Transcript Expressing_CENH3_Orthologs

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Ron M. G. Menorca • Pak Kwong • Ravi Maruthachalam • Simon Chan
College of Biological Sciences: Section of Plant Biology
University of California Davis, 95616
Faithful chromosome segregation is mediated by the centromere and the
kinetochore (1). The main protein that recruits other kinetochore proteins and
assembles a functional centromere is the centromere-specific histone H3 (CENH3)
which replaces the canonical histone H3 in centromeric
chromatin (2).
Conventional histone H3 is highly conserved due to its importance in DNA
packaging. In contrast, the CENH3 primary sequence has been shown to evolve
rapidly. We hypothesize that there is a conserved structure that underlies CENH3
function, despite the lack of sequence conservation. To test this hypothesis, we
have replaced Arabidopsis thaliana CENH3 with orthologs from other organisms
to determine which sequences are essential for a functional CENH3. We have
found that only the closely related CENH3 from Arabidopsis arenosa localized
correctly while the others from distantly related species did not. Further
experiments will test CENH3s from other closely related species, strengthening
our knowledge about the properties of the centromere histone and its influence on
chromosome segregations.
INTRODUCTION
The centromere is the locus on the chromosome
where the kinetochore, which will be bound by the
spindle microtubules, assembles. There are three
broadly defined types of centromere DNA
sequences :
•
•
*Nature Reviews Neuroscience
Normal histone H3 is highly conserved due to its importance in DNA packaging. In contrast, CENH3 primary sequence has evolved rapidly even between
species with similar centromere structure.
We hypothesize that there is a conserved structure that underlies CENH3
function, despite the lack of sequence conservation.
GFP-CENH3/+
Selecting the organisms for this study
Complementation
Point and holocentric centromeres:
Regional centromeres:
O. sativa
A. arenosa
S. cerevisiae
How far across evolutionary distance
is function maintained?
L. nivea
C. elegans
Can CENH3 that interacts with point and
holocentric centromeres function in a regional centromere?
S. cerevisiae (point)
L. nivea (holocentric)
C. elegans (holocentric)
Z. mays
O. sativa
H. sapiens
Native CENH3
Promoter
The centromere
contains the centromere specific
histone (CENH3)
which recruits kinetochore proteins.
Seed Counting Assay of cenh3/+ O.sativa CENH3
/cenh3/+
Self-fertilizes
Cloning strategy
Regional -Mb of tandem repeats (A.thaliana &
H.sapiens)
Point -125 bp sequence (S.cerevisiae)
Holocentric-microtubules bind along chromosome
(C.elegans).
Rapid evolution of CENH3
O. sativa genotyping results: complementation unlikely
Test whether CENH3 orthologs localize correctly in Arabidopsis
thaliana and complement a mutation in the endogenous CENH3
A. arenosa
B. stricta
B. holboellii
What is the centromere?
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METHODS
% Lethality
ABSTRACT
CENH3
Terminator
6x Glycine
1/16=6.25%
1/4=25%
Lethality only in
cenh3 homozygote
lacking GFP CENH3
Lethality in all
cenh3
homozygote
Plant Number
Five siliques were collected from thirteen heterozygotes with the O. sativa
GFP-tagged CENH3.
Although two plants exhibited low percentage of lethality (15.5 & 17.7),
it is not close enough to the expected percentage for complementation.
CONCLUSION
The correct localization of A. arenosa CENH3 and the non-localization of H.
sapiens CENH3 suggest that a conserved structure exist only between closely
related orthologs.
Diffused fluorescence and seed counting assay of cenh3/+ with O. sativa
suggest that complementation unlikely.
pCAMBIA
GFP
CENH3 Variant
Non-localization of S. cerevisiae and C. elegans CENH3s suggest that CENH3
from interact with holocentric and point
FUTURE DIRECTIONS
Continue with A. arenosa genotyping
Agrobacterium in sol’n
Transformed by dipping
Hygromycin selection
Image root tips
RESULTS
Fluorescent microscopy results
A. thaliana
(regional)
A. arenosa
(regional)
C. elegans
At this time, transformants are still
too young to collect tissue from for
genotyping.
Homozygote mutants found in the
T1, will be examined for any
phenotypic variations from the wildtype.
(holocentric)
For CENH3 orthologs that lack localization: Are they expressed?
•
•
RTPCR of CENH3 mRNA to determine if transgene was transcribed
Western blot to determine if mRNA was expressed
Further examination of rice CENH3:
•
(Figure above compares only half of the histone gene sequence from A.thaliana, A.arenosa, O.sativa, and H.sapiens)
O. sativa
H. sapiens
S. cerevisiae
(regional)
(regional)
(point)
Progress of other orthologs: Cloning ongoing
Boechera stricta, Boechera holboellii, Zea mays:
and Brassica rapa (new on list):
•A. thaliana with transgene
•In process of cloning
currently growing on ms plate
Luzula nivea:
•Construct will be sent to us
A. thaliana CENH3 can be tagged with GFP
Wild-type
Genotype progeny from plant 5 and 13 for complementation
cenh3/CENH3
REFERENCES
A. arenosa showed fluorescence similar to A. thaliana CENH3, proving that
the protein can correctly localize despite sequence differences.
Wild-type
cenh3 +
GFP-CENH3 transgene
Null mutation is embryo lethal allowing us to test complementation. Transgene
with A.thaliana CENH3 showed kinetochore localization as well as
complementation (plants had a wild-type phenotype).
O. sativa CENH3 showed diffuse fluorescence within the nucleus, but
localization in centromere is not clear. Possible over-expression or lack of
protein degradation. No fluorescence shown with H. sapiens GFP-tagged
CENH3.
CENH3s from non-regional centromeres did not show fluorescence. Protein
might be unrecognizable by the A.thaliana CENH3 loading machinery.
1) Cleveland, D.W., Mao, Y., and Sullivan, K.F. (2003). Centromeres and kinetochores:
from epigenetics to mitotic checkpoint signaling. Cell 112, 407-421.
2) Black, B.E., and Bassett, E.A. (2008). The histone variant CENP-A and centromere
specification. Curr Opin Cell Biol 20, 91-100
4) Images were from: Napsal, CS Kuoh, Masur, ESA , BBC.co.uk
ACKNOWLEDGEMENTS
This research was supported in part by an NIH-IMSD award to UC Davis (GM-56765) and by a Howard
Hughes Medical Institute grant (#52005892) in support of the Biology Undergraduate Scholars Program
(BUSP)
Special thanks to all the members of the Chan lab for their kindness and guidance.
Thanks also to BUSP and the UC LEADS program for all of the support and mentorship.