DOI: 10.5281/zenodo.17456258
title: Transcription Represses Origin Activity in a Late-Replicating Fragile Site

description: Genome-wide analyses in vertebrates suggest that transcription can negatively regulate replication origin activation, thereby shaping the global landscape of replication initiation. In this study, we investigated how transcription and late replication influence replication origin activity by inserting an efficient minimal model origin into the avian DMD common fragile site (CFS) and a second transcriptionally silent late-replicating region. The DMD gene, which is neither transcribed nor fragile in wild-type DT40 cells, became fragile following transcriptional activation in a genetically engineered DT40 cell line. Previous molecular combing experiments have shown that transcription represses origin firing within the 500 kb 5' half of the gene. Here, we demonstrate that when a minimal origin is inserted into this repressive region, it remains partially active under basal transcription conditions but is fully inactivated upon transcriptional induction. In contrast, inserting the efficient β-actin promoter/origin at the same site retains origin functionality despite transcriptional activation. Introducing these two origins into a late-replicating, transcriptionally silent locus reveals the same functional hierarchy. Notably, this contrasts with their comparable activity in a mid-late replicating region. These results define a class of replication origins capable of maintaining functionality across diverse chromosomal contexts. Our findings support the model in which CFSs arise due to transcription-dependent repression of replication origin initiation across large, late-replicating genes. Moreover, our results reinforce the idea that replication initiation in late-replicating genomic compartments is intrinsically less efficient than earlier-replicating regions.
description: We analysed a DMDTet/Tet DT40 chicken clonal cell line and a DMDTet/Tet DT40 chicken clonal cell line containing the 90 bp minimal origin sequence derived from the βA-globin origin fused to the IL2R gene and SV40 PolyA sequence, flanked with two USF binding sites inserted into the centre of the DMD termination zone on one chromosome (insertion site chr1:114.796.392-114.798.322, galGal5). 

DT40Cre1 has been described previously (Arakawa et al. 2001), the DMDTet/Tet cell line has been described previously (Blin et al. 2019).

The DMDTet/tet gene was induced by treatment with 1 µg/mL Tetracycline hydrochloride.

5-ethynyl uridine incorporated nascent RNAs were captured using the Click-iT Nascent RNA Capture Kit (Thermo Fischer Scientific, #C10365) and reverse transcribed using Superscript VILO cDNA Synthesis Kit (Thermo Fischer Scientific, #11754050). Sequencing libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760S) following the manufacturer’s instructions for RT-nascent RNAs. Libraries were paired-end sequenced on an Illumina NovaSeq X Plus.

Paired-end were aligned to the Gallus gallus genome assembly galGal5 using bowtie2 (version 2.5.1) with default parameters. Processed BAM files were converted into BigWig coverage file using the Galaxy France platform for visualisation in the UCSC Genome Browser. 
description: Library: Nascent RNA -seq

Raw files (fastq):

- DMDTet/Tet  +Tet CD9_MKDL250007194-1A_2323JFLT3_L8_1.fq CD9_MKDL250007194-1A_2323JFLT3_L8_2.fq

- DMDTet/Tet +minimal origin -Tet#1 CD5_MKDL250007190-1A_2323JFLT3_L8_1.fq CD5_MKDL250007190-1A_2323JFLT3_L8_2.fq

- DMDTet/Tet +minimal origin -Tet#2 CD7_MKDL250007192-1A_2323JFLT3_L8_1.fq CD7_MKDL250007192-1A_2323JFLT3_L8_2.fq

- DMDTet/Tet +minimal origin +Tet#1 CD6_MKDL250007191-1A_2323JFLT3_L8_1.fq CD6_MKDL250007191-1A_2323JFLT3_L8_2.fq

- DMDTet/Tet +minimal origin +Tet#2 CD8_MKDL250007193-1A_2323JFLT3_L8_1.fq CD8_MKDL250007193-1A_2323JFLT3_L8_2.fq

Processed data files BamCoverage file (bam and BigWig files):

- DMDTet/Tet  +Tet CD9_TetO-Tet_bamCoverage.bigwig  

- DMDTet/Tet +minimal origin -Tet CD5+CD7_bamCoverage.bigwig 

- DMDTet/Tet +minimal origin +Tet CD6+CD8_bamCoverage.bigwig 

UCSC Browser view:

https://genome.ucsc.edu/s/Doncarli_Caroline/Transcription_Represses_Origin_Activity_in_a_Late_Replicating_Fragile_Site