Drosophila melanogaster consist of compact “black” bands alternating with less compact “grey” bands and interbands. We developed a comprehensive approach that combines cytological mapping data of FlyBase-annotated genes and novel tools for predicting cytogenetic features of chromosomes on the basis of their protein composition and determined the genomic coordinates for all black bands of polytene chromosome 2R. By a PCNA immunostaining assay, we obtained the replication timetable for all the bands mapped. The results allowed us to compare replication timing between polytene chromosomes in salivary glands and chromosomes from cultured diploid cell lines and to observe a substantial similarity in the global replication patterns at the band resolution level. In both kinds of chromosomes, the intervals between black bands correspond to early replication initiation zones. Black bands are depleted of replication initiation events and are characterized by a gradient of replication timing; therefore, the time of replication completion correlates with the band length. The bands are characterized by low gene density, contain predominantly tissue-specific genes, and are represented by silent chromatin types in various tissues. The borders of black bands correspond well to the borders of topological domains as well as to the borders of the zones showing H3K27me3, SUUR, and LAMIN enrichment. In conclusion, the characteristic pattern of polytene chromosomes reflects partitioning of the Drosophila genome into two global types of domains with contrasting properties. This partitioning is conserved in different tissues and determines replication timing in Drosophila.Morphologically, polytene chromosomes of  The polytene chromosomes of dipterans are giant entities showing a distinct banding pattern. Interbands are the most decompacted regions of polytene chromosomes, with a DNA packing ratio of 3–15. According to their morphology, bands can be classified into “grey” and “black.” The former are relatively thin decompacted structures with a DNA packing ratio of 54–63. The latter are normally thicker and more tightly packed, with the DNA packing ratio at times exceeding 200 . As demoIdentification of the genomic coordinates of 32 interbands has led to the conclusion that the chromatin organization and protein composition of interbands in polytene chromosomes are conserved across tissues. These interbands are characterized by open chromatin, and many of them correspond to 5' ends of housekeeping genes , 13–15. Drosophila genome into four chromatin types formerly referred to as “cyan,” “blue,” “green,” and “magenta”  have been defined for cultured cells, whole embryos, and salivary gland cells [D. melanogaster, active TADs are normally short. They are detectable at a mapping resolution of 4–10 kb [D. melanogaster are largely the same as the features typical of interbands [D. melanogaster embryos and cell cultures are located in aquamarine and lazurite but not ruby chromatin. Hence, it has been concluded that TADs may correspond to black bands, whereas TAD boundaries tend not to [New insights into how polytene chromosome morphology is linked to the organization of the hromatin , 25–28. nd cells –31. In and cells  have spl 4–10 kb , 30 but  4–10 kb , 27. At terbands . Most TAd not to . Althougd not to  nevertheSuURES mutants [3H-thymidine labeling patterns specific for different S phase substages in salivary gland polytene chromosomes were first described in the 1950s [otu11 mutants. When replication is seen virtually everywhere along the chromosome arms, the large black bands have not even started, but some regions of lightly packed chromatin have completed replication [D. melanogaster tissues. The order of replication completion by these bands is conserved too, as demonstrated by analysis of replication patterns in polytene chromosomes from different tissues in Drosophila [Anopheles stephensi [3H-thymidine is used, analysis of the order of replication completion by bands becomes possible only after ≥40–45 sites on each chromosome arm have been labeled. These loci always contain quite large black bands of polytene chromosomes [The main marker of IH regions is under-replication in polytene chromosomes. These regions, whether in polytene chromosomes or in cultured cells, are distinguished by the very late completion of replication and the manner in which replication proceeds: from the boundaries toward the center , 18, 33. mutants , by the  mutants , 34, and mutants . In salihe 1950s , 35–37. he 1950s . In recelication , 40. Thuosophila , 40 and tephensi . When 3Homosomes . What reDrosophila genome into two global types of domains with contrasting properties. For direct testing of this hypothesis, it is necessary to identify the genomic coordinates of all black bands in an extended genomic region.It has long been thought that IH regions are special genomic regions that are more heterochromatic than the surrounding material of the chromosome arms , 18. PutThe aims of this work were to find the genomic coordinates of all black bands on one whole arm of polytene chromosome 2R, to analyze the properties of these bands, to infer their detailed replication schedule, and to compare replication timing profiles between polytene chromosomes and diploid cells.We wanted to map all black bands of polytene chromosome 2R, under the following assumptions: 1) all black bands contain ruby chromatin, and 2) any band is flanked by an interband that is located in aquamarine chromatin.Fig 1A and 1B). Next, we carried out rounds of more detailed localization (S1 Text).According to published data, we presumed that all aquamarine segments that contain CHRIZ-binding peaks .To roughly localize ruby-containing polytene chromosome bands (rb-bands) on the genome map, we selected zones that contain ruby chromatin and are flanked by aquamarine chromatin (ODE data ) in the ODE data  in both S1 Fig). To assign names to bands according to the polytene chromosome map, we used Saura’s and Sorsa’s electron microscopic maps [To match up the predicted black bands and black bands on Bridges’ map, we proceeded as follows. First, we assigned the predicted genomic fragment to a particular chromosomal region by means of the FlyBase map . Becausepic maps , 46, on pic maps .S1 Table. When the pattern of the predicted bands was not consistent with the morphology or experimental mapping data, we refined the boundaries on the basis of the data about gene expression, TADs [S1 Text). It should be noted that, in each case, the boundary localized to an aquamarine chromatin segment.When looking for the correspondence between the predicted bands and the bands observed in polytene chromosomes, we introduced the following correction criteria for the locations and boundaries of the predicted rb-bands. When the morphology of the polytene chromosomes was fully consistent with predictions, and the positions of the predicted boundaries did not contradict experimental gene mapping data, the coordinates of the boundary were considered final and an entry was made in on, TADs , distribon, TADs  in salivFig 1G). This region of chromosome 2R stained with aceto-orcein is presented in Fig 1H. This staining discriminates between black and grey bands quite well. All the black bands seen in Fig 1H correspond to rb-bands that we predicted. Moreover, thicker bands typically map to more extended genomic intervals.Thus, in region 43F-46A of chromosome 2R, we identified 13 genomic intervals (rb-bands) and matched each with one or several bands on Bridges’ map .D. melanogaster, polytene chromosomes suitable for cytological mapping are present in the pseudonurse cells of otu11 mutants, these cells being germline cells by their origin. Fig 1I illustrates the same polytene chromosome region in pseudonurse cells of otu11 mutants   and the intervals between them (INTs throughout) , respectively. The bands varied from 15 to 500 kb in size (S1 Table).The genomic coordinates of all rb-bands on chromosome 2R were identified via the approach described above. A flow chart showing the main steps of the approach is given in in situ hybridization of P inserts from the BDGP project [in situ hybridization does not always allow the probe to be localized accurately to a thin band, all the photographs were fully consistent with our mapping .To additionally test the correspondence between our predictions and their homonyms on Bridges’ map for accuracy, we used data on  project . AlthougWe compared the positions of rb-bands with the FlyBase coordinates of the homonymous bands. The genomic regions occupied by most of our rb-bands (142 out of 159) overlap with FlyBase mapping predictions. Nevertheless, even when bands overlap, FlyBase is seen to grossly misplace band boundaries and to mostly underestimate band sizes. In total, the FlyBase bands contain 30% less DNA than our bands do (9.3 vs. 11.9 Mb). In particular, 80% of the material in the FlyBase bands overlaps with our bands and only 64% of DNA in our bands overlaps with their FlyBase homonyms.S1 Table). Rb-bands contain 97% of material from the under-replication zones, whereas the FlyBase counterparts contain only 70%. This observation implies that the quality of our mapping is much higher than that of FlyBase.DNA under-replication in polytene chromosomes invariably occurs within bands. This is best seen in electron micrographs of the chromosome breaks in regions of IH . On chroFig 3illustrates some typical staining patterns for region 43F-46B of chromosome 2R in the order of appearance in the S phase. Chromosomes in Fig 3A are in the early S phase. Signals are not yet coming from individual bands but are emitted from “zones” composed of groups of grey bands and interbands. Most of the black bands are not yet replicating. The pattern in Fig 3B appears to be an inversion of the previous one. All black bands yield bright PCNA signals. The thickest bands, 44C4-5 and 44D1-2, got labeled only peripherally. As the S phase proceeds through its substages, increasingly fewer bands have signals, and at the end of the S phase, only 44C4-5 does . 