- Email: [email protected]
Gene Amplification: Trophoblast Giant Cells Use All the Tricks
Current Biology
Dispatches 10. Reid, A.J., Blake, D.P., Ansari, H.R., Billington, K., Browne, H.P., Bryant, J., Dunn, M., Hung, S.S., Kawahara, F., Miranda-Saavedra, D., et al. (2014). Genomic analysis of the causative agents of coccidiosis in domestic chickens. Genome Res. 24, 1676–1685. 11. Gardner, M.J., Bishop, R., Shah, T., de Villiers, E.P., Carlton, J.M., Hall, N., Ren, Q., Paulsen, I.T., Pain, A., Berriman, M., et al. (2005). Genome sequence of Theileria parva, a bovine pathogen that transforms lymphocytes. Science 309, 134–137. 12. Matsuzaki, M., Misumi, O., Shin-i, T., Maruyama, S., Takahara, M., Miyagishima, S.Y., Mori, T., Nishida, K., Yagisawa, F., Nishida, K., et al. (2004). Genome sequence of the ultrasmall unicellular red alga
Cyanidioschyzon merolae 10D. Nature 428, 653–657. 13. Giovannoni, S.J., Tripp, H.J., Givan, S., Podar, M., Vergin, K.L., Baptista, D., Bibbs, L., Eads, J., Richardson, T.H., Noordewier, M., et al. (2005). Genome streamlining in a cosmopolitan oceanic bacterium. Science 309, 1242–1245. 14. Derelle, E., Ferraz, C., Rombauts, S., Rouze´, P., Worden, A.Z., Robbens, S., Partensky, F., Degroeve, S., Echeynie´, S., Cooke, R., et al. (2006). Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features. Proc. Natl. Acad. Sci. USA 103, 11647–11652. 15. Cornillot, E., Hadj-Kaddour, K., Dassouli, A., Noel, B., Ranwez, V., Vacherie, B., Augagneur,
Y., Bre`s, V., Duclos, A., Randazzo, S., et al. (2012). Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti. Nucleic Acids Res. 40, 9102–9114. 16. Haag, K.L., James, T.Y., Pombert, J.F., Larsson, R., Schaer, T.M., Refardt, D., and Ebert, D. (2014). Evolution of a morphological novelty occurred before genome compaction in a lineage of extreme parasites. Proc. Natl. Acad. Sci. USA 111, 15480–15485. 17. Martin, W., and Koonin, E.V. (2006). Introns and the origin of nucleus–cytosol compartmentalization. Nature 440, 41–45. 18. Martin, W., and Mu¨ller, M. (1998). The hydrogen hypothesis for the first eukaryote. Nature 392, 37–41.
Gene Amplification: Trophoblast Giant Cells Use All the Tricks James C. Cross Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, Alberta, T1S 1A2 Canada Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2015.12.030
Evolutionary gene duplication, developmental endoreduplication and selective gene amplification are alternative strategies for increasing gene copy number. When these processes occur together, things get really interesting, and new work shows that is the lifestyle of cells in the placenta. Genomes are rapidly changing during evolution through mutations, as well as through gene-duplication events that create gene families. It has become clear that somatic cells also undergo genomic changes that are associated with new functions. Amplification of oncogenes is a common occurrence in cancer cells [1], and is presumably selected for by giving a growth advantage to cells. Endoreduplication is widely recognized during development of some cell types in plants, insects and mammals [2], and represents a more widespread amplification of genes, and results in polytene chromosomes. In mammals, trophoblast giant cells of the rodent placenta show extensive endoreduplication, doubling in DNA content for each round of DNA replication, and can reach a DNA content equivalent of over 1000 copies of the genome (Figure 1) [3,4],
and it had been assumed for years that endoreduplication in trophoblast giant cells covered the entire genome. However, it was recently shown using new-generation genome analytical tools that, while the entire genome is polyploid, about 5% of the genome is relatively under-replicated [5]. In a report recently published in Current Biology, Roberta Hannibal and Julie Baker extend their analysis with even more sensitive methods and identify regions that are relatively over-replicated, representing less than 0.2% of the genome [6]. These regions include genes that are highly expressed in trophoblast cells but, amazingly, these regions encompass large multi-gene families that arose through gene duplication. The mouse placenta contains several subtypes of trophoblast giant cells that differ in location within the placenta, cell lineage origin and the extent of
polyploidy [7]. Hannibal and Baker focused their analysis on the parietal subtype of trophoblast giant cells, which are the first to form in the placenta, achieve the highest ploidies, and line the embryo implantation site and so are the easiest to isolate. Relative gene copy number across the genome was assessed at high resolution by using whole-genome sequencing, and then confirmed by digital droplet PCR, which is a probabilistic method for quantification, as it divides each PCR reaction into 20,000 droplets. Over-replicated parts of the genome occurred in five regions covering 4.7 million base pairs of the mouse genome. All five amplified regions contain gene families expressed in the placenta and thought to be important for normal pregnancy. One region is on mouse chromosome 6 and includes the NK/CLEC complex involved in interactions of natural killer cells in the
Current Biology 26, R157–R179, February 22, 2016 ª2016 Elsevier Ltd All rights reserved R177
Current Biology
Dispatches Mitosis DNA
n
catio
repli
2C
4C
2C
2C DNA
repli
catio
2C = diploid
n
Endoreduplication 8C
4C
up to 1000C
Overreplicated
Repeated endocycles
Underreplicated
Current Biology
Figure 1. Regulation of DNA content during development of trophoblast giant cells. Trophoblast progenitor cells exit the mitotic cell cycle and undergo rounds of DNA replication without intervening mitoses (endoreduplication), leading to increased ploidy. High-resolution quantification of DNA content shows that small regions of the genome are relatively under-replicated (5% of genome in 47 loci) and others over-replicated (0.2% of genome in 5 loci). Genome is not drawn to scale.
innate immune system. In humans, polymorphisms in the equivalent system (KIR/HLA-C) are associated with risk of preeclampsia and intrauterine growth restriction [8,9]. Interestingly, the other four regions occur on chromosome 13 and encompass two regions encoding prolactin-like hormones, and the others encoding proteins similar to serine protease inhibitors (serpins) and cysteine proteases (cathepsins). The function of placental serpins and cathepsins is poorly understood, but the placental prolactin-related genes have been extensively studied with respect to expression and function [7,10,11]. There are several mechanistic questions raised by the findings of Hannibal and Baker. There must be sequence or epigenetic signatures that define the regions to be over-amplified and something must define the boundaries of the regions that are 1 megabase in size. The latter was investigated by assessing copy number from several genes within a single amplified region and, in general, the genome was more amplified in the central regions. For the larger prolactin-related
gene locus, at least, parietal trophoblast giant cells express genes from all across the locus and the central region includes genes not expressed in those cells or their progenitors [10]. Temporal or spatial expression of the placental prolactin-related genes does not correlate with position within the locus. Interestingly, Hannibal and Baker found evidence for a single enhancer with the center of the large prolactin-related gene locus from ChIP-seq data assessing the histone modifications H3K4me1 and H3K27ac. The finding of both over- and under-replicated regions of the genome in trophoblast giant cells begs the question as to whether or not the amplification and under-replication are regulated by a common mechanism. Analysis of Rif1 mutant mice indicated that the timing of DNA replication was important for under-replicated regions but not over-replicated regions. The excess firing of origins of DNA replication at amplified regions likely requires more complex mechanisms, especially given that trophoblast giant cells endoreduplicate their entire genomes. Determining how adding a round of DNA
