Mapping of bands and interbands in the 9F13-10B13 region of nonpolytene chromosomes
According to the combined cytogenetic and molecular-genetic analysis, the region of polytene chromosome band 10A1–2 is marked with two genes, vermilion
which is located at its distal end 
, and sevenless
, at its proximal side 
. Therefore, we used positions of these two genes on the physical map as starting points for mapping the region in nonpolytene chromosomes.
shows map of the DNA features and protein localization profiles in the region of interest in nonpolytene chromosomes; these data have been generated by fly modENCODE consortium 
as well as in experiments of genome-wide analysis of chromosome proteins 
Localization of proteins and DNA elements in 9F13 – 10B3 region of nonpolytene chromosomes (according to data of modENCODE).
Much like as was observed with the EM mapping data of polytene chromosomes, one can subdivide this region into three distinct domains. First, there are two zones on both flanks of the region (), which were previously shown to lack any interband-associated proteins. The DNA sequences that map to these zones are very large and are flanked with regions displaying interband-like features (). According to the physical map, the leftmost domain may correspond to the 10A1–2 band, as it comprises vermilion and sevenless genes (). The other region showing similar properties should correspond to the large band 10B1–2. Mimicking the EM pattern observed in polytene chromosomes, the region in between 10A1–2 and 10B1–2 appears as a paling of alternating interband-like zones and regions lacking interband features (middle of the ). This region is composed of faint bands intermingled with interbands which corresponds well to the EM mapping data (see ).
So, in nonpolytene chromosomes of mitoticaly dividing cells one can find DNA fragments demonstrating features characteristic to polytene chromosomes, i.e. bands and interbands.
Identical positions of bands and interbands in polytene and nonpolytene chromosomes on genome map
We asked whether interbands have the same borders in nonpolytene and polytene chromosomes. To do so, we first selected two interband fragments on both sides of the 10A1–2 and 10B1–2 bands of nonpolytene chromosomes. The large sizes of both bands allow accurate mapping of said DNA fragments using FISH and immunostaining.
A short chromosome fragment with interband feature which is found immediately distal to the tentative 10A1–2 in nonpolytene chromosomes (arrow 1 on ) has been mapped on polytene chromosomes using three DNA probes ( in Materials and Methods
, spas 9F
(hereafter the names of FISH probes correspond to the gene names that they map to). Given that the 10A1–2 band frequently fuses with the neighboring distal 9F11-12 and 9F13 bands (), we only analyzed rare polytene chromosome spreads where all the bands appeared separate from each other. The three above-mentioned probes were mapped to the interval between the faint band 9F13 and the distal edge of 10A1–2 (, data shown for CG15208
only), i.e. in the expected interband 9F13/10A1–2.
Coordinates and descriptions of probes, selected for FISH mapping on polytene chromosomes in the region 9F13 – 10B3 (coordinates correspond to the version dm3 (r5.24), FlyBase).
Localization in polytene chromosomes of DNA fragments, which according to modEncode data are located in the predicted interbands flanking the 10A1–2 and 10B1–2 bands of the nonpolytene chromosomes.
The region found on proximal side of the 10A1–2 band of nonpolytene chromosomes, has been mapped using two DNA probes, Vago and CG2076 (arrow 2 on ). The signal maps to the proximal edge of 10A1–2 in polytene chromosomes ( show the data for Vago probe only), i.e. to the interband 10A1–2/10A3.
The CG32668 and l(1)10Bb genes (arrows 3 and 4 on ) located distally and proximally to the tentative 10B1–2 band of the nonpolytene chromosomes, map by FISH to the interbands flanking this band in polytene chromosomes (). So, the four probes essentially hybridize to interband regions immediately flanking the two dark bands of the region 10A–B.
To address the question of whether Chriz/CHRO-associated DNA fragments are the same in polytene and nonpolytene chromosomes, we performed simultaneous FISH for these interband sequences and immunodetection of Chriz/CHRO in polytene chromosomes. shows that there is only one Chriz/CHRO-positive region between the bands 9F13 and 10A1–2 (marked with an asterisk on ), i.e. Chriz/CHRO mapped in the 9F13/10A1–2 interband in nonpolytene chromosomes does map to the 9F13/10A1–2 interband in polytene chromosomes. At the same time, the FISH probe of CG15208 which bind this protein in nonpolytene chromosomes () displays perfect co-localization with Chriz/CHRO in this region of polytene chromosomes ().
Colocalization of DNA probes limiting the 10A1–2 band with the Chriz/CHRO protein.
We successfully mapped Vago
probe on stretched polytene chromosomes which, as accepted [2, for more details]
, provided greater resolution. demonstrate that upon stretching, both the length of large bands and their spacing have dramatically increased, which, in turn, resulted in that Chriz binding pattern in between 10A1–2 and 10B1–2 is now seen as comprising a series of relatively distinct fluorescent bands reminiscent of the EM map for this region (). We also observed that, first, Vago
probe located in interband of the nonpolytene chromosome also completely co-localizes with one of these Chriz/CHRO -positive regions (), and this probe itself mapped to the same interband in the proximal edge of the 10A1–2 band of polytene chromosome. Similarly, CG32668
that in nonpolytene chromosomes flanked 10B1–2 from both sides, displayed extensive co-localization with Chriz/CHRO -bound fragments in the 10B1–2 band in polytene chromosomes ().