44C4-5 is devoid of “classic” IH bands, with their under-replication fate, and nevertheless, at the end of the S phase, most nuclei have this weak signal in 44CD. At the substage corresponding to the pattern in Fig 3B, virtually perfect correspondence is observed between rb-bands and the bands that get labeled at this substage . Because the intervals between these bands at this time emit virtually no signals, it can be assumed that all the rb-bands in this particular genomic region complete replication later than the intervals between them.To address replication timing on chromosome 2R, we stained salivary gland polytene chromosomes with antibodies to PCNA. S3 Fig). As in 43F-46A, here we identified six distinct PCNA staining patterns, which we designated ER (early replication) and LR1–LR5 . Each band listed in S1 Table was assigned to one of the substages (LR1–5), meaning that this is the substage at which the band completes replication. The bands that had completed replication before LR1 were assigned to LR0.Next, we analyzed the order in which all the rb-bands of chromosome 2R are replicated . On average, these INTs were replicated significantly earlier than the rb-bands. The time of replication completion was also significantly different among LR0–1, LR2–3, and LR4–5 bands. The rb-bands that are the last to complete replication in polytene chromosomes are the last to complete replication in Kc cells . It can be concluded that, at the level of rb-bands and intervals between them, chromosomal regions are replicated in the same order in polytene chromosomes as in cell cultures.Once we profiled the replication timing of rb-bands, we could compare replication timing between polytene chromosomes and diploid cells. oworkers  for all Fig 4B and 4C) indicates that in Kc and BG3 cells, the portions of the replication curves corresponding to rb-bands assume a U-shape, suggesting that the central part of each band is replicated considerably later than its peripheries. Moreover, most rb-bands were found to have positive replication scores at their peripheries, while the bulk of each band has a negative score. A similar tendency, albeit less pronounced, is seen in the Cl8 and S2 profiles.A visual comparison of the rb-bands and the replication profiles published previously for the four cell cultures . Comoglio and Paro [S4 Fig).These data are consistent with the fact that rb-bands coincide well with the zones that are virtually devoid of ORC2-binding peaks in Kc cells and in salivary glands . In all three cases, a gradient of the replication score from the periphery to the center is seen, with the minimum at ~75 kb for LR4–5, at ~50 kb for LR2–3, and at less than 50 kb for LR0–1. This behavior of the averaged replication profile is consistent with size differences between bands in different groups . The coefficients of correlation between band size and the mean replication score corresponding to this band were found to be -0.47 (p < E-10) for Kc cells and -0.51 (p < E-10) for C18 cells.We generated an averaged replication profile for the regions surrounding bands from each of three groups  in Kc and C18 cells .It can be concluded that the local minima in the replication profiles for diploid cells fall within rb-bands, while the local maxima fall outside them, although quite close to their boundaries. This replication profile is consistent with the theoretically predicted U-shaped profile, where the region deprived of early origins is flanked by extended replication initiation zones . This coFig 6A and 6B indicates, all rb-bands show significantly lower densities of 5' untranslated regions (UTRs) and entire gene loci than INTs do.We analyzed gene density in the LR0–1, LR2–3, and LR4–5 rb-bands and INTs. As Fig 6C, all the LR0–LR5 rb-bands show lower median expression levels in all tissues in comparison with INTs. The gene expression profiles look similar in all tissues, except for the testes, in which a relatively large group of genes manifested very high expression. This finding is consistent with the fact that male-specific genes represent a considerable proportion of genes in under-replication regions [Fig 6D). Incidentally, the genes in the bands are virtually not expressed in the ovaries.We analyzed gene expression in LR0–LR5 bands and INTs from 13 larval and adult tissues. As illustrated in  regions  and blacD. melanogaster, some of them grouped into multigene clusters [Fig 6E). Notably, it is the most late-replicating LR4–5 rb-bands that are enriched with multigene clusters among the others. It is noteworthy that all the genes in each cluster lie entirely within one (and only one) rb-band. This distribution is significantly different from random . According to the cytological map in FlyBase, four of these 16 clusters fall within intervals between late-replicating bands. There are eight clusters and each corresponds to more than one band, with intervals between them. This is another occasion to compare our mapping with that in FlyBase and to stress that ours much more precisely reflects the functional organization of the Drosophila genome.A work by Shevelev and coworkers provides a list of testis-specific genes in clusters . The regFig 6F shows, at 0, most INT genes are expressed in all 13 tissues. At 0.1 and 1, the median value is equal to 13 too. In rb-bands, however, the median values are substantially lower. It can be concluded that the INTs are enriched with the genes that are expressed ubiquitously at relatively high levels, whereas the rb-bands are enriched with genes that are expressed at lower levels and in fewer tissues.For each gene, we then identified how many tissues out of the 13 express this gene beyond each preset threshold level . As S3 Table). According to FlyBase, the fragment being analyzed on chromosome 2R contains 2753 protein-coding genes. GO annotations [Next, we checked whether there were differences between the genes in rb-bands and INTs by means of Gene Ontology . An average rb-band is 66% ruby, 22% malachite, 6% lazurite, and 2% aquamarine. The rb-bands contain most of unannotated fragments that offer no predictions according to the 4-state model. The prevalent chromatin types in INTs are aquamarine (32%), lazurite (39%), and malachite . If we look at the proportions of these four chromatin types in the LR5–LR0 bands, then we will see the following tendency: the later the bands are replicated, the higher is the percentage of ruby chromatin in them . Examination of the distribution of chromatin types in all rb-bands  and the averaged distribution of chromatin types around the boundaries of the LR0–1, LR2–3, and LR4–5 bands  suggests that lazurite largely localizes to the edges of the rb-bands and does not occur in them deeper than 10 kb from the provisional boundary. In all the three late-replication groups, the most prevalent chromatin type deeper than 5 kb from the provisional boundary is ruby. At 1–2 kb outside each band, the most prevalent chromatin type is aquamarine, in line with our boundary criterion.We named our bands rb-bands because all of them contain ruby chromatin. Ruby makes up 42% of DNA in the genomic region in question . Our band prediction technique initially indicated that ruby chromatin is always confined to rb-bands. Nonetheless, only those predictions were included in the final table that could be assigned to a particular cytological structure (a band or a group of bands). Thus, ~4% of ruby chromatin was outside our bands . Most bands consist of alternating blue and black chromatin domains. Among the 159 bands, only 14 do not include black chromatin . A large number of rb-bands have, in their boundary regions, some open-type yellow chromatin, with its average distribution there being identical to that of open-type lazurite chromatin . Intervals in turn appear as alternations of different chromatin types, with open-type yellow and red as the commonest varieties . A glance at  reveals that the most tightly packed chromatin tends to be located near the central parts of the bands, whereas the intermediate types in the boundary regions and shorter bands.A comparison of rb-bands with the distribution of three chromatin types notable for compactness in S2 cells  showed tFig 7M, 1) rb-bands are enriched with SUUR in all groups; 2) the later the bands are replicated, the higher is their SUUR score; 3) INTs are virtually devoid of SUUR. The averaged SUUR distribution profile for salivary glands and Kc cells  shows a sharp increase from a minimum at the boundary of the band to a maximum deeper into the band.We analyzed the mean SUUR score in salivary glands  for LR0. The following tendency was observed: the later the band is replicated, the higher the LAMIN score it has. The averaged LAMIN distribution profile in the boundary regions of the LR4–5, LR2–3, and LR0–1 bands  shows a sharp increase, and the latest-replicating bands have the highest LAMIN scores. The genomic region being studied is reported to include 67 lamina-associated domains (LADs) [S4 Table). Seventy percent of LR3, 60% of LR2, and 30% of LR0–1 bands overlap with LADs. Thus, LADs lie in the longer bands, although not all of the shorter, earlier-replicating bands correspond to LADs. It can be concluded that LAMIN is a component of chromatin in all rb-bands, but its overall low binding values and small band sizes make the corresponding regions to meet the statistical criteria used to define LADs.The mean LAMIN score  too is s (LADs) . Only tw and displays a sharp increase on the band/interband boundary .An averaged distribution profile for yet another component of silent chromatin types, histone