The TP73 gene gives rise to transactivation domain-p73 isoforms (TAp73) as well as DeltaNp73 variants with a truncated N terminus. Although TAp73alpha and -beta proteins are capable of inducing cell cycle arrest, apoptosis, and differentiation, DeltaNp73 acts in many cell types as a dominant-negative repressor of p53 and TAp73. It has been proposed that p73 is involved in myeloid differentiation, and its altered expression is involved in leukemic degeneration. However, there is little evidence as to which p73 variants (TA or DeltaN) are expressed during differentiation and whether specific p73 isoforms have the capacity to induce, or hinder, this differentiation in leukemia cells. In this study we identify GATA1 as a direct transcriptional target of TAp73alpha. Furthermore, TAp73alpha induces GATA1 activity, and it is required for erythroid differentiation. Additionally, we describe a functional cooperation between TAp73 and DeltaNp73 in the context of erythroid differentiation in human myeloid cells, K562 and UT-7. Moreover, the impaired expression of GATA1 and other erythroid genes in the liver of p73KO embryos, together with the moderated anemia observed in p73KO young mice, suggests a physiological role for TP73 in erythropoiesis.
The formation of haploid gametes from diploid germ cells requires the regulated two-step release of sister chromatid cohesion (SCC) during the meiotic divisions. Here, we show that phosphorylation of cohesin subunit REC-8 by Aurora B promotes SCC release at anaphase I onset in C. elegans oocytes. Aurora B loading to chromatin displaying Haspin-mediated H3 T3 phosphorylation induces spatially restricted REC-8 phosphorylation, preventing full SCC release during anaphase I. H3 T3 phosphorylation is locally antagonized by protein phosphatase 1, which is recruited to chromosomes by HTP-1/2 and LAB-1. Mutating the N terminus of HTP-1 causes ectopic H3 T3 phosphorylation, triggering precocious SCC release without impairing earlier HTP-1 roles in homolog pairing and recombination. CDK-1 exerts temporal regulation of Aurora B recruitment, coupling REC-8 phosphorylation to oocyte maturation. Our findings elucidate a complex regulatory network that uses chromosome axis components, H3 T3 phosphorylation, and cell cycle regulators to ensure accurate chromosome segregation during oogenesis.
Membrane contact sites (MCSs) are areas of close proximity between organelles that allow the exchange of material, among other roles. The endoplasmic reticulum (ER) has MCSs with a variety of organelles in the cell. MCSs are dynamic, responding to changes in cell state, and are therefore best visualized through inducible labeling methods. However, existing methods typically distort ER-MCSs, by expanding contacts or creating artificial ones. Here we describe a new method for inducible labeling of ER-MCSs using the Lamin B receptor (LBR) and a generic anchor protein on the partner organelle. Termed LaBeRling , this versatile, one-to-many approach allows labeling of different types of ER-MCSs (mitochondria, plasma membrane, lysosomes, early endosomes, lipid droplets and Golgi), on-demand, in interphase or mitotic cells. LaBeRling is non-disruptive and does not change ER-MCSs in terms of the contact number, extent or distance measured; as determined by light microscopy or a deep-learning volume electron microscopy approach. We applied this method to study the changes in ER-MCSs during mitosis and to label novel ER-Golgi contact sites at different mitotic stages in live cells.
The biology of a cell is the sum of many highly dynamic processes, each orchestrated by a plethora of proteins and other molecules. Microscopy is an invaluable approach to spatially and temporally dissect the molecular details of these processes. Hundreds of genetically encoded imaging tools have been developed that allow cell scientists to determine the function of a protein of interest in the context of these dynamic processes. Broadly, these tools fall into three strategies: observation, inhibition and activation. Using examples for each strategy, in this Cell Science at a Glance and the accompanying poster, we provide a guide to using these tools to dissect protein function in a given cellular process. Our focus here is on tools that allow rapid modification of proteins of interest and how observing the resulting changes in cell states is key to unlocking dynamic cell processes. The aim is to inspire the reader's next set of imaging experiments.