Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation

Abstract

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADPribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.Chromatin is widely held to be a barrier to the execution of nuclear functions including DNA replication, DNA repair, and RNA transcription. Thus, it is important to define the mechanisms that regulate chromatin structure to orchestrate and execute these functions. The molecular delineation of cellular events occurring at the sites of DNA strand breaks has revealed an intricate network of sensing, signaling, and repair proteins that coordinate the accurate correction of DNA damage (1, 2). Accompanying this DNA strand break repair factor network is a remodeling of chromatin structure (3, 4). For example, modification of the histone H2A variant, H2AX, through phosphorylation of serine 139 is so characteristic of DNA double-strand break (DSB) 4 signaling that it has been used as a quantitative marker to measure the number of DSBs in the cell (5).In addition to biochemical modifications of chromatin proteins, morphological changes in chromatin structure have also been reported. In a landmark paper, Kruhlak and colleagues (6) used laser microirradiation to introduce DSBs at defined sites in the interphase nucleus while simultaneously photoactivating a GFP-tagged histone H2B. This enabled visualization of the changes in the organization of chromatin in response to the introduction of DSBs by virtue of the photoactivation of GFPhistone H2B solely in the region of the nucleus where DSBs were introduced into the chromatin. The large-scale decondensation of irradiated chromatin observed in this study was found to be extremely rapid, occurring within seconds of damage, and was ATM-independent but ATP-dependent. This chromatin relaxation was observed in both H2AX null and ATM null cells indicating that the phosphorylation of histone H2AX or any process dependent on either it or ATM kinase activity were not required for the observed decondens...

The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage

“…In agreement with recent reports (Khurana et al ., 2014; Strickfaden et al ., 2016), we observed that chromatin relaxation at DNA lesions is PARylation dependent (Figures 2A and Supplemental Figure S1, A–D). Interestingly, inhibiting PARylation not only abolished chromatin relaxation at DNA damage sites but also induced a small but significant chromatin overcompaction upon laser microirradiation (Figure 2A).…”

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

“…Our data support this proposed mode of recruitment given that: (1) 53BP1 strongly parallels the pattern observed with γH2AX and the FK2 antibody (although the broad specificity of this antibody toward many ubiquitinated substrates precludes a definitive conclusion regarding the nature of the modified protein) and (2) 53BP1 association with DSB is minimal in G2, in agreement with the proposed dilution of H4K20me2 on post-replicative chromatin (Pellegrino et al., 2017). H1, on the other hand, was recently shown to be displaced from sites of DNA damage (Sellou et al., 2016, Strickfaden et al., 2016). Interestingly, RNF8-mediated H1 ubiquitination loosens its interaction with chromatin (Thorslund et al., 2015), providing potential mechanisms for H1 eviction from megabase-wide γH2AX domains.…”

The nucleosomal surface is the main target of histone ADP-ribosylation in response to DNA damage

“…This automodification has been linked to relaxation of chromatin, recruitment of DNA damage repair factors, and subsequent dissociation of the enzyme from chromatin. 1,40–42 Conformational changes within the enzyme and electrostatic repulsion aid in this dissociation from DNA. 43 …”