CenH3: in haploid induction technology

CenH3: An emerging player in haploid induction technology
CENH3 (called CENP-A in humans) is a centromere-specific histone H3 variant that replaces histone H3 in eukaryotic centromeric nucleosomes. CENH3 plays an essential role in the epigenetic formation of kinetochore and is sufficient to determine centromere identity, at least in Drosophila's.26-Apr-2019

1. Cenh3: An Emerging Player in Haploid Induction Technology
Presented by
HajraSarfrazFA22-RBT-006
SafaMushtaq FA22-RBT-007
Course title :
Advances in Plant Biotechnology

3. Introduction
´CENH3(CentromereHistone3)isaspecializedhistone
variantcrucialforcentromerefunctionineukaryoticcells.
´Centromeresareessentialforproperchromosome
segregationduringcelldivisionandarepivotalfor
maintaininggenomestability.
´Haploidinductioninvolvesgeneratinghaploidplantsfrom
diploidones,crucialinplantbreedingandgenetic
research.
´CENH3playsapivotalroleinhaploidinduction.

4. The Variant Histone CENH3 is Required for
Centromere Localization
´EukaryoticDNAwrapsaroundnucleosomes,which
enablesthecompactionofthegenomewithinthe
cellnucleus.
´Thelocationofthecentromereisdeterminedbythe
presenceofnucleosomescarryingthevarianthistone
CENH3
´CENH3containsanN-terminaltailandaC-terminal
HistoneFoldDomain.ItsN-terminaltailisrapidly
evolving,whilethehistonefolddomainisrelatively
conserved.

5. Mutations in
CENH3
´CENH3isessentialforproperchromosome
segregation.MutationsoralterationsinCENH3
have been linkedtochromosome
segregationerrorsandlethalityinvarious
organisms.
´Inyeast,CENH3mutations cause
chromosome non-disjunctionandmitotic
arrest.
´ErrorsinreloadingCENH3atcentromeres
derivedfromCENH3-modifiedlinescould
contributetohaploidinduction.

6. Importance of haploid induction
Doubled haploids streamline breeding by creating plants
with a genome from a single gamete, dramatically reducing
the time needed to establish stable homozygous lines.
Haploid lines and genetics expedite genetic research and
applications, benefiting both breeding programs and
research laboratories.
Haploids occur naturally in some species and can also be
generated through techniques like anther culture and
hybridization.
Recent progress in haploid induction technology involves
modifying CENH3, a histone variant present in all plants,
which holds promise for extending the technique to a wide
array of crop species.

7. CENH3 MEDIATED HAPLOID INDUCTION
´RaviandChan’sstudyin2010revealedthatmodifying
CENH3inArabidopsisledtotheinductionofhaploids.
´Asuccessfulhaploidinducercalled“GFP-tailswap”was
createdbyreplacingtheN-terminaltailofCENH3withGFP
fusedtoanH3variant’staildomain.
´Surprisingly,the“tailswap”didnotaffectCENH3’smitotic
functionality,thoughitcompromiseditsmeioticfunction,
resultinginmaleandpartialfemalesterility.
´CrossbreedingGFP-tailswapfemaleswithwild-typeCENH3
malesyielded25-45%paternalhaploidprogeny,losingthe
maternalgenome.
´AddingGFPormodifyingtheN-terminaltailweakenedthe
centromere'scompetitivestrengthforreloadingCENH3
comparedtowild-typecentromeres.

8. Genome elimination induced by modification of
centromerichistone H3(CENH3).

9. Effect of various modifications of transgenic

10. CENH3 FUNCTION IS CONSERVED ACROSS
WIDE VARIETY OF SPECIES
StudiesusingyeastCENH3(Cse4)complemented human
CENH3(CENPA)deficiency,highlightingconservation.
TaggedversionsofCENH3fromcertainplantspeciescould
targetthecentromeresofArabidopsisthaliana,whileothers
couldn’t.
GFP-taggingcanimpactfunctionality.WhileC-terminal
tagginglocalizedCENH3tothecentromere,N-terminal
taggingrestoredfunctionalityinArabidopsis.
UntaggedversionsofCENH3fromvariousplantspecies
couldcomplement Arabidopsiscenh3nullfunction,
indicatinghighconservationofessentialCENH3functions.