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Wapl induces cohesin dissociation from DNA throughout the mitotic cell cycle, modulating sister chromatid cohesion and higher-order chromatin structure. Cohesin complexes containing meiosis-specific kleisin subunits govern most aspects of meiotic chromosome function, but whether Wapl regulates these complexes remains unknown. We show that during C. elegans oogenesis WAPL-1 antagonizes binding of cohesin containing COH-3/4 kleisins, but not REC-8, demonstrating that sensitivity to WAPL-1 is dictated by kleisin identity. By restricting the amount of chromosome-associated COH-3/4 cohesin, WAPL-1 controls chromosome structure throughout meiotic prophase. In the absence of REC-8, WAPL-1 inhibits COH-3/4-mediated cohesion, which requires crossover-fated events formed during meiotic recombination. Thus, WAPL-1 promotes functional specialization of meiotic cohesin: WAPL-1-sensitive COH-3/4 complexes modulate higher-order chromosome structure, while WAPL-1-refractory REC-8 complexes provide stable cohesion. Surprisingly, a WAPL-1-independent mechanism removes cohesin before metaphase I. Our studies provide insight into how meiosis-specific cohesin complexes are regulated to ensure formation of euploid gametes. https://doi.org/10.7554/eLife.10851.001 eLife digest Most of the genetic material of plant and animal cells is stored in structures called chromosomes. Nearly all the cells in the body contain two copies of each chromosome, one inherited from the mother and the other from the father, but sex cells – such as egg and sperm – contain just one copy of each. If eggs or sperm contain the wrong number of copies of a chromosome, genetic disorders such as Down syndrome can occur. New sex cells form in a process called meiosis, which begins with a cell that contains two copies of each chromosome duplicating each of these copies. The duplicated copies are known as sister chromatids, and are held together by a ring-like protein complex called cohesin. In addition to tethering sister chromatids, cohesin affects the ‘higher-order’ organization of chromosome structure and promotes the recruitment of other proteins that are essential for different aspects of chromosome behavior during meiosis. Therefore, regulating cohesin binding during meiosis is key to ensuring that sex cells contain the correct number of chromosomes. Cohesin is ultimately removed from chromosomes in two steps during the consecutive cell divisions at the end of meiosis, resulting in the formation of sex cells containing a single copy of each chromosome. However, whether cohesin is actively removed from chromosomes during early meiosis, when chromosomes undergo dramatic structural changes, is not known. Using a combination of microscopy and genetic techniques to study the developing egg cells of the worm Caenorhabditis elegans, Crawley et al. investigated how a protein called WAPL-1 affects cohesin binding to chromosomes during early meiosis. This revealed that WAPL-1’s effects depend on the identity of a particular subunit of the cohesin complex. If this subunit is a protein called COH-3 or COH-4, WAPL-1 reduces the ability of cohesin to bind to chromosomes during the early stages of meiosis. However, WAPL-1 does not affect cohesin complexes that instead feature a protein called REC-8 as this subunit. By preventing excessive binding of COH-3 and COH-4 cohesin, WAPL-1 regulates chromosome structure and sister chromatid cohesion during early meiosis. Crawley et al. further observed that during the stage preceding the first meiotic division, cohesin is removed from chromosomes by a mechanism that does not involve WAPL-1. The next challenge is to work out why cohesin containing the REC-8 protein is protected from being released by WAPL-1. Whether defects in this protection can trigger the premature separation of sister chromatids is also an important question to answer. https://doi.org/10.7554/eLife.10851.002 Introduction Structural maintenance of chromosome (SMC) proteins take part in complexes that associate with DNA to promote key events of the cell cycle, such as chromosome condensation and segregation, DNA repair, and gene expression (Jeppsson et al., 2014). The cohesin complex, which mediates sister chromatid cohesion (SCC) between S-phase and chromosome segregation at anaphase (Michaelis et al., 1997), consists of two SMC proteins (Smc1 and Smc3) plus a kleisin subunit (Scc1/Rad21), forming a tripartite structure that topologically embraces DNA molecules (Haering et al., 2002; Haering et al., 2008). A fourth cohesin subunit (Scc3) binds to the kleisin and is also required for the functionality of the complex, while other proteins associate temporarily with cohesin to regulate its binding to DNA (Haarhuis et al., 2014). Cohesin is loaded to chromosomes by the Scc2/4 complex (Ciosk et al., 2000), and SCC is established during DNA replication in a process that involves acetylation of Smc3 (Ben-Shahar et al., 2008; Unal et al., 2008; Zhang et al., 2008). SCC is ultimately dissolved at anaphase onset, when cleavage of the kleisin subunit by the protease separase triggers the segregation of sister chromatids to opposite poles of the spindle (Uhlmann et al., 1999). Proper establishment and release of SCC is also essential for chromosome segregation during meiosis, the specialized cell division program that produces haploid gametes from diploid germ cells (Petronczki et al., 2003). In addition to the separase-dependent removal of cohesin at anaphase onset, a pathway dependent on the Wapl protein removes cohesin from chromosomes at earlier stages of the cell cycle in somatic cells (Gandhi et al., 2006; Kueng et al., 2006). Wapl is thought to destabilize the interaction between the kleisin and Smc3 subunits, allowing the release of cohesin from DNA without catalytically cleaving any subunit (Chan et al., 2012; Eichinger et al., 2013; Huis in 't Veld et al., 2014). Cohesin complexes in which the Smc3 subunit is acetylated during DNA replication become resistant to Wapl and remain stably bound to DNA, thereby providing persistent SCC (Ben-Shahar et al., 2008; Nishiyama et al., 2010; Lopez-Serra et al., 2013). However, acetylated cohesin is also removed from chromosome arms during prophase and early prometaphase in mammalian cells, when phosphorylation of Scc3 and Sororin, a protein that antagonizes Wapl, renders these complexes sensitive to Wapl (Hauf et al., 2005; Nishiyama et al., 2010; Nishiyama et al., 2013). This mode of cohesin removal is known as the prophase pathway and its failure in cells lacking Wapl causes increased arm cohesion in metaphase chromosomes and defects in chromosome segregation (Waizenegger et al., 2000; Haarhuis et al., 2013; Tedeschi et al., 2013). Removal of Wapl before S-phase causes a large increase in chromatin-associated cohesin and dramatic changes in chromosome organization (Tedeschi et al., 2013), demonstrating that Wapl is a key regulator of SCC and chromatin organization throughout the mitotic cell cycle. Cohesin is an essential component of meiotic chromosomes, not only by mediating SCC, but also by promoting the acquisition of structural features required for meiotic chromosome function (McNicoll et al., 2013). Meiotic chromosomes are organized as linear arrays of chromatin loops, which are attached at their base to cohesin-containing proteinaceous axial elements (Kleckner, 2006). Proper assembly of axial elements during early prophase is required for subsequent pairing and recombination between homologous chromosomes. Crossovers formed during recombination, together with SCC, form attachments between homologous chromosomes (chiasmata) that are responsible for the correct orientation of chromosomes on the first meiotic spindle and ultimately for their correct partitioning during the meiotic divisions (Petronczki et al., 2003). Crucially, these events require the formation of axial elements containing meiosis-specific cohesin complexes in which the mitotic kleisin Scc1 is substituted by Rec8 (Klein et al., 1999; Watanabe and Nurse, 1999). Moreover, additional meiosis-specific kleisins beyond Rec8 have been identified in mouse (Rad21L) (Herrán et al., 2011; Ishiguro et al., 2011; Lee and Hirano, 2011) and in C. elegans, where the highly homologous and functionally redundant COH-3 and COH-4 kleisins associate with SMC-1 and SMC-3 to form cohesin complexes that associate with meiotic chromosomes independently of REC-8 cohesin (Severson et al., 2009; Severson and Meyer, 2014). Although Rad21L and COH-3/4 are not essential for SCC, these kleisins are required for pairing and recombination between homologous chromosomes (Ishiguro et al., 2014; Severson and Meyer, 2014). Interestingly, large amounts of Rad21L and COH-3/4 are removed from chromosomes before metaphase I (Herrán et al., 2011; Ishiguro et al., 2011; Lee and Hirano, 2011; Severson and Meyer, 2014), and a prophase pathway has recently been proposed to operate during late meiotic prophase in plants (De et al., 