Eukaryotic genomes are packaged through multiple steps into higher levels of chromatin structure. It is now well established that remodeling of local chromatin structure is a key step common to the initiation of multiple chromosomal functional events, including transcription, DNA replication, repair, and recombination (Elgin and Workman, 2000
; Fyodorov and Kadonaga, 2001
). Whereas intense research in the past decade has provided a wealth of information regarding the biochemical basis for chromatin remodeling at the nucleosome level, much less is known about reorganization of higher levels of chromatin structure. It remains unclear whether the known modifications of nucleosome organization are sufficient for changes in large-scale chromatin organization, or whether novel mechanisms acting at higher levels of chromatin structure are responsible for changes in large-scale chromatin organization.
A major difficulty in distinguishing these two possibilities is that most assays for identifying transcriptional activators or coactivators have used transcriptional activity as a final readout. Direct assays for changes in higher order chromatin structure have not been used previously. Because BRCA1 had been functionally implicated in a range of nuclear processes, it was reasonable to postulate that the regulation of these multiple nuclear events might occur through a general chromatin remodeling activity of BRCA1. The lac repressor–tethering system, while artificial in many aspects, provided an excellent assay to pursue this research direction.
Our findings in this current study strongly suggest that BRCA1 recruits COBRA1, a novel factor, to the lac operator–containing chromatin region. Within the constraints of the lac repressor–tethering assay, BRCA1-dependent unfolding of higher levels of chromatin structure appears to be at least partially mediated through recruitment of COBRA1. Notably, BRCA1-mediated chromatin decondensation is distinct from transcriptional activation per se and histone hyperacetylation. It is unclear how unique the histone acetylation–independent chromatin unfolding is. Although the chromatin unfolding produced by VP16, E2F1, and p53 is accompanied by histone hyperacetylation, no causal relationship between histone acetyltransferases recruitment and chromatin unfolding has been demonstrated. Moreover, preliminary data suggests that large-scale decondensation produced by estrogen receptor does not correlate with histone hyperacetylation (A. Nye and A. Belmont, personal communication).
Whereas the lac-based chromatin-unfolding assay provides a new tool for visualizing chromatin dynamics and in vivo protein–protein interactions in mammalian cells, it is important to point out that the molecular and biochemical basis for BRCA1-mediated chromatin decondensation is yet to be understood. Furthermore, utilization of a long tandem array of lac binding sites may raise the concern that the observed chromatin unfolding could simply be due to steric effects of the proteins/protein complexes that are brought to the lac binding sites. However, we believe this possibility is unlikely because our work does not indicate an obvious correlation between the potency of chromatin unfolding and the size or charge of the tethered protein fragments. For example, the minimal chromatin-unfolding domain defined in our study is only 50 aa long (BRCA1C). In contrast, several other BRCA1 fragments that range in size from 324 to 740 aa do not display any chromatin-unfolding activity ( B). In addition, BRCT1 and BRCT2 have a net charge of +5 and –6, respectively, yet both demonstrate strong chromatin-unfolding activity. On the other hand, BRCT1N carries more positive charges (+5) than BRCT1C (+1), but only the latter can induce chromatin decondensation. Finally, in previous work using either lac repressor tetramer, or lac repressor fused to several other protein domains up to ~350 aa in size (i.e., GFP), no effect on large-scale chromatin structure has been observed (Robinett et al., 1996
, and A. Belmont, personal communication).
In our minds, a more serious caveat concerning the lac repressor–tethering system is the question of whether the observed effects produced by BRCA1 and other proteins on large-scale chromatin unfolding are physiologically relevant given the high numbers of lac operator repeats involved. In fact, the exact number of lac repressors binding per lac operator has not been determined and there is reason to believe that lac repressor binding may be significantly limited by steric constraints and phasing of lac operators relative to the nucleosome linker DNA. However, we note that a recent study on a transgene array containing a viral promoter with several glucocorticoid hormone response elements observed a very similar type of large-scale chromatin decondensation produced by glucocorticoid receptor (Muller et al., 2001
). Ultimately, validation of the physiological significance of our observations of BRCA1-dependent large-scale chromatin unfolding will depend on the outcome of future experiments exploring the mechanisms of unfolding and identifying the biological functions of other transacting factors involved, such as COBRA1.
