesis only. PPK-1 kinase localizes to the plasma membrane and is strongly expressed in the neuronal system of C. elegans
. The purified PPK-1 kinase is able to generate PIP2 in vitro and the PPK-1 overexpression increases the PIP2 level in vivo [31
Another described role of PPK-1 is in the C. elegans
ovulation. PPK-1 localization was observed in somatic tissues, such as neuronal cells [31
] and also in gonad sheath and distal tip cells, spermatheca, and uterine and vulva muscles [32
]. The reduced PPK-1 expression results in the depletion of PIP2, which causes worms sterility, defective ovulation, reduced contractility of sheath cells, and disorganization of myosin filament.
Here, in this study, we observed PIP2 localization in the nuclei in the distal gonad of C. elegans
hermaphrodite, and in the nuclei of the early embryos. Thus, we focused on the PIP2 functions connected with C. elegans
chromatin organization and their physiological importance. Interestingly, we discovered that the depletion of PIP2 results in an altered chromosome structure, an impaired chromosome pairing with a consequent defect in crossover formation. Strikingly, these defects were accompanied with increased levels of DNA transcription. As the DNA transcription in the germ cells is tightly regulated [5
], the increased localization of RNA polymerase I and II in the germ cell nuclei and the increased 5-Fluorouridine (5-FU) incorporation are probably signals of DNA-damage-driven apoptosis in the C. elegans
gonad. Moreover, we identified PIP2 interacting proteins in the gonadal nuclei, pointing to its involvement in a ubiquitin–proteasome pathway.
PIP2 is implicated in various physiological processes therefore it is very challenging to address its individual functions. Most studies on PIP2 and its synthetizing enzymes were performed in mammalian cells, and only a few studies take advantage of the C. elegans
simpler phosphoinositides metabolism. We decided to address specifically the PIP2 function, and thus we benefit from using C. elegans
as a model organism for this study. Here, we present that PIP2 influences the chromosomestructure, and is involved in processes such as transcriptional regulation, DNA-damage-driven apoptosis, chromosome condensation with subsequent pairing, and the ubiquitin–proteasome degradation pathway. Based on our data we concluded that the observed C. elegans
phenotypes might be caused by (i) the altered chromosome structure; (ii) impaired ubiquitin-proteasome pathway; or (iii) disrupted binding of PIP2 to some essential proteins. Based on our data presented here and already published data in mammalian cell lines [16
], we propose that PIP2 is involved in the regulation of the molecular processes at multiple levels, and may be a part of many protein complexes in the C. elegans
germ cells nuclei.
PIP2 and PPK-1 kinase have important functions in many physiological processes in C. elegans
, which are crucial in early development [31
]. Here, we described the PIP2 localization in the C. elegans
gonad and embryo by an anti-PIP2 antibody directly. PIP2 has a strong nuclear staining during prophase I, which differs from the PPK-1 staining in the whole the gonad. On the other hand, during C. elegans
embryogenesis, we observed a nuclear, cytoplasmic, and membrane localization of PIP2. Panbianco et al. suggested that PIP2 might be asymmetrically generated in the C. elegans
embryos, because PPK-1 is enriched at the posterior part of the embryo [45
]. However, we did not observe any obvious asymmetrical localization of PIP2 in the embryos, and thus provide more support for another proposal, that PPK-1 might influence the spindle positioning independently of its function as a PIP2 producer. Suggestions about the independent role of PI5PK and PIP2 were previously published by us [18
] and Chakrabarti et al. for the regulation of the RNA polymerase I transcription in mammalian cells [46
We observed an increased incidence of males in the C. elegans
progeny upon PIP2 depletion (Figure 1
g). The C. elegans
male genotype is represent by only one X chromosome (X0), therefore, we suggest that the increased incidence is dependent on the observed presence of univalent DAPI stained bodies in ppk-1(RNAi) (Figure 2
]. Moreover, we observed that the mislocalisation of the oocytes and altered cthe hromosome structure in diakinesis oocytes. The altered chromatin shape was also described after the depletion of ZTF-8 and RAD-51 proteins, and both were shown to be important for the C. elegans
germ line genomic stability [40
The presence of univalents and the altered chromosome structurein nuclei of C. elegans
oocytes are connected with defects in the synaptonemal complex formation and DNA damage [40
]. The depletion of PIP2 resulted in defective SC formation, pointing to the altered chromosome structure in our experiments. This altered chromosome structure probably causes a synapsis defect, which subsequently results in the increased presence of univalents. This is additionally supported by the impaired crossover formation.