2014). However, whether Wapl induces cohesin removal at any stage of meiotic prophase in animals, and whether Wapl may regulate some of the functions of different meiosis-specific cohesin complexes is not known. Using the C. elegans germ line, which contains a complete time course of meiotic prophase, we demonstrate that WAPL-1 antagonizes cohesin binding from the onset of meiosis, and show that cohesin complexes containing the COH-3/4 kleisins are specifically targeted by WAPL-1. By antagonizing the binding of COH-3/4 complexes to axial elements, WAPL-1 acts as a regulator of meiotic chromosome structure and SCC. Moreover, we also show that SCC is modulated by WAPL-1 and recombination during the chromosome remodeling process that starts at the end of pachytene, and report that a WAPL-1-independent mechanism removes cohesin during the oocyte maturation process preceding metaphase I. Results WAPL-1 is required for fertility In order to investigate the role of WAPL-1 during meiotic prophase, we used a deletion allele, wapl-1(tm1814), that removes the first two exons of the C. elegans Wapl homolog (Figure 1A). Western blot analysis on whole-worm protein extracts showed that the WAPL-1 protein is absent in wapl-1(tm1814) mutants, confirming that the tm1814 deletion is a null allele of wapl-1 (Figure 1B). Homozygous wapl-1(tm1814) mutants (referred from now on as wapl-1 mutants) are viable, but display a reduction in brood size and high levels of embryonic lethality (Figure 1C). In addition to these reproductive defects, wapl-1 mutants also displayed somatic defects, as demonstrated by the high incidence of larval arrest amongst the hatched wapl-1 embryos (Figure 1C) and by the presence of an egg laying defect in adult worms. In order to prevent the accumulation of somatic defects, all the analysis presented here was performed in homozygous wapl-1 worms derived from heterozygous mothers. Figure 1 with 1 supplement see all Download asset Open asset WAPL-1 localizes to germ line nuclei and promotes viability. (A) Structure of the wapl-1 gene, red bar indicates the region deleted in the tm1814 allele. (B) Western blot demonstrates that wapl-1(tm1814) is a null allele and that a protein of the expected size is present in worms carrying a GFP::wapl-1 transgene. (C) wapl-1 mutants display reduced fertility and larval lethality, numbers in parenthesis indicate total number of embryos analysed per genotype . (D) Projections of whole-mounted germ lines stained with DAPI, the different stages of meiotic prophase are noted above the WT germ line, with transition zone containing nuclei in leptotene and zygotene. Note that overall germ line organization in wapl-1 mutants is similar to WT. (E) Projections of diakinesis oocytes stained with DAPI, six bivalents are present in both WT and wapl-1 mutants. (F) Whole-mounted germ line from a transgenic worm homozygous for the wapl-1(tm1814) deletion and for a GFP::wapl-1 single copy transgene stained with DAPI and anti-GFP antibodies. Note that the intensity of GFP::WAPL-1 decreases in transition zone and peaks again during late pachytene. (G) Insets from germ line shown in F showing GFP::WAPL-1 staining in transition zone and pachytene nuclei, note that GFP::WAPL-1 intensity is very high in transition zone nuclei that do not display chromosome clustering (arrowheads). Figure 1—figure supplement 1 shows quantification of GFP::WAPL-1 intensities along the germ line. Scale bars in E and G = 5 µm. https://doi.org/10.7554/eLife.10851.003 The overall organization of wapl-1 mutant germ lines appears largely normal, with clearly defined mitotic and meiotic compartments in which the different stages of meiotic prophase can be easily identified (Figure 1D). In fact, observation of diakinesis oocytes (the last stage of meiotic prophase) showed that both wild type and wapl-1 mutant oocytes displayed 6 DAPI-stained bodies, demonstrating that WAPL-1 is not required for chiasma formation (Figure 1E). Nonetheless, the reduced fertility of wapl-1 mutants suggested that WAPL-1 may play important roles in the germ line. Thus, we investigated the staining pattern of WAPL-1 during meiosis by creating transgenic worms homozygous for the tm1814 deletion and for a single-copy insertion of a transgene that expresses a GFP::WAPL-1 fusion protein using the 5’ and 3’ UTRs from the wapl-1 locus. Expression of this transgene largely rescued the fertility defects of wapl-1(tm1814) mutants (Figure 1C), and western blot analysis confirmed the