H3K4 demethylase activities repress proliferative and postmitotic aging

Homeostasis of postmitotic and proliferating cells is maintained by pathways that repress stress. We found that the Caenorhabditis elegans histone 3 lysine 4 (H3K4) demethylases RBR‐2 and SPR‐5 promoted postmitotic longevity of stress‐resistant daf‐2 adults, altered pools of methylated H3K4, and promoted silencing of some daf‐2 target genes. In addition, RBR‐2 and SPR‐5 were required for germ cell immortality at a high temperature. Transgenerational proliferative aging was enhanced for spr‐5; rbr‐2 double mutants, suggesting that these histone demethylases may function sequentially to promote germ cell immortality by targeting distinct H3K4 methyl marks. RBR‐2 did not play a comparable role in the maintenance of quiescent germ cells in dauer larvae, implying that it represses stress that occurs as a consequence of germ cell proliferation, rather than stress that accumulates in nondividing cells. We propose that H3K4 demethylase activities promote the maintenance of chromatin states during stressful growth conditions, thereby repressing postmitotic aging of somatic cells as well as proliferative aging of germ cells.     

Processing bodies and germ granules are distinct RNA granules that interact in C. elegans embryos

In somatic cells, untranslated mRNAs accumulate in cytoplasmic foci called processing bodies or P-bodies. P-bodies contain complexes that inhibit translation and stimulate mRNA deadenylation, decapping, and decay. Recently, certain P-body proteins have been found in germ granules, RNA granules specific to germ cells. We have investigated a possible connection between P-bodies and germ granules in Caenorhabditis elegans. We identify PATR-1, the C. elegans homolog of the yeast decapping activator Pat1p, as a unique marker for P-bodies in C. elegans embryos. We find that P-bodies are inherited maternally as core granules that mature differently in somatic and germline blastomeres. In somatic blastomeres, P-bodies recruit the decapping activators LSM-1 and LSM-3. This recruitment requires the LET-711/Not1 subunit of the CCR4-NOT deadenylase and correlates spatially and temporally with the onset of maternal mRNA degradation. In germline blastomeres, P-bodies are maintained as core granules lacking LSM-1 and LSM-3. P-bodies interact with germ granules, but maintain distinct dynamics and components. The maternal mRNA nos-2 is maintained in germ granules, but not in P-bodies. We conclude that P-bodies are distinct from germ granules, and represent a second class of RNA granules that behaves differently in somatic and germline cells.     

Genital primordium of the free-living marine nematode Halichoanolaimus sonorus (Chromadoria,Chromadorida)

Summary

The genital primordium of the first stage juvenile (J1) of the free-living marine nematode Halichoanolaimus sonorus (Chromadorida: Selachinematidae) was studied using transmission electron microscopy. The primordium consists of four undifferentiated cells: two primordial germ cells (PGC) 5–6 μm in diameter and two somatic cells. The PGC have a large nucleus with nucleolus. The centriole was detected in close vicinity of the PGC nucleus. Most of the cell mitochondria are in close contact with the nuclear envelope. The mitochondria are interspersed by 0.2–0.3 μm particles of an electron-dense diffuse substance devoid of surrounding membrane. Both PGC are closely attached to each other and to the neighboring somatic cells of the genital primordium. The elongated somatic cells contain nuclei devoid of nucleoli; the cytoplasm is filled with free ribosomes and contains occasional cisternae of rough endoplasmatic reticulum (RER), Golgi bodies, mitochondria, and transparent vesicles. The genital primordium is separated by a narrow space from of the intestine (dorsally) and the somatic muscles (ventrally). The PGC of H. sonorous are devoid of typical P granules known for previously studied nematodes as distinct markers of germ line cell lineage. Perinuclear particles of dense diffuse substance found in PGC of H. sonorous could be considered as germ determinants analogous to P granules.     

Evidence in a nematode for regulation of transposon excision by tissue-specific factors

Summary The transposable element Tc1 in Caenorhabditis elegans undergoes an excision reaction, which can be detected in a Southern hybridization as the appearance of empty chromosomal insertion sites. This excision reaction is under tissue-specific regulation in that it occurs at much higher frequency in somatic cells than in the germ line. We show here that this regulation is likely to be due to the action of tissue-specific factors that either promote excision in somatic tissues or repress it in the germ line. The rate of excision of elements at five distinct chromosomal sites has been measured by a method that avoids ambiguities due to cell division. All these elements are found to undergo excision at closely similar rates during the L1 larval stage. No distinct difference exists among the elements at different sites that would suggest regulation by flanking sequences.     