The transcription of the germ cell specific, but not somatic genes is ongoing in the germ cell nuclei of C. elegans
. The germ line transcription represents around ~21% of all known C. elegans
genes’ transcription, and the ribosomal genes transcription is ~4-fold higher in the germ than in the somatic cell nuclei [5
]. Our investigation of the altered chromosome structure after the PIP2 depletion revealed an increased localization of the RNA polymerase I, and a phosphorylated form of RNA polymerase II and a 5-fluorouracil (FU) incorporation in the nascent RNA, both indicat intensified transcription. As transcription is tightly regulated, the increased transcription rate together with the altered chromosome structure is presumably a cause for DNA-damage-driven apoptosis.
In the mammalian cells, it was proven that defects in the nuclear lamina may lead to misshapen nuclei [48
]. Another important protein complex is SUN/KASH, which is known to transmit forces from the cytoplasm through the nuclear envelope to the lamina and the chromatin [49
]. Penkner et al. showed that the active involvement of the nuclear envelope is inevitable for the homologous chromosome pairing, and that the SUN-1 mutation leads to the disruption of early meiosis chromatin reorganization. On the other hand, they proved that SUN-1 is not essential for SC formation [50
]. However, we did not observe any change in the lamin localization or SUN-1 localization (data not shown) upon the PIP2 depletion in the C. elegans
gonad, thus it is improbable that the observed phenotypes could be caused by cytoplasmic signaling or membranous PIP2.
We hypothesize that these observed impaired processes might originate from the disruption of PIP2 interactions with protein partners. To discover more details, we performed a mass-spectrometry analysis of the immunoprecipitated proteins in complex with PIP2. We identified 45 proteins as potential PIP2 binding partners, where the most striking partners are involved in the ubiquitin proteasome pathway. We showed that PIP2 interacts and co-localizes with the LRR-1 protein in C. elegans
germ cell nuclei. LRR-1 is a described binding partner of CRL2 E3-ligase [43
], and is important for proliferation and progression through C. elegans
germline development. Burger et al. concluded that HTP-3 is likely a direct target for CRL-2LRR-1
E3 ligase, as they observed its accumulation in distally located germ cells. We did not observe this accumulation (data not shown). However, they also showed that CRL-2LRR-1
acts at multiple levels of germ cell development [44
]. Based on different observed phenotypes upon the PIP2 depletion, we hypothesize that the PIP2 interaction with CRL-2LRR-1
plays a role at different levels than the CRL-2LRR-1
and HTP-3 complex. Probably, the PIP2 interaction targets the CRL-2LRR-1
to RNA polymerase II, as we observed an increased RNA pol II localization in the ppk-1(RNAi) and CUL-2 depleted worms [43
]. Thus, this supports our suggestion that PIP2 may target CRL-2LRR-1
as well as other proteins connected with the disrupted phenotypes upon ppk-1(RNAi). Interestingly, Jungmichel et al. identified Cullin-1 as a possible specific interactor of nuclear PIP2, together with other proteins, such as ubiquitin–protein ligase, ubiquitin-like modifier-activating enzyme, and ubiquitin-associated protein 2 in mammalian cells [51
]. PIP2 has multiple protein binding partners (e.g., splicing factors) based on our mass-spectrometry data, and therefore, may play a role at various levels of C. elegans