presence of a band of the expected molecular weight for the GFP::WAPL-1 fusion protein, although the overall intensity of this band was reduced compared to the endogenous WAPL-1 protein (Figure 1B). Staining of germ lines from these transgenic worms with anti-GFP antibodies demonstrated that the GFP::WAPL-1 protein is present, with a diffuse staining pattern, in both mitotic and meiotic nuclei (Figure 1F). Interestingly, the intensity of the GFP::WAPL-1 signal decreased drastically as transition zone nuclei acquired the chromosome clustering characteristic of early meiotic prophase stages (leptotene and zygotene), peaking again at late pachytene and then remaining at similar high levels in diplotene and diakinesis oocytes (Figure 1F–G and Figure 1—figure supplement 1). WAPL-1 promotes timely repair of meiotic DSBs and correct polar body extrusion during the meiotic divisions Despite normal formation of chiasmata, the high incidence of embryonic lethality among the progeny of wapl-1 mutants (Figure 1C) suggested the existence of meiotic defects. Moreover, the presence of developmental defects among the progeny of wapl-1 mutants could be a consequence of defects in DNA repair during meiosis, so we monitored the progression of meiotic recombination by visualizing the appearance and disappearance of the RAD-51 recombinase, which labels early meiotic recombination intermediates (Colaiácovo et al., 2003), and of COSA-1 and ZHP-3 foci, two proteins that are required for crossover formation and that localize specifically to crossover-fated recombination events in late pachytene nuclei (Bhalla et al., 2008; Yokoo et al., 2012). We observed 6 COSA-1 and 6 ZHP-3 foci in both wapl-1 mutants and wild-type controls (Figure 2A and Figure 2—figure supplement 1), consistent with the normal presence of chiasmata in wapl-1 diakinesis oocytes. Despite this, RAD-51-positive recombination intermediates accumulate in mid pachytene nuclei of wapl-1 mutants (Figure 2B). Furthermore, while RAD-51 foci were no longer detected in 98% of late pachytene nuclei of wild-type controls, 52% percent of nuclei in the same region of wapl-1 mutant germ lines displayed RAD-51 foci. Thus, although crossover precursors are successfully formed, the repair of a subset of DSBs is delayed in wapl-1 mutants. Figure 2 with 1 supplement see all Download asset Open asset WAPL-1 affects DNA repair during meiotic prophase. (A) Projections of late pachytene nuclei from worms expressing COSA-1::GFP, note that both wapl-1 mutants and WT controls display 6 COSA-1 foci per nucleus. Graph showing quantification of COSA-1 foci (126 nuclei from wapl-1 mutants and 100 nuclei from WT). (B) Projections of pachytene nuclei stained with anti-RAD-51 antibodies and DAPI, note increased RAD-51 foci in wapl-1 mutant panel. Quantification of RAD-51 foci in germ lines of WT and wapl-1 mutants. Each germ line was divided into 7 equal-sized regions, with regions 4 to 7 representing early to late pachytene. The X axis indicates the seven regions along the germ line, while the Y axis indicates the percentage of nuclei with a given number of RAD-51 foci (as indicated in the color key). wapl-1 mutants accumulate RAD-51 in mid and late pachytene nuclei. Number of nuclei analyzed (WT, wapl-1 mutant): Zone 1 (133, 138), zone 2 (246, 195), zone 3 (137, 135), zone 4 (154, 101), zone 5 (122, 90), zone 6 (114, 69), zone 7 (93, 61). https://doi.org/10.7554/eLife.10851.005 Next, we investigated if the meiotic divisions proceeded normally in the absence of WAPL-1, as defects in this process could induce aneuploidy even if chiasma formation is not affected. During oogenesis, each meiotic division results in the formation of a polar body that contains a full complement of homologs (meiosis I) or sister chromatids (meiosis II). Polar bodies are extruded away from the egg pronucleus, localizing on the cortex and not contributing to the genetic content of the developing embryo (Figure 3A). All wapl-1 mutant embryos analyzed formed two polar bodies, however, only 22% of post meiotic embryos (up to the two-cell embryo) displayed both polar bodies at the cortex, with most embryos (61%) displaying one polar body located away from the cortex and the remaining (11%) displaying both polar bodies away from the cortex (Figure 3A–B). In addition, we noticed that chromatin morphology in the polar bodies was altered in wapl-1 mutants, often displaying separated DNA masses within the polar body (Figure 3—figure supplement 1). To gain better