Colocalization of DNA probes limiting the band 10B1–2, and Chriz/CHRO protein.
Taking into account that Chriz/CHRO is a protein found both in the “open” chromatin of nonpolytene chromosomes and in polytene chromosome interbands tagged with P-element insertions 
, we conclude that DNA sequences associated to Chriz/CHRO proteins correspond to interbands of polytene chromosomes. This, in turn, indicates that Chriz/CHRO is invariably bound to interbands in chromosomes of various cell types. Thus, we can map all the interband borders in a given region of a physical map, using the localization borders of various interband-specific proteins ().
Mapping the band/interband borders on physical map and molecular-genetic characterization of bands and interbands
In order to determine the span of DNA sequences in each interband in this region, we first plotted the localization profiles for proteins on nonpolytene chromosomes and those we previously selected as markers for the transposon-tagged interbands of polytene chromosomes: this list included Chriz/CHRO, WDS, ORC, BEAF as well as DNase I hypersensitive sites and histone H1 density dips (see ). Using these data, we identified the limits of interbands (). Within the region of interest, there are nine such interband regions, which is exactly the number of interbands on the Bridges 
and EM (see ) maps. shows the coordinates of interband borders and the length of interband DNAs. Distances between interband borders, thus, correspond to the lengths of bands, totaling eight (in agreement with both Bridges and EM maps. Two out of these bands can be classified as late replicating bands, while the rest six are regular early-replicating (). The chromosome region from 9F13 to 10B3 encompasses 428,307 bp, nine interbands account for 20,100 bp, i.e. 4.69% of DNA length. Interbands range from 1,336 to 4,181 bp in size, with average size about 2,233 bp.
Coordinates and sizes of bands and interbands on physical map of the 9F13 – 10B3 region.
Lengths of DNA sequences mapping within bands vary in much broader range. Eight bands concerned here account for 408,207 bp DNA, i.e. average length of DNA per band is roughly 51 kb. The largest IH bands 10A1–2 and 10B1–2, span 189 kb and 170 kb, respectively, whereas the smallest bands from around the 10A6 region are as short as 2761 bp, which is about 69 times less than found in the largest band 10A1–2. The region of faint bands at 10A3–10A11 encompasses about 52 kb DNA, and these bands show much more constrained variation, from 2761 to 14634 bp, being 8725 bp on average.
Accurate mapping of band and interband borders on the physical map and proper identification of the band/interband material, allows us to describe and characterize two types of polytene chromosome bands: late- and early-replicating bands, and give new insight into the structure of interbands.
Late-replicating IH bands 10A1–2 and 10B1–2 composed of compacted material, as is seen by cytology 
. Degree of DNA compaction in them, i.e. the ratio of visual length of the bands to their actual DNA length, is highest (158- and 204-fold compaction, respectively) (). These bands are late replicated in both polytene 
and nonpolytene 
DNA compaction ratio of the bands in the 9F13 -10B3 region.
The DNA in such bands displays several common features, such as noticeably decreased level of ORC2 localization (), complete absence of any of the “open chromatin” ensemble of proteins which are normally found in interbands (Chriz/CHRO, BEAF-32, RNA polymerase II, BRE-1, WDS, NURF, TRX) (), and low frequency of intergration of P-element based transgenes, insertions of which are also characteristic for open chromatin ().
Recently, by integrative analysis of genome-wide binding maps 53 broadly selected chromatin components in Drosophila
cells it was shown that the genome is segmented into five principal chromatin types that are defined by unique combinations of proteins and form specific domains. Each of these chromatins were conditionally labeled with a color: BLUE and BLACK – repressive chromatins, RED and YELLOW – transcriptionally active chromatins, GREEN – heterochromatic domain (see Filion et al., 2010 
for details and protein compositions of each domains). In other work the genome-wide chromatin landscape based mainly on eighteen histone modifications and several non-histone chromatin proteins was summarized by 30 combinatorial patterns or states 
Bands like 10A1–2 and 10B1–2 can be categorized as having the chromatin state 30, which is described as lacking any active chromatin marks in the fly modENCODE 
. In both 10A1–2 and 10B1–2 bands, strong enrichment for SUUR, D1 and lamin B is observed 
() which is characteristic for BLACK chromatin, with depletion for H3.3 () and low level of newly synthesized histone subunits (). These regions show low gene density, which is characteristic of late replicating regions of the genome 
(), they show no depletion for histone H1, which is necessary for higher-order nucleosome packaging ().
Yet, these bands display several distinct features. The band 10A1–2 is virtually homogeneous in terms of its principal chromatin “color” - it is BLACK 
throughout, showing pronounced enrichment in SUUR, lamin B, and D1 proteins. Only its proximal-most part shows some contribution of BLUE and YELLOW chromatins ().