development regulation. Consistently, it was shown in mammalian cell lines, that nuclear PIP2 regulates the RNA polymerase I transcription at multiple levels, by the interaction with several proteins [16
On the other hand, PPK-1 per se may contribute to the regulation of these processes independently of its function as a PIP2 producer, and possibly by the interaction with different protein binding partners, too. Previously, a distinct role of PIP2 and its kinase has been described, based on the binding partners for mammalian cell lines [18
]. Partially, some of the observed phenotypes might be indirect, caused by the decrease of the second messengers produced by the PIP2 cleavage. However, here, we provide evidence about the PIP2 binding partners that are involved in the regulation of various processes; thus, we are inclined to conclude that the observed phenotypes are connected directly with PIP2. The overall data confirmed the PIP2 involvement and importance in the regulation of crucial processes in the C. elegans
4. Materials and Methods
4.1. Strains and Culture Conditions
All of the strains were maintained and cultured under standard conditions at 20 °C using E. coli
OP50 as a food source [52
], except when subjected to RNAi treatment. The used worm strains were obtained from the CGC (Ceanorhabditis Genetics Centre; Minnesota University, Minneapolis, MN, USA): N2 (C. elegans
var Bristol), NL2098 (rrf-1(pk1417) I.), AV630 (pie-1p::GFP::cosa-1 + unc-119(+)), MD701 (bcIs39 (lim-7p::ced-1::GFP + lin-15(+))), EU640 (cul-2(or209) III.; cultured at 15 °C). We obtained H2B::GFP/HIM-8::mCherry (him-8(tm611) IV; ttTi5605 vv Is17 II; unc-119(ed3) III); H2B::GFP vvIs17(pie-1p::mCherry::him-8::unc-54ter, unc-119(+)) strain from Monique Zetka (McGill University, Montreal, QC, Canada). We obtained CED-1::GFP/CEP-1 (cep-1(ep347) I; bcIs39((lim-7)ced1p::GFP + lin-15(+)) V) strain from Susan Gasser (FMI, Basel, Switzerland).
4.2. RNA Interference
For the RNAi of ppk-1
, we used the full coding sequence of 1836 bp (F55A12.3) from C. elegans
cDNA. The primers used were as follows: forward 5′-CCCGGGATGGCTTCTCGGTCCAC-3′ and reverse 5′-CCATGGTCAAGCGACAGGTGTGT-3′. The sequence was cloned to a L4440 feeding vector (pPD129.36) [53
]. The resulting plasmid was transformed into the HT115(DE3) RNase Ill-deficient E. coli
strain. Transformed bacteria were spread on an nematode growth medium (NGM) plate with ampicillin (50 mg/mL) and isopropyl β-d
-1-thiogalactopyranoside (IPTG; 1 mM). Hermaphrodite worms in the L3/L4 stage were transferred onto the seeded plates and incubated at 20 °C for 48 h. As a mock control, we used empty L4440 plasmid transformed in E. coli
HT115(DE3) bacteria, and the worms were incubated under the same conditions.
The following primary antibodies were used in this study: anti-PIP2 mouse monoclonal (Echelon Biosciences Inc., Salt Lake City, UT, USA, clone 2C11, Z-A045), anti-Polymerase I rabbit polyclonal (RPA116; gift from I. Grummt), anti-Polymerase II mouse monoclonal (gift from H. Kimura), anti-BrdU mouse monoclonal (Sigma, St. Louis, MO, USA, 094M4821V), anti-Lamin rabbit polyclonal (Abcam, Cambridge, UK, ab16048), anti-PPK-1 rabbit polyclonal (gift from D. Weinkove), anti-fibrillarin rabbit monoclonal (2639S, Cell signaling, Danver, MS, USA), anti-HTP3 rabbit polyclonal (gift from M. Zetka), anti-SYP-1 mouse monoclonal (gift from M. Zetka), control mouse anti-IgM (Abcam, ab18401), control rabbit anti-IgG (Abcam, ab46540), anti-PBS-4 goat polyclonal (Abcam, ab166792), and anti-LRR-1 rabbit monoclonal (gift from L. Pintard) antibody.