understanding of the effect of WAPL-1 on polar body extrusion, we performed live imaging of the meiotic and early mitotic divisions in wild type and wapl-1 mutant embryos expressing histone H2B::mcherry (Figure 3D; Videos 1–3). These experiments confirmed that most wapl-1 mutant embryos display defects in the extrusion of the second polar body (Figure 3C–D; Videos 2–3). We also observed that in 2 out of 11 wapl-1 mutant embryos, the second polar body, which failed to migrate to the cortex, underwent chromatin decondesation and condensation cycles, mimicking the changes observed in the mitotic nuclei of the embryo (Figure 3D and Videos 2–3). Moreover, in 1 out of 11 filmed embryos the second polar body eventually fused with one of the mitotic nuclei generated after the first mitotic division (Figure 3D and Video 3), demonstrating that the failure in polar body extrusion of wapl-1 mutants can lead to aneuploidy in the embryo. We also used fluorescence in situ hybridization (FISH) to investigate if chromosome non-disjunction occurs during the meiotic divisions of wapl-1 mutants. Labeling of the 5S rDNA locus on chromosome V in early embryos demonstrated that the oocyte pronucleus contained one single signal for the 5S rDNA in all embryos analyzed from wild-type (17) and wapl-1 mutants (17) (Figure 3E), suggesting that chromosome V segregates properly during the meiotic divisions in the absence of WAPL-1. This analysis demonstrates that WAPL-1 is required to ensure proper polar body extrusion during the meiotic divisions, and suggests that defects in this process may contribute to the embryonic lethality observed in wapl-1 mutants. Figure 3 with 1 supplement see all Download asset Open asset WAPL-1 is required for polar body extrusion. (A) Projections of fixed embryos at the 1- or 2-cell stage stained with DAPI, arrowheads point to the position of polar bodies generated during the meiotic divisions. Polar bodies are found near the cortex in WT control, but one (middle panel) or both (right-hand side panel) polar bodies localize away from the cortex in wapl-1 mutant embryos. (B) Quantification of the percentage of embryos with zero, one, or two polar bodies localized at the cortex (36 embryos scored in wapl-1 and 34 in WT). (C) Quantification of polar body behavior in videos from live WT and wapl-1 mutant embryos expressing a histone H2B::mcherry fusion protein. Note that 7 out of 11 wapl-1 mutant embryos displayed defects in polar body extrusion. Examples of videos used for the quantification are shown in Video 1 (WT), Videos 2–3 (wapl-1). (D) Selected frames from the WT embryo shown in Video 1 and from the wapl-1 mutant embryo shown in Videos 2–3. Time is indicated on top-left corner, starting from metaphase I. Abbreviations: PB I (first polar body), PB II (second polar body), OP (oocyte pronucleus), SP (sperm pronocleus), P0 (first mitotic metaphase following fusion of OP and SP), P1 and AB (cells resulting from the first mitotic division). Note that in the WT embryo PB II remains highly condensed and locates close to PB I on the cortex. In the wapl-1 mutant embryo, PB II becomes decondensed and fails to move to the cortex, first remaining close to the OP and then close to the AB cell produced after the first mitotic division. Chromosomes from AB and PB II appear to mix together before the second mitotic division of the embryo. (E) Projections of fixed embryos following the completion of the second meiotic division and labeled with a FISH probe against the 5S rDNA locus on chromosome V and DAPI. Note that in both WT and wapl-1 mutant embryos the oocyte pronucleus (OP) and the second polar body (PB II) contain a single FISH signal, even when PB II is not localized on the cortex and chromatin appears decondensed (wapl-1 example on right-hand side). https://doi.org/10.7554/eLife.10851.007 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live imaging of a WT embryo expressing histone H2B::mcherry. Filming covers the interval between the first meiotic metaphase and the end of the first mitotic division. Each meiotic division results in the production of a polar body and both polar bodies remain highly condensed and located at one end of the embryo, away from the oocyte and sperm pronuclei. Figure 3D contains individual images from this video in which specific meiotic and mitotic events are labeled (images in Figure 3D are rotated with respect to the video). https://doi.org/10.7554/eLife.10851.009 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live