Apparent microRNA-target-specific histone Apparent microRNA-target-specific histone modification inmodification in

mammalian spermatogenesismammalian spermatogenesis

Y-h. TaguchiY-h. Taguchi

Dept. Phys.Dept. Phys.

Chuo Univ.Chuo Univ.

IntroductionIntroduction

Epigenetic regulation of gene expression is Epigenetic regulation of gene expression is known to be important nowadays.known to be important nowadays.

e.g.e.g.

- Development and Cellular Differentiation- Development and Cellular Differentiation- Diseases including Cancer - Diseases including Cancer - Aging- Aging

Example of Epigenetic regulation of genesExample of Epigenetic regulation of genes

- Transcription Factor Bindings to - Transcription Factor Bindings to promoter regionpromoter region- Histon modification- Histon modification- Promoter Methylation- Promoter Methylation- miRNA regulation of target genes.- miRNA regulation of target genes.

etc. etc.etc. etc.InteractionInteractionRarely Rarely DiscussedDiscussed

Both gene regulationBoth gene regulation

miRNA

cell nucleus

Gene

3'UTR

cytoplasm

histone modification ↔ cell nucleusmiRNA ↔ cytoplasm

histone modification

Previous ResearchPrevious Research

Su et al (2011, BMC Genomics)Su et al (2011, BMC Genomics)““miRNAs tended tomiRNAs tended to targettarget the genesthe geneswith awith a lowlow DNA methylation level in their DNA methylation level in their promoter regions”promoter regions” Promoter methylation ↔ miRNA targeting Promoter methylation ↔ miRNA targeting

Purpose of Purpose of mymy study: study:Histone modification is miRNA-targeting-specific or not?Histone modification is miRNA-targeting-specific or not?

(In other words, histone modification is affected by being (In other words, histone modification is affected by being targeted by individual miRNA or not?)targeted by individual miRNA or not?)

significantly up/downregulated?

(t test, Wilcoxon test, KS test)

MiRaGE :MiRNA Ranking by Gene Expression

miRNA

target gene

target gene

VS

gene

considered miRNA

MiRaGE Method

Histone modification

modified histone?

My Previous Results

“Substantial number of miRNAs have target genes with significantly hyper/hypomethylated promoters”

References: - Y-h. Taguchi (2012) IPSJ SIG Tech. Rep. 2012-BIO-31(1) pp.1-6- Y-h. Taguchi, (2013) F1000Research [v1; ref status: approved 1, not approved 2, http://f1000r.es/wv]

This Study

Comparison between miRNA and histone modification during spermatogenesis in miRNA-centric manner.

NGS:Spermatogonia(SG)→Spermatocytes(SC)→Spermatids(ST) vs Controls

H3K4me1, H3K27me3, H3K4me3, H3K27ac, H2AZ

Calculation - Many miRNAs with significant (*) histone modification among multiple miRNAs for all SC, ST, SG vs Controls

(*) BH adjusted P <0.05

SC

SG

ST SCSG ST

less modificated more modificated

Differenciation

Cluste

Clustering using negative signed correlation coefficient of P-valuesP-values

Biologically feasible

Clustering using negative signed correlation coefficient of logarithmic ratiologarithmic ratio

Less biologically feasible

P-values computed by MiRaGE are more biologically

feasible than raw values (logarithmic ratio)

less modificated

more modificated

Stemness Mouse ES cell specific

hsa-

↑during differentiation

hsa-

Critical roles in spermatogenesis

hsa-

Sperm abnormaly

hsa-

Expressed in abnormal semen

Selected miRNAs are biologically reasonableSelected miRNAs are biologically reasonable

less modificated more modificated

Is it artifact ? → No, since essentially no miRNAs with Is it artifact ? → No, since essentially no miRNAs with significant histone modification after gene shufflingsignificant histone modification after gene shuffling

Samples →

mR

NA

(g ene ) →

Test 2

Simultaneous shuffling with keeping correlation coefficients of logarithmic ratio

Clustering using negative signed correlation coefficient of P-values P-values after simultaneous shuffling

More similar to cluster obtained by raw values (logarithmic ratio)

Thus, converting logarithmic ratio to P-values by MiRaGE enphances

biological feasibility

less modificated more modificated

Human sperm exhibits similar miRNA specific histone modification

Too simple model? → comparison with lassoToo simple model? → comparison with lasso

differential expressionof i th mRNA and j th histone

modification

11 if k th miRNA targets ith mRNA 00 otherwise

Predicted activity of k th miRNA

β

k

P-v

alu

es

MiRaGE is well coincident with lasso. Thus, biological feasibilities obtained by miRNA specific histone modification will

not be likely fake.

ConclusionsConclusions

・ P-values computed by MiRaGE are highly tissue/histone modification specific.・ Most significant miRNAs are stem cell or sperm specific. Thus, biologically feasible.・ Since gene order shuffling destroys miRNA specific significant histone modification, it is not an artifact.・ Since it is coincident with lasso, biological feasibility is not due to too simple algorithm・ Biological mechanism that causes miRNA specific histone modification should be cleared.