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Expression and function of Suppressor of zeste 12 in Drosophila melanogaster
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). (Åsa Rasmuson-Lestander)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The development of animals and plants needs a higher order of regulation of gene expression to maintain proper cell state. The mechanisms that control what, when and where a gene should (or should not) be expressed are essential for correct organism development. The Polycomb group (PcG) is a family of genes responsible for maintaining gene silencing and Suppressor of zeste 12 (Su(z)12) is one of the core components in the PcG. The gene is highly conserved in organisms ranging from plants to humans, however, the specific function is not well known. The main tasks of this thesis was to investigate the function of Su(z)12 and its expression at different stages of Drosophila development.

In polytene chromosomes of larval salivary glands, Su(z)12 binds to about 90 specific euchromatic sites. The binding along the chromosome arms is mostly in interbands, which are the most DNA de-condensed regions. The binding sites of Su(z)12 in polytene chromosomes correlate precisely with those of the Enhancer-of-zeste (E(z)) protein, indicating that Su(z)12 mainly exists within the Polycomb Repressive Complex 2 (PRC2). However, the binding pattern does not overlap well with Histone 3 lysine 27 tri-methylations (H3K27me3), the specific chromatin mark created by PRC2. The Su(z)12 binding to chromatin is dynamically regulated during mitotic and meiotic cell division. The two different Su(z)12 isoforms: Su(z)12-A and Su(z)12-B (resulting from alternative RNA splicing), have very different expression patterns during development. Functional analyses indicate that they also have different functions he Su(z)12-B form is the main mediator of silencing. Furthermore, a neuron specific localization pattern in larval brain and a giant larval phenotype in transgenic lines reveal a potential function of Su(z)12-A in neuron development.  In some aspects the isoforms seem to be able to substitute for each other.

The histone methyltransferase activity of PRC2 is due to the E(z) protein. However, Su(z)12 is also necessary for H3K27me3 methylation in vivo, and it is thus a core component of PRC2. Clonal over-expression of Su(z)12 in imaginal wing discs results in an increased H3K27me3 activity, indicating that Su(z)12 is a limiting factor for silencing. When PcG function is lost, target genes normally become de-repressed. The segment polarity gene engrailed, encoding a transcription factor, is a target for PRC2 silencing. However, we found that it was not activated when PRC2 function was deleted. We show that the Ultrabithorax protein, encoded by another PcG target gene, also acts as an inhibitor of engrailed and that de-regulation of this gene causes a continued repression of engrailed. The conclusion is that a gene can have several negative regulators working in parallel and that secondary effects have to be taken into consideration, when analyzing effects of mutants.

PcG silencing affects very many cellular processes and a large quantity of knowledge is gathered on the overall mechanisms of PcG regulation. However, little is known about how individual genes are silenced and how cells “remember” their fate through cell generations.

Place, publisher, year, edition, pages
Umeå University, 2009. , 92 p.
Keyword [en]
Epigenetics, histone, polycomb, Drosophila, Suppressor of zeste 12
National Category
Genetics
Research subject
Genetics
Identifiers
URN: urn:nbn:se:umu:diva-18483ISBN: 978-91-7264-729-9 (print)OAI: oai:DiVA.org:umu-18483DiVA: diva2:159903
Public defence
2009-03-06, Major Groove, , Molecular Biology Department, Umeå University, Byggnad 6L, 10:00 (English)
Opponent
Supervisors
Available from: 2009-02-13 Created: 2009-02-10 Last updated: 2009-02-20Bibliographically approved
List of papers
1. In vivo analysis of Drosophila SU(Z)12 function
Open this publication in new window or tab >>In vivo analysis of Drosophila SU(Z)12 function
2007 (English)In: Molecular Genetics and Genomics, ISSN 1617-4615, E-ISSN 1617-4623, Vol. 279, no 2, 159-170 p.Article in journal (Refereed) Published
Abstract [en]

Polycomb group (PcG) proteins are required to maintain a stable repression of the homeotic genes during Drosophila development. Mutants in the PcG gene Supressor of zeste 12 (Su(z)12) exhibit strong homeotic transformations caused by widespread misexpression of several homeotic genes in embryos and larvae. Su(z)12 has also been suggested to be involved in position effect variegation and in regulation of the white gene expression in combination with zeste. To elucidate whether SU(Z)12 has any such direct functions we investigated the binding pattern to polytene chromosomes and compared the localization to other proteins. We found that SU(Z)12 binds to about 90 specific eukaryotic sites, however, not the white locus. We also find staining at the chromocenter and the nucleolus. The binding along chromosome arms is mostly in interbands and these sites correlate precisely with those of Enhancer-of-zeste and other components of the PRC2 silencing complex. This implies that SU(Z)12 mainly exists in complex with PRC2. Comparisons with other PcG protein-binding patterns reveal extensive overlap. However, SU(Z)12 binding sites and histone 3 trimethylated lysine 27 residues (3meK27 H3) do not correlate that well. Still, we show that Su(z)12 is essential for tri-methylation of the lysine 27 residue of histone H3 in vivo, and that overexpression of SU(Z)12 in somatic clones results in higher levels of histone methylation, indicating that SU(Z)12 is rate limiting for the enzymatic activity of PRC2. In addition, we analyzed the binding pattern of Heterochromatin Protein 1 (HP1) and found that SU(Z)12 and HP1 do not co-localize.