imaging of a wapl-1 mutant embryo expressing histone H2B::mcherry. Filming covers the interval between the first meiotic division and the end of the first mitotic division. Note that the second polar body does not migrate to the cortex, instead it follows the movement of the oocyte pronucleus towards the middle of the embryo and the chromosomes appear decondensed. As chromosomes in the sperm and oocyte pronuclei condense in preparation for the first mitotic division, condensation of chromatin also occurs in the second polar body. Figure 3D contains individual images from this video in which specific meiotic and mitotic events are labeled, and Video 3 shows continued filming from the same embryo. https://doi.org/10.7554/eLife.10851.010 Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Continuation of live imaging of the wapl-1 mutant embryo shown in Video 2. Filming covers the interval between the end of the first mitotic division and up to the four cell embryo. Following the completion of the first mitotic division, chromatin in the second polar body (PB II) undergoes decondensation. PB II localizes to the vicinity of the AB mitotic nucleus, and as chromosomes in the AB nucleus condense so do chromosomes in PB II. At this point condensed chromosomes from the AB nucleus and PB II appear to mix up before dividing into two daughter nuclei. Figure 3D contains individual images from this video in which specific mitotic events are labeled. https://doi.org/10.7554/eLife.10851.011 WAPL-1 regulates axial element morphogenesis at meiosis onset and axis compaction during pachytene During the initial observation of wapl-1 mutant germ lines we noticed that pachytene chromosomes appeared more widely spaced within the nucleus and somewhat thicker than in wild-type controls, suggesting that WAPL-1 may regulate the shape of meiotic chromosomes, as it does in mitotic cells (Tedeschi et al., 2013). Meiotic chromosomes are organized around axial elements containing cohesin and meiosis-specific HORMA-domain proteins (Kleckner, 2006). Thus, we used antibodies against HTP-3, a HORMA-domain protein that is an essential component of axial elements in C. elegans (Goodyer et al., 2008; Severson et al., 2009), to investigate chromosome organization in wapl-1 mutants. Projections made from late pachytene nuclei of wild-type germ lines demonstrated ample overlap between different HTP-3-labeled axial elements, making it difficult to follow individual HTP-3 tracks along their whole length (Figure 4A and Figure 4—figure supplement 1). In contrast, the overlap of HTP-3 tracks was reduced in wapl-1 mutants, with some nuclei displaying six distinctive HTP-3 tracks (one per homolog pair) that could be clearly traced along their full length (Figure 4A and Figure 4—figure supplement 1). Measuring of total HTP-3 track length per nucleus demonstrated a 28% decrease in axial element length in late pachytene nuclei of wapl-1 mutants (Figure 4B). Figure 4 with 6 supplements see all Download asset Open asset WAPL-1 regulates chromosome organization during meiotic prophase. (A) Projections of late pachytene nuclei stained with anti-HTP-3 antibodies (axial elements) and DAPI. Axial elements are shorter in wapl-1 mutants, arrowheads point to 6 HTP-3 tracks that can be individually traced along their whole length (each track represents a pair of aligned homologous chromosomes). (B) Quantification of total HTP-3 length per late pachytene nucleus in WT controls and wapl-1 mutants. Error bars represent standard deviation, differences are significant (p<0.0001, t-test). (C) Projections of late pachytene nuclei stained with HTP-3 antibodies (axial elements) and DAPI. The number of HTP-3 tracks appears larger in syp-1 mutants than in wapl-1 syp-1 double mutants, where some short HTP-3 tracks are seen. (D) Projections of mid pachytene nuclei stained with anti-HTP-3 antibodies (axial elements), DAPI, and labeled with a probe against the 5S rDNA locus on chromosome V. Most nuclei in syp-1 and wapl-1 syp-1 mutants display 2 5S foci, showing that homologs are not associated (quantification shown in Figure 4—figure supplement 2). (E) Projection of a late pachytene nucleus stained with DAPI, anti-HTP-3 antibodies and anti-HIM-8 antibodies (binding to a single end of the X chromosome). Both HIM-8 signals are located at the end of a short HTP-3 track, each one representing a highly compacted X chromosome. Shortening of X chromosome axial elements in late pachytene nuclei was seen in 3 out of 3 