Gene regulation by epigenetics and chromosomal interactions

The Stam subgroup studies the role of epigenetic mechanisms and chromosomal interactions in gene regulation and in targeted mutagenesis using maize and Arabidopsis as a model system. Both Epigenetic gene regulation and chromosomal interactions are essential for normal growth and development. Epigenetic gene regulation refers to mitotically or meiotically heritable changes in gene expression that do not involve DNA sequence changes, but changes in DNA methylation and chromatin structure instead. Recent studies have shown that, besides gene control by nearby cis -acting regulatory elements, gene regulation in higher eukaryotes also involves physical interactionsbetween chromosomal regions up to hundreds of kilobase pairs apart on thesame chromosome ( cis ), or between regions located on different chromosomes ( trans ).

In the context of epigenetics and chromosomal interactions different studies are being performed

1) Unraveling the mechanisms underlying paramutation at the maize B-I and B' alleles

We investigate gene regulation in trans . As a modelsystem we study a proces called paramutation, a mitotically and meiotically heritable change in gene expression induced by allele interactions in trans . We examine paramutation at b1 , a regulatory gene of the maize pigmention pathway. With b1 paramutation, the low-expressing B' epiallele communicates in trans with the high-expressing B-I epiallele, changing B-I into B' in a mitotically and meiotically heritable manner. B' and B-I have the same DNA sequence, but differ in DNA methylation and chromatin structure and are therefore called epialleles. The expression of the b1 gene is controlled by regulatory sequences up to at least 110 kb upstream. Recent data indicate a role for RNA in paramutation, but also suggest that siRNAs may not be not sufficient.  Seven tandem repeats, ~100 kb upstream of the b1 coding region, are essential for trans-inactivation and for high b1 expression. High b1 expression is tissue-specific and associated with H3ac and nucleosome depletion at the B-I hepta-repeat (Haring et al. 2010 Plant J). The repeats of B' are DNA methylated in a tissue-independent manner. DNA methylation appears strongly associated with the mitotic heritability and paramutagenicity of B' .   3C technology showed a role for physical interactions between the hepta-repeat and b1 promoter in tissue-specific regulation of b 1 (Louwers et al 2009, Plant Cell). High b1 expression appears mediated by multiple physical interactions; besides the hepta-repeat, other sequence regions physically interact with the transcription start site as well, and these interactions are epiallele- and expression level-specific. FAIRE and ChIP uncovered multiple of these regions as potentially regulatory.  Rechien Bader (research technician) investigates the epigenetic mechanisms underlying paramutation.       

2) Quantitative relationships between chromatin looping and gene activity

The functional relationship between gene activity and long-distancechromosomal interactions is investigated using Arabidopsis thaliana as a model organism. This study is a collaboration with Dr. Paul Fransz (NOG) and employs Chromatin Conformation Capture technology and 3D-FISH. Mathematical modeling will be used to establish quantitative relationships between chromatin folding and gene activity (collaboration with Prof. D. Heermann, Heidelberg). Iris Hövel (PhD student) is appointed on this project.

3) The role of epigenetic regulation and chromosomal interactions in hybrid vigour

The aim of this project, which is performed in collaboration with the groups of Prof. Angenent, Dr. Keurentjes and Dr Sanchez Perez at Wageningen University, is to enhance the understanding of the role of epigenetic mechanisms and chromosomal interactions in hybrid vigour, which has been a major innovation improving the yield of food crops. To further improve yield, it is essential to understand the mechanisms underlying hybrid vigour. Recent data obtained by other groups indicate a role for epigenetic regulation in hybrid vigour. Kathrin Lauss (PhD student) is appointed on this project.

4) Epigenetics meets targeted mutagenesis

To meet the global food challenge, plant breeders are continuously searching for new sources of genetic variation and are consequently interested in novel technologies for precise genome engineering, e.g. oligonucleotide-directed mutagenesis (ODM) . Such technologies allow the introduction of small, targeted modifications, but are hampered by low efficiencies. This 4-year project, funded by STW and industrial partners, aims at identifying epigenetic drugs and/ormutations that significantly enhance the efficiency of ODM. Arabidopsis will be used as model system; promising results will be evaluated in tomato. The project will be performer by a Post-doc and technician, in close collaboration with Dr. Michiel de Both and Dr. Franck Lhuissier of KeyGene (Wageningen), and with four international seed companies. 

