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Novel insights into gene silencing mechanisms in Zea mays and Arabidopsis
thaliana
Hövel, I.
Publication date
2016
Document Version
Final published version
Link to publication
Citation for published version (APA):
Hövel, I. (2016). Novel insights into gene silencing mechanisms in Zea mays and Arabidopsis
thaliana.
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Novel insights into
gene silencing mechanisms
in
Zea mays
and
The research described in this thesis was funded by the Research Priority Area Systems
Biology of the University of Amsterdam and was carried out at the laboratory of Sythetic
Systems Biology and Nuclear Organization, Swammerdam Institute for Life Sciences,
University of Amsterdam, The Netherlands.
Title:
A picture of Arabidopsis thaliana seedlings with a light microscopy image of Arabidopsis
“The idea was fantastically, wildly improbable.
But like most fantastically, wildly improbable ideas
it was at least as worthy of concideration as a more mundane one
to which the facts had been strenuously bent to fit.”
Novel insights into gene silencing mechanisms
in Zea mays and Arabidopsis thaliana
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof. dr. D.C. van den Boom
ten overstaan van een door het College voor Promoties ingestelde commissie,
in het openbaar te verdedigen in de Aula der Universiteit
op woensdag 08 Juni 2016, te 13:00 uur door
Iris Hövel
Promotiecommissie
Promotor:
prof. dr. W.J. Stiekema,
Universiteit van Amsterdam
Copromotor:
dr. M.E. Stam,
Universiteit van Amsterdam
Overige leden:
prof. dr. G.C. Angenent,
Wageningen Universiteit
dr. A.T. Groot,
Universiteit van Amsterdam
prof. dr. J.F. Gutierrez-Marcos,
University of Warwick, United Kingdom
prof. dr. R.E. Koes,
Universiteit van Amsterdam
dr. R.J.T.S.. Palstra,
Erasmus MC Rotterdam
prof. dr. H.V. Westerhoff,
Universiteit van Amsterdam
Table of Contents
Chapter 1: Introduction - Gene silencing in Zea Mays and Arabidopsis thaliana via
different repressive mechanisms ... 1
Gene activity is determined by different levels of chromatin structure and organization... 2
DNA and histone modifications ... 2
Long-range chromosomal interactions ... 3
Functional chromatin domains ... 4
Distinct transcriptional silencing machineries are dedicated to different types of chromatin... 5
RdDM silencing ... 5
Polycomb silencing ... 8
Outline of this thesis ... 9
Chapter 2: Cis-acting determinants of paramutation ... 13
Abstract ... 14
Introduction ... 15
Defining paramutation ... 16
Canonical RdDM model in relation to paramutation ... 18
The various paramutation phenomena display different behavior ... 19
Cis-acting determinants of paramutation ... 21
Genetic features of cis-acting sequences ... 21
Differential DNA methylation and chromatin features of cis-acting sequences required for paramutation ... 26
A refined model for siRNA-mediated paramutation... 29
Outlook ... 32
Chapter 3: The B’ epiallele is activated in the maize RNA-dependent RNA polymerase mutant mop1-1 despite the presence of repressive epigenetic marks ... 35
Abstract ... 36
Introduction ... 37
Results ... 39
Approach... 39
Regulatory sequences at the b1 locus are activated in B’ mop1-1 ... 39
mop1-1 allows the formation of multiple chromatin loops at b1 ... 40
mop1-1 causes an intermediate level of regulatory activity in B’ ... 41
Hepta-repeat DNA methylation level remains high in B’ mop1-1 ... 43
Mainly symmetric DNA methylation at differentially methylated repeat region ... 44
Genome-wide variation in mCHH levels associated with MOP1 activity ... 46
In mop1-1 the B’ hepta-repeat retains H3K9me2 and H3K27me2 ... 48
mop1-1 does not significantly affect nucleosome occupancy at the B’ repeats... 49
Discussion ... 50
Materials and Methods ... 56
Chapter 4: Two mutants in the RdDM pathway, affecting Pol IV and Pol V, have
distinct effects on the chromatin structure of the paramutagenic B’ allele ... 75
Abstract ... 76
Introduction ... 77
Results ... 80
Approach... 80
b1 expression levels are increased in mop3, but not mop2 mutants ... 80
The regulatory hepta-repeat is activated in a mop3, but not in mop2 mutants ... 82
B’ mop3 loses, while B’ mop2 retains repressive histone marks at the hepta-repeat ... 84
Repressive histone marks at B’ locus in mop3-1 mutants are reduced before transcriptional activation of the b1 gene ... 86
B’ transcript levels do not correlate with DNA methylation levels at repeat junction region in both mop2-1 and mop3-1 mutants ... 86
Activated regulatory sequences of the b1 locus are associated with the formation of a multi-loop structure ... 89
Discussion ... 92
Material and methods ... 96
Supporting Information ... 99
Chapter 5: A maize paramutagenic transgenic locus partially repressed by RdDM is marked with H3K27me2, but not with H3K9me2 ... 109
Abstract ... 110
Introduction ... 111
Results ... 114
NYR expression is variegated in vNYR and mostly uniform in aNYR tissue ... 114
The NYR transgene is not marked with H3K9me2 ... 114
H3K27me2 levels at the vNYR transgene decrease after reversion to aNYR ... 115
Activation of vNYR in rmr1-1 is associated with increased H3ac at the locus ... 115
Discussion ... 118
vNYR is an RdDM locus devoid of H3K9me2 ... 118
Chromatin states of vNYR and B’ are distinct from each other ... 118
H3ac and H3K27me2 enrichment at vNYR may be explained by a variegated and bivalent state ... 119
Does the neighboring Grande TE repress transcription of NYR as well? ... 120
Material and Methods ... 121
Supporting Information ... 123
Chapter 6: 3C Technologies in plants ... 129
Abstract ... 130
1. Introduction ... 131
2. Practicalities of 3C in plants ... 134
3. Method ... 137
4. Materials ... 144
5. Hints for troubleshooting ... 144
Chapter 7: Changes in chromosomal interaction patterns of FLC upon vernalization ... 153
Abstract ... 154
Introduction ... 155
Results and Discussion ... 157
Experimental approach... 157
Preference for chromosomal interactions in cis is reduced during vernalization ... 159
Full vernalization induces a redistribution of preferential interactions of FLC within Chr5L ... 160
Upon vernalization frequencies of specific distant interactions increase, while frequencies of specific short-range interactions increase or decrease ... 162
Only changes in short-range interaction frequencies correlate with H3K27me3 enrichment of FLC upon vernalization ... 164
Interactions of the FLC gene sequence with directly adjacent sequences decrease significantly upon vernalization ... 167
The average nuclear size is elevated during cold treatment ... 168
The average DNA content of nuclei is elevated during cold treatment ... 170
Conclusion and perspectives ... 171
Material and methods ... 172
Supporting Information ... 176
Chapter 8: General discussion ... 187
DNA methylation and chromatin structure of two different paramutagenic loci in maize are distinct ... 188
Is RdDM sufficient for paramutation?... 188
Tandem repeats act as cis-determinants of paramutation ... 188
Distinct DNA methylation patterns displayed by different RdDM-targeted paramutagenic loci ... 189
The chromatin structure of the B’ and vNYR paramutagenic loci is different ... 190
An inverse gradient of mCHH and H3K9me2 emphasizes the variability of RdDM loci ... 191
Chromosomal interaction pattern of a polycomb target in Arabidopsis ... 192
Final remarks ... 194
References ... 197
Summary ... 217
Samenvatting in het Nederlands ... 219
Publications ... 221
