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SINE retrotransposons link replication timing with higher-order genome organization by recruiting H2Bub to gene body

ABSTRACT: The mammalian genome is spatially segregated into euchromatin-associated A and heterochromatin-associated B compartments that replicate early, and late in S-phase respectively. The molecular links connecting higher-order genome organization with replication timing, however, remain unclear. Here, we show that H2Bub (histone H2B lysine 120 monoubiquitination), a transcription-coupled epigenetic mark enriched over the gene body, regulates early replication. Furthermore, enforced recruitment of H2Bub promotes early replication in cis. Consistent with these observations, genetic ablation of RNF20, an E3 ubiquitin ligase that mediates H2Bub deposition, leads to reduced firing of early replication origins and suppression of cellular and embryonic growth. We also find a role of H2Bub for promoting proximal chromatin contacts at the gene body, and in gene rich A compartments, indicating that H2Bub may connect replication timing with higher-order genome organization. Surprisingly, H2Bub-marked regions in the genome coincide with SINEs (short interspersed nuclear elements). SINE retrotransposon enrichment in transcribed gene bodies correlates with H2Bub levels, indicating that these elements may direct H2Bub deposition at chromatin. Indeed, ectopic insertion of SINEs promotes local recruitment H2Bub. Taken together, our results suggest an unexpected role for SINE retrotransposons in linking higher-order genome organization with replication timing via targeting of the transcription-coupled epigenetic mark H2Bub.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Jafar Sharif

PROVIDER: PXD067536 | JPOST Repository | Wed Aug 27 00:00:00 BST 2025

REPOSITORIES: jPOST

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			Action	DRS
	001_WT_ESC_RNF20_CST_IP.wiff	Wiff
	001_WT_ESC_RNF20_CST_IP.wiff.scan	Wiff
	001_WT_ESC_RNF20_CST_IP_SW.wiff	Wiff
	001_WT_ESC_RNF20_CST_IP_SW.wiff.scan	Wiff
	002_Rnf20_cKO_ESC_RNF20_CST_IP.wiff	Wiff

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Project description:Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1’s role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4∆ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1∆ cells, and checkpoint-defective mec1∆ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres. 4 samples BrdU-IP-seq in HU, 2 strains with 2-replicates each (strains:WT and rif1 delta)

Project description:Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1’s role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4∆ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1∆ cells, and checkpoint-defective mec1∆ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres. 30 total samples: (6 samples - BrdU- HU arrest 45min with 2 replicates, strains: WT, rif1 delta, pfa4 delta) (12 samples -S-phase BrdU time course with 2 replicates at 25 and 35 min, strains: WT, rif1 delta, mec1_100) (12 samples - S-phase BrdU time course with 2 replicates at 25 and 35 min, strains: sml1 delta, sml1 delta rif1 delta, sml1 delta mec1 delta)

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SINE retrotransposons link replication timing with higher-order genome organization by recruiting H2Bub to gene body

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