Bre1-dependent H2B ubiquitination promotes homologous recombination by stimulating histone eviction at DNA breaks

Abstract

Repair of DNA double-strand breaks (DSBs) requires eviction of the histones around DNA breaks to allow the loading of numerous repair and checkpoint proteins. However, the mechanism and regulation of this process remain poorly understood. Here, we show that histone H2B ubiquitination (uH2B) promotes histone eviction at DSBs independent of resection or ATP-dependent chromatin remodelers. Cells lacking uH2B or its E3 ubiquitin ligase Bre1 exhibit hyper-resection due to the loss of H3K79 methylation that recruits Rad9, a known negative regulator of resection. Unexpectedly, despite excessive single-strand DNA being produced, bre1Δ cells show defective RPA and Rad51 recruitment and impaired repair by homologous recombination and response to DNA damage. The HR defect in bre1Δ cells correlates with impaired histone loss at DSBs and can be largely rescued by depletion of CAF-1, a histone chaperone depositing histones H3-H4. Overexpression of Rad51 stimulates histone eviction and partially suppresses the recombination defects of bre1Δ mutant. Thus, we propose that Bre1 mediated-uH2B promotes DSB repair through facilitating histone eviction and subsequent loading of repair proteins.

Regulation of Histone Ubiquitination in Response to DNA Double Strand Breaks

“…Overall, loss of RNF20-RNF40 gives rise to increased damage and renders cells sensitive to DSB-inducing agents [ 90 ]. These findings mimic those in yeast depleted for Bre1, the homolog to RNF20-RNF40 [ 93 , 94 , 95 ]. Similarly, use of H2BK120/125R, an H2B mutant that cannot be ubiquitinated (K125 can be ubiquitinated in the event that K120 is unavailable) also delayed resolution of DSB foci.…”

“…Furthermore, promoting chromatin decondensation by treatment with chloroquine, trichostatin A, or hypotonic buffer restores IRIF formation in RNF20-depleted cells [ 90 ]. It has been proposed in yeast that monoubiquitination of H2B facilitates the accumulation of downstream repair factors at sites of breaks through altering histone dynamics and promoting nucleosome disassembly, reminiscent of its known role in transcriptional elongation [ 94 , 98 , 99 ]. In support of this, H2B ubiquitination has been shown to influence crosstalk with other histone marks including H4K20 methylation and H4K9 methylation that in turn may impact the downstream recruitment of DSB factors [ 94 , 95 ].…”

Repression of a large number of genes requires interplay between homologous recombination and HIRA

“…Our results indicate that nucleosome peaks around the TSS are slightly increased in the dbl2Δ mutant compared with the wild-type (Figure 7A ), indicating that Dbl2 could be involved in removing excess nucleosomes. In support of this notion, Rad51 overexpression stimulates the histone eviction at DSBs in bre1Δ mutant ( 123 ). In wild-type cells, Bre1 stimulates histone eviction at DSBs by H2B ubiquitination ( 123 ).…”

“…In support of this notion, Rad51 overexpression stimulates the histone eviction at DSBs in bre1Δ mutant ( 123 ). In wild-type cells, Bre1 stimulates histone eviction at DSBs by H2B ubiquitination ( 123 ). Furthermore, a recent study analysing regulators of heterochromatin spreading in different chromatin contexts demonstrated that the HIRA histone chaperone and genes involved in HR pathway, such as rad50 and sfr1 , act in opposite ways ( 124 ).…”

RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer

“…RNF20 is known to be required to regulate chromosome structure by monoubiquitinating histone H2B at lysine 123 (H2BK123) in budding yeast and at lysine 120 (H2BK120) in humans (12,13). H2BK120ub is a key histone modification that plays critical roles in gene transcriptional regulation and higher order chromatin organization in many species (8,(14)(15)(16)(17)(18)(19)(20). Ubiquitination of histone H2B (H2Bub) has been reported to be associated with highly expressed active genes in human cells (15,21).…”