Genetic activity of interphase chromosomes is intimately linked to the properties of chromatin organization. At a very basal level, chromatin is organized in nucleosomes, histone octamere/DNA complexes. These, in turn, form higher-order structures, such as chromomeres, loops, domains, etc. Clearly, key to this organization are the chromatin proteins: histones, their post-translational modifications, and non-histone proteins. Modern methods help reliably address the question of interphase chromatin organization at a nucleosomal level, however details of higher-order chromatin organization still remain obscure. This is largely due to our inability to directly visualize the supra-nucleosomal structures in diploid interphase nuclei. Giant polytene chromosomes from dipterans, in particular from Drosophila, allow one to mitigate this problem.
"Classic" polytene chromosomes from larval salivary glands of D. melanogaster
are composed of bundles of one to two thousand tightly synapsed chromosomal strands, which are formed via multiple rounds of endoreplication of just two starting chromatids. As all the homologous chromomeres from all chromatids are aligned to each other, this results in the formation of a thick "cable" with transverse stripes of compacted chromatin (bands) alternating with decompacted interchromomeric regions (interbands). Local differences in size and compaction of banded material form a unique banding pattern that can be used to accurately map any polytene chromosome region. This in turn allows one to link a particular DNA sequence, genes and proteins to the specific chromosomal region, and so to spatially analyze the genetic processes taking place in the interphase nucleus (for review: [1
According to different estimates, there are 3500-5000 bands and interbands in Drosophila melanogaster
polytene chromosomes; these comprise about 95% and 5% of euchromatic DNA, respectively. On average this corresponds to 30 kb of genomic material per band and 2 kb per interband [2
]. Obviously, the vast majority of genes are situated in bands, as they encompass most of the DNA. As a rule, the degree of chromatin compaction in bands correlates with their transcriptional activity. This is manifested most clearly in case of puffing, i.e. when upon gene activation bands form puffs. Despite the fact that interbands are also represented by decompacted chromatin, their genetic organization and functions are still largely enigmatic. Several hypotheses regarding the functions of interbands were put forward in the literature (for review: [4
]), but can be essentially reduced to just two alternatives. Namely, the interbands correspond to active genes. Or, interbands harbor regulatory regions for genes that are found in the neighboring bands. Neither of these scenarios had been adequately addressed experimentally.
In light microscope, many decompacted regions appear as interbands, however upon closer examination at an EM-level they in fact comprise series of faint bands. Thus, of the regions typically considered interband-like, only some are true interbands. Presently it is well-known that numerous "open chromatin" proteins are typically found in such decompacted regions. For instance, these are different forms of RNA polymerase II [5
], including the paused RNA polymerase II [7
] which is necessary for transcription initiation; these are proteins and protein complexes involved in transcriptional elongation: SPT4, SPT5, SPT6, TFIIH, dMEDIATOR, dELL [8
]. Likewise, these regions frequently contain nucleosome remodeling and histone-modifying proteins: CHD1 [12
], JIL-1 [13
], BRM [14
], COHESIN [15
], TRX [16
], WDS [17
], H2B monoubiquitinating enzyme BRE1 [18
], and NURF, which increases accessibility of chromatin templates [19
]; they harbor histone variants: H4K16ac [20
], H3K9ac, H3K14ac [21
], H3K4me3 [22
]. Furthermore, insulator proteins BEAF-32 [23
] and GAF [24
] as well as pre-replication complex protein ORC6 [25
] are also found in many decompacted regions of polytene chromosomes. Finally, there are at least two interband-specific and interacting proteins, Z4 and CHRIZ (CHROMATOR), however their functions in interbands are presently unknown [26
Despite this plethora of interesting chromatin proteins linked to interbands, their very cytological mapping is not accurate enough, as it is quite challenging to reliably map the protein localization signal to a fine structure of an interband, at least at the resolution level of light microscopy.
Clearly, in order to address the functions of interbands, it is important to be able to accurately map interband regions on a physical map and then to analyze the protein binding profiles and chromatin features in these regions. Unfortunately, using standard mapping techniques, it is close to impossible to precisely map DNA sequences to interbands as their axial lengths are quite small (0.12 mkm on average) [2
]. To solve this problem, one must develop new approaches to mark and identify interband regions. P-element insertions could serve as such useful "markers". Using electron microscopy (EM) analysis of polytene chromosomes from stocks with P-element-based insertions, our group has previously shown that such insertions can be visualized on polytene chromosomes as distinct cytological structures [28
]. In most cases, transcriptionally silent chromatin in such transgenes becomes compacted and forms novel bands, provided that insertions occurred into interbands. When inserted into bands, the compacted material from a transgene typically fuses with the neighboring material and does not form a separate band (Figure ). As the transgene sequence is known, cloning the DNA sequence adjacent to the transgene insertion is straightforward, and so one can unambiguously identify the sequences that belong to interbands [30
Figure 1 Morphology of P-element insertions in polytene chromosomes. Possible scenarios: A - transgenic insertion into the interband results in formation of a novel band; B - electron microscopy image of the region 84E from chromosome arm 3R of wild-type (top) (more ...)
Using this approach, we mapped and cloned the DNA from 13 interband regions. We found that these interbands were mainly composed of non-coding intergenic regions and 5'-UTRs. Also, many of the interbands were rich in DNase I hypersensitive sites (DHSs), which turned out to behave as "hot spots" for integration of P-element based transgenes [33
With these observations in hands, we decided to further explore the question of functional organization of interbands. First of all, we wanted to establish which proteins were specific to the interbands' open chromatin, and then to ask whether localization of some of these proteins could be correlated on a genome-wide scale. Obviously it was of utmost importance also to understand whether the interbands from polytene chromosomes were "mirrored" by analogous regions in chromosomes from cell lines. Also, in order to address the question of existence of a defined molecular border between bands and interbands, it was interesting and necessary to estimate the length of DNA sequences associated with such proteins. To tackle all these questions, we analyzed the data from Drosophila
genome-wide protein mapping databases, mostly those from NHGRI modENCODE project [34
] and from Filion with co-authors [35
]. These projects included comprehensive genome-wide analysis of a wide array of chromatin proteins and histone modifications from D. melanogaster
cell lines. As a result, 5 [35
], 9 and even 30 [36
] distinct chromatin types were identified, which were characterized by specific combinations of classes of genes and associated proteins.
Using the abovementioned data obtained on interphase chromosomes of cell lines, in the present work we performed comparative analysis of thirteen interband regions from polytene chromosomes searching for the proteins specifically enriched in interbands. Vast majority of interbands studied was found to associate with a set of proteins that is typically found in open chromatin. These open chromatin proteins tended to localize to low nucleosome density and histone H1-depleted regions and to correlate with binding of ORC2, a pre-replication complex protein. Our data suggest that regions possessing most of these features combined are typically smaller than 3-4 kb in length, and that the number of such regions closely matches the estimated number of cytologically distinct interbands in polytene chromosomes. Furthermore, our data demonstrate that interband chromatin is similarly organized in different cell types, thereby suggesting its participation in general processes that serve to form and maintain the functional architecture of interphase chromosomes.