With these caveats in mind, we find it particularly intriguing that a subset of cancer-predisposing mutations of BRCA1 lead to increased chromatin unfolding and recruitment of COBRA1. Although the genotype–phenotype relationship in cancer-predisposing mutations of BRCA1 remains to be understood, it is generally assumed that most, if not all, BRCA1 mutations lead to loss of the biological functions of the protein. However, the behaviors of the BRCA1 mutants in the chromatin-unfolding assay clearly demonstrate an allele-specific effect. Consistent with this finding, it has been reported that mutations at different locations along the coding sequence of BRCA1 differentially affect the penetrance of BRCA1-dependent breast and ovarian cancer (Gayther et al., 1995
; Risch et al., 2001
). It remains to be determined whether the three groups of mutations that cause differential effects on chromatin unfolding () may indeed lead to distinct clinical consequences in terms of risks, types, or prognosis of BRCA1-associated cancers. In particular, it will be interesting to see whether those 3′ mutations that enhance chromatin unfolding exhibit any dominant or semidominant phenotype in cancer genetics. It is conceivable that constitutive decondensation of large-scale chromatin structure may cause additional deleterious effects on genome stability and thus result in more severe clinical consequences in cancer development.
Our study also indicates that BRCT-mediated chromatin unfolding may be tightly regulated. As shown in and , a single BRCT motif is more potent in chromatin unfolding than the larger fragments of the protein that contain both BRCT repeats. Furthermore, the full-length wild-type BRCA1 only exhibits a moderate chromatin-unfolding activity, whereas the cancer-predisposing mutations in group c ( B) that affect the integrity of the BRCT repeats significantly enhance the chromatin-unfolding activity and COBRA1 binding. These results lead us to the following two models that could explain negative regulation of BRCA1-mediated chromatin unfolding. In a “trans-inhibition” model, we speculate that binding of a putative inhibitor (i.e., CtIP) to AD2 region of BRCA1 may prevent BRCA1 from interacting with its cofactors for chromatin unfolding (i.e., COBRA1). In an alternative, “cis-inhibition” model, the two BRCT tandem repeats may form an intramolecular dimer. This in turn may reduce the affinity of both BRCT repeats for their corresponding cofactors. Conceivably, the “superactivating” mutations in group c may prevent binding of the putative inhibitor or the intra-molecular interaction between the two BRCT motifs, thus rendering the protein constitutively active for binding to the cofactors that mediate chromatin unfolding.
It is plausible that BRCT-mediated chromatin unfolding may lead to a novel nuclear function of BRCA1 in global reorganization of the genome. However, in light of the known function of the COOH-terminal region of BRCA1 in transcription and DNA repair, the observed chromatin decondensation may represent the first step in BRCA1-mediated regulation of these two nuclear processes ()
. In such a model, higher order chromatin decondensation may be followed by BRCA1-mediated chromatin modification at the nucleosomal level (i.e., histone hyperacetylation) and recruitment of the transcription or repair machineries. As shown in , nonsense mutations that result in truncation of the entire COOH-terminal region (5′ mutations) may abolish BRCA1 functions in all three steps, resulting in a completely inactive mutant protein. On the other hand, mutations located at the 3′ end of the gene (3′ mutations) may render BRCA1 incompetent at the second and third steps, but still allow constitutive chromatin decondensation at the first step. This could then lead to accumulation of extensively unfolded chromatin structure as seen in our study. Consistent with this model, many 3′ cancer-predisposing mutations abolish BRCA1 interactions with RNA pol II holoenzyme and the histone modifying enzymes (Scully et al., 1997a
; Neish et al., 1998
; Yarden and Brody, 1999
), as well as nucleosome remodeling in yeast (Hu et al., 1999
). Thus, chromatin unfolding may be a necessary but not sufficient step for BRCA1-dependent transcriptional activation. Additional steps such as histone modification and recruitment of the basal machinery may also be required for fulfilling BRCA1 function in transcription and DNA repair.
Figure 7. Model for BRCA1-mediated nuclear functions. Inhibitory and stimulatory effects of the 5′ and 3′ mutations on the three steps are indicated by bars and arrows on the sides, respectively. Factors in parentheses are those that may be targeted (more ...)