H1, in Kc cells (DamID data. ) revealeS8 Fig shows, the behavior of protein distributions and the profiles of replication on D/E domain boundaries match up perfectly with those on the rb-band/INT boundaries. On the other hand, the averaged profile of the H3K27me3 distribution in primary spermatocytes shows a typical sharp increase on the rb-band/INT boundary . Many of the TADs correspond to groups of closely spaced bands (band coverage >90%): only as few as 32 TADs overlapped with one rb-band each, and 27 with 2–4 bands (regardless of how many of these rb-bands are in fact groups of closely spaced bands). Examination of band pairs  indicated that the bands lie within a single TAD; however, the Hi-C interaction map (heatmap) shows a distinct “barrier” that partitions TADs into smaller TADs. Electron microscopic section of polytene chromosomes reveals multiple fibers of tightly packed material in the paired bands . This result is consistent with a relatively high probability of contacts within a single TAD according to the heatmap.We compared rb-bands and salivary-gland TADs . TADs coS4 Table). At the same time, most of the shorter bands did not overlap with TADs. The only explanation we can think of is that the mapping of TADs in salivary glands was performed at too low resolution to resolve TADs shorter than 75 kb.Any rb-band longer than 85 kb had more than a 70% overlap with TADs . Between them, there are zones where short-range interactions predominate. The predominance of interactions at distances shorter than 1 bin (1 bin amounts to 15 kb) makes all the points along the heatmap diagonal in the intervals between ruby bands look very dark. Their darkness makes them easily distinguishable from the bins that fall into ruby bands. The boxplot in S9B Fig supports the view that the distribution itself of contact probabilities along the diagonal  is quite a marker of the 3D chromatin organization and allows the genomic coordinates of large black bands in polytene chromosomes to be predicted. As readers can see in the boxplot, this distribution in rb-bands is similar to that of TADs, whereas INTs resemble interTADs . This is an impressive piece of indirect evidence that all rb-bands correspond to TADs.Visual comparison of rb-bands and the salivary gland Hi-C interaction map revealed that not only the longest but also some shorter rb-bands corresponded to the triangles on the heatmap, which indicate the regions with a high probability of internal interactions in them . This variability can probably be due to the Hr3 gene, with its high expression shortly before the prepupal stage (according to modENCODE). The heatmap interval corresponding to this variable region revealed alternations of bins with different properties and looked contrasting against extended regions with relatively uniform patterns of interactions. This observation provides further support to the notion that salivary-gland Hi-C allows various morphological features of polytene chromosomes—black bands, intervals between them, and morphologically complex regions—to be predicted.In region 46F, the morphology of polytene chromosomes varies: it sometimes features a group of prominent black bands and sometimes a series of decompacted bands on the left of the large prominent band 47A1-2 . By analysis of the 3D chromatin organization in embryos at high resolution, not only extended but also shorter domains were resolved. It was found that TADs in embryos cover 98% of the region under study on chromosome 2R [S9C and S9B Fig).It has previously been demonstrated that the 3D chromatin organization corresponding to TADs is well conserved across cell types. A visual comparison of rb-bands and the heatmaps generated for interactions along chromosomes 2R in different cell cultures suggested that all our bands correspond to the triangles on the heatmaps that indicate regions with a high probability of internal interactions in them ; 53% of TAD boundaries fall within 10-kb intervals centered around each boundary of black bands . This finding provides further support to the hypothesis that TAD boundaries are in fact the boundaries of black bands in polytene chromosomes.The rb-bands are substantially depleted of TAD boundaries .We analyzed the patterns of distribution of proteins specific for the silent chromatin type as well as replication timing around the boundaries between different TAD types as identified elsewhere . S8 Fig Consequently, it can be concluded that TADs correspond to rb-bands or groups of closely spaced rb-bands in polytene chromosomes.S1 Fig).For the most part, our band-mapping approach depends on the data coming from cell lines. The best way to verify our mapping is by comparing the results with the distribution of the proteins in salivary gland polytene chromosomes. Good candidate markers of rb-bands are SUUR and H3K27me3 because they show a sharp change at band boundaries in a profile averaged for all such boundaries. Data on the distribution of SUUR and H3K27me3 in polytene chromosomes were published recently . The resS1 Table). Of 159 rb-bands, 131 showed exact coincidence (with a possible shift not exceeding 3 kb) between the bands and zones with high SUUR scores. For 19 bands, the high-SUUR-score zone in part coincided with a band. We believe that in these cases, the shifts of boundaries are due to the inaccuracy of our prediction. There were only six bands for which no local increase in SUUR was observed; it was found that all these bands are low on ruby chromatin. It is possible that the chromatin type in these bands is different from that in black bands.We visually assessed the correspondence between bands and zones with increased levels of these markers  and show very good correspondence with local increases in SUUR and H3K27me3. The accuracy of our mapping for these bands is, by our estimate, up to 2 kb.In The question how the presence of a transcriptionally active gene at the band boundary influences the morphology of this boundary requires special research. Detailed cytogenetic analysis of small locus 61C7-61C8 on chromosome 3L by high-resolution microscopy has been reported in ref. . Those aThus, the distribution of SUUR and H3K27me3 in polytene chromosomes is, in our opinion, a very good criterion for predicting rb-band boundaries; however, the resolution of this prediction can be affected by local transcription.In this work, we used the four-state chromatin model, previously published data on the chromatin localization of proteins, and in situ hybridization of annotated genes and identified the locations of all black bands of polytene chromosome 2R on a genome map.The special feature of the four-state chromatin model is the generalization of data obtained from four cell lines. This generalization resulted in identification of two chromatin types—aquamarine and ruby—which show stable properties in all these cell lines . StartinTo take into account the tissue-specific features of chromatin organization in salivary gland polytene chromosomes, we analyzed the morphology of polytene chromosomes, an extensive pool of “experimental cytology” data, and data on gene expression in salivary glands and, whenever deemed necessary, introduced corrections into initial predictions. The results of our work reveal good performance of this approach.Yet another unique feature of the four-state chromatin model is that it reveals the chromatin type  that is enriched with interband-specific proteins; no other models of clustering chromatin proteins can do this. According to the most recent high-resolution Hi-C data from embryos , TAD bouFig 2can in our opinion be conveniently used to identify the coordinates of specific polytene-chromosome black bands with high accuracy. For most black bands, accuracy of 2–10 kb is attainable.The choice of aquamarine chromatin as potential band boundaries is supported well by comparing our coordinates with the distribution of SUUR and H3K27me3 on polytene chromosomes: as we showed, these two are markers of black bands and display sharp changes on their boundaries. Thus, the approach used in this work and schematically detailed in With the band coordinates inferred, we performed a detailed comparison of replication profiles from diploid cells and replication patterns observed in polytene chromosomes and analyzed the properties of black bands.We demonstrated a considerable degree of similarity in replication timing between salivary gland polytene chromosomes and diploid cells. In both object types, the zones between black bands correspond to early replication initiation zones. This result is consistent with the observation that most ORC2-binding sites are in aquamarine chromatin corresponding to interbands . Rb-bandD. nasuta polytene chromosomes [Drosophila species irrelevant to us and the typical sizes and genomic distances may be different to some extent [Drosophila species [The averaged boundary replication profiles in INTs that we built from previously published cell culture data  are consomosomes  has reveomosomes . Althouge extent , we prop species .Fig 8. The schematic illustrates how replication patterns change in polytene chromosomes and how these patterns are linked to events in DNA sequences. At the beginning of the S phase, replication is initiated in INTs, which may contain a large number of potential replication initiation sites. In each DNA strand, an initiation event occurs only once per INT in a random interband. Initiation events in different interbands can occur in asynchrony, either in different INTs or in the same INT on the parallel DNA strands of a polytene chromosome. Replication forks move through INTs in the opposite directions from the site of replication initiation and eventually enter the nearest rb-bands . After all INTs have completed replication, the replication fork should be detectable only in rb-bands. This situation is consistent with the observed inverted PCNA pattern, when all black bands produce the signal that the intervals do not.A schematic derived from original and previously published data is presented in D. nasuta polytene chromosomes, it has been demonstrated that the rates of replication fork movement in polytene chromosomes during the late S phase are on average one-tenth of those in the early S phase [Fig 8B–8D). In D. melanogaster, replication rates depend on SUUR [By analysis of stretched DNA fibers in  S phase . In our  on SUUR , 66. We  on SUUR , local aDrosophila genome. The genome consists of alternations of domains capable of initiating early replication (INTs) and domains with the potential to initiate replication late in the S phase. These late domains vary in size, but seldom are they longer than a few hundred kilobases. In diploid cells, these relatively short domains are replicated by replication forks coming from border origins of replication and complete replication before the classic late S phase, which is when late-firing origins activate. Thus, replication of a large portion of a euchromatic arm is, in the classic sense, early replication. Only the most extended bands and regions of pericentric heterochromatin initiate replication in the late S phase . The question of whether replication initiation events occur in the bands is not easy to answer. Schwaiger and coworkers [D. melanogaster [Evidence exists that the S phase of the endocycle is quite different from that in diploid cells. The former is distinguished by under-replication of a large part of the genome and low expression of genes involved in replication. The presence of the intra-S checkpoint in salivary gland cells is questionable, and so is activation of any late-firing origins , 68–70. oworkers  have anaoworkers , 71. Thenogaster .Multiple published comparisons of replication profiles for different tissues of the same organism suggest that each cell type has its own schedule of origin activation. One study on individual IH regions indicates that all the 60 analyzed regions are late replicating in cultured cells , but insS5 Fig). Among all the rb-bands, only two had early-replication zones spanning them from end to end. It can be theorized that in different tissues, most bands similar in size undergo replication within a similar time interval in the S phase, and gene activation in these bands makes the corresponding fragment of the band earlier replicating.In cell cultures, early-replication zones within rb-bands can be identified by analysis of the outliers in the boxplots of the averaged boundary replication profile . We demonstrated that all rb-bands, including small ones, share a large number of properties with IH regions. This is direct evidence that among all genomic regions, IH regions do not stand out as some special type of sequences. Genes in any rb-band tend to be expressed in a limited number of tissues and, according to GO analysis, these regions are enriched with tissue-specific genes. By contrast, the intervals between black bands are enriched with genes that are highly expressed in most tissues chosen for analysis here. Each rb-band appears as a combination of repressed chromatin types; however, open chromatin can be found in its boundary regions, pointing to a similarity between bands and TADs . We confribution –80. Thesribution , 78–80.  mammals , 82.Drosophila polytene chromosomes can serve as vivid visualization of the organization of the eukaryotic genome, which is conserved between Drosophila and mammals. The characteristic pattern of polytene chromosomes—the compacted black bands alternating with less compact grey bands and interbands—reflects the partitioning of the Drosophila genome into domains with contrasting properties.The results of our work suggest that Drosophila cornmeal–yeast–agar medium at 18°C and 25°C. The Oregon R stock from the Bloomington Drosophila Stock Center was used as wild-type control. The SuURES mutation was first described by Belyaeva and the co-workers [Flies were raised on standard -workers .2PO4 and 2 mM KH2PO4), transferred to aceto-orcein solution (1% orcein in 45% acetic acid) for 10–15 min then to 55% lactic acid for 1–2 min and squashed. Phase-contrast images were captured with an Olympus BX51 microscope using a DP52 camera with a 100× oil immersion objective lens.For acetic orcein staining of polytene chromosomes, glands were dissected in PBS . Polytene chromosome morphology was analyzed using previously published electron microscopic photographs of separate regions of polytene chromosome 2R [We relied on the detailed cytological map of chromosome 2R  and an eosome 2R , 84–86, D. melanogaster Release 5 assembly. The UCSC Genome Browser [http://modencode.org/>. The list of genes with in situ hybridization data published for them was extracted from FlyBase [All the genomic coordinates used in this work are provided in  Browser  was used Browser  and the  Browser . CHRIZ e FlyBase  using th FlyBase . For ban FlyBase , as well FlyBase  (data fr FlyBase  release S1 Table).We determined the naming accuracy for each band against Bridges’ map  by how min situ hybridization of P inserts to chromosome 2R from the BDGP project [To verify the mapping of large bands, we used 186 photographs (the most relevant from among 2255 kindly provided by Todd Laverty) resulting from  project . The namSuURES wandering third instar larval polytene chromosomes with antibodies to PCNA. Salivary glands were dissected in Ephrussi-Beadle solution  supplemented with 0.1% Tween-20. Glands were then transferred into a formaldehyde-based fixative  for 1 min. Next, the salivary glands were placed in an acetic acid–formaldehyde mix  for 1 min and squashed in 45% acetic acid. Squashes were snap-frozen in liquid nitrogen; the coverslips were removed. Slides were incubated in 70% ethanol for 5 min twice and stored in 70% ethanol at −20°C. Slides were washed three times in PBST  for 5 min. The primary mouse anti-PCNA  antibody were added in a blocking solution (0.1% BSA in PBST) and incubated in a humid chamber for 2 h at room temperature. The squashes were washed three times for 5 min in PBST, and the secondary Texas Red-labeled goat anti-mouse IgG specific conjugates  in blocking solution were applied. Incubation lasted for 1 h and was followed by washing in PBST three times for 5 min each. DNA was visualized by DAPI staining  for 5 min.To detect replication events in salivary gland polytene chromosomes, we immunostained squashes of Oregon R and SuURES. All whole 2R chromosomes were analyzed on the preparations and to each chromosome a particular staining pattern  was assigned. All these patterns were classified into six designated ER and LR1 through LR5. To each rb-band, the substage at which its replication ends was assigned. Although the order of band replication is overall consistent, there was a slight variability to it. That is why substaging assignments were finalized after all preparations were analyzed.The total number of preparations analyzed was 35 (more than 1000 nuclei) for Oregon R and 12 (more than 500 nuclei) for SuURES mutants than in the wild-type flies. As a result, late S phase reveals replication patterns that suggest replication only in the chromocenter and few bands, which is untypical of the wild type [SuURES is clearer than in the wild type, because underreplication is not there [SuURES was used.It was previously demonstrated that IH bands complete replication earlier in ild type . At the ot there . The figData processing, plotting and visualization were achieved using statistical language R  and Micrhttp://www.replicationdomain.com/. DamID data were retrieved from GEO: LAMIN (GSE20313) in Kc cells [Replication timing data for diploid Kc and Cl8 cells  were dowKc cells ; SUUR  and INTs by LOESS regression with R.When constructing plots with replication score data normalized to median length of rb-bands Intersect tool in the Operate on Genomic Intervals section.The percentage of each of the different chromatin types in rb-bands and INTs was determined using Galaxy  using thGene expression in bands and INTs was analyzed using Expression data generated by modENCODE , 90  values [SUUR, SG: a SUUR DamID profile in salivary glands. Data are presented as log10(P) units, where P is the significance level assessed with Fisher’s exact test [Replication timing: grey-shaded rb-bands, from latest (black) to earliest (light-grey) replicating bands. Hi-C SG: a normalized Hi-C interaction map (15-kb bins) from salivary gland cells [Expression, SG: color-coded gene expression levels  values . SUUR, Sact test . Replicand cells . ExpressodENCODE , 90): gr(TIF)Click here for additional data file.S2 Fig(A–C) Region 51F-52C of chromosome 2R. This region is difficult to map because it contains a large number of closely spaced morphologically similar bands. (D–F) Region 60A–60F of chromosome 2R. (A) In the photographs: results of in situ hybridization of probes corresponding to the P element in the following lines: 1) l(2)k11320, 2) l(2)k06203, 3) l(2)k03617, 4) l(2)k16004, 5) l(2)14707, 6) l(2)k08015, 7) l(2)k06403, 8) l(2)k04218, 9) l(2)k17029, 10) l(2)k03308, and 11) l(2)k07201. Each photograph is linked to the region on the genome map in which the insert was located. On the map: rb-bands and chromatin types according to the 4-state model. (B) A part of Bridges’ detailed map of chromosome 2R (regions 50D–52C) [(C) A part of Bridges’ detailed map of regions 50D–52C (B). Blue normal and T-headed arrows indicate hybridization loci according to our mapping prediction. Red normal and T-headed arrows indicate their locations according to the photographs (A). (D) In the photographs: results of in situ hybridization of probes corresponding to the P element in the following lines: 1) l(2)k13108 site2, 2) l(2)k11035, 3)l(2)k07623, 4) l(2)k04201, 5) l(2)k04405 site1, 6) l(2)k01208, 7) l(2)01296, 8) l(2)k12101, 9) l(2)k05318, 10) l(2)03267, 11) l(2)k00808, 12) l(2)04012, 13) l(2)k10502, 14) l(2)k04809, 15) l(2)k03704, 16) l(2)k15609, 17) l(2)10481, and 18) l(2)k05826. Each photograph is linked to the region on the genome map in which the insert was located. On the map: rb-bands and chromatin types according to the 4-state model.  