Question 4: Genomic Integrity and Transgenerational Inheritance The lecturer describes how ddm1 mutants in Arabidopsis appear relatively normal in the first generation but show severe developmental defects by the fifth generation (F5). • Task: Explain the causal link between the loss of DDM1-mediated DNA methylation and the accumulation of physical DNA mutations across generations. • Logic Challenge: If you were to reintroduce a functional DDM1 gene into an F5 mutant line through crossing, would you expect the offspring to immediately return to a wild-type phenotype? Justify your answer by distinguishing between epimutations and transposon-induced DNA mutations

What phenotype do ddm1 mutants show in the first generation?

They appear relatively normal.

What phenotype do ddm1 mutants show by the fifth generation (F5)?

Severe developmental defects and can barely survive.

What is DDM1?

A chromatin remodeler that facilitates access of DNA methyltransferases to transposable elements located within tightly packed heterochromatin.

What happens to DNA methylation in ddm1 mutants?

DNA methylation is lost across all sequence contexts (CG, CHG, and CHH).

What immediate consequence does loss of DNA methylation have on transposable elements?

Transcriptional activation of TEs (they become expressed).

What do activated transposable elements do?

They can excise themselves or copy themselves into new chromosomal locations (transposition).

Why are ddm1 mutants relatively normal in the first generation?

Because only a few random transposition events have occurred, so the phenotypic impact is minimal.

Why do ddm1 mutants show severe defects by the fifth generation?

The mutational load accumulates with each generation. By F5, TEs have randomly inserted into and disrupted enough developmentally important genes to cause severe defects.

If you reintroduce a functional DDM1 gene into an F5 mutant line, will the offspring immediately return to a wild-type phenotype?

No, they will not immediately return to wild-type.

What is an epimutation?

A heritable change in gene expression that does not involve a change in the DNA sequence itself.

What is a transposon-induced DNA mutation?

An actual physical change to the DNA sequence where a TE has inserted into a gene.

What can reintroducing DDM1 restore?

It can re-establish DNA methylation and silence active TEs, preventing further transposition. It might also allow reversion of certain unstable epimutations.

What cannot DDM1 fix?

It cannot fix the permanent physical DNA sequence changes (insertions) that have already occurred from TE activity.

Why does the genomic integrity of the F5 line remain compromised even after restoring DDM1?

Because the severe defects are caused by permanent DNA mutations in essential genes from five generations of TE activity, and sequence changes are permane