The packaging of DNA into chromatin is critical for the organization and expression of eukaryotic genes 
. The basic unit of chromatin structure, the nucleosome, contains the core histones H2A, H2B, H3 and H4. The association of nucleosomes with histone H1 and other linker histones facilitates their packaging into 30 nm fibers, which in turn are packaged into increasingly compact higher-order structures. Nucleosomes and other components of chromatin can repress transcription by blocking the access of regulatory proteins and the basal transcriptional machinery to DNA. There is growing evidence that levels of chromosome organization above the level of the nucleosome – including chromosome folding, pairing and looping – also play important roles in the regulation of gene expression. For example, condensin and cohesin, which were initially identified by their roles in mitosis and meiosis, modulate transcription by promoting long-range chromosomal interactions and DNA looping in interphase cells 
The repressive effects of nucleosomes on transcription are modulated by two general mechanisms: the covalent modification of nucleosomal histones and ATP-dependent chromatin remodeling 
. By altering the structure or positioning of nucleosomes, ATP-dependent chromatin-remodeling factors play critical roles in transcription and other nuclear processes. Dozens of chromatin-remodeling factors, including members of the SWI/SNF, ISWI, CHD and INO80 families, have been identified in organisms ranging from yeast to humans. By contrast, relatively little is known about how higher-order chromatin structure is regulated and exploited to control gene expression and other nuclear processes.
A major barrier to the identification of factors that regulate higher-order chromatin structure is the difficulty of visualizing the decondensed interphase chromosomes of diploid cells. This barrier can be overcome through the use of Drosophila melanogaster
as a model organism. During Drosophila
development, many tissues undergo multiple rounds of DNA replication in the absence of cytokinesis, leading to the formation of huge polytene chromosomes containing hundreds of aligned sister chromatids. These transcriptionally active chromosomes are indistinguishable from the interphase chromosomes of diploid cells in most respects. Genetic studies in Drosophila
have identified numerous factors that regulate polytene chromosome structure, including ISWI, an ATP-dependent chromatin-remodeling factor. The loss of ISWI
function leads to the decondensation of salivary gland polytene chromosomes, possibly due to failure to assemble chromatin containing the linker histone H1 
. This striking phenotype led us to investigate the potential involvement of another ATP-dependent chromatin-remodeling factor, Drosophila
Mi-2 (dMi-2), in the regulation of higher-order chromatin structure.
dMi-2 functions as the ATPase subunit of multiple chromatin-remodeling complexes, including the NuRD (Nucleosome Remodeling and Deacetylase) complex and dMec (Drosophila
MEP-1 containing complex) 
. NuRD is highly conserved in metazoans and is thought to repress transcription via its chromatin-remodeling and histone deacetylase activities 
. dMec is the most abundant dMi-2 complex in Drosophila
and has been implicated in SUMO-dependent transcriptional repression 
. Mi-2 plays an important role in cell fate specification in organisms ranging from nematodes to vertebrates. For example, Mi-2 helps maintain the distinction between the germline and soma during C. elegans
; regulates the terminal differentiation of B lymphocytes into plasma cells in mammals 
; and participates in the transcriptional repression of HOX genes by Hunchback and Polycomb in Drosophila
. dMi-2 is also required for the efficient expression of heat-shock genes in Drosophila
, indicating that its function is not limited to transcriptional repression 
Here we report an unanticipated role for Mi-2 in the regulation of higher-order chromatin structure in Drosophila. The loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, the increased expression of dMi-2 in the salivary gland disrupts interactions between sister chromatids and triggers the decondensation of polytene chromosomes. Consistent with these findings, dMi-2 disrupts the association of cohesin with polytene chromosomes. Our studies reveal that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells.