Identical Functional Organization of Nonpolytene and Polytene Chromosomes in Drosophila melanogaster

Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes

Late Replication Domains in Polytene and Non-Polytene Cells of Drosophila melanogaster

In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed genes. IH displays distinctly low gene density, indicative of their particular regulation. Genes embedded in IH replicate late in the S phase and become underreplicated. We asked whether localization and organization of these late-replicating domains is conserved in a distinct cell type. Using published comprehensive genome-wide chromatin annotation datasets (modENCODE and others), we compared IH organization in salivary gland cells and in a Kc cell line. We first established the borders of 60 IH regions on a molecular map, these regions containing underreplicated material and encompassing ∼12% of Drosophila genome. We showed that in Kc cells repressed chromatin constituted 97% of the sequences that corresponded to IH bands. This chromatin is depleted for ORC-2 binding and largely replicates late. Differences in replication timing between the cell types analyzed are local and affect only sub-regions but never whole IH bands. As a rule such differentially replicating sub-regions display open chromatin organization, which apparently results from cell-type specific gene expression of underlying genes. We conclude that repressed chromatin organization of IH is generally conserved in polytene and non-polytene cells. Yet, IH domains do not function as transcription- and replication-regulatory units, because differences in transcription and replication between cell types are not domain-wide, rather they are restricted to small “islands” embedded in these domains. IH regions can thus be defined as a special class of domains with low gene density, which have narrow temporal expression patterns, and so displaying relatively conserved organization

Physical map and molecular features of the band 59D1-2.

<p>Vertical lines delimit the borders of this IH band. Data on protein profiling and replication timing are from: (1) – Belyakin et al., 2005 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone.0030035-Belyakin1" target="_blank">[14]</a>; (2) – Kharchenko et al., 2011 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone.0030035-Kharchenko1" target="_blank">[30]</a>; (3) –Filion et al., 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone.0030035-Filion1" target="_blank">[32]</a>; (4) – Belyakin et al., 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone.0030035-Belyakin2" target="_blank">[15]</a>; (5) – Kharchenko et al., 2011 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone.0030035-Kharchenko1" target="_blank">[30]</a>; (6) – MacAlpine et al., 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone.0030035-MacAlpine2" target="_blank">[29]</a>; (6)* - Eaton et al., 2011 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone.0030035-Eaton1" target="_blank">[37]</a>; (7) – Nordman et al., 2011 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone.0030035-Nordman1" target="_blank">[31]</a>; [8] – Schwaiger et al., 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone.0030035-Schwaiger1" target="_blank">[22]</a>.</p

Physical map and molecular features of the region 79E1-4.

<p>Legends are the same as on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone-0030035-g001" target="_blank">Fig. 1</a>. IH band has an early-replicating region, which corresponds to two active (RED) fragments.</p

Correlation of sizes of open chromatin (RED and YELLOW) fragments with the length of early replication areas.

<p>Correlation of sizes of open chromatin (RED and YELLOW) fragments with the length of early replication areas.</p

Nomenclature, sizes of IH regions and overlapping with LADs.

<p>*% overlap was calculated as a ratio between the length of overlapping region and the length of IH band.</p

Proportion of various chromatin types in IH regions.

<p><b>A</b> – 5 color chromatin types by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone.0030035-Filion1" target="_blank">[32]</a>. <b>B</b> – 9 chromatin states as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone.0030035-Kharchenko1" target="_blank">[30]</a> (states 6–9 correspond to repressed chromatin).</p

DNA replication in 10A-11A region of the polytene chromosome.

<p><b>A–C</b> – Immunostaining for pre-replication complex component DUP/CDF1. Pre-replication complex is not detected in IH bands 10A1-2, 10B1-2, 11A6-9. <b>A</b> – phase contrast; <b>B</b> – immunolocalization; <b>C</b>- merge; <b>D–G</b> – immunostaining for PCNA at consecutive replication steps (further description in text).</p

Proportion of the DNA sequences covered by the corresponding proteins (%).

<p>Proportion of the DNA sequences covered by the corresponding proteins (%).</p

Physical map and molecular features of the region 12E.

<p>Legends are the same as on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030035#pone-0030035-g001" target="_blank">Figure 1</a>. The region consists of two bands, 12E1-2 (left) and 12E8-9 (right).</p