Biology subjects

Villano, D. J.

Publications and source records attributed to Villano, D. J..

3 recordsLinked to original sources

bioRxiv2026

A PIWI protein-dependent DNA N6-adenine methylation pathway in Oxytricha protects genomic sequences from deletion

The ciliate Oxytricha undergoes massive genome rearrangements during development to produce a functional nucleus from an encrypted zygotic genome. PIWI-interacting small RNAs protect DNA regions against deletion, but how that protective mark is established has been a mystery. Recently our lab discovered MTA1, a methyltransferase that catalyzes DNA N6-adenine (6mA) methylation. Both MTA1 and the Oxytricha Piwi protein, Otiwi1, are required for development, and Otiwi1 mutation eliminates 6mA signal. To examine the role of 6mA, we analyzed its genome-wide distribution across development in wild-type and MTA1 mutant backcrossed cells. We find specific and abundant enrichment on retained sequences, suggesting a protective role for this epigenetic mark. Furthermore, programmed retention of a DNA region that is normally deleted leads to accumulation of new 6mA marks on the ectopically retained DNA sequence. Together, these results suggest that piRNA-guided 6mA DNA methylation leads to protection of DNA sequences against deletion during nuclear differentiation. HighlightsO_LIDNA N6-methyladenine accumulates on retained DNA regions during Oxytricha development. C_LIO_LImta1 mutant backcrosses have disrupted DNA methylation and a developmental delay. C_LIO_LIDNA N6-adenine methylation during genome rearrangement requires the presence of Otiwi1. C_LIO_LIProgrammed retention of a germline-limited region leads to developmental methylation. C_LI

molecular biology↗

bioRxiv2025

Relaxed DNA substrate specificity of transposases involved in programmed genome rearrangement

During post-zygotic development, the ciliate Oxytricha trifallax undergoes massive programmed genome rearrangement that involves over 225,000 DNA cleavage and joining events. An Oxytricha family of Tc1/mariner transposons, known as Telomere-Bearing Elements (TBEs), encodes a transposase that has been implicated in rearrangement, but its high copy number (>34,000 paralogs) has precluded genetic strategies to investigate its DNA recognition properties directly in Oxytricha. Here, we developed a heterologous strategy to assay TBE transposase expression and activity in E. coli, revealing highly promiscuous DNA cleavage properties. Systematic ChIP-seq experiments allowed us to define the DNA binding specificities of multiple distinct transposase subfamilies, which exhibited a binding and cleavage preference for short, degenerate sequence motifs that resemble features present within the TBE transposon ends. The relaxed sequence preference is striking for autonomous transposases, which typically recognize their end sequences with strict specificity to avoid compromising host fitness. Finally, we developed a custom antibody to investigate TBE transposases in their native environment and found that they precisely localize to the developing nucleus exclusively during the rearrangement process. Collectively, this work establishes a robust heterologous workflow for the biochemical investigation of enzymes that have been repurposed for large-scale genome rearrangements.

molecular biology↗

bioRxiv2025

Widespread 3D genome reorganization precedes programmed DNA rearrangement in Oxytricha trifallax

Genome organization recapitulates function, yet ciliates like Oxytricha trifallax possess highly-specialized germline genomes, which are largely transcriptionally silent. During post-zygotic development, Oxytrichas germline undergoes large-scale genome editing, rearranging precursor genome elements into a transcriptionally-active genome with thousands of gene-sized nanochromosomes. Transgenerationally-inherited RNAs, derived from the parental somatic genome, program the retention and reordering of germline fragments. Retained and eliminated DNA must be distinguished and processed separately, but the role of chromatin organization in this process is unknown. We developed tools for studying Oxytricha nuclei and apply them to map the 3D organization of precursor and developmental states using Hi-C. We find that the precursor conformation primes the germline for development, while a massive spatial reorganization during development differentiates retained from eliminated regions before DNA rearrangement. Further experiments suggest a role for RNA-DNA interactions and chromatin remodeling in this process, implying a critical role for 3D architecture in programmed genome rearrangement.

genomics↗