replication in small regions is accomplished will require different approaches. It is important to know if over-replication occurs at the same time, as more widespread endoreduplication, or if it begins after endoreduplication stops. Gene amplification is very interesting in its own right. However, it is even more interesting, if not baffling, to think about the functional significance of amplifying regions of the genome that already contain gene duplications, and in cells that have already committed to replicate their entire genomes to become polyploid. Clearly there is an evolutionary advantage to increasing gene copy number, but are these three mechanisms simply accomplishing the same thing? For me, the fact that they occur together in one cell type suggests not. Gene duplication events have functional significance over evolutionary time, whereas endoreduplication and gene amplification occur in developmental time. The NK/CLEC, prolactin, serpin and cathepsin gene families are rapidly evolving and indeed are rodent specific. With respect to the placental prolactin-related hormones, the duplicated genes have diversified functions, as not all can bind the prolactin receptor [12]. Entry into endoreduplication appears to occur in the G2 phase of an otherwise normal start of a mitotic cell cycle but, instead of progressing through mitosis, the cells go through another round of DNA synthesis [4]. This appears to be regulated as a normal developmental event occurring in all cells. By contrast, the under- and over-replication is on average less than one round of DNA replication per cell — 28–54% less for under-replicated [5] and 22–37% more for over-replicated [6] regions compared with the rest of the genome. It will be important to know how much variation occurs between cells, why some cells over-replicate and others don’t, and how much of a difference copy number makes in gene expression. This sort of variation suggests that selective gene amplification is fine-tuning to suit the needs of some cells. Like all great discoveries, the sophisticated bag of tricks that trophoblast giant cells use to alter gene copy number forces us to think about
R178 Current Biology 26, R157–R179, February 22, 2016 ª2016 Elsevier Ltd All rights reserved
Current Biology
Dispatches gene copy number in a different way. The surprises likely aren’t over. REFERENCES 1. Albertson, D.G. (2006). Gene amplification in cancer. Trends Genet. 22, 447–455. 2. Fox, D.T., and Duronio, R.J. (2013). Endoreplication and polyploidy: insights into development and disease. Development 140, 3–12. 3. Zybina, T.G., and Zybina, E.V. (2000). Genome multiplication in the tertiary giant trophoblast cells in the course of their endovascular and interstitial invasion into the rat placenta decidua basalis. Early Preg. 4, 99–109. 4. MacAuley, A., Cross, J.C., and Werb, Z. (1998). Reprogramming the cell cycle for endoreduplication in rodent trophoblast cells. Mol. Biol. Cell 9, 795–807.
5. Hannibal, R.L., Chuong, E.B., Rivera-Mulia, J.C., Gilbert, D.M., Valouev, A., and Baker, J.C. (2014). Copy number variation is a fundamental aspect of the placental genome. PLoS Genet. 10, e1004290. 6. Hannibal, R.L., and Baker, J.C. (2016). Selective amplification of the genome surrounding key placental genes in trophoblast giant cells. Curr. Biol. 26, 230–236. 7. Simmons, D.G., Fortier, A.L., and Cross, J.C. (2007). Diverse subtypes and developmental origins of trophoblast giant cells in the mouse placenta. Dev. Biol. 304, 567–578. 8. Hiby, S.E., Apps, R., Chazara, O., Farrell, L.E., Magnus, P., Trogstad, L., Gjessing, H.K., Carrington, M., and Moffett, A. (2014). Maternal KIR in combination with paternal HLA-C2 regulate human birth weight. J. Immunol. 192, 5069–5073.
9. Nakimuli, A., Chazara, O., Hiby, S.E., Farrell, L., Tukwasibwe, S., Jayaraman, J., Traherne, J.A., Trowsdale, J., Colucci, F., Lougee, E., et al. (2015). A KIR B centromeric region present in Africans but not Europeans protects pregnant women from pre-eclampsia. Proc. Natl. Acad. Sci. USA 112, 845–850. 10. Simmons, D.G., Rawn, S., Davies, A., Hughes, M., and Cross, J.C. (2008). Spatial and temporal expression of the 23 murine Prolactin/Placental Lactogen-related genes is not associated with their position in the locus. BMC Genom. 9, 352. 11. Soares, M.J., Konno, T., and Alam, S.M. (2007). The prolactin family: effectors of pregnancy-dependent adaptations. Trends Endocrinol. Metab. 18, 114–121. 12. Wiemers, D.O., Shao, L.J., Ain, R., Dai, G., and Soares, M.J. (2003). The mouse prolactin gene family locus. Endocrinology 144, 313–325.