The following secondary antibodies were used in this study: goat anti-mouse IgM (μ-chain specific) antibody conjugated with Alexa Fluor 555 (Invitrogen, Carlsbad, CA, USA, A21426), goat anti-mouse IgG (H+L) antibody conjugated with Alexa Fluor 488 (Invitrogen, A21202), goat anti-rabbit IgG (H+L) antibody conjugated with Alexa Fluor 488 (Invitrogen, A11034), goat anti-rabbit IgG (H+L) antibody conjugated with Alexa Fluor 555 (Invitrogen, A21429), IRDye 680 donkey anti-mouse IgG (H+L) antibody (LI-COR Biosciences, 926-68072), IRDye 800 donkey anti-mouse IgG (H+L) antibody (LI-COR Biosciences Lincoln Nebraska USA, 926-32212), IRDye 800 donkey anti-rabbit IgG (H+L) antibody (LI-COR Biosciences, 925-32213), IRDye 680 donkey anti-rabbit IgG (H+L) antibody (LI-COR, Biosciences, 926-68073), IRDye 800 Goat anti-Mouse IgM (µ chain specific) antibody (LI-COR Biosciences 926-32280), IRDye 800 donkey-anti Goat IgG (H+L) antibody (LI-COR Biosciences, 926-32214), and IRDye 680 donkey-anti Goat IgG (H+L) antibody (LI-COR Biosciences, 926-68074).
4.4. Indirect Immunofluorescence, 5-FU Treatment, and Actinomycin D Inhibition
Immunofluorescence staining was performed according to the standard protocol [54
]. The images were acquired at the Delta Vision Image Restoration System (Applied Precision, Santa Clara, CA, USA), with stacks of approximately 10 optical sections with 0.3 μm using 60×, 1.2 NA U Plan Apochromat objective (Olympus, Hamburg, Germany). We deconvolved the obtained images using the soft-WoRX 3.0 software (Issaquah Washington, DC, USA). The same imaging conditions were used for the WT and RNAi samples.
The rrf-1(pk1417) strain was subjected to RNAi feeding for 48 h. After 48 h, the adult worms, ppk-1
) or WT, were dissected and incubated in 8 mM 5-FU in M9 (22 mM KH2
, 42 mM Na2
, 86 mM NaCl, 1 M MgSO4
) for 5, 10, and 15 min. After treatment, the standard protocol for indirect immunofluorescence was followed [54
For the AMD inhibition, NGM plates for the RNAi knockdown, with addition of different AMD concentrations (0.01, 0.05, 1, and 2 µg/mL) were prepared. After 46 h of RNAi, the rrf-1(pk1417) worms were placed in fresh dishes, to continue the RNAi effect with supplement of AMD. The worms were left on the AMD containing plates for 2 h at 20 °C, then were subjected to 5-FU treatment and, subsequently, indirect immunofluorescence protocol.
For the mass-spectrometry (MS) analysis, we used material immunoprecipitated by the anti-PIP2 or control anti-mouse antibody from synchronized adults. The gonads from at least 50 N2 worms were dissected from the body and collected to tubes. The nuclear fraction was prepared according to Singh et al. [55
]. After immunoprecipitation, the washed beads were resuspended in 100 mM tetraethylammonium tetrahydroborate (TEAB) containing 1% sodium deoxycholate (SDC). The cysteines were reduced with a 5 mM final concentration of tris(2-carboxyethyl)phosphine TCEP (60 °C for 60 min) and were blocked with a 10 mM final concentration of methyl methanethiosulfonate (MMTS);for 10 min at room temperature). The samples were cleaved on beads with 1 µg of trypsin at 37 °C overnight. After digestion, samples were centrifuged and the supernatants were collected and acidified with trifluoroacetic acid (TFA) to a 1% final concentration. The SDC was removed by extraction to ethyl acetate [56
]. The peptides were desalted on a Michrom C18 column. A nano reversed phase column (EASY-Spray column, 50 cm × 75 µm ID, PepMap C18, 2 µm particles, 100 Å pore size) was used for liquid chromatography–mass spectrometry (LC/MS) analysis. The mobile phase buffer A was composed of water and 0.1% formic acid. The mobile phase B was composed of acetonitrile and 0.1% formic acid. The samples were loaded onto the trap column (Acclaim PepMap300, C18, 5 µm, 300 Å wide pore, 300 µm × 5 mm, five cartridges) for 4 min at 15 μL/min. The loading buffer was composed of water, 2% acetonitrile, and 0.1% trifluoroacetic acid. The peptides were eluted with mobile phase B gradient from 4% to 35% B in 60 min. The eluting peptide cations were converted to gas-phase ions by electrospray ionization, and were analysed on a Thermo Orbitrap Fusion (Q-OT-qIT, Thermo). The survey scans of the peptide precursors from 400 to 1600 m
