Most studies of ISWI complexes in Drosophila
and other organisms have focused on their ability to alter the structure or spacing of nucleosomes, the fundamental unit of chromatin structure. Our findings reveal that ISWI also plays a global role in the regulation of higher-order chromatin structure. The Iswi
mutations used in this study eliminate the function of multiple chromatin-remodeling complexes, including ACF, NURF, and CHRAC [11
]. Which of these complexes are required for the formation of higher-order chromatin structure? Loss of function mutations in Acf1—
which encodes a subunit protein shared by ACF and CHRAC—do not cause obvious defects in higher-order chromatin structure [27
]. By contrast, loss of function mutations in E(bx)—
which encodes a subunit specific to NURF—cause male X chromosome defects similar to those observed in Iswi
]. These findings suggest that ISWI modulates higher-order chromatin structure within the context of NURF, as opposed to ACF or CHRAC.
We observed a striking correlation between the severity of the chromosome defects resulting from the loss of ISWI function and the loss of the linker histone H1. This correlation suggests that ISWI regulates higher-order chromatin structure by promoting the association of histone H1 with chromatin. Histone H1 and other linker histones influence higher-order chromatin structure in vitro by stabilizing interactions between nucleosomes and chromatin fibers [29
]. Although the ability of histone H1 to promote chromatin compaction in vitro is well established, its function in vivo has been a topic of considerable debate [30
]. A protein with biochemical properties reminiscent of linker histones—HHO1—is present in budding yeast; surprisingly, HHO1 is not essential for viability in yeast, and hho1
mutations have little effect on either gene expression or chromatin structure [32
]. Genetic studies in Tetrahymena
have suggested roles for linker histones in chromatin condensation and gene expression [34
], but the relevance of these studies to histone H1 function in higher eukaryotes remains unclear. Studies of histone H1 function in higher eukaryotes have been complicated by the presence of redundant genes encoding histone H1 or histone H1 subtypes [36
]. In spite of these difficulties, recent studies have revealed important roles for histone H1 in chromosome compaction in Xenopus
and mice [37
]. Thus, the chromosome defects observed in Iswi
mutants could easily result from inefficient incorporation of histone H1 into chromatin.
How might ISWI promote the association of histone H1 with chromatin? Since ISWI is not required for histone H1 synthesis, ISWI may directly promote the assembly of chromatin containing histone H1 following DNA replication. Recent biochemical studies provide support for this possibility: ACF promotes the ATP-dependent assembly of H1-containing chromatin in vitro [26
]. Loss of ACF1 function does not cause obvious changes in chromosome structure, however, suggesting that ACF either does not regulate higher-order chromatin structure in vivo or plays a redundant role in this process [27
]. It remains possible that ISWI promotes the assembly of histone-H1-containing chromatin within the context of NURF or another chromatin-remodeling complex.
The ability to promote histone H1 assembly is not a common property of all chromatin-remodeling factors, as illustrated by recent biochemical studies of CHD1 [26
]. Like ACF and other ISWI complexes, the CHD1 ATPase promotes the assembly of regularly spaced nucleosomes in vitro [26
]. By contrast, CHD1 does not promote the incorporation of histone H1 during chromatin assembly in vitro [26
]. These biochemical studies provide a plausible explanation for why the loss of ISWI function leads to the loss of histone H1 without causing dramatic changes in nucleosome assembly in vivo.
In other organisms, depletion of histone H1 leads to a significant decrease in the nucleosome repeat length [29
], presumably because of the failure to efficiently incorporate histone H1 during replication-coupled chromatin assembly. By contrast, the loss of ISWI function in salivary gland nuclei leads to a decrease in the amount of histone H1 associated with chromatin without causing dramatic changes in nucleosome repeat length (B). It is therefore tempting to speculate that ISWI promotes histone H1 incorporation via a replication-independent process. The association of histone H1 with chromatin is far less stable than that of core histones; histone H1 undergoes dynamic, global exchange throughout the cell cycle [41
]. Photobleaching experiments in Tetrahymena
and vertebrates have suggested that the majority of histone H1 molecules associated with chromatin are exchanged every few minutes [37
], but little is known about the factors that regulate this process. Based on our findings, ISWI is an excellent candidate for a factor that regulates the dynamic exchange of histone H1 in vivo. Further work will be necessary to determine whether ISWI promotes histone H1 incorporation via replication-dependent or -independent mechanisms.
Our findings, together with previous studies, suggest that acetylation of H4K16 may regulate the association of linker histones with chromatin in vivo. The histone H4 tail is required for the nucleosome-stimulated ATPase activity of ISWI, and for its ability to slide nucleosomes and alter their spacing in vitro [21
]. The region of the H4 tail that is critical for ISWI function in vitro is a DNA-bound basic patch (R17
) adjacent to H4K16, the residue that is acetylated by the MOF histone acetyltransferase [21
]. The acetylation of H4K16 interferes with the ability of ISWI to interact with the histone H4 tail and alter the spacing of nucleosome arrays in vitro [19
]. Consistent with these findings, dosage compensation is necessary and sufficient for the decondensation of the X chromosome in Iswi
mutant larvae, and genetic studies have revealed a strong functional antagonism between ISWI and MOF [20
]. Thus, H4K16 acetylation may function as a switch that regulates the histone H1 assembly mediated by ISWI.
Our microarray studies revealed that ISWI is required for the proper expression of a large number of genes. These findings are consistent with numerous studies implicating ISWI in transcriptional regulation in vitro and in vivo [11
]. Does ISWI modulate transcription by altering higher-order chromatin structure? We suspect that ISWI regulates transcription and higher-order chromatin structure via distinct mechanisms, since we observed no obvious correlation between the magnitude of the changes in gene expression and chromosome structure observed in Iswi
mutant larvae. This is consistent with genetic studies in other organisms that have revealed that the loss of histone H1 does not cause dramatic changes in gene expression [33
]. We also failed to observe a correlation between the magnitude of transcriptional derepression and gene size in Iswi
mutant larvae (data not shown), as would be expected if ISWI relieved a general block to transcriptional elongation by Pol II. It should be noted, however, that relatively subtle, but biologically important, changes in gene expression may have escaped detection in our microarray studies. Further work will be necessary to clarify this issue and to determine whether ISWI regulates transcription and higher-order chromatin structure via distinct or related mechanisms.