A part of Bridges’ detailed map of regions 60A–60F [in situ hybridization does not always allow the probe to be located accurately to a thin band, all the photographs are fully consistent with our mapping.(TIF)Click here for additional data file.S3 Fig(A) Region 42-46 of chromosome 2R. (B) Regions 46-50. (C) Regions 50–56. (D) Regions 56-60.Top to bottom: six S-phase substages designated as ER (early replication) and LR0 to LR5 (late replication). Because this substaging is somewhat arbitrary and because there is variation in replication observability in separate bands at each substage, bands were assigned to substages following analysis of a large number of preparations. For this reason, the names of some substages end with more than one number. (PDF)Click here for additional data file.S4 Fig(A) Density of ORC2 peaks (peaks/100 kb)  Density of replication origins (peaks/100 kb) identified in Bg3 (B) and S2 (C) cells by next-generation sequencing of small nascent leading strands [rding to ) in Bg3  strands .(TIF)Click here for additional data file.S5 Fig(A) and Cl8 (B) cells for all sequences within 10-kb windows at the same position relative to the band boundary (shown as a red line). Right boundary data were mirrored and combined with left boundary data. Within-band regions are to the right of the border; outside regions are to the left. On the x-axis: distances from the border. On the y-axis: replication time scores (data were taken from [Boxplots of replication time scores in Kc ken from ), with +On the right: all bands containing DNA sequences with replication scores corresponding to the outliers in the boxplots. For each band, the presence in a group from LR0 to LR5, the number of the windows with replication times in the outliers, and the size are indicated. To be noted most outliers correspond to the windows found at rb-band/INT borders. In Kc cells, an extended (~80 kb) outlier zone is observed in band 47D1-6 . Of note, 47D1-6 is not a solid band, but a cytologically complex region, in which our interband criteria failed to identify individual bands. Extended outlier zones are observed in bands 49A1-2  and 50A1-4 . In C18 cells, extended outlier zones are observed in bands 45C1-2 and 45E1-2. Here the zones span the bands and continue beyond them. In all other bands, the outlier zones are normally not longer than 20 kb each. It can be concluded that zones of changes in replication timing normally correspond to band fragments, not to entire bands.(TIF)Click here for additional data file.S6 Fig(A) Distribution of four chromatin types [(B) Distribution of five chromatin types in Kc cells [(C) Distribution of differently compacted chromatin types in S2 cells (after [(D) Distribution of four TAD types  is indicated below. in types . (B) DisKc cells . (C) Diss (after ). (D) Dis (after ).(TIF)Click here for additional data file.S7 Fig(A) An averaged distribution of four chromatin types (after [(B) Averaged distribution of five chromatin types in Kc cells (after [(C) An averaged distribution of differently compacted chromatin types in S2 cells [s (after ) at locas (after ) at locaS2 cells  at locat(TIF)Click here for additional data file.S8 FigAveraged profiles of the distribution of scores for repressed chromatin markers close to the boundaries between rb-bands and INTs, D- and E-domains (E domains less than 100 kb were removed), TADs of different types , and TADs of the same type . Right-boundary data were mirrored and combined with left boundary data. Markers: SUUR in salivary glands (data were taken from ), SUUR i(TIF)Click here for additional data file.S9 Fig(A) Hi-C data from different sources on the region 45F-46B of chromosome arm 2R (approximately 2 Mb). Top to bottom: rb-bands; a normalized Hi-C interaction map (15-kb bins) from salivary gland cells [(B) Chromatin packaging in all rb-bands corresponds to chromatin packaging in TADs. Boxplots of contact probabilities inside bins along the diagonal on the normalized Hi-C interaction map published by Eagen et al. [U test). (C) The ratio of the overall lengths of genomic intervals corresponding to LR0–LR5 bands (black) and INTs (grey) in TADs of different types. In all inactive TAD types, the prevalent chromatin is the one corresponding to bands. (D) The proportion ratio of chromatin types corresponding to TADs of different types according to [(E) Rb-bands are depleted of TAD boundaries in embryos. The observed and the expected number  of TAD boundaries (after [(F) Coefficients of correlation  between fragment size and such measures as replication timing in Kc and Cl8 cells, SUUR binding in Kc and salivary gland cells, and Lamin binding in Kc cells.nd cells ; TADs innd cells ; TADs innd cells : null tynd cells ; Hi-C innd cells . (B) Chrn et al.  for salirding to , in LR0-s (after ) within (TIF)Click here for additional data file.S10 Fig#according to Bridges, 1939 [$aceto-orcein staining, phase-contrast); the heatmap of Hi-C data on salivary glands [(A) Each pair of closely spaced bands, 57B2-3/57B4-6 and 56A1-2/56B1-2, forms a single TAD. The internal interband manifests a very special feature on the heatmap: within the triangles with nearly evenly distributed interactions, the interband bin shows strong interactions between internal sequences and weak interactions between external. (B) In region 46F, polytene chromosome morphology is variable: what is seen to the left of the large and always prominent band 47A1-2 is sometimes a group of distinct black bands and sometimes a series of decompacted grey bands. This variability is probably associated with the activity of the Hr3 gene (the orange rectangle on the expression map), which, according to modENCODE, starts to be highly expressed shortly before the prepupal stage. The heatmap interval corresponding to this variable region showed alternations of bins with different properties and looked contrasting against extended regions with relatively uniform patterns of interactions.Top to bottom: polytene chromosome morphology . (A) Ea(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Acholeplasma laidlawii, Spiroplasma melliferum, and Mycoplasma gallisepticum) to perform promoter mapping and activity quantitation. We revealed that Mollicutes evolved towards a promoter architecture simplification that correlates with a diminishing role of transcription regulation and an increase in transcriptional noise. Using the identified operons structure and a comparative genomics approach, we reconstructed the transcription control networks for these three species. The organization of the networks reflects the adaptation of bacteria to specific conditions and hosts.Bacteria of the class Mollicutes have significantly reduced genomes and gene expression control systems. They are also efficient pathogens that can colonize a broad range of hosts including plants and animals. Despite their simplicity, Mollicutes demonstrate complex transcriptional responses to various conditions, which contradicts their reduction in gene expression regulation mechanisms. We analyzed the conservation and distribution of transcription regulators across the 50 Mollicutes species. The majority of the transcription factors regulate transport and metabolism, and there are four transcription factors that demonstrate significant conservation across the analyzed bacteria. These factors include repressors of chaperone HrcA, cell cycle regulator MraZ and two regulators with unclear function from the WhiA and YebC/PmpR families. We then used three representative species of the major clades of Mollicutes ( Mycoplasma genitalium to nearly 2 Mb in Acholeplasma brassicae. Most known Mollicutes are parasites, and some are saprophytes. Despite their reduced genome, Mollicutes demonstrate high adaptive potential within a parasitic niche. The range of their hosts is wide and includes plants  mapping at a single-nucleotide resolution may significantly contribute to understanding gene expression organization and control. However, there are few data on TSS mapping across the bacteria despite numerous transcriptomics projects . Recent Acholeplasma laidlawii and Spiroplasma melliferum and used our previous data on Mycoplasma gallisepticum (A. laidlawii represents a clade of Mollicutes that underwent less reduction (1.5 Mb genome), whereas S. melliferum and M. gallisepticum are average Mollicutes with a genome size of approximately 1 Mb. A. laidlawii is likely a saprophyte or a facultative plant pathogen. S. melliferum has two hosts (angiosperm plants and honeybees) and is predominantly pathogenic in the latter. M. gallisepticum is a bird pathogen closely related to the human pathogens M. pneumoniae and M. genitalium. Mapping TSSs enabled us to study the promoters’ structure and to identify potential transcription factor binding sites and regulons in respective species.In the current work, we carried out whole-genome mapping of transcription start sites (TSSs) of septicum  for a coMycoplasma gallisepticum S6 was cultivated in liquid medium containing tryptose 20 g/L, Tris 3 g/L, NaCl 5 g/L, KCl 5 g/L, yeast dialysate (5%), horse serum (10%), and glucose 1% at pH 7.4 and 37°C in aerobic conditions and exposed to heat stress conditions (46°C for 15 min) as described previously (eviously . For theAcholeplasma laidlawii PG-8A was cultivated in modified Edward’s medium (liquid): tryptose 20 g/L, NaCl 5 g/L, NaOAc 5 g/L, KCl 1.3 g/L, Tris 3 g/L, yeast dialysate 5%, horse serum 6%, glucose 0.5%, and pH 7.6 at 37°C in aerobic conditions (nditions . For theSpiroplasma melliferum KC3 was cultivated in SP4 medium (liquid): tryptone 10 g/L, peptone 10 g/L, sorbitol 70 g/L, yeast extract 7 g/L, brain-heart infusion 2.5 g/L, NaCl 4.5 g/L, sucrose 1%, fructose 1%, glucose 0.8%, horse serum 10%, and pH 7.6 at 30°C in aerobic conditions  and lysed in TRIzol LS reagent (Life Technologies) at a 1:3 ratio of resuspended cells:TRIzol LS (v/v). The lysates were extracted with chloroform, and the aqueous phase was purified with a PureLink RNA Mini Kit (Ambion) to remove tRNA or used directly to precipitate RNA with the addition of an equal volume of isopropanol.Preparation of 5′-ERS (5′-end Enriched RNA Sequencing) libraries was performed as previously described . Cells f4, 100 mM Tris, pH = 7.0 at 70°C for 15 min). The fragmentation reaction was stopped with 20 mM EDTA (pH = 8.0). The fragmented RNA was end-repaired with T4 polynucleotide kinase according to the manufacturer’s protocol (Thermo).Approximately 20 μg of total RNA was fragmented into 200 bp via chemical fragmentation (100 mM ZnSOFragmented end-repaired RNA was treated with Terminator exonuclease (Epicentre). This process resulted in the degradation of the non-primary 5′-end RNA fragments, whereas the primary 5′-fragments were protected by the tri-phosphate groups on their 5′-ends. As chemical fragmentation leaves phosphates randomly on fragment ends, an end-repair procedure was used to enhance the degradation of non-primary 5′-end fragments , which otherwise undergo adapter ligation and cDNA synthesis (if they have 3′-OH) and to rescue primary 5′-end fragments with 3′-phosphate (via 3′-phosphatase activity of T4 PNK). The addition of an end-repair procedure increases signal  and decreases background. Then, the RNA was precipitated with isopropanol and treated with tobacco acid phosphatase (Epicentre) to remove the pyrophosphate groups. Next, the RNA was precipitated via isopropanol and used for strand-specific ds-cDNA preparation according to the standard protocol for SOLiD libraries. The sample cDNA was normalized in one round as described above and used to prepare SOLiD libraries according to the standard protocol. The quality of the RNA, fragmented RNA and cDNA libraries was assayed with an Agilent 2100 Bioanalyzer system (Agilent).Ortholog clusters for 50 Mollicute species were identified using protein BLAST. To exclude paralogs, we calculated thresholds to sequence identity for all pairs of the analyzed genomes. We scanned proteomes of all species against a fully conserved domain database  and extrFrom ortholog clusters, we extracted proteins conserved between all analyzed species. All clusters were aligned with MUSCLE  and thenM. gallisepticum strain S6 genome (CP006916.3 assembly) using the Bowtie software . The strength of the promoters was calculated as the number of reads covered by TSS normalized to library size. We manually verified all TSSs, removed false-positives within coding regions or appended those missed.To identify TSSs, we searched for a local maximum in the read coverage that was supported by at least five reads. We then modeled the coverage at each local maximum while considering 5 nt in each direction as background using a GLM  with a quasi-binomial distribution and controlling the overdispersion parameter to be no lower than 1. We used a quasi-log likelihood test to identify significant coverage peaks . To identify transcription factors (TFs), we comprehensively searched for the genes with high homology with known TFs or proteins with a helix-turn-helix DNA-binding domain. The general genome reduction in Mollicutes involves a massive loss of transcriptional regulators. In Escherichia coli and Bacillus subtilis, core cellular functions, including genome replication and protein translation, require nearly 5 and 6% of the proteome, respectively. In Mollicutes, this fraction progressively increases with genome reduction: 12.6% in A. laidlawii, 14% in S. melliferum, and 18.7% in M. gallisepticum, which indicates a progressive loss of dispensable functions (Table 1). The average genomic repertoire of TFs in bacteria is 131 proteins per species. For example, 290 transcription factors were described for E. coli and 238 for B. subtilis (Mycoplasma hominis (0.07%). In general, the number of TFs demonstrates a nearly linear decrease in the reduction of the genome size until approximately 1 Mb (the S. melliferum genome size was estimated from the contigs’ length). The number of TFs plateaus at 5-10 TFs per genome . The studied set of species retains a significant number of non-conserved regulators, which also decreases with the genome reduction.We studied the distribution and conservation of transcriptional regulators in a representative group of 50 species of Mollicutes  and the distribution of genome sizes and number of TFs . The first represents acholeplasmas and shows a relatively high number of conserved TFs. The second group includes spiroplasmas with an average TFs number of 27. The two latter groups feature group-specific TFs, which are conserved in one and absent in another. The third group consists of mycoplasmas, ureaplasmas, and phytoplasmas. This group demonstrates a high number of TF mosaicism and an extremely low number of TFs conserved within the group and with other Mollicutes. There are only four conserved TFs within more than one group of Mollicutes: the repressor of chaperones of HrcA, which are poorly studied TFs from the WhiA and YebC/PmpR families, as well as MraZ, a regulator of the cell cycle. However, the latter is lost in several species including most acholeplasmas. We propose that in these cases, the functions of MraZ were taken by a non-orthologous protein. These TFs are of particular interest because two of them (HrcA and MraZ) control core cellular function. The two others most likely control important but as yet unknown functions. The binding sites of HrcA . However, the functions of these TFs were substantially different according to the prediction. Taking into account the ubiquitous distribution of WhiA homologs, we propose that this prediction may be incorrect and that WhiA homologs control more core cellular functions. Alternatively, Streptomyces. Hence, we searched for common conserved motifs in the promoters of the respective genes in Mollicutes. We identified common motifs for acholeplasmas and spiroplasmas, which were similar in both clades . We did not identify conserved motifs common for mycoplasmas in either a reference-independent search or by searching for the identified acholeplasma-spiroplasma motif.The binding site of the WhiA-family TF was predicted using two approaches. The first, as described earlier, resulted in the identification of putative binding sites in Figure 1). For GRs, we denoted proteins that regulate a broad spectrum of genes through the modulation of RNA-polymerase activity. We included in this group both alternative sigma factors and other proteins that alter promoter recognition. We identified a set of alternative sigma factors in the Mollicutes genomes and SpxA and SpoT  proteins, which regulate transcription through different mechanisms. SpxA binds to RNA-polymerase alpha subunits and interacts with the region upstream of the core promoter. SpxA is involved in the host-pathogen interaction and controls oxidative stress response in bacteria  (bacteria . The pribacteria . Mollicubacteria . Hpr kinA. laidlawii (Supplementary Table S1), 240 for S. melliferum (Supplementary Table S2) and 430 for M. gallisepticum (Supplementary Table S3). The core promoter structure is similar in all species . The most conserved elements are the -10 box (TAWAAT), EXT element (TRTG) and initiator nucleotide (G or A). The most frequent spacer between the TSS and -10 box is 6 nt; however, 5 and 7 nt spacers also exist. One of the determinants for a spacer that is other than 6 nt in length is the non-optimal initiator nucleotide (C or T) at an optimal TSS position. In A. laidlawii and S. melliferum, there is also a pronounced -35 box (TTGACA), whereas in M. gallisepticum it is rare and less conserved. In general, A. laidlawii demonstrates the most pronounced promoter structure, regardless of whether M. gallisepticum shows promoter degeneration.Algorithmic TSSs identification with subsequent manual curation resulted in 607 TSSs for Figure 3B). Transcriptional noise correlates with promoter complexity and is calculated as the number of nucleotides with high informational content . We assume that promoters simplified in Mollicutes along with the genome reduction and the loss of transcriptional regulators. For instance, we observed an extremely high number of spurious promoters within cassettes of vlhA hemagglutinins of M. gallisepticum. Approximately 40% of TSSs in vlhA cassettes emerge from the promoters of this type. The expansion of antisense transcription could compensate for TF deficiency and improve adaptive capabilities in Mycoplasmas.Bacteria demonstrate a significant level of transcriptional noise arising at spurious promoters . The numFigure 3C). A relative reduction in mycoplasma promoters does not change the dynamic range of the promoters’ strength of protein coding genes on a genome scale. Notably, M. gallisepticum shows a relatively increased number of weak promoters. In addition to overall promoter simplification, M. gallisepticum demonstrates a phenomenon of tandem promoters. These promoters consist of two -10 boxes located near each other with a short spacer or even overlapping. These promoters have two TSSs, respectively, for example, promoters of rpmH and tsf genes . Tandem promoters likely evolved as an alternative to optimal promoters with pronounced Ext and -35 elements.The 5′-ERS (5′-end Enriched RNA Sequencing) method enables not only the identification of TSSs positions but also the ability to