The emerging concept of gene bookmarking unifies potential mechanisms by which genes are marked for post-mitotic re-activation or re-silencing so that their transcriptional state is accurately passed on to progeny cells [1]. In a recent elegant study, Zhao et al. [2] employed real-time quantitative fluorescence microscopy to assess the kinetics of transcriptional reactivation after mitosis. Their findings support a bookmarking mechanism centered on bromodomain protein 4 (BRD4), a member of the bromodomain and extraterminal (BET) family of proteins, and a promising target for cancer therapeutics.

Importantly, the Tet-On activator was lost from chromatin at the onset of mitosis, precluding the possibility that the bookmarking and enhanced re-activation kinetics were due to its continued association with the locus. This observation suggests the presence of an activator-independent memory system for gene bookmarking.

In addition to the data presented by Zhao et al., BRD4 has several features rendering it a promising candidate for an efficient bookmark: it is a member of several transcription complexes and acts predominantly as a transcriptional coactivator (through its interaction with positive transcription elongation factor b (pTEFb) [4]), and its tandem bromodomains bind to acetylated histone tails, preferentially those of H4 acetylated at two or more lysines. Most tellingly, one of the defining characteristics of BRD4 is its retention on mitotic chromosomes in a range of cell types. This is perhaps surprising given the chromatin binding specificity of BRD4, since overall histone acetylation levels are markedly reduced on mitotic chromosomes; however, certain transcriptional start sites retain acetylation throughout mitosis. Intriguingly, BRD4 has been shown to localize to these regions during mitosis [5]. In addition, binding of BRD4 to these sites rapidly increased at the onset of telophase, concurrent with enhanced acetylation [5]. Both phenomena are likely to be mediated by interaction between BRD bromodomains and acetyl-lysines, suggesting an active role for BRD proteins and acetylated lysines in a bookmarking system.

The observation that the transgene locus, visualized by the fluorescent lacI fusion protein, takes up a larger volume in the nucleus when bound by BRD4 may point to BRD4 exerting its bookmark function by engaging H4K5ac at the locus and establishing a less compact and more accessible chromatin environment, facilitating re-activation of the locus. The first bromodomain of BRD4 is both necessary and sufficient for chromatin decompaction, and both full-length BRD4 and the isolated bromodomain can establish a decondensed structure at the reporter when artificially recruited as lacI fusions. Further studies are necessary to determine whether decompaction is the predominant mode of action for BRD4 in bookmarking; other mechanisms, such as the efficient recruitment of transcription complexes, are likely to exist in parallel with, or alternatively to, decompaction. Moreover, it cannot be ruled out that the highly repetitive architecture of the reporter, featuring arrays of hundreds of binding sites, may distort kinetics and chromatin environments. Although technically challenging, it would be fascinating to observe in real time post-mitotic reactivation of loci that more closely resemble actual gene structures. Despite those technical limitations, the work of Zhao et al. provides us with novel insights into the kinetics of BRD4 recruitment and its role in gene bookmarking.

The finding by Zhao et al. that BRD4 functions as a gene expression bookmark prompts the question of whether the mode of action of BET inhibitors extends to the disruption of gene bookmarking in addition to the impairment of transcriptional elongation. Zhao et al. addressed this question by examining the impact of JQ1 on the output of their assay, finding that JQ1 treatment generally increased the mobility of BRD4 at actively transcribing reporter cassettes [2]; this is in agreement with previous studies [7] and indicative of a weakened interaction with chromatin. Notably, post-mitotic reactivation was markedly slowed down in the presence of JQ1, whereas interphase induction kinetics remained unaffected [2]. These results are consistent with a pronounced impact of JQ1 (and likely other BET inhibitors) on gene bookmarking.