Cells are constantly exposed to environmental insults that may cause DSB of DNA. Cells respond to DSB by activating repair pathways that depend on alterations of chromatin structure (38
). Histone modifications play a key role in mediating these chromatin remodeling events. Previous studies have identified some of the histone modifications that occur in response to DSB in mammalian cells. For e.g., phosphorylation of S14 in histone H2B, phosphorylation of S139 in histone H2AX, and poly(ADP-ribosylation) of glutamate residues in histone H2A (20
). The present study expands the list of damage-dependent histone modifications by unveiling a temporal pattern of K12-biotinylation in histone H4 in response to DSB in human cells.
This is the first time that a correlation has been shown between the biotinylation of a distinct lysine residue in a human histone and a physiological event that requires chromatin remodeling. In previous studies, streptavidin peroxidase has been used as a probe for biotin to provide circumstantial evidence for biological functions of histone biotinylation in cell proliferation and UV-induced DNA damage (12
). Note that binds to biotin in general and does not permit pinpointing distinct biotinylation sites in histones, e.g., K8 and K12 in histone H4 (13
). In contrast, the site-specific antibody to K12-biotinylated histone H4 as a probe used in this study indicates the physiological relevance of biotinylation of K12 in histone H4 in DNA repair signaling.
Specifically, this study provides evidence (i) that a transient decrease of K12-biotinylation in histone H4 is an early signaling event in response to DSB; (ii) that signaling by biotinylation of K12 is a universal rather than a tissue-specific mechanism in humans; (iii) that biotinylation of K12 in histone H4 decreases specifically in response to DSB but does not decrease in response to single-strand breaks or formation of thymine dimers; and (iv) that biotin deficiency is associated with decreased cell survival in response to DSB.
The decreased biotinylation of K12 in histone H4 in response to DSB occurs before phosphorylation of S14 in histone H2B and potentially ahead of or in concert with poly(ADP-ribosylation) of glutamate residues in histone H2A. Currently it is unknown whether phosphorylation of S14 and poly(ADP-ribosylation) of glutamate residues directly depend on biotinylation of K12 in histone H4. This uncertainty is being addressed in ongoing investigations in our laboratory. Moreover, it is uncertain as to whether biotinylation of K12 itself depends on other damage-induced modifications of histones such as phosphorylation of H2AX (40
). Note that histone H2AX also is a potential target for biotinylation (15
). Theoretically, biotinylation of K12 in histone H4 might mediate either DNA repair or apoptosis in response to DSB. We favor the former explanation, given that biotin deficiency was associated with decreased survival of etoposide-treated cells compared with biotin-sufficient controls in the present study. In this context the following question needs to be addressed. Why would biotin deficiency decrease cell survival in response to DSB, although the damage-related signaling event actually causes a decrease in K12-biotinylation? We speculate that a sufficient supply of biotin is essential to allow for an efficient re-biotinylation of K12 following its rapid transient debiotinylation. We further speculate that biotinylation of K12 plays a role in the reassembly of nucleosomes following DNA repair (41
Current models of chromatin remodeling in response to DSB are consistent with the findings reported here. Specifically, it has been proposed that DSB are associated with a rapid, yet transient, relaxation of chromatin structures mediated by histone modifications (42
). Relaxation of chromatin gives DNA repair factors access to sites of DNA breaks. Subsequently, chromatin re-condensation prevents dissociation of the two broken ends of DNA before repair is mediated by ligases. Note that K12-biotinylated histone H4 is known to be associated with chromatin condensation such as in pericentromeric heterochromatin (A.M. Oommen and J. Zempleni, submitted for publication). Hence, a rapid decrease in K12-biotinylation in response to DSB is likely associated with an opening of chromatin structures, giving repair proteins access to sites of damage. Subsequent re-biotinylation of histone H4 is likely associated with re-condensation of chromatin. Note, however, that this is an untested hypothesis.
Is the response to DSB breaks comparable in transformed human cell lines and in primary human cells? Previous studies suggested that the frequency of DNA breaks in response to X-rays is similar in transformed and primary cells (43
). Also, the kinetics of joining DNA ends is similar in transformed and primary cells. These observations are consistent with the notion that transformed human cells maintain a normal response to DSB breaks. Nevertheless, it remains to be formally demonstrated that pathways of DSB repair are identical in the choriocarcinoma cells used in this study and in primary placental cells.
Taken together, the findings presented here are important for health professionals, based on the following lines of observation. Dietary biotin deficiency is relatively rare in humans, but certain subgroups in the general population are at increased risk for developing biotin deficiency. First, catabolism of biotin is increased during pregnancy (44
); consistent with this observation is the fairly high prevalence of marginal biotin deficiency in pregnant women (45
). Second, some drugs are known to interfere with biotin metabolism. For example, certain anti-convulsants and lipoic acid impair biotin transport and distribution, potentially causing biotin deficiency (35
). Third, biotin transporter deficiency may cause intracellular biotin depletion (47
). Fourth, mutations of the two enzymes biotinidase and holocarboxylase synthetase that mediate biotinylation of histones are relatively prevalent in humans. For example, the prevalence of biotinidase deficiency is 1 in 60,000 live births in humans (48
). Currently it is unknown whether dietary biotin deficiency or drug-induced biotin deficiency, and mutations of genes coding for biotin transporters, biotinidase, and holocarboxylase synthetase are associated with an impaired ability to repair damaged DNA in humans. Likewise, it is unknown whether supplementation with pharmacological dose of biotin enhances an individual’s DNA repair capabilities.