5) Epigenetic regulation of economically important plant traits (EpiTRAITS - www.epitraits.eu)

Maike Stam is coordinator of the EU FP7 Marie Curie ITN EpiTRAITS (12 full partners, 3 associated partners; 11 PhD and 3 Post-doc positions, start date October 2012). The mission of EpiTRAITS is to train young researchers in epigenetic gene regulation and flowering in the model plant Arabidopsis thaliana and the crop plants maize ( Zea mays ) and barley ( Hordeum vulgare ). EpiTRAITS will focus on one of the key plant traits, flowering, which is controlled by various epigenetic mechanisms. The scientific program aims to bridge the gap between fundamental and applied research by translating results from epigenetic research in model organisms to improved technologies for crop breeding and molecular diagnostic tools.

Nuclear Organisation Group

I am one of the PIs of the Nuclear Organisation Group (NOG) of the Swammerdam Institute for Life Sciences (SILS)

Student Projects

There are possibilities for students to do an internship in our group. Please enquire for the projects currently available. Every internship will involve a range of techniques. Techniques to be used are amongst others recombinant DNA technology, regular and quantitative PCR, DNA blot analyses, RNA blot analyses, plant transformation, fluorescent in situ hybridization (FISH) and microscopy

Peer reviewed journal publications

1. Van der Meer, I.M., Stam, M.E., van Tunen, A.J., Mol, J.N.M. and Stuitje, A.R. (1992) Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility. Plant Cell 4, 253-262.  
2 . Van Aarssen, R., Soetaert, P., Stam, M., Dockx, J., Gosselé, V., Seurinck, J, Reynearts, A. and Cornelissen (1995) cry IA(b) transcript formation in tobacco is inefficient. Plant Mol. Biol.   28, 513-524.  
3 .Fransz, P.F., Stam, M.,Montijn, B., TenHoopen, R., Wiegant, J., Kooter, J.M/, Oud, O. and Nanninga, N (1996) Detection of single copy genes and chromosome rearrangements in Petunia hybrida byfluorescence in situ hybridization. Plant J. 9, 767-774.  
4 . Kunz, C., Schöb, H., Stam, M., Kooter, J.M. and Meins, Jr. (1996) Developmentally regulated silencing and reactivation of tobacco chitinase transgene expression. Plant J. 10, 437-450.  
5. Stam, M. , Mol, J.N.M and Kooter, J.M. (1997) The silence of genes in transgenic plants. Annals of Botany 79, 3-12.  
6. Stam, M. , de Bruin, R., Kenter, S., van der Hoorn, R.A.L., van Blokland, R., Mol, J N.M. and Kooter, J.M. (1997) Post-transcriptional silencing of endogenous genes in Petunia by inverted transgene repeats. Plant J. 12, 63-82.  
7. Stam, M. , Viterbo, A., Mol, J. N.M. and Kooter, J.M. (1998) Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats. Implications for posttranscriptional silencing of homologous host genes in plant. Mol Cell Biol 18, 6165-6177.  
8 . Jorgensen, R. A., Que, Qiudeng and Stam, M. (1999) Do unintended antisense transcripts contribute to sense co-suppression in plants? Trends in Genet. 15, 11-12.  
9. Stam, M ., de Bruin, R., Van Blokland, R., Van der Hoorn, R.A.L., Mol, J.N.M. and Kooter, J. (2000) Distinct features of post-transcriptional gene silencing by antisense transgenes in single copy and inverted T-DNA repeat loci. Plant J. 21, 27-42.  
10 Stam, M. , Belele, C., Dorweiler, J and Chandler, V.L. (2002) Differential chromatin structure within a tandem array 100 kb upstream of the maize b1 locus is associated with paramutation. Genes and Development 16, 1906-1918.  
11 Stam, M. , Belele, C., Ramakrishna, W., Dorweiler, J., Bennetzen, J. andChandler, V.L. (2002) The regulatory regions required for B' paramutation and expression are located far upstream of the maize b1 transcribed sequences. Genetics 162, 917-930.  
12 Chandler V.L. , Stam M. (2004) Chromatin conversations: mechanisms and implications of paramutation. Nat Rev Genet. 5, 532-44.  
13 Stam M., Mittelsten Scheid O. (2005) Paramutation: an encounter leaving a lasting impression. TIPS 10, 283-290.  
14 Haring M , Offermann S , Danker T , Horst I , Peterhaensel C , Stam M . (2007) Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods, 3, 11. 
15 . Louwers M, Bader R, Haring M, van Driel R, de Laat W and Stam M (2009) Tissue- and expression level-specific chromatin looping at maize b1 epialleles. Plant Cell, 21, 832.  
16. Stam M. (2009) Paramutation: A heritable change in gene expression by allelic interactions in trans. Molecular Plant 2009 2(4):578-588;doi:10.1093/mp/ssp020.  
17 . Louwers, M, Splinter, E. van Driel R, de Laat, W and Stam, M (2009) Studying physical chromatin interactions in plants using Chromosome Conformation Capture (3C). Nature Protocols, 4 (8): 1216-1229.  