syp-1 wapl-1 germ lines. (F) Projections of transition zone nuclei from worms carrying a GFP tag on the endogenous smc-1 gene (generated by CRISPR) stained with DAPI, anti-GFP antibodies, and anti-PLK-2 antibodies. Appearance of PLK-2 aggregates on the nuclear envelope marks the onset of meiotic prophase (leptotene stage). In the WT germ line, SMC-1::GFP tracks are only observed in nuclei with PLK-2 aggregates, while pre-leptotene nuclei display diffuse SMC-1::GFP staining (arrows). SMC-1::GFP tracks are present in pre-leptotene nuclei of wapl-1 mutants (arrows). (G) Projections of late pachytene and diplotene nuclei from worms carrying a GFP tag on the endogenous smc-1 gene (generated by CRISPR) stained with DAPI and anti-GFP antibodies. A large accumulation of nuclear soluble SMC-1::GFP is present in wild-type nuclei, but not in wapl-1 mutants (see quantification on Figure 4—figure supplement 5). Note that axial elements become elongated, twisted and with a more diffuse appearance in wild-type nuclei compared with wapl-1 mutant nuclei (insets show magnification of the indicated nuclear region). Scale bar = 5 µm in all panels. https://doi.org/10.7554/eLife.10851.012 The organization of meiotic chromosomes in pachytene nuclei is not only determined by axial elements, but also by the synaptonemal complex (SC), a proteinaceous structure that glues together homologous axial elements to promote stable pairing and inter-homolog recombination (MacQueen et al., 2002). In order to understand better the effect of WAPL-1 on the organization of axial elements, we investigated chromosome structure in pachytene nu
Abstract Metamorphic HORMA domain proteins (HORMADs) nucleate protein complex formation by refolding their mobile safety belt region to bind short motifs on interactors. Meiotic HORMADs (mHORMADs) bind proteinaceous axial elements to orchestrate complex chromosomal events that underpin fertility, including pairing and recombination between homologous chromosomes. However, the mechanisms supporting the diverse roles of mHORMADs remain unclear. Here, we show that mHORMADs have a second structurally mobile region, the β5-αC loop, which controls mHORMAD conformation and function. Molecular dynamics and in vivo approaches show that functional specialisation of C. elegans paralogs HTP-1 and HTP-2 depends on the interplay between their β5-αC loop and safety belt. The β5-αC loop can interact with the same HORMA core surface as the safety belt, forming a “loop engaged” conformation. The β5-αC loop HORMA core interaction is essential for axis loading of HTP-1 and its paralog HTP-3, and is also present in yeast, plant, and mammalian mHORMADs, suggesting that it represents a conserved functional feature of mHORMADs. Our study reveals that mHORMADs have expanded the bimodal folding landscape first identified in Mad2, paving the way to elucidate how non-canonical HORMAD conformations control meiotic chromosome function to ensure fertility.
Inhibition of differentiation has been proposed as an important mechanism for Myc-induced tumorigenesis, but the mechanisms involved are unclear. We have established a genetically defined differentiation model in human leukemia K562 cells by conditional expression of the cyclin-dependent kinase (Cdk) inhibitor p27 (inducible by Zn2+) and Myc (activatable by 4-hydroxy-tamoxifen). Induction of p27 resulted in erythroid differentiation, accompanied by Cdk inhibition and G1 arrest. Interestingly, activation of Myc inhibited p27-mediated erythroid differentiation without affecting p27-mediated proliferation arrest. Microarray-based gene expression indicated that, in the presence of p27, Myc blocked the upregulation of several erythroid-cell-specific genes, including NFE2, JUNB, and GATA1 (transcription factors with a pivotal role in erythropoiesis). Moreover, Myc also blocked the upregulation of Mad1, a transcriptional antagonist of Myc that is able to induce erythroid differentiation. Cotransfection experiments demonstrated that Myc-mediated inhibition of differentiation is partly dependent on the repression of Mad1 and GATA1. In conclusion, this model demonstrates that Myc-mediated inhibition of differentiation depends on the regulation of a specific gene program, whereas it is independent of p27-mediated cell cycle arrest. Our results support the hypothesis that differentiation inhibition is an important Myc tumorigenic mechanism that is independent of cell proliferation.
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