Identifiers
urn:nbn:se:umu:diva-7290 (URN)10.1007/s00438-007-0304-3 (DOI)18034266 (PubMedID)
Available from: 2008-01-07 Created: 2008-01-07 Last updated: 2011-04-08Bibliographically approved
2. Regulation of the Drosophila engrailed gene by Polycomb repressor complex 2
Open this publication in new window or tab >>Regulation of the Drosophila engrailed gene by Polycomb repressor complex 2
2009 (English)In: Mechanisms of Development, ISSN 0925-4773, E-ISSN 1872-6356, Vol. 126, no 5-6, 443-448 p.Article in journal (Refereed) Published
Abstract [en]

Suppressor-of-zeste-12 (Su(z)12) is a core component of the Polycomb repressive complex 2 (PRC2), which has a methyltransferase activity directed towards lysine residues of histone 3. Mutations in Polycomb group (PcG) genes cause de-repression of homeotic genes and subsequent homeotic transformations. Another target for Polycomb silencing is the engrailed gene, which encodes a key regulator of segmentation in the early Drosophila embryo. In close proximity to the en gene is a Polycomb Response Element, but whether en is regulated by Su(z)12 is not known. In this report, we show that en is not de-repressed in Su(z)12 or Enhancer-of-zeste mutant clones in the anterior compartment of wing discs. Instead, we find that en expression is down-regulated in the posterior portion of wing discs, indicating that the PRC2 complex acts as an activator of en. Our results indicate that this is due to secondary effects, probably caused by ectopic expression of Ubx and Abd-B.

Place, publisher, year, edition, pages
Amsterdam: Elsevier, 2009
Keyword
Gene regulation, Su(z)12, Engrailed, Wing disc, Drosophila
National Category
Cell and Molecular Biology Developmental Biology
Identifiers
urn:nbn:se:umu:diva-18646 (URN)10.1016/j.mod.2009.01.004 (DOI)000266975100014 ()
External cooperation:
Available from: 2009-02-20 Created: 2009-02-20 Last updated: 2017-12-13Bibliographically approved
3. The role of Suppressor of zeste 12 in cell cycle regulation
Open this publication in new window or tab >>The role of Suppressor of zeste 12 in cell cycle regulation
(English)Manuscript (Other academic)
Abstract [en]

Polycomb group (PcG) proteins control a large amount of target genes and are essential for genomic programming and differentiation. Many members in the PcG family have been shown to be upregulated in different types of cancers. Suppressor of zeste 12 (Su(z)12) is an essential component in PcG silencing and is necessary for histone 3 lysine 27 tri-methylation in vivo. To unravel a possible role of Su(z)12 in cell cycle regulation, we first investigate the localization pattern of Su(z)12 in Drosophila wildtype testes and embryos by immunohistochemical staining. We found that Su(z)12 was dynamically regulated during cell division. Further investigation of the function of Su(z)12 in cell division was done by cell number counting, apoptosis and proliferation marker staining in Su(z)12 somatic knockout clones in wing discs. The conclusion from the small wing phenotype in Su(z)12 knockout wing discs is that Su(z)12 may increase apoptosis and decrease cell proliferation rate.

Keyword
Su(z)12, apoptosis, proliferation, cell cycle, mitosis
Identifiers
urn:nbn:se:umu:diva-18651 (URN)
Available from: 2009-02-20 Created: 2009-02-20 Last updated: 2010-01-14Bibliographically approved
4. In vivo analysis of Suppressor of zeste 12´s different isoforms
Open this publication in new window or tab >>In vivo analysis of Suppressor of zeste 12´s different isoforms
Show others...
(English)Manuscript (Other academic)
Abstract [en]

Polycomb Group (PcG) genes are known to encode a large chromatin-associated family of proteins which are involved in genomic regulation of many cellular processes. Su(z)12 is a key component in PcG silencing. It is needed for three levels of methylation of histone 3 lysine 27 in vivo in Drosophila. Here, we report that Su(z)12 may exist in different isoforms and that these isoforms are spatially and temporally regulated. The biological function of the Su(z)12-A and -B isoforms seems to be very different. For instance the transgenic Su(z)12-B and the human homolog SUZ12, but not Su(z)12-A, rescue Su(z)12 mutants. Furthermore, transgenic flies over-expressing Su(z)12-B show typical homeotic transformation phenotypes, while over-expression of Su(z)12-A does not. However, the two isoforms appears to be able to substitute for each other in some aspects. During larval and pupal stages, Su(z)12-A seems to play the main role. 

Keyword
Su(z)12, expression, isoforms, Drosophila
Identifiers
urn:nbn:se:umu:diva-18656 (URN)
Available from: 2009-02-20 Created: 2009-02-20 Last updated: 2010-01-14Bibliographically approved

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