Recent advances enabled by the Hi-C technique have unraveled many principles of 
chromosomal folding that were subsequently linked to disease and gene 
regulation. In particular, Hi-C revealed that chromosomes of animals are 
organized into topologically associating domains (TADs), evolutionary conserved 
compact chromatin domains that influence gene expression. Mechanisms that 
underlie partitioning of the genome into TADs remain poorly understood. To 
explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C 
and poly(A)(+) RNA-seq in four cell lines of various origins (S2, Kc167, 
DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between 
TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly 
enriched with the insulator protein dCTCF, while another insulator protein 
Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs 
harbor active chromatin and constitutively transcribed (housekeeping) genes. 
Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) 
predicts TAD boundaries much worse than active chromatin marks do. 
Interestingly, inter-TADs correspond to decompacted inter-bands of polytene 
chromosomes, whereas TADs mostly correspond to densely packed bands. 
Collectively, our results suggest that TADs are condensed chromatin domains 
depleted in active chromatin marks, separated by regions of active chromatin. We 
propose the mechanism of TAD self-assembly based on the ability of nucleosomes 
from inactive chromatin to aggregate, and lack of this ability in acetylated 
nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and 
find that TAD partitioning may be explained by different modes of 
inter-nucleosomal interactions for active and inactive chromatin.