Maike StamGroup leader Maike Stam

tel.: 020-5257655
e-mail: m.e.stam@uva.nl

Gene regulation in trans

Regulation of gene activity via the binding of protein factors to sequence elements in cis is being studied in great detail. In contrast, little is known about gene regulation in trans, i.e. how sequences on one chromosome control the activity of a gene on another chromosome. There is mounting evidence that such trans-regulatory systems are important in higher eukaryotes, including flies, plants and mammals. Our research is aimed at unravelling molecular mechanisms underlying gene regulation in trans.

We concentrate on a naturally occurring trans-regulatory system: the genetically well-studied maize b1 paramutation system. Paramutation is a mitotically and meiotically heritable change in the expression of one allele caused by a trans-interaction with another (epi)allele of the same gene. With b1 paramutation, the low expressed epiallele (B’;resulting in light colored plants) imposes its low transcription rate onto the high expressed epiallele (B-I; resulting in dark colored plants) in trans, thereby changing the B-I into a B’ epiallele. Paramutation-like phenomena have not only been observed in plants, but also in fungi and mammals, emphasizing its significance. The underlying molecular mechanism involves RNA-dependent RNA polymerase (RdRP; Alleman (2006) Nature 442:295), but may in addition also involve physical contact between chromosomal regions

Figure 1. b1 paramutation.  b1 encodes a transcription factor activating the maize pigmentation pathway. With b1 paramutation, a low (B’; light purple plant) and high (B-I; dark purple plant) expressing b1 epiallele interact in trans, resulting in the heritable change of the B-I into the B’ epiallele. Seven 853 bp repeats (hepta-repeat: black triangles), located 100 kb upstream of the b1 coding region, are required for the trans-interaction (yellow arrow), and also for enhancing b1 expression (green arrow; 3,4). We propose the repeats mediate trans-inactivation in the B', and enhancer activity in the B-I epigenetic state. Keys: red circles = silencing protein complex; purple oval = enhancer protein complex.

Role of DNA methylation and chromatin structure in b1 paramutation and expression
Seven 853 bp tandem repeats (hepta-repeat), located about 100 kb upstream of the b1 coding region, are essential for the trans-interaction (Stam et al., 2002, Genes & Dev. 16:1906 & Genetics 162:197), and in addition required for the high B-I expression level. Paramutation at the b1 locus involves changes in DNA methylation and chromatin structure at the hepta-repeat sequences. We investigate the role of DNA methylation and chromatin structure in the epigenetic regulation of the b1 alleles, using DNA blot analyses and chromatin immunoprecipitation (ChIP; Rechien Bader, Research technician; Max Haring, PhD student). The regulatory hepta-repeat is located 100 kb upstream of the b1 transcription start site, indicating long-range in cis interactions. We have recently implemented 3C technology to study the role of physical in cis interactions in the regulation of b1 expression (Marieke Louwers, PhD student). To study cause and effect relationships we make use of trans-acting mutations affecting various aspects of paramutation.

Figure 2. Chromosome Conformation Capture (3C). To identify spatial, long-distance in cis interactions within the ~110 kb b1 chromatin domain, we use the Chromosome Conformation Capture technique (3C). Maize tissue is fixed in formaldehyde, crosslinking protein complexes and DNA. The nuclei are then isolated and permeabilized. The crosslinked DNA-protein network is subjected to restriction digestion, followed by a ligation step under low DNA concentration. After reversal of the crosslinks, the ligation products are detected by quantitative PCR (qPCR). 3C analysis thus provides information about the spatial organization of chromosomal regions in vivo.

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 by fluorescence 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. and Chandler, 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. and Sidorenko, L.V. (2002) Long distance cis and trans interactions mediate paramutation. In: Advances in Genetics, Vol. 46. J.C. Dunlap and C.-ting Wu. Academic Press, San Diego, USA, pp. 215-234.

13 Chandler V.L., Stam M. (2004) Chromatin conversations: mechanisms and implications of paramutation. Nat Rev Genet. 5, 532-44.

14 Stam M., Mittelsten Scheid O. (2005) Paramutation: an encounter leaving a lasting impression. TIPS 10, 283-290.

15. 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.

16 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.

17 Haring M, Bader R, Louwers M, van Driel R, Stam M (2008) The role of DNA methylation, nucleosome positioning and histone modifications in paramutation in maize, submitted.

18 Stam, M, Louwers, M. (2008) Paramutation: Heritable in Trans Effects. In maize handbook. In Press.

Techniques

  • Recombinant DNA technology
  • regular and quantitative PCR
  • DNA and RNA analyses
  • plant transformation
  • Chromatin immunoprecipitation (ChIP)
  • Chromosome conformation capture (3C)
  • Genetics

Educational contributions

  • Bachelorcourse BW08K eukaryote genregulatie (lecturer)
  • Mastercourse Frontiers in Plant Sciences (Lecturer)
  • Mastercourse 001LS Biotechnology (coordinator and lecturer)
  • Supervision internships
  • Supervision literature projects

Collaborations

  • Our collaboration with Christoph Peterhaensel and his group at the Institute for Biology I, Aachen University, Germany resulted in the optimization of the ChIP technique for plants tissue and especially maize.
  • Our collaboration with Wouter de Laat and his group at the Erasmus University in Rotterdam and soon the Hubrecht Institute in Utrecht, The Netherlands, resulted in the optimization of the 3C technique for plants tissue.
  • We have a long-term collaboration with Vicki Chandler and her group at the University of Arizona, Tucson, Arizona, USA, on paramutation.

Visiting address
Maike Stam
Swammerdam Institute for Life Sciences
Universiteit van Amsterdam
Kruislaan 318
1098 SM Amsterdam
The Netherlands
Tel: +31-20-5257655
FAX: +31-20-5257935
m.e.stam@uva.nl