Remodeling of the endoplasmic reticulum in Caenorhabditis elegans oocytes is regulated by CGH‐1

A key aspect of development in all metazoans is remodeling at the cellular level. During the development of gametes, remodeling occurs throughout the germ line. When Caenorhabditis elegans hermaphrodites become depleted of sperm after 4 days of adulthood, significant cellular remodeling occurs within the meiotically‐arrested oocytes, including the formation of ribonucleoprotein granules. Since major remodeling of the endoplasmic reticulum (ER) occurs in early embryos, we investigated the extent of ER remodeling in meiotically‐arrested oocytes. We found, using a combination of fluorescence reporters and transmission electron microscopy, that the ER in arrested oocytes accumulates in patches and sheets that are enriched at the cortex. Our findings suggest this remodeling is not due to simple displacement by large amounts of yolk that accumulate in arrested oocytes, and instead may be genetically regulated. We further identified the Ddx6 RNA helicase, CGH‐1, as a key regulator of ER in the germ line. In cgh‐1(tn691) oocytes, we detected cortical ER patches as well as aberrant granules of the RNA‐binding proteins, PAB‐1, MEX‐3, and CGH‐1. Taken together, our results suggest the possibility that the spatial organization of RNA binding proteins may regulate the translation of mRNAs associated with the ER that in turn, controls the organization of the ER in the adult germ line.     

The impact of mitotic versus interphase chromatin architecture on the molecular flow of EGFP by pair correlation analysis

Here we address the impact nuclear architecture has on molecular flow within the mitotic nucleus of live cells as compared to interphase by the pair correlation function method. The mitotic chromatin is found to allow delayed but continuous molecular flow of EGFP in and out of a high chromatin density region, which, by pair correlation function analysis, is shown as a characteristic arc shape that appears upon entry and exit. This is in contrast to interphase chromatin, which regulates flow between different density chromatin regions by means of a mechanism which turns on and off intermittently, generating discrete bursts of EGFP. We show that the interphase bursts are maintained by metabolic energy, whereas the mitotic mechanism of regulation responsible for the arc is not sensitive to ATP depletion. These two distinct routes of molecular flow were concomitantly measured in the Caenorhabditis elegans germ line, which indicates a conservation of mechanism on a scale more widespread than cell type or organism.     

A mutation of <Emphasis Type="Italic">cdc</Emphasis>-25.1 causes defects in germ cells but not in somatic tissues in <Emphasis Type="Italic">C. elegans</Emphasis>

By screening C. elegans mutants for severe defects in germline proliferation, we isolated a new loss-of-function allele of cdc-25.1, bn115. bn115 and another previously identified loss-of-function allele nr2036 do not exhibit noticeable cell division defects in the somatic tissues but have reduced numbers of germ cells and are sterile, indicating that cdc-25.1 functions predominantly in the germ line during postembryonic development, and that cdc-25.1 activity is probably not required in somatic lineages during larval development. We analyzed cell division of germ cells and somatic tissues in bn115 homozygotes with germline-specific anti-PGL-1 immunofluorescence and GFP transgenes that express in intestinal cells, in distal tip cells, and in gonadal sheath cells, respectively. We also analyzed the expression pattern of cdc-25.1 with conventional and quantitative RT-PCR. In the presence of three other family members of cdc-25 in C. elegans defects are observed only in the germ line but not in the somatic tissues in cdc-25.1 single mutants, and cdc-25.1 is expressed predominantly, if not exclusively, in the germ line during postembryonic stages. Our findings indicate that the function of cdc-25.1 is unique in the germ line but likely redundant with other members in the soma.     

Transgenerational inheritance of centromere identity requires the CENP-A N-terminal tail in the C. elegans maternal germ line

Centromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes, CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is reestablished on chromatin during diplotene of meiosis I. Here, we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but then its presence becomes dispensable for centromere maintenance during development. Worms homozygous for a CENP-A tail deletion maintain functional centromeres during development but give rise to inviable offspring because they fail to reestablish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2 and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.