measure the activity of the respective promoters. The difference in the degree of promoter reduction between more and less reduced species may result in an alteration of the dynamic range of the promoters’ activities. However, all three species demonstrated comparable dynamic ranges of promoter strength (Supplementary Tables S1–S3) to determine the regulatory potential of the studied species. We were primarily focused on transcription factors; there are also homologs of global transcriptional regulators in Mollicutes. Transcription factors (TFs) can be divided into two groups: TFs located within the operon and isolated TFs. The operon structures were inferred from the TSS map. We then studied the conservation of the operon structure. In this approach, we assumed that the conservation of a TF within the operon is due to a functional link between them rather than due to a random event. For isolated TFs, we performed essentially the same procedure with the reference to the potential target operon’s structure. We assumed that there is a chance that isolated TFs belong to their own regulon in at least some species. To predict TF binding sites, we used sequences of closely related bacteria. We searched for homologs of transcription factors with no less than 60% identity between protein sequences in at least three bacteria. We presumed that regulators within operons are regulated by TFs within this operon because autoregulation is a very frequent phenomenon across bacteria High informational content  in combi(2)Presence of palindromes or repetitive sequences .(3)Similar words with known motifs: we used a manually curated database of regulons and transcription factors (RegPrecise) . For exaBased on the predicted motifs, we constructed Positional Weighted Matrices and scanned genomes to predict the putative targets of the regulators.A. laidlawii. There are 53 conserved TFs in A. laidlawii; 39 of these are located within operons including the only TF with a known binding site: HrcA. There is also one case of the fusion of a TF with an enzyme (ACL_RS02790). The search for potential regulatory sequences using the simultaneous conservation of TF, operon and promoter resulted in the identification of 28 potential binding sites (Supplementary Table S4). The rest demonstrated relatively low homology with known proteins. Most of the identified putative regulatory sequences showed a regular structure, e.g., inverted or direct repeats, which is in accordance with the organization of known binding sites in bacteria. Only five were irregular. The regulons of 21 TFs were limited by their own operon. The conservation approach also revealed several potential binding sites of isolated TFs. As we expected, their regulons encompassed multiple operons. We identified potential binding sites for 6 isolated TFs. In total, our approach enabled the identification of 34 of the 52 potential binding sites. For the rest, we were not able to identify conserved motifs most likely due to the low conservation of the respective TFs. An analysis of TF conservation showed that most are conserved among acholeplasmas; however, four have close homologs outside Mollicutes.In general, Acholeplasmas have a broad range of transcriptional regulators compared with other clades of Mollicutes. We constructed a transcriptional regulatory network as an example for that of Figures 4 and 5. The spectrum of functions controlled by TFs in A. laidlawii includes membrane transport and metabolism, the modulation of translation, protein folding, DNA supercoiling and a toxin-antitoxin system. Twenty TFs control operons involved in membrane transport and metabolism. Seven of these TFs control solely membrane transporters. One TF is likely involved in the modulation of translation. Only HrcA controls stress-related functions (protein folding). The rest of the TFs control regulons with an unclear function. Some of their targets appear to be membrane proteins; they may thus be involved in nutrient uptake.The identified regulons are summarized in laidlawii Two regulatory systems appear to form cascades (ACL_RS03205 - ACL_RS07030 and ACL_RS01290 - ACL_RS05980).The most represented group of TF-controlled metabolic pathways is the metabolism of membrane components and carbohydrates and the catabolism of cell wall components (8 TFs). Enzymes of these groups are often combined within one regulon. For example, ACL_RS01290 controls enzymes related to the glycolysis and metabolism of murein and ribose. Acholeplasma laidlawii demonstrates horizontal gene transfer of TFs . Two TFs were transferred with respective operons; however, one ACL_RS05490 controls multiple operons and resides outside them. The spectrum of TF-donor bacteria includes Carnobacterium, Blautia, Eubacterium, Lachnospiraceae, and Olsenella. Isolated and low-conserved TFs in Mollicutes pose a theoretical problem. If they were recently acquired from yet unknown species, they are useless without respective target genes. These TFs may represent an evolutionary source for the subsequent development of new regulatory pathways and adaptation to new environmental conditions , 11 of which are located within operons . One of the problems with the identification of putative binding sites in S. melliferum is the relatively high number of isolated TFs.Spiroplasmas occupy an intermediate position between TF-rich acholeplasmas and reduced mycoplasmas. S. melliferum lives in two hosts and feeds on substances with very different nutritional compositions: insect hemolymph and phloem sap of vascular plants. Insect hemolymph is rich in monosaccharides and trehalose  with identified binding sites control metabolic functions. In particular, they control the uptake and metabolism of carbohydrates. Other TF-controlled genes code for proteins involved in pantothenate/coenzyme A metabolism, polyamines transport, and protein export. We note that the putative regulator of pantothenate/coenzyme A metabolism is a member of an ambiguous protein family that encompasses both enzymes and transcriptional repressors (ROK-family IPR000600). Thus, this regulator may not be a TF but rather an enzyme of the CoA biosynthetic pathway. rehalose  and contrehalose , whereasrehalose , containrehalose , does norehalose  and is drehalose . We idenSpiroplasma melliferum demonstrates a gradual loss of HrcA binding sites. There are 3 HrcA-controlled promoters of the dnaK operon, clpB, and lon. Promoters of the dnaK operon and clpB have two copies of CIRCE. However, both copies in the dnaK promoter have four mutations and one copy in clpB promoter has three mutations; in each case, this disrupts one of the repeats. Comparison with M. gallisepticum shows that functional CIRCE have no more than two mutations, which leaves one in the clpB promoter and one in the lon promoter.S. melliferum and M. gallisepticum have interesting commonalities. They avoid using G and C nucleotides on the same strand (in the same repeat). However, the phenomenon of compositional bias is widespread in bacteria, especially among pathogenes and endosymbionts . The binding sites of HrcA and MraZ are known. Other TF are significantly less conserved. Close homologs were found only in M. imitans (and once solely in M. hominis), which significantly reduces the reliability of the conservation approach because many intergenic regions in M. imitans show extreme homology with M. gallisepticum. Eight potential TFs of M. gallisepticum reside within operons. Regularly conserved structures were found in the promoters of three: Fur-family TF, WhiA-family TF and XRE-family TF . A functional analysis of the respective operons showed that the Fur-family protein may regulate AP-endonuclease and respond to oxidative stress. Transcriptomics data are in accordance with this hypothesis. The WhiA-family protein may regulate thioredoxin-reductase and the SecG subunit of the protein export system. The XRE-family TF and the whole subsequent operon of restriction-methylation system is common in M. gallisepticum and M. hominis and was acquired by either species through horizontal transfer. Thus, the XRE-family TF is most likely a regulator of its own operon and may play a role in the global regulator controlling the methylation of the genomic DNA.Mycoplasma gallisepticum and related mycoplasmas have unique genomic regions (gene clusters of membrane hemagglutinins of the VlhA family). The regulation of their expression is tightly controlled by repetitive GAA sequences upstream of the genes. The length of GAA was shown to be an important determinant of their expression level . VlhA genes can be classified into three groups on the basis of their expression level. There are two major, three medium and other minor vlhA. The transcription level between groups differs by at least one order of magnitude.on level . Due to ous work . Due to vlhA promoter regions revealed conserved sequences including a deviant -10 box of a GCGAAAAT sequence . This type of -10 box was identified in 29 genes including the major ones. Because this sequence is non-optimal for the sigma-70 but major vlhA transcription is extremely high, we propose that an alternative sigma factor may be involved in the expression of vhlA genes. We identified two additional conserved regions in addition to the GAA tract and -10 box. One is located upstream of the GAA tract and another in 5′-UTR. Disruption of the GAA tract is associated with the loss of other motifs and the reorganization of promoters from alternative to regular promoters. We identified that RNAs of medium-transcribed vlhA genes originate from promoters with a disrupted GAA tract and regular -10 boxes. We conclude that these genes left the control of the specific vlhA-regulating system.An analysis of vlhA has a 12-GAA tract, which is in accordance with previous data  and have one of the lowest levels of antisense transcription