18.Haring M, Bader R, Louwers M, van Driel R, StamM(2010) The role of DNA methylation, nucleosome positioning and histone modifications in paramutation in maize. The Plant Journal 63(3), 366-378. doi: 10.1111/j.1365-313X.2010.04245.x. Recommended by Faculty of 1000 Biology (http://www.f1000biology.com/article/id/3908956)

19.Hövel, I., Louwers, M.L.D., and Stam, M. (2012). 3C technologies in plants. METHODS, 58, 204-211. doi.org/10.1016/j.ymeth.2012.06.010.

20. Belele C*, Sidorenko L*, Stam M*, Bader R, Arteaga-Vazquez MAand Chandler VL (2013) Specific tandem repeats are sufficient for paramutation-inducedtrans-generational silencing. PLoS Genetics 9(10), e1003773.doi: 10.1371/journal.pgen.1003773.* first co-authors.

21.Gent J.I., Madzima T.F., BaderR., Kent M.R., Zhang X., Stam M., McGinnis K.M., and Dawe R.K. (2014) Accessible DNA and relative depletion of H3K9me2 at maize RdDM loci. The Plant Cell 26, 4903-4917. www.plantcell.org/cgi/doi/10.1105/tpc.114.130427

22.Hövel, I., Pearson, N.A. and Stam, M.(2015).Cis-acting determinants of paramutation.Semin Cell Dev Biol. pii: S1084-9521(15)00156-1. doi: 10.1016/j.semcdb.2015.08.012.

23.Weber, B.*, Oka, R.*, Zicola, J.*, Stam, M.(2016). Plant Enhancers: A Call for Discovery.Invited review by Trends in Plant Science 21, pp. 974-987. doi: 10.1016/j.tplants.2016.07.013.*co-first authors.

24.Anggoro, D.T., Tark-Dame, M., Walmsley, A., Oka, R., de Sain, M.,Stam, M.(2017) BIBAC-GW-based vectors for generating reporter lines for site-specific genome editing in planta. Plasmid 89, pp. 27-36. http://dx.doi.org/10.1016/j.plasmid.2016.12.002

25.Oka^*, R., Zicola^*, J., Weber^*, B., Anderson, S.N., Hodgman, C., Gent, J.I., Wesselink, J.J., Springer, N.M., Hoefsloot, H.C.J., Turck, F.,Stam, M.(2017) Genome-wide Mapping of Transcriptional Enhancers Using Chromatin Features in Zea mays. Genome Biology 18:137. doi: 10.1186/s13059-017-1273-4. *co-first authors.

26.Suraj Jamge,Maike E. Stam, Richard G.H. Immink, Gerco C. Angenent. (2017) A cautionary note on the use of chromosome conformation capture in plants. Plant Methods 13: 101.DOI 10.1186/s13007-017-0251-x.

27. Kathrin Lauss, René Wardenaar, Rurika Oka, Marieke H.A. van Hulten, Damar T. Anggorro, Frank Becker, Victor Guryev, Joost J.B. Keurentjes, Maike Stam*, Frank Johannes*. (2018) Parental DNA Methylation States Are Associated with Heterosis in Epigenetic Hybrids. Plant Physiology. 176: 1627-1645. doi: 10.1104/pp.17.0105.*Corresponding co-last authors.

28 Jihed Chouaref,  Esther de Boer,Paul Fransz,and Maike Stam (2018) Protocol for Chromatin Immunoprecipitation of Meiotic-Stage-Specific Tomato Anthers. Current Protocols in Plant Biology.3(3):e20074. doi: 10.1002/cppb.20074.

29. Marieke van Hulten, Maria-Joāo Paulo, Willem Kruijer, Hetty Blankestijn-De Vries, Brend Kemperman, Frank F. M. Becker, Jiaming Yang, Kathrin Lauss, Maike E. Stam, Fred A. van Eeuwijk, Joost J.B. Keurentjes (2018).Assessment of heterosis in two Arabidopsis thalianacommon-reference mapping populations. PLoS One Oct 12;13(10):e0205564. doi: 10.1371/journal.pone.0205564.

