Expressing Centromere Histone (CENH3) Orthologs in Arabidopsis thaliana to Test Functional Conservation

1
Expressing Centromere Histone (CENH3) Orthologs
in Arabidopsis thaliana to Test Functional
Conservation
9206 Delair Wy. Elk Grove, CA 95616 (916) 479
4544 rgmenorca_at_ucdavis.edu

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.
ABSTRACT
METHODS
O. sativa genotyping results complementation
unlikely
Test whether CENH3 orthologs localize correctly
in Arabidopsis thaliana and complement a mutation
in the endogenous CENH3
Seed Counting Assay of cenh3/ O.sativa CENH3
/cenh3/
GFP-CENH3/
Self-fertilizes
Selecting the organisms for this study
Complementation
Lethality
1/425
1/166.25
Lethality in all cenh3 homozygote
Lethality only in cenh3 homozygote lacking GFP
CENH3
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.
INTRODUCTION

• Z. mays
• O. sativa
• H. sapiens

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. Non-localization of S. cerevisiae and
C. elegans CENH3s suggest that CENH3 from
interact with holocentric and point
Cloning strategy
The centromere contains the centro-mere
specific histone (CENH3) which recruits
kine-tochore proteins.
Rapid evolution of CENH3
Nature Reviews Neuroscience
Normal  histone H3 is highly conserved due to its
importance in DNA packa-ging. 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.
RESULTS
Fluorescent microscopy results
(Figure above compares only half of the histone
gene sequence from A.thaliana, A.arenosa,
O.sativa, and H.sapiens)
REFERENCES
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
A. arenosa showed fluorescence similar to A.
thaliana CENH3, proving that the protein can
correctly localize despite sequence differences.
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.
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.
CENH3s from non-regional centromeres did not
show fluorescence. Protein might be
unrecognizable by the A.thaliana CENH3 loading
machinery.