Current Biology 26, R157–R179, February 22, 2016 ª2016 Elsevier Ltd All rights reserved R179
Dispatches 10. Reid, A.J., Blake, D.P., Ansari, H.R., Billington, K., Browne, H.P., Bryant, J., Dunn, M., Hung, S.S., Kawahara, F., Miranda-Saavedra, D., et al. (2014). Genomic analysis of the causative agents of coccidiosis in domestic chickens. Genome Res. 24, 1676–1685. 11. Gardner, M.J., Bishop, R., Shah, T., de Villiers, E.P., Carlton, J.M., Hall, N., Ren, Q., Paulsen, I.T., Pain, A., Berriman, M., et al. (2005). Genome sequence of Theileria parva, a bovine pathogen that transforms lymphocytes. Science 309, 134–137. 12. Matsuzaki, M., Misumi, O., Shin-i, T., Maruyama, S., Takahara, M., Miyagishima, S.Y., Mori, T., Nishida, K., Yagisawa, F., Nishida, K., et al. (2004). Genome sequence of the ultrasmall unicellular red alga
Cyanidioschyzon merolae 10D. Nature 428, 653–657. 13. Giovannoni, S.J., Tripp, H.J., Givan, S., Podar, M., Vergin, K.L., Baptista, D., Bibbs, L., Eads, J., Richardson, T.H., Noordewier, M., et al. (2005). Genome streamlining in a cosmopolitan oceanic bacterium. Science 309, 1242–1245. 14. Derelle, E., Ferraz, C., Rombauts, S., Rouze´, P., Worden, A.Z., Robbens, S., Partensky, F., Degroeve, S., Echeynie´, S., Cooke, R., et al. (2006). Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features. Proc. Natl. Acad. Sci. USA 103, 11647–11652. 15. Cornillot, E., Hadj-Kaddour, K., Dassouli, A., Noel, B., Ranwez, V., Vacherie, B., Augagneur,
Y., Bre`s, V., Duclos, A., Randazzo, S., et al. (2012). Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti. Nucleic Acids Res. 40, 9102–9114. 16. Haag, K.L., James, T.Y., Pombert, J.F., Larsson, R., Schaer, T.M., Refardt, D., and Ebert, D. (2014). Evolution of a morphological novelty occurred before genome compaction in a lineage of extreme parasites. Proc. Natl. Acad. Sci. USA 111, 15480–15485. 17. Martin, W., and Koonin, E.V. (2006). Introns and the origin of nucleus–cytosol compartmentalization. Nature 440, 41–45. 18. Martin, W., and Mu¨ller, M. (1998). The hydrogen hypothesis for the first eukaryote. Nature 392, 37–41.
Gene Amplification: Trophoblast Giant Cells Use All the Tricks James C. Cross Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, Alberta, T1S 1A2 Canada Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2015.12.030
Evolutionary gene duplication, developmental endoreduplication and selective gene amplification are alternative strategies for increasing gene copy number. When these processes occur together, things get really interesting, and new work shows that is the lifestyle of cells in the placenta. Genomes are rapidly changing during evolution through mutations, as well as through gene-duplication events that create gene families. It has become clear that somatic cells also undergo genomic changes that are associated with new functions. Amplification of oncogenes is a common occurrence in cancer cells [1], and is presumably selected for by giving a growth advantage to cells. Endoreduplication is widely recognized during development of some cell types in plants, insects and mammals [2], and represents a more widespread amplification of genes, and results in polytene chromosomes. In mammals, trophoblast giant cells of the rodent placenta show extensive endoreduplication, doubling in DNA content for each round of DNA replication, and can reach a DNA content equivalent of over 1000 copies of the genome (Figure 1) [3,4],
and it had been assumed for years that endoreduplication in trophoblast giant cells covered the entire genome. However, it was recently shown using new-generation genome analytical tools that, while the entire genome is polyploid, about 5% of the genome is relatively under-replicated [5]. In a report recently published in Current Biology, Roberta Hannibal and Julie Baker extend their analysis with even more sensitive methods and identify regions that are relatively over-replicated, representing less than 0.2% of the genome [6]. These regions include genes that are highly expressed in trophoblast cells but, amazingly, these regions encompass large multi-gene families that arose through gene duplication. The mouse placenta contains several subtypes of trophoblast giant cells that differ in location within the placenta, cell lineage origin and the extent of
polyploidy [7]. Hannibal and Baker focused their analysis on the parietal subtype of trophoblast giant cells, which are the first to form in the placenta, achieve the highest ploidies, and line the embryo implantation site and so are the easiest to isolate. Relative gene copy number across the genome was assessed at high resolution by using whole-genome sequencing, and then confirmed by digital droplet PCR, which is a probabilistic method for quantification, as it divides each PCR reaction into 20,000 droplets. Over-replicated parts of the genome occurred in five regions covering 4.7 million base pairs of the mouse genome. All five amplified regions contain gene families expressed in the placenta and thought to be important for normal pregnancy. One region is on mouse chromosome 6 and includes the NK/CLEC complex involved in interactions of natural killer cells in the
Current Biology 26, R157–R179, February 22, 2016 ª2016 Elsevier Ltd All rights reserved R177
Current Biology
Dispatches Mitosis DNA
n
catio
repli
2C
4C
2C
2C DNA
repli
catio
2C = diploid
n
Endoreduplication 8C
4C
up to 1000C
Overreplicated
Repeated endocycles
Underreplicated
Current Biology
Figure 1. Regulation of DNA content during development of trophoblast giant cells. Trophoblast progenitor cells exit the mitotic cell cycle and undergo rounds of DNA replication without intervening mitoses (endoreduplication), leading to increased ploidy. High-resolution quantification of DNA content shows that small regions of the genome are relatively under-replicated (5% of genome in 47 loci) and others over-replicated (0.2% of genome in 5 loci). Genome is not drawn to scale.
innate immune system. In humans, polymorphisms in the equivalent system (KIR/HLA-C) are associated with risk of preeclampsia and intrauterine growth restriction [8,9]. Interestingly, the other four regions occur on chromosome 13 and encompass two regions encoding prolactin-like hormones, and the others encoding proteins similar to serine protease inhibitors (serpins) and cysteine proteases (cathepsins). The function of placental serpins and cathepsins is poorly understood, but the placental prolactin-related genes have been extensively studied with respect to expression and function [7,10,11]. There are several mechanistic questions raised by the findings of Hannibal and Baker. There must be sequence or epigenetic signatures that define the regions to be over-amplified and something must define the boundaries of the regions that are 1 megabase in size. The latter was investigated by assessing copy number from several genes within a single amplified region and, in general, the genome was more amplified in the central regions. For the larger prolactin-related
gene locus, at least, parietal trophoblast giant cells express genes from all across the locus and the central region includes genes not expressed in those cells or their progenitors [10]. Temporal or spatial expression of the placental prolactin-related genes does not correlate with position within the locus. Interestingly, Hannibal and Baker found evidence for a single enhancer with the center of the large prolactin-related gene locus from ChIP-seq data assessing the histone modifications H3K4me1 and H3K27ac. The finding of both over- and under-replicated regions of the genome in trophoblast giant cells begs the question as to whether or not the amplification and under-replication are regulated by a common mechanism. Analysis of Rif1 mutant mice indicated that the timing of DNA replication was important for under-replicated regions but not over-replicated regions. The excess firing of origins of DNA replication at amplified regions likely requires more complex mechanisms, especially given that trophoblast giant cells endoreduplicate their entire genomes. Determining how adding a round of DNA