Invited peer reviewed book chapters/proceedings

1. Van der Meer, I.M., Stam, M.E., van Tunen, A.J., Mol, J.N.M. and Stuitje, A.R. (1992) Inhibition of   flavonoid biosynthesis in Petunia anthers by antisense RNA: a novel way to engineer nuclear male sterility. In: Angiosperm pollen and ovules, basic and applied aspects. E. Ottaviano, D.L. Mulcahy, M. Sari Gorla and G. Bergamini Mulcahy, eds. Springer-Verlag New York, Inc., 175 Fifth Avenue, New York, NY 10010, USA, pp 22-27.  
2. Kooter, J.M., Van Blokland, R., de Lange, P., Stam, M. and Mol, J.N.M. (1993). The use of antisense and sense genes to generate mutant phenotypes: Suppression of flower pigmentation in Petunia. Proceedings XVI Journées Internationales du Groupe Polyphenols. 
3. Mol, J.N.M., van Blokland, R., de Lange, P., Stam, M. and Kooter, J.M. (1994) Post-transcriptional inhibition of gene expression: sense and antisense genes. In J Paszkowski, ed, Homologous Recombination and Gene silencing in Plants. Kluwer, Dordrecht, The Netherlands, pp 309-334.  
4. Van Blokland, R., Van der Geest, N., De Lange, P., Stam, M., Mol, J.N.M. and Kooter, J.M. (1996)   Post-transcriptional suppression of chalcone synthase genes in Petunia hybrida and the accumulation of unspliced pre-mRNAs. In: Mechanisms and Applications of Gene Silencing. D. Grierson, G.W. Lycett and G.A. Tucker. Nottingham: Nottingham University Press, pp. 57-69.  
5. Stam, M., T. Lavin and V. Chandler (2000) Npi402 and ncsu1 are identical; inra1 (tmp) maps upstream of the b promoter. Maize Genetics Coop Newsl. 74 : p. 66-67.  
6. Chandler, V.L., Stam, M. and Sidorenko, L.V. (2002) Long distance cis and trans interactions mediate paramutation. In: Advances in Genetics, Vol. 46. J.C.Dunlapand C.-ting Wu. Academic Press, San Diego, USA, pp. 215-234.  
7. Louwers, M., Haring, M. and Stam, M. ( 2005) When alleles meet: Paramutation. In P. Meyer, ed, Plant Epigenetics. Blackwell Publishing Ltd, Oxford, UK, 134-173.  
8. Stam, M, Louwers, M. (2009) Paramutation: Heritable in Trans Effects. In Handbook of Maize; Genetics and Genomics. Jeff Bennetzen and Sarah Hake (eds). Springer New York. pp 405-427. 
9. Hövel, I., Louwers, M.L.D., and Stam, M. (2012). 3C technologies in plants. METHODS, in press http://dx.doi.org/10.1016/j.ymeth.2012.06.010.

10. Weber, B.*, Jamge. S.*, Stam, M.E.3C in Maize and Arabidopsis (2017). Marian Bemer and Celia Baroux (eds.), Plant Chromatin Dynamics: Methods and Protocols, Methods in Molecular Biology, vol. 1675, pp 247-270. DOI 10.1007/978-1-4939-7318-7_15. *co-first authors.

Patent applications

"MODIFIED BT GENE II"- Invention: Cornelissen, Soetaert, Stam, Dockx- Plant Genetic Systems n.v., filed in USA and Canada, PCT/EP 91/00733; EP 91402920.2; EP 92400820.4; PCT/EP92/02547; PCT/EP92/02547.

Rechien Bader, research technician

Title Project: Unraveling the mechanisms underlying paramutation at the maize B-I and B' alleles

r.bader@uva.nl

Iris Hövel, PhD student

Title Project: Quantitative relationships between chromatin looping and gene activity 

Systems Biology Project University of Amsterdam

I.Hovel@uva.nl

Kathrin Lauss, PhD student

Title project: The role of Epigenetic regulation and chromosomal interactions in hybrid vigour 

CIPY project, http://www.cipy.nl 

K.Lauss@uva.nl

Mariliis Tark-Dame, Post-doc

Title project: Epigenetics meets targeted mutagenesis

Open Technology Program of STW, project in close collaboration with KeyGene (Wageningen, The Netherlands) 

m.tark@uva.nl

Damar Anggoro, Research Technician

Title project: Epigenetics meets targeted mutagenesis

Open Technology Program of STW, project in close collaboration with KeyGene (Wageningen, The Netherlands)

D.T.Anggoro@uva.nl

Picture will follow soon 

Blaise Weber, PhD student (starts March 1, 2013)

Title project: Epigenetic regulation of economically important plant traits

EU-FP7 Marie Curie ITN EpiTRAITS

Picture will follow soon 

Former PhD students

Max Haring 
Marieke Louwers

Former MSc Students

Joke van Bemmel (2004-2005) 
Yan Yang (2005) 
Mara de Sain (2007-2008) 
Gerben Marsman (2008) 
Michael Ignarski (2008-2009) 
Pegah Poorfaraj (2009-2010) 
Bas Jansen (2011)

Former HLO students

Erica Geers (2005-2006) 
Elisa Teunissen (2006) 
Annemarie Castricum (2011-2012)