THE ROLE OF FMRP IN DROSOPHILA CHROMOSOME SEGREGATION DURING

MEIOSISUdo Onwubiko - Rotation 2

Dr. McKee’s Lab

Image: National Fragile X Foundation — www.nfxf.org

1 in 3600 boys, 1 in 4000-6000 females, every year born with FMR1 full mutation - Fragile X association of Australia

Symptoms range from minor to severe

Impaired brain development – caused by a mutation in a single gene

Fragile X Syndrome

FMRP BINDING CAPABILITIES

2Methyl histone bindingRNA, protein binding

RNA binding

RNA, protein bindingRNA binding, protein binding?

Human missense mutation in functional domain

*Methyl substrate binding ability Y98A substitution in dFMRP

Agenet domain plays a major role in FMRP ability to bind to methylated histone sites on DNA

*

FMRP in the nucleus is associated with- DNA damage and Meiotic Chromosomes

Agenet domain binds methylated histones

Alpatov et al, Cell, 2014

Mouse spermatocytes- prophase

LOCALIZATION OF FMR IN MEIOTIC CHROMOSOMES

• dFMR is spread out through the cell

DAPI dFMR CID Merge

Immunostaining: Drosophila Spermatocytes- late prophase 1

HOW CAN WE STUDY EFFECTS OF FMR ON DNA?

Observe FMR effects on meiotic chromosomes • Study defects in synaptonemal complex – Most likely at the level of DNA

repair• Look at non-disjunction during meiosis

Approach: Conducted 4 different crosses, and observed phenotypesGenotypic outcome of crosses a. Homozygous Null mutation b. Hemizygous Agenet domain

mutation Y98Ac. Hemizygous-Point mutation in KH

domain I307Nd. Heterozygous control – has one

functional FMR gene

INVESTIGATING FMR ROLE IN MEIOSIS BY STUDYING CHROMOSOME SEGREGATION

• FMR effects were assessed by observing phenotypes for non-disjunction(NDJ) in the XY Chromosome pairs during Meiosis in male flies.

• NDJ marked by: Bar shaped eye in Males, and Normal shaped eyes in females

  X^Xy2 O

y+X X^X XDead

XOMale-sterile

y+YBar X^X y+YBar

Female-bar-eyedYODead

 

Proper chromosome segregation in Meiosis

Improper segregation in Meiosis (NDJ)  X^Xy

2 O

y+X y+YBar X^Xy

2 XYDead

X y+YBar Omale-bar-eyed

O X^X Ofemale-

round-

eyed

OODead

XX XXX^XDead

XXO

HYPOTHESISFMR might have an important role in meiotic chromosome segregation

FINDINGS: % OF NDJ PER MUTATION

0.0000%

0.2000%

0.4000%

0.6000%

0.8000%

1.0000%

1.2000%

1.4000%

1.6000%

0.0069%

1.0900%

1.4800%

0.5540%

%NDJ

Het. Control KH domain AG domain Null

Axis

Tit

le

n=4328

n=4332n=288

0

n=total population size

n=3297

Interestingly 58 out of the 66 total supposed NDJ events were Females: 88%Only 8 out of the 66 were NDJ males : 12%

Question: is this really NDJ that we were seeing?

An outlier in Null mutation: A Jackpot NDJ – suggesting mitotic NDJ in spermatogonia stem cells

Comparison Null vs control

I307N vs control

Y98A vs control

Chi-square statistic

11.2329 26.9321 37.2613

p-value 0.0008 p < 0.001 p< 0.001These results are significant at p< 0.05Chi square calculator: http://www.socscistatistics.com/tests/chisquare/

THE STORY OF 4 SAVED NDJ FEMALES

4 NDJ suspect females (X^X O)were crossed with new males with phenotype (Xy2ct6 Y)

We need to go back and repeat the cross and determine ratio of true NDJ to other events ( cross overs, deletions, loss)

Female Phenotype of X^X O females

Outcomes so far when crossed to males

1 y+ True NDJ (Meiosis II all males fertile)

2 y+ False NDJ (y+, ct6 -males sterile)3 y+ False NDJ (y+, ct6- males sterile)

4 y2 True NDJ ( all sterile males- probably Meiosis I)

Why did Bs marker disappear?

Data : Dr. T. Dockendorff, University of Tennessee

Recall: Normal outcome females: X^X y+YBs

One possible outcome-NDJ females: X^Xy2

ONE POSSIBILITY FOR A FALSE NDJ EVENT

  X^Xy2 Yy+ ( Bar-S marker missing?)

Xy2ct6 X^X Xy2ct6

DeadXy2ct6 Yy+

Males- Sterile?

Y X^Xy2Yfemale-fertile

YYDead

 

A False NDJ event

How could FMR mutations cause chromosome breaks?

Where did the yellow+ come from? Recall: Normal Female from the first cross: X^X y+YBs

FMRP IS PART OF THE PIWI-RNA PATHWAYGenome guardians- Silence transposons and repetitive sequences in meiotic and somatic cells

Nature review article: Maartje J. Luteijn1 and René F. Ketting

- piRNA pathways occur in mammalian, drosophila, c.elegans, yeast cells

FMRp?

A reasonable explanation for our False NDJ events?

CONCLUSIONS• FMRp localizes in centromeres in Meiotic chromosomes • Both KH and Agenet Domain of FMR protein are somewhat critical

for FMR role in meiotic chromosome segregation• While Piwi interaction with FMR has been reported, we

demonstrated with actual evidence through our crosses that chromosomal alterations are occurring possibly as a result of FMR mutations.

• The observed phenotypes (i.e.. False NDJ, NDJ, DNA breaks, etc. ) could be contributing factors to phenotypic variances seen in the fragile X syndrome patients. The jackpot is critical because it supports that NDJ events can occur in somatic stem cells (mitotic division), which suggests that the same events could be happening in neuronal stem cells of Fragile X patients.

FUTURE DIRECTIONS• Track frequencies of true NDJ events compared to other events such as

deletions, insertions, etc. • Analyze male NDJ events as well• Look at recombination events in females with agenet domain mutations

ACKNOWLEDGEMENTS

• Dr. Bruce McKee• Dr. Thomas Dockendorff ( Rotation Mentor)• Quitao He• Elsie Adams• Xiaofei ( Dr. Bembeneck’s Lab)• 2016 Cohort ( Go Vols )

Any questions?