This study of the nematode Caenorhabditis elegans shows that centromere identity is set in the maternal germ line and passed on to the progeny via an epigenetic mechanism that requires the N-terminal tail of the centromeric histone H3 variant CENP-A.     

Membraneless organelles: P granules in Caenorhabditis elegans

Membraneless organelles are distinct compartments within a cell that are not enclosed by a traditional lipid membrane and instead form through a process called liquid‐liquid phase separation. Examples of these non‐membrane‐bound organelles include nucleoli, stress granules, P bodies, pericentriolar material and germ granules. Many recent studies have used Caenorhabditis elegans germ granules, known as P granules, to expand our understanding of the formation of these unique cellular compartments. From this work, we know that proteins with intrinsically disordered regions (IDRs) play a critical role in the process of phase separation. IDR phase separation is further tuned through their interactions with RNA and through protein modifications such as phosphorylation and methylation. These findings from C elegans, combined with work done in other model organisms, continue to provide insight into the formation of membraneless organelles and the important role they play in compartmentalizing cellular processes.     

Highlights from this issue

Germline P granules are specialized protein/RNA aggregates that are found exclusively in germ cells in C. elegans. During the early embryonic divisions that generate germ blastomeres, aggregate-prone P granule components PGL-1 and PGL-3 that remain in the cytoplasm destined for somatic daughters are selectively removed by autophagy. Loss-of-function of components of the autophagy pathway, including the VPS-34/BEC-1 complex, causes accumulation of PGL-1 and PGL-3 into aggregates in somatic cells (termed PGL granules). Formation of PGL granules depends on SEPA-1, which is an integral component of these granules. SEPA-1 is preferentially degraded by autophagy and is also required for the autophagic degradation of PGL-1 and PGL-3. SEPA-1 functions as a bridging molecule in mediating degradation of P granule components by directly interacting with PGL-3 and also with the autophagy protein LGG-1/Atg8. The defect in embryonic development in autophagy mutants is suppressed by mutation of sepa-1, suggesting that autophagic degradation of PGL granule components may provide nutrients for embryogenesis and/or also prevent the formation of aggregates that could be toxic for animal development. Our study reveals a specific physiological function of selective autophagic degradation during C. elegans development.     

Dynamic Regulation of Autophagy and Endocytosis for Cell Remodeling During Early Development

Fertilization triggers cell remodeling from each gamete to a totipotent zygote. Using Caenorhabditis elegans as a model system, it has been revealed that lysosomal degradation pathways play important roles in cellular remodeling during this developmental transition. Endocytosis and autophagy, two pathways leading to the lysosomes, are highly upregulated during this period. A subset of maternal membrane proteins is selectively endocytosed and degraded in the lysosomes before the first mitotic cell division. Autophagy is also induced shortly after fertilization and executes the degradation of paternally inherited embryonic organelles, e.g. mitochondria and membranous organelles. This mechanism underlies the maternal inheritance of the mitochondrial genome. Autophagy is also required for the removal of extra P‐granule (germ granules in C. elegans) components in somatic cells of early embryos and thereby for the specific distribution of P‐granules to germ cells. This review focuses on recent advances in the study of the physiological roles and mechanisms of lysosomal pathways during early development in C. elegans.      

On the Organization of the Naked Filose Amoeba,Nuclearia moebiusi Frenzel, 1897 (Sarcodina,Filosea) and Its Implications1

The ultrastructural organization of Nuclearia moebiusi is described. The organism has filose pseudopodia without supportive microtubules; it has mitochondria with flattened cristae, lacks extrusomes, microtubule-organizing centers, and cytoplasmic microtubules. During cell division, microtubules appear only within the nucleus, and reorganization of the nuclear envelope occurs late in the nuclear division cycle. Ultrastructural studies reveal that Nuclearia spp. have a pattern of cellular organization distinct from that of other amoebae. Comparison of ultrastructural features suggests that this organism is only distantly related to other rhizopod amoebae (including other filose amoebae) and to the heliozoa.     

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.     

