Ron M. G. Menorca Pak Kwong Ravi Maruthachalam Simon Chan College of Biological Sciences: Section of Plant Biology University of California Davis, 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. RESULTS INTRODUCTION 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. Regional centromeres: Point and holocentric centromeres: How far across evolutionary distance is function maintained? Can CENH3 that interacts with point and holocentric centromeres function in a re- gional centromere?  A. arenosa  B. stricta  B. holboellii  S. cerevisiae (point)  L. nivea (holocentric)  C. elegans (holocentric) A. arenosa O. sativa L. niveaS. cerevisiaeC. elegans Rapid evolution of CENH3 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 : What is the centromere? Regional -Mb of tandem repeats ( A.thaliana & H.sapiens) Point -125 bp sequence ( S.cerevisiae ) Holocentric-microtubules bind along chromosome ( C.elegans ). Selecting the organisms for this study 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 CONCLUSION  Z. mays  O. sativa  H. sapiens 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. O. sativa genotyping results: complementation unlikely REFERENCES 1) Cleveland, D.W., Mao, Y., and Sullivan, K.F. (2003). Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell 112, ) Black, B.E., and Bassett, E.A. (2008). The histone variant CENP-A and centromere specification. Curr Opin Cell Biol 20, ) 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 (# ) 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. A. thaliana CENH3 can be tagged with GFP 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). Wild-type cenh3 + GFP-CENH3 transgene Wild-typecenh3/CENH3 LOCALIZATION COMPLEMENTATION (Figure above compares only half of the histone gene sequence from A.thaliana, A.arenosa, O.sativa, and H.sapiens) METHODS Test whether CENH3 orthologs localize correctly in Arabidopsis thaliana and complement a mutation in the endogenous CENH3 Cloning strategy Native CENH3 Promoter GFP 6x Glycine CENH3 Variant CENH3 Terminator pCAMBIA Agrobacterium in sol’n Hygromycin selection Image root tips Transformed by dipping Fluorescent microscopy results A. arenosa showed fluorescence similar to A. thaliana CENH3, proving that the protein can correctly localize despite sequence differences. CENH3s from non-regional centromeres did not show fluorescence. Protein might be unrecognizable by the A.thaliana CENH3 loading machinery. 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. ABSTRACT A.thaliana (regional) O. sativa (regional) A.arenosa (regional) S. cerevisiae (point) C. elegans (holocentric) H. sapiens (regional) /cenh3/+ GFP-CENH3/+ Self-fertilizes Complementation 1/4=25%1/16=6.25% Lethality only in cenh3 homozygote lacking GFP CENH3 Lethality in all cenh3 homozygote FUTURE DIRECTIONS Progress of other orthologs: Cloning ongoing  Zea mays: A. thaliana with transgene currently growing on ms plate  Boechera stricta, Boechera holboellii, and Brassica rapa (new on list): In process of cloning  Luzula nivea: Construct will be sent to us 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 wild- type. Further examination of rice CENH3: Genotype progeny from plant 5 and 13 for complementation 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 Continue with A. arenosa genotyping Seed Counting Assay of cenh3/+ O.sativa CENH3 % Lethality Plant Number We hypothesize that there is a conserved structure that underlies CENH3 function, despite the lack of sequence conservation Delair Wy. Elk Grove, CA (916) The centromere contains the centro- mere specific histone (CENH3) which recruits kine- tochore proteins. *Nature Reviews Neuroscience