The band 10B1–2 appears as a more complex body. In both polytene and nonpolytene chromosomes, this band always appears as a single unit, flanked by interbands from both sides. However, depending on the differentiation stage, the chromatin state within 10B1–2 can change. For instance, in diploid cells, the RED chromatin typical of interbands is present, or some features of chromatin state #1 become apparent (). Despite the fact that overall gene density throughout 10B1–2 is decreased, the corresponding late completion of replication and SUUR binding in Kc cells is only observed in the distal 40% of this band (). In Kc cells, Lamin B is found associated with both a fraction of late-replicating sequences and all of the early-replicating sequences of 10B1–2 (). Notably, one of the bands - 10A1–2 – has all its genes replicating late, whereas only a fraction of genes from within 10B1–2 appears late-replicating (). In this latter case, one can view the 10B1–2 band as only partially composed of late-replicating material.
And, finally, the band 10B1–2 is mosaic in terms of “colored” chromatin types 
. Even though it is mostly composed of BLACK and BLUE chromatins, it also encompasses YELLOW chromatin at its distal edge, which correlates with localization of histone H1 dips ().
Very thin (ca 9 kb/band on average, see above) early-replicating bands are harbored in the region 10A3–10A11 ( and ). These bands are distinct from IH bands in many ways. They complete replication early, do not contain BLACK or BLUE chromatin, however, and much like the IH bands, they show poor enrichment (if any) for ORC2 (). Morphology-wise, they appear less dense, the degree of DNA compaction in them was found to be 16–38 fold, which is much higher than in interbands, but lower than in IH bands. Notably, two bands, 10A3 and 10A4–5, that are adjacent to 10A1–2 display the level of compaction somewhat higher, around 54–63 fold (). These bands do not associate with RNA polymerase II, interband-specific proteins, although they are composed of YELLOW, i.e. transcriptionally-active, chromatin 
; according to 
they contain chromatin states ## 22–24 and 30. It must be emphasized that these bands lack DNase I hypersensitive sites.
Interbands. appear decondensed in terms of their morphology. The minimal DNA compaction according to data of , is observed in interbands (3- to 15- fold. Additionally, they display major features of “open” chromatin, such as DNase I hypersensitive sites (DHS), interband-specific proteins Chriz/CHRO, BEAF, PolII, various transcription factors, histone variant H3.3, nucleosome-remodelling factors such as WDS and BRE-1, histone H1 dips. In all interbands, the chromatin is YELLOW or RED (see ), which is indicative of their participation in transcriptional activity 
According to the 30 chromatin state model 
, interbands fall into states #1–6. These are exactly the same properties that we previously found specific for 13 interbands mapped throughout the analysis of P-transgene insertions 
. In that work we showed, that 11 of the 13 transgene-tagged interbands corresponded to either intergenic regions or 5′-ends of genes. Moreover, chromatin state #1 is enriched in TSSes, 5′UTRs and start codons 
. As the demonstrates the comparison of gene localization from FlyBase with chromatin state #1 
, interbands/bands, enrichment profiles for Chriz/CHRO, WDS, ORC2 and a nucleosome density plot. The dashed lines that mark the 9F13/10A1–2 interband, maps to the intergenic region between the 5′-ends of CG1582
. The interband 10A1–2/10A3 comprises the 5′-half of the CG2076
gene (see Flybase). The interband 10A3/10A4–5 maps to the intergenic region between CG42249
. Likewise, interband 10A4–5/10A6 is found between divergently transcribed CG11122
. The central part of the 10A6/10A67 interband corresponds to the 5′-end of Gtp-bp
; center of 10A7/10A8–9 interband maps to the 5′-end of Klp10A
, the interband 10A8–9/10A10–11 corresponds to the common upstream regulatory regions for ran
. The 5′-end of Dlic2
makes up the 10A10–11/10B1–2 interband, whereas 10B1–2/10B3 interband occupies the 5′-ends of l(1)10Bb
(). Thus, of the nine interbands studied in this work, eight map to the 5′-ends of genes, intergenic regions or first exons of genes.
Relation of genetic map, and band/interband pattern in the region 9F13 – 10B3.
DNA sequences that bind ORC2 in the nonpolytene chromosomes are unevenly distributed along this region 
. For example, the band 10A1–2 lacks any ORC2 binding (), whereas 10B1–2 shows four regions or ORC2 enrichment in Kc cells, but not in other cell types (). ORC2 appeared virtually uniformly present throughout the region 10A3–10A11, with all origin recognition complexes being invariably found in interbands ( and ). For instance, this 68 kb-long region (between 10A1–2 and 10B1–2 bands) encompasses 5–7 ORC2 binding sites(13.6 – 9.7 kb per ORC2), whereas 10A1–2 region shows no ORC2 binding over 190 kb, in the 10B1–2 band the ORC2 density varies from 42.5 kb/ORC2 to complete absence in 168 kb in chromosomes of some cell lines.