Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (SLC34A1/PFN3)

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• Natural antisense transcripts (NATs) are long noncoding RNAs involved in gene regulation, with prominent expression in testes but also found in other organs.
• NATs are typically transcribed at low levels and co-expressed with their sense counterparts, but their direct interference is often overlooked.
• The regulatory roles of NATs may differ between germ and somatic cells, necessitating further investigation into their mechanisms.

Purpose of the Study:

• To investigate the interplay between sense and antisense transcription at the SLC34A1/PFN3 locus using a cellular model.
• To explore the epigenetic mechanisms, including DNA methylation and histone modifications, that regulate bidirectional transcription.
• To elucidate the cis-acting nature of NATs and their influence on the expression of their sense counterparts.

Main Methods:

• Induction of sense and antisense transcription at the SLC34A1/PFN3 locus in HEK293 and HKC-8 renal cell lines using epigenetic modifiers and CRISPR-Cas9.
• Treatment with dexamethasone to stimulate sense and antisense transcript levels.
• Analysis of promoter methylation and histone marks to assess epigenetic changes.
• Use of modified transcripts to study the effects of constitutive expression and knock-in strategies.

Main Results:

• Dexamethasone exposure significantly increased SLC34A1 sense transcript levels and minimally PFN3 antisense transcript levels in HEK293 cells, associated with reduced promoter methylation and increased activating histone marks.
• Constitutive expression of truncated sense or antisense transcripts enhanced the expression of the opposite transcript, demonstrating concordant expression.
• The observed effects were cis-acting, as transient transfections did not influence the expression of the opposite transcript.

Conclusions:

• Bidirectional transcription of the SLC34A1/PFN3 locus stimulates the expression of both sense and antisense transcripts, mediated by epigenetic modifications.
• NATs may function differently in somatic and germ cells, with somatic NATs potentially acting as lncRNAs due to their proximity to target genes.
• The findings support a model where NATs play distinct roles in different cell types, requiring RNA complementarity and double-stranded RNA formation in germ cells.