among vlhA. We propose that vlhA transcription is controlled by both GAA length (positive regulation) and antisense activity for their respective genes (negative regulation). GCW_01940 appears to be the primary major vlhA. The high transcription of GCW_01940 drains antisenses from highly homologous GCW_03350, which results in its derepression.One of the major ous data . HoweverA. laidlawii, 30 in S. melliferum, and 195 in M. gallisepticum. Strikingly, in the well-annotated TF network of A. laidlawii, heat stress induced nine known regulons in addition to the regulon of heat-shock repressor HrcA. Two of these additional regulons included more than one operon. The induced regulons covered a broad range of functions including membrane transport, peptidases, and the metabolism of aminosugars and nucleic acids. However, the upregulation of these promoters was not comparable with the activation of HrcA-dependent promoters, which was approximately 100-fold. The maximum upregulation of other TF-controlled regulons was 10-fold (ABC-transporter controlled by ACL_RS02085). This observation raises the question of whether this response was non-specific but mediated by TFs.Previous observations demonstrated that particular stresses may result in the differential expression of numerous genes. This response may be stochastic or specifically controlled. After the reconstruction of the transcription control networks in the studied species, we applied a heat stress model to study the transcriptional response of the known system of regulators. Using quantitative 5′-ERS data, we measured the differential activity of promoters. We identified a total of 157 differentially expressed promoters in M. gallisepticum leads to the differential expression of numerous genes. Although regulatory mechanisms of some of these were proposed, there remain numerous genes with unexplained upregulation. The example of A. laidlawii demonstrates that particular stress may impact numerous components of the regulatory network . A similar effect of multiple regulon induction in heat stress was observed for M. gallisepticum, which induced regulons of HrcA (twofold), MraZ (twofold), and GCW_02350 (fivefold).We previously demonstrated that heat stress in Acholeplasma and reduced Mycoplasma. The first demonstrates substantially greater difference (orders of magnitude) between an adaptive and a noise-like response. Better transcriptional control is likely a result of the more pronounced promoter structure of A. laidlawii; in M. gallisepticum, any increased or reduced promoter-resembling sequence may initiate transcription.Heat stress induces multiple promoters in addition to those directly responsible for the heat stress defense in all three species. Promoters controlled by the same TF behave coherently. However, there are multiple upregulated promoters lacking a predicted TF. The behavior of some of these promoters may be explained by a core-promoter structure, where heat stress favors to non-optimal promoters with a high activation barrier. However, others are likely controlled by yet unknown mechanisms. The amount of overall transcriptional noise during both logarithmic growth and heat stress increases with genome reduction. Generally, there is a great difference in the overall heat stress response in TF-rich S. melliferum produced different results. None of the identified regulons were induced. The most upregulated genes in S. melliferum represent the groESL operon (10-fold). However, a lack of CIRCE in the respective promoter indicates the presence of an alternative regulator. This observation is in accordance with the degeneration of multiple CIRCE in S. melliferum. Alternatively, the parameters of heat stress for S. melliferum could be not optimal for this particular experiment.Heat stress in The combination of experimental whole-genome promoter identification and cross-species conservation analysis resulted in the identification of a substantial number (>50%) of the transcriptional control network components in the studied species of Mollicutes. Gene expression regulation can be divided into two types based on its aims: regulation in response to the external conditions and regulation of the housekeeping processes. A fundamental question remains regarding whether the cell’s functional core requires any regulation of gene expression or if its stability may be solely achieved through a perfect balance of promoter and RBS strength, codon usage and secondary structures within RNA as well as optimal protein-protein interactions within this core. Attempts to derive minimal gene content of the cell using a comparative genomics approach resulted in a set of genes that is too small to sustain the living cell . Hence, Streptomyces coelicolor , most likely the RuvC subunit  has a homolog of the carbon catabolite repressor CcpA (ACL_RS05870). Its regulon consists of three operons involved in the transport and metabolism of carbohydrates. In contrast, spiroplasmas and mycoplasmas appear to lack a respective transcriptional control system. The function of CcpA in these species appears to be played by an unknown TF; alternatively, Hpr kinase may solely regulate carbohydrates transport.All studied species have Hpr kinase, a component of the global regulation of carbon metabolism, but only Figure 6C). A. laidlawii demonstrates a substantial number of multi-operon regulons; however, they are rare in other species. The overall reduction of Mollicutes likely resulted not solely in the reduction of the number of TFs but also in a decrease in the operons amount per regulon. However, the most obscure aspect of the studied networks includes isolated TFs. Their regulons are unknown and can be very broad. A reduction in the regulatory network in Mollicutes correlates with an increased amount of antisense and noise-like transcription. The number of noise transcripts increases with AT content, which increases the fraction of AT-rich promoter-like sequences across the genome [21] and with the decay of core-promoter determinants such as -35 and Ext elements. This phenomenon is also observed in Helicobacter pylori, another genome-reduced bacterium with genome-wide antisense transcription [8]. The structure of the H. pylori promoter is similar to that of S. melliferum, and the number of transcriptional regulators in genomes that comprises  is very similar. Noise-like transcription appears to impose a negative effect on fitness because it wastes resources; however, the degenerative evolution of Mollicutes led to its increase. In pathogenic bacteria, asRNA have diverse functions and often regulate their virulence and host interactions   and is rich in TFs controlling various transporters and membrane components. S. melliferum lives in two hosts: plants and insects, where various carbohydrates are the primary nutrients either in the phloem or hemolymph. Consequently, S. melliferum have a number of TFs controlling the transport and utilization of a broad range of carbohydrates and the specialized TF-controlling catabolism of pantothenate during persistence in the phloem. Similar to other mycoplasmas, M. gallisepticum lives in an animal host rich in all nutrients. Most TFs with identified binding sites in the three species are involved in metabolism control. The progressive loss of the conserved TFs from A. laidlawii to S. melliferum and M. gallisepticum is associated with a drastic loss of metabolic pathways. The simple metabolism of M. gallisepticum likely does not require any transcription-level regulation. Instead, at least two TFs in M. gallisepticum were predicted to control functions involved in the response to oxidative stress. The induction of reactive oxygen species is an important mechanism of antimicrobial response in the immune system; thus, it may be crucial to M. gallisepticum and other vertebrate pathogens in regulating respective defense systems. Additionally, M. gallisepticum developed a specialized system (M. gallisepticum has specific regulators of oxidative damage. We hypothesize that response to various stresses in other species may be mediated by alternative sigma factors or global regulators such as SpxA (From the functional point of view, the transcription control networks of the studied bacteria reflect their lifestyle. d system  includin as SpxA . Low TFsM. pneumoniae and M. gallisepticum did not result in the elucidation of novel TFs. We propose that the TF-based approach may partially resolve this situation. TFs of reduced Mollicutes tend to control single operons and even single genes. However, there are numerous genes with unknown functions in their genomes; some of these may represent TFs. Thus, the transcriptional control network in these bacteria may consist of broad range of TF with narrow regulons. The identification of MraZ TF provides grounds for the potential existence of novel TF families that are non-homologous to those currently known.The identification of the regulatory network is based on two approaches. The first is based on the identification of genes coregulated in a panel of conditions and the subsequent identification of genetic determinants in their promoters. The second starts with the identification of binding sites of potential TFs. The first is relatively easier, especially for a small genome. However, a high-throughput analysis of stress panels of GF: Study design, conduction of the experiments, data interpretation, drafting of the manuscript. IG: Conduction of the experiments, data interpretation, drafting of the manuscript. DE: Conduction of the experiments, data interpretation, drafting of the manuscript. TS: Conduction of the experiments, data interpretation, drafting of the manuscript. AN: Conduction of the experiments, drafting of the manuscript. VG: Study design, data interpretation, drafting of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.