Puff, the polytene chromosome: Visualizing transcription elongation control.

In this issue, Versluis et al.1 use a highly sensitive live-cell imaging system to examine transcription dynamics and functions of various key transcription elongation regulators at the Hsp70 loci.

Live-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation.

RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.

Phosphorylated histone variant γH2Av is associated with chromatin insulators in Drosophila.

Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of Topologically Associating Domains (TADs). Here, we demonstrate an association between gypsy insulator proteins and the phosphorylated histone variant H2Av (γH2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome, in polytene chromosomes and in diploid cells in which Chromatin IP data shows it is enriched at TAD boundaries. Mutation of insulator components su(Hw) and Cp190 results in a significant reduction in γH2Av levels in chromatin and phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av, but not H2Av, is a component of insulator bodies, which are protein condensates that form during osmotic stress. Phosphatase activity is required for insulator body dissolution after stress recovery. Together, our results implicate the H2A variant with a novel mechanism of insulator function and boundary formation.

ZFH-2 is required for Drosophila ovarian follicle development and is expressed at the band/interband boundaries of polytene chromosomes.

The transcription factor ZFH-2 has well-documented roles in Drosophila neurogenesis and other developmental processes. Here we provide the first evidence that ZFH-2 has a role in oogenesis. We demonstrate that ZFH-2 is expressed in the wild-type ovary and that a loss of zfh-2 function produces a mutant ovary phenotype where egg chambers are reduced in number and fused. We also show that a loss of zfh-2 function can suppress a daughterless loss-of-function ovary phenotype suggesting a possible genetic relationship between these two genes in the ovary. We also show that ZFH-2 is located at the boundary between bands and interbands on polytene chromosomes and that at a subset of these sites ZFH-2 colocalizes with the insulator/promoter cofactor CP190.

Different Protein Groups Involved in Transcription Regulation in Development and Housekeeping Genes in Drosophila.

Antibodies to histone modifications and an insulator protein involved in the processes of transcription initiation and elongation are mapped in Drosophila polytene chromosomes. The CHRIZ protein