It is known that the SWI/SNF complex employs one of two different core ATPases, Brg1 or Brm, to remodel chromatin structure by repressing and facilitating transcription. In this study, the individual function of Brg1 and Brm on chromatin organization, nuclear structure, and mitotic division was determined.
The SWI/SNF complex is thought to use the Brg1 and Brm subunits interchangeably to mediate the ATPase function critical for chromatin remodeling (Roberts and Orkin, 2004
). In terms of biochemical activity, there is significant functional redundancy between these core ATPases (Phelan et al., 1999
). Furthermore, several transcriptional processes can be mediated via the activity of either ATPase. However, there are clear distinctions between Brg1 and Brm related to tissue-specific dependence and overall organismal survivability (Muchardt and Yaniv, 2001
; Kadam and Emerson, 2003
). Our data show that deficiency of Brg1, but not Brm, leads to the dissolution of discrete pericentromeric heterochromatin domains. This finding suggests that the Brg1 ATPase either represents a larger fraction of the total ATPase protein in the cell or that it has distinct functions from Brm. However, this effect on chromatin is readily apparent via multiple approaches and results in the structural dispersion of heterochromatin domains. These structures are known to be highly enriched in trimethylated histone H3 lysine 9 and histone H4 lysine 20. Correspondingly, Brg1 deletion results in a significant dissemination of these modifications. Interestingly, our data reveal that loss of SNF5, another core subunit of the SWI/SNF complex, does not elicit dissolution of heterochromatin domains, nor does it affect the localization of trimethylation on histone H4 lysine 20 or histone H3 lysine 9 (Supplemental Figure S2). It is well established that these histone markers are not required for the structural maintenance of the heterochromatin domains, because deletion of Suv39H1/H2 and retinoblastoma (RB)-related family members results in the loss of trimethylation of histone H3 lysine 9 and histone H4 lysine 20 at heterochromatin domains, respectively, without compromising the integrity of the overall chromatin domain structure (Peters et al., 2001
; Gonzalo et al., 2005
). Thus, the maintenance of pericentromeric heterochromatin domains is hierarchical, with an underlying Brg1-dependent function that is critical for structural integrity.
The regulation of transcription and modulation of chromatin structure are critically involved in cellular proliferation, and aberrations associated with these processes are implicated in tumorigenesis. The effect of SWI/SNF ATPase deficiency on cellular proliferation remains the subject of controversy, as there are tumor cell lines which harbor discrete loss of both ATPases (Strobeck et al., 2002
; Reisman et al., 2003
). These cell lines actively proliferate and are, in fact, compromised for the appropriate response to growth inhibitory signals as elicited through the RB pathway. Although such tumor cell models are important for interrogating pathways, it is not possible to determine the cellular requirement for ATPases because other genetic events could obviate their necessity. Thus, the analyses of cultured cells from gene-targeted animals afford an opportunity to define their intrinsic role in proliferation. Loss of Brm has minimal effect on proliferation, and primary and immortalized lines lacking Brm function can be readily propagated. However, Brg1 deletion resulted in a substantial reduction in cellular proliferation and BrdU incorporation and was selected against during culture. Moreover, this effect was observed in both primary cell culture and cultures specifically deficient in canonical p53 function. Thus, loss of Brg1 is not tolerated even in the context of rapidly proliferating immortalized populations. This finding is supportive of previous analyses in embryonal carcinoma cells (Sumi-Ichinose et al., 1997
). Cell cycle analyses strongly suggest that the principle negative impact of Brg1 deficiency on proliferation is manifest during mitotic progression. Our data support the notion that Brg1 deficiency can be overcome by virtue of additional stochastic events; however, this process was highly sporadic even in the context of immortalized 3T3 populations and selection for Brg1 deletion. Thus, tumor cell lines and potentially other Brg1-deficient cell types, presumably use compensatory mechanisms to bypass the requirement that we observed.
Findings from multiple laboratories have suggested that deletion/loss of Brg1 may contribute to the genesis of cancer (Murphy et al., 1999
; Lee et al., 2002
); however, the underlying mechanism for this process is unclear. The data presented here indicate that loss of Brg1 results in aberrant mitotic progression and provides evidence of genomic instability. These findings are supported by previous studies that show both the localization of SWI/SNF to mitotic chromosomes (Xue et al., 2000
), and the requirement for related complexes for proper mitosis and chromosome maintenance (Baetz et al., 2004
; Campsteijn et al., 2007
). This phenomenon is also similar to that observed with the knockout of a critical centromeric protein, inner centromere protein, which disrupts chromatin structure and leads to genomic instability (Cutts et al., 1999
). Importantly, normal mitotic progression is dependent upon proper centromeric function, and the loss of Brg1 seems to result in disassembly of these pericentromeric heterochromatin domains. This suggests a mechanism for the observed mitotic abnormalities, because disruption of such domains compromises mitotic fidelity. It has been recently reported that tumors arising in Brg1+/−
mice, although not mimicking specific pathways, are best characterized by genomic instability (Bultman et al., 2008
). Importantly, this study also concludes that tumor formation in Brg1+/−
mice occurs due to haploinsufficiency rather than loss of heterozygosity (Bultman et al., 2008
), suggesting a lack of selection or proliferative advantage with the complete ablation of Brg1. Thus, in the context of Brg1 deficiency, resultant dispersion of pericentric heterochromatin domains and mitotic dysfunction could potentially represent the underlying key etiological feature relevant to tumorigenesis.