replication in small regions is accomplished will require different approaches. It is important to know if over-replication occurs at the same time, as more widespread endoreduplication, or if it begins after endoreduplication stops. Gene amplification is very interesting in its own right. However, it is even more interesting, if not baffling, to think about the functional significance of amplifying regions of the genome that already contain gene duplications, and in cells that have already committed to replicate their entire genomes to become polyploid. Clearly there is an evolutionary advantage to increasing gene copy number, but are these three mechanisms simply accomplishing the same thing? For me, the fact that they occur together in one cell type suggests not. Gene duplication events have functional significance over evolutionary time, whereas endoreduplication and gene amplification occur in developmental time. The NK/CLEC, prolactin, serpin and cathepsin gene families are rapidly evolving and indeed are rodent specific. With respect to the placental prolactin-related hormones, the duplicated genes have diversified functions, as not all can bind the prolactin receptor [12]. Entry into endoreduplication appears to occur in the G2 phase of an otherwise normal start of a mitotic cell cycle but, instead of progressing through mitosis, the cells go through another round of DNA synthesis [4]. This appears to be regulated as a normal developmental event occurring in all cells. By contrast, the under- and over-replication is on average less than one round of DNA replication per cell — 28–54% less for under-replicated [5] and 22–37% more for over-replicated [6] regions compared with the rest of the genome. It will be important to know how much variation occurs between cells, why some cells over-replicate and others don’t, and how much of a difference copy number makes in gene expression. This sort of variation suggests that selective gene amplification is fine-tuning to suit the needs of some cells. Like all great discoveries, the sophisticated bag of tricks that trophoblast giant cells use to alter gene copy number forces us to think about
R178 Current Biology 26, R157–R179, February 22, 2016 ª2016 Elsevier Ltd All rights reserved
Current Biology
Dispatches gene copy number in a different way. The surprises likely aren’t over. REFERENCES 1. Albertson, D.G. (2006). Gene amplification in cancer. Trends Genet. 22, 447–455. 2. Fox, D.T., and Duronio, R.J. (2013). Endoreplication and polyploidy: insights into development and disease. Development 140, 3–12. 3. Zybina, T.G., and Zybina, E.V. (2000). Genome multiplication in the tertiary giant trophoblast cells in the course of their endovascular and interstitial invasion into the rat placenta decidua basalis. Early Preg. 4, 99–109. 4. MacAuley, A., Cross, J.C., and Werb, Z. (1998). Reprogramming the cell cycle for endoreduplication in rodent trophoblast cells. Mol. Biol. Cell 9, 795–807.
5. Hannibal, R.L., Chuong, E.B., Rivera-Mulia, J.C., Gilbert, D.M., Valouev, A., and Baker, J.C. (2014). Copy number variation is a fundamental aspect of the placental genome. PLoS Genet. 10, e1004290. 6. Hannibal, R.L., and Baker, J.C. (2016). Selective amplification of the genome surrounding key placental genes in trophoblast giant cells. Curr. Biol. 26, 230–236. 7. Simmons, D.G., Fortier, A.L., and Cross, J.C. (2007). Diverse subtypes and developmental origins of trophoblast giant cells in the mouse placenta. Dev. Biol. 304, 567–578. 8. Hiby, S.E., Apps, R., Chazara, O., Farrell, L.E., Magnus, P., Trogstad, L., Gjessing, H.K., Carrington, M., and Moffett, A. (2014). Maternal KIR in combination with paternal HLA-C2 regulate human birth weight. J. Immunol. 192, 5069–5073.
9. Nakimuli, A., Chazara, O., Hiby, S.E., Farrell, L., Tukwasibwe, S., Jayaraman, J., Traherne, J.A., Trowsdale, J., Colucci, F., Lougee, E., et al. (2015). A KIR B centromeric region present in Africans but not Europeans protects pregnant women from pre-eclampsia. Proc. Natl. Acad. Sci. USA 112, 845–850. 10. Simmons, D.G., Rawn, S., Davies, A., Hughes, M., and Cross, J.C. (2008). Spatial and temporal expression of the 23 murine Prolactin/Placental Lactogen-related genes is not associated with their position in the locus. BMC Genom. 9, 352. 11. Soares, M.J., Konno, T., and Alam, S.M. (2007). The prolactin family: effectors of pregnancy-dependent adaptations. Trends Endocrinol. Metab. 18, 114–121. 12. Wiemers, D.O., Shao, L.J., Ain, R., Dai, G., and Soares, M.J. (2003). The mouse prolactin gene family locus. Endocrinology 144, 313–325.
Current Biology 26, R157–R179, February 22, 2016 ª2016 Elsevier Ltd All rights reserved R179