11. A ROLE FOR N-TERMINAL DOMAIN
MUTATIONS IN HAPLOID INDUCTION
´ChangestoCENH3’sstructurecanleadtohaploid
induction(HI),andsurprisingly,itsN-terminal
domainisn’tnecessaryformitoticfunction.
´ReplacementofArabidopsisN-terminaltailwith
thatofLepidiumoleraceumledtoviable,fertile
plants,butextensiveseeddeathandHIwhen
crossedbywild-typelines.;
´Tailswapexperimentsindicatedthatthesequence
oftheN-terminaltailinfluencesthe“strength”of
CENH3incompetitivecrosses.

12. Alignment of Arabidopsis thaliana histone3.3 vs. CENH3
sequences

13. Point Mutations in CENH3 Histone Fold Domain Induce Haploids
´TheCENH3tailregionevolvesrapidlyinlengthand
sequence,whiletheHFDiswell-conserved.
´Specificpointmutations,resultinginsingleamino
acidsubstitutionsintheHFD,caninduceuniparental
genome elimination,leadingtohaploidyor
aneuploidy.
´Thesemutationsinconservedpartsoftheprotein's
structureshowedthatthesechangesresultedin
viableandfertileplants.
´Thesemutationscanbeintroducedbymutagenesis
methodslikeethylmethanesulfonate(EMS),it
suggeststhepossibilityofnon-transgenichaploid
inducersincropspecies.

14. HI line in female parent
´CENH3 mediated genome elimination is
most efficient when the HI line is the female
parent
´There are three ways to create single sets of
chromosomes (haploids) using changes to a
protein called CENH3:
1.by swapping parts of the protein and adding a
glowytag,
2.by mixing CENH3 from different species,
3.by changing specific parts of the protein.

15. Conti…..
´Forinstance,ifweusetheglowytagmethod,
thesuccessrateofmakinghaploidsismuch
higher(40%)whenthechangedproteinisin
thefemaleplant,comparedtowhenit'sin
themaleplant(5%).
´Whentheychangedonespecificpartofthe
proteincalledL130F,itonlyworkedinmaking
haploidswhenusedinfemaleplants.
´Althoughit'spossibletousethechanged
CENH3inmaleplants,it'sclearthatthe
changesintheproteinhaveastrongereffect
onmakinghaploidswhenusedinfemale
plants.

16. SEED ABORTION IS CORRELATED WITH HAPLOID INDUCTION
´AllCENH3-mediatedhaploidinductionmethods
exhibitasharedfeatureofseedabortion,where
inviable,small,andwrinkledseedsarefound
alongsidehealthyseedsinthesamesilique.
´A GFP marker under 2S3 promoter control (At2S3:
GFP) in GFP-tailswaplines allowed visualization of
haploid seeds. Mottled GFP expression indicated
haploids, while fully green seeds indicated diploids or
aneuploids.
´Notably, no GFP-free seeds were observed,
suggesting these events are rare that likely lead to
seed abortion, particularly of haploids.

17. REGULATORY ISSUES AND POSSIBLE ALTERNATIVES
´HI using modified CENH3 can be viewed as GMO or non-GMO based
on interpretation of local laws.
´Labelingof GMOs can be based on product or generation process.
´GFP-tailswapHI could be non-GMO based on product, but GMO based
on process.
´Point mutations via EMS are accepted as non-GMO under current
regulations.

19. Conclusion
´Thesemethodssavetimeandeffortincreatingtrue-
breedingvarieties.
´CENH3-mediatedgenome eliminationhasbeen
appliedintraitmapping,reversebreeding,and
more,benefitingcropbreeding.
´AdoptionwaslimitedduetothelackofCENH3
knockoutsinvariousspecies,nowaddressedby
CRISPR-basedgenetargeting.
´Understandingthismechanismnotonlyshedslighton
CENH3’sfunctionbutalsocouldrevealnewtargets
forfuturehaploidinducers.