Molecular characterization of Ascaris suum DNA and of chromatin diminution

A technique for the extraction of pure somatic (post-diminution) and germ line (pre-diminution) DNA from the parasitic nematode Ascaris is described. Uncontaminated post- and pre-diminution DNAs were sheared and reassociated to different C₀t values. Computer analysis of the complete reassociation kinetics determined that 33% of the germ line genome is eliminated during the process of chromatin diminution. The eliminated DNA is comprised of repetitive and unique sequences in an approx. 1:1 ratio.     

Chromatin diminution in nematodes

The process of chromatin diminution in Parascaris and Ascaris is a developmentally controlled genome rearrangement, which results in quantitative and qualitative differences in DNA content between germ line and somatic cells. Chromatin diminution involves chromosomal breakage, new telomere formation and DNA degradation. The programmed elimination of chromatin in presomatic cells might serve as an alternative way of gene regulation. We put forward a new hypothesis of how an ancient partial genome duplication and chromatin diminution may have served to maintain the genetic balance in somatic cells and simultaneously endowed the germ line cells with a selective advantage.     

Germ cell survival in C. elegans and C. remanei is affected when the dead box rna helicases Vbh‐1 or Cre‐VBH‐1 are silenced

The Vasa family of proteins comprises several conserved DEAD box RNA helicases important for mRNA regulation whose exact function in the germline is still unknown. In Caenorhabditis elegans, there are six known members of the Vasa family, and all of them are associated with P granules. One of these proteins, VBH‐1, is important for oogenesis, spermatogenesis, embryo development, and the oocyte/sperm switch in this nematode. We decided to extend our previous work in C. elegans to sibling species Caenorhabditis remanei to understand what is the function of the VBH‐1 homolog in this gonochoristic species. We found that Cre‐VBH‐1 is present in the cytoplasm of germ cells and it remains associated with P granules throughout the life cycle of C. remanei. Several aspects between VBH‐1 and Cre‐VBH‐1 function are conserved like their role during oogenesis, spermatogenesis, and embryonic development. However, Cre‐vbh‐1 silencing in C. remanei had a stronger effect on spermatogenesis and spermatid activation than in C. elegans. An unexpected finding was that silencing of vbh‐1 in the C. elegans caused a decrease in germ cell apoptosis in the hermaphrodite gonad, while silencing of Cre‐vbh‐1 in C. remanei elicited germ cell apoptosis in the male gonad. These data suggest that VBH‐1 might play a role in germ cell survival in both species albeit it appears to have an opposite role in each one. genesis 1–18 2012. © 2012 Wiley Periodicals, Inc.     

Open and closed domains in the mouse genome are configured as 10‐nm chromatin fibres

The mammalian genome is compacted to fit within the confines of the cell nucleus. DNA is wrapped around nucleosomes, forming the classic ‘beads‐on‐a‐string’ 10‐nm chromatin fibre. Ten‐nanometre chromatin fibres are thought to condense into 30‐nm fibres. This structural reorganization is widely assumed to correspond to transitions between active and repressed chromatin, thereby representing a chief regulatory event. Here, by combining electron spectroscopic imaging with tomography, three‐dimensional images are generated, revealing that both open and closed chromatin domains in mouse somatic cells comprise 10‐nm fibres. These findings indicate that the 30‐nm chromatin model does not reflect the true regulatory structure in vivo.     

Observations on the extent and temporal stability of latitudinal clines for alcohol dehydrogenase allozymes and four chromosome inversions in Drosophila melanogaster

The chromosomes of embryos within thelytokous females of Forda spp. were studied, in samples from galls on Pistacia in the Middle East, and from roots of Poaceae in the Middle East, Europe and North America. The nuclei of oogonial cells, oocytes and early cleavage stages have consistently more chromosomes than the nuclei of dividing cells in the somatic tissues of young embryos from the same mothers. Elimination of the extra germ line chromosomes apparently occurs in late cleavage. In F. marginata Koch the germ line chromosome number varies from 25 to 40 in different populations and the somatic cell number varies from 17 to 20; in F. formicaria the germ line has 21–23 chromosomes and somatic nuclei have 18 or 20. In both species variation occurs between samples from galls on Pistacia as well as between populations on roots of Poaceae. The numbers and relative sizes of the eliminated chromosomes also differ between populations. Comparable phenomena in other insects are briefly discussed.