investigated using meta-polycentric chromosomes of Pisum sativum. Jasper Manning1, Veit Schubert2, Pavel Neumann1, Andreas Houben2 and Jiri Macas1.
Epigenetic Epigenetic modifications modifications of of plant plant centromeric centromeric chromatin chromatin investigated investigated using using meta-polycentric meta-polycentric chromosomes chromosomes of of Pisum Pisum sativum sativum Jasper Jasper Manning Manning11,, Veit Veit Schubert Schubert22,, Pavel Pavel Neumann Neumann11,,Andreas Andreas Houben Houben22and and Jiri Jiri Macas Macas11 11
Institute Institute of of Plant Plant Molecular Molecular Biology, Biology, Biology Biology Centre Centre ASCR, ASCR, Ceske Ceske Budejovice, Budejovice, Czech Czech Republic; Republic; 22 Leibniz Institute of Plant Genetics and Crop Research (IPK), Gatersleben, Germany Leibniz Institute of Plant Genetics and Crop Research (IPK), Gatersleben, Germany Background Background
Pea Pea (Pisum (Pisum sativum sativum L., L., 2n=14) 2n=14) chromosomes chromosomes are are exceptional exceptional in in the the structure structure of of their their centromeric centromeric regions regions which which appear appear to to be be intermediates intermediates between between monocentric monocentric and and polycentric polycentric types. types. The The so-called so-called meta-polycentric meta-polycentric chromosomes chromosomes show show extended extended primary primaryconstrictions constrictions which which contain contain 3-5 3-5 separate separate clusters clusters of of the the centromeric centromeric histone histone variant variant CenH3. CenH3. Large Large pericentromeric pericentromeric regions regions and and multiple multiple CenH3 CenH3 loci loci make make pea pea chromosomes chromosomes suitable suitable for for analyzing analyzing the the distribution distribution of of various various epigenetic epigenetic modifications modifications that that are are supposed supposed to to play play an an important important role role in in the the determination determination of of peri/centromeric peri/centromeric chromatin. chromatin.
Panel 1 Methods Methods Immunodetection Immunodetection of of various various epigenetic epigenetic modifications modifications was was accomplished accomplished using using specific specific antibodies antibodies to to histones histones H2A H2Aand and H3. H3. This This technique technique was was combined combined with with fluorescent fluorescent in in situ situ hybridization hybridization (FISH) (FISH) using using PisTR-B, PisTR-B, aa tandem tandem repeat repeat that that is is used used to to distinguish distinguish individual individual chromosomes. chromosomes. Pea Pea CenH3 CenH3 antibody antibody was was employed employed to to visualize visualize the the position position of of functional functional centromere centromere domains. domains.
Chr1
Chr2
Chr3
Chr4
Chr5
Chr6
Chr7
CenH3
PisTR-B
Panel 2
Panel 3 H3Ser28ph CENH3
H3K4me2 PisTR-B
H3K4me2 CENH3
H3K4me3 PisTR-B
H3K4me3 CENH3
H3K9me2 CENH3
H3K9me2 PisTR-B
H3Thr3ph CENH3
H3K27Ac CENH3
H3K27Ac PisTR-B
H3K27me2 CENH3
H3K27me2 PisTR-B
H3Ser10ph CENH3
H3Ser10ph PisTR-B H2AThr120ph CENH3
H3Ser28ph CENH3
H3Ser28ph PisTR-B
H3Thr3 CENH3
H3Thr3ph PisTR-B
H2AThr120 CENH3
H2AThr120ph PisTR-B
Panel 1. FISH detection of the tandem repeat PisTR-B (Green) combined with immunocytochemical detection of histone modifications (Red) on metaphase chromosomes. Panel 2. Combined immunodetection using antibodies to CenH3 and various histone modifications.
Panel 3. Structured illumination Microscopy (SIM) visualization of immundetection staining of the histone variant CENH3 in combination with histone marks H3Thr3, H2AThr120ph and H3Ser28ph on metaphase chromosomes.
References: References: 1. 1.Neumann NeumannP,P,Pozarkova PozarkovaD, D,Vrana Vrana J, J,Dolezel DolezelJJand andMacas MacasJ. J. Chromosome Chromosomesorting sortingand andPCR-based PCR-basedphysical physicalmapping mappingin inpea pea (Pisum (Pisumsativum sativumL.) L.)Chromosome ChromosomeRes. Res.2002;10(1):63-71. 2002;10(1):63-71. 2. 2.Neumann NeumannP,P,Navratilova NavratilovaA, A,Schroeder-Reiter Schroeder-ReiterE, E,Klobizkova KlobizkovaA, A, Steinbauerova SteinbauerovaV, V,Chocholova ChocholovaE, E,Nova NovaP, P,Wanner WannerGGand andMacas MacasJ. J. Stretching Stretchingthe therules: rules:monocentric monocentricchromosomes chromosomeswith withmultiple multiple centromere centromeredomains. domains.PLoS PLoSGenet. Genet.2012;8(6):e1002777. 2012;8(6):e1002777.
Summary Summary
Due Dueto tothe theextended extendedprimary primaryconstriction constrictionand andthe themultiple multipleCenH3 CenH3loci lociwithin withinthe thecentromere centromere of of P. P.sativum, sativum,the thepericentromere/centromere pericentromere/centromereis isideal idealfor forthe thestudy studyof ofepigenetic epigeneticmodifications. modifications. Signals from histone modifications were distinctly different in the pericentromere and on the chromosome arms Signals from histone modifications were distinctly different in the pericentromere and on the chromosome arms Immunodetection analysis of CenH3 combined with the selected histone modifications did not show Immunodetection analysis of CenH3 combined with the selected histone modifications did not show colocalization colocalizationof ofthe thetwo twosignals. signals. SIM microscopy showed that signals of H2AThr120ph were intermingled with CENH3. SIM microscopy showed that signals of H2AThr120ph were intermingled with CENH3. A detailed picture using SIM demonstrated there was no colocalization between phosphorylated marks and A detailed picture using SIM demonstrated there was no colocalization between phosphorylated marks and CenH3 CenH3within withincentromere. centromere.