were performed at a 120 K resolution (at 200 m
), with a 5 × 105
ion count target. The tandem MS was performed by isolation at 1.5 Th with the quadrupole, higher-energy collisional dissociation (HCD) fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS 2 ion count target was set to 104
and the max injection time was 35 ms. Only those precursors with a charge state of two–six were sampled for MS. The dynamic exclusion duration was set to 45 s, with a 10-ppm tolerance around the selected precursor and its isotopes. The monoisotopic precursor selection was turned on. The instrument was run at top speed mode with 2 s cycles [57
]. All of the data were analysed and quantified with the MaxQuant software (version 18.104.22.168, Martinsried, Germany) [58
]. The false discovery rate (FDR) was set to 1% for both the proteins and the peptides, and we specified a minimum length of seven amino acids. The Andromeda search engine was used for the MS/MS spectra search against the Caenorhabditis elegans
database (downloaded from Uniprot on April 2015, containing 25,527 entries). The enzyme specificity was set as the C-terminal to Arg and Lys, also allowing for the cleavage at the proline bonds and a maximum of two missed cleavages. The dithiomethylation of cysteine was selected as the fixed modification and the N-terminal protein acetylation and methionine oxidation were used as variable modifications. The ‘match between runs’ feature of MaxQuant was used to transfer the identifications to other LC-MS/MS runs, based on their masses and retention time (maximum deviation 0.7 min), and this was also used in the quantification experiments. The quantifications were performed with the label-free algorithms, described recently. The data analysis was performed using Perseus 22.214.171.124 software.
4.6. Immunoprecipitation and Dot Blot
The nuclear fraction was prepared according to Singh et al. [55
]. For the precipitation, anti-PIP2 (2 µg), control anti-mouse IgM antibody (2 µg), and protein L magnetic beads (50 µL of slurry) were used according to the manufacturer’s protocol (Pierce, Thermo Scientific, Waltham, MA, USA). For the dot blot, the N2 worms were subjected to the ppk-1
) or the control RNAi for 48 h. After the RNAi treatment, the single worms were lysed in a lysis buffer (50 mM KCl; 10 mM Tris (pH 8.3); 2.5 mM MgCl2
; 0.45% NP-40; 0.45% Tween-20) in a total volume of 10 µL. The whole volume was spotted on a polyvinylidene difluoride (PDVF) membrane and the anti-PIP2 antibody was used for detection.
4.7. Characterization of Brood Sizes and Embryonic Lethality
The L3–L4 hermaphrodites were individually plated onto NGM plates seeded with E. coli HT115(DE3) transformed by L4440 or ppk-1(RNAi) at 20 °C. After 24 h, the adult worms were removed from the plates and the progeny (laid eggs, L1) was counted. Subsequently, we counted the progeny (non-hatched eggs, L1, L2, and L3) after 48 and 72 h. We preformed the experiment on at least for 30 worms in three independent experiments.
4.8. Quantitation of COSA-1::GFP, HIM-8::mCherry and RAD-51 Foci
The foci were quantified from deconvolved three-dimensional (3D) data stacks of C. elegans
WT or ppk-1
) germ cell nuclei. We counted the foci in at least 150 germ cell nuclei I, from at least 10 gonads each. The quantification of the RAD-51 foci was performed in the whole gonad composing the mitotic tip to the late pachytene regions [39
]. COSA-1::GFP was quantified in late pachytene [42
]. HIM-8::Cherry was quantified in transition zone and late pachytene [59
]. The statistical comparisons between the genotypes were performed using the Student t
4.9. Detection of DAPI Stained Bodies in Oocyte Nuclei
The oocyte chromosomes were fixed with 4% formaldehyde and were stained with DAPI [60
]. In some of the oocyte nuclei, the individual univalents or bivalents lie too close to each other so as to be resolved unambiguously, thus, this method tends to underestimate the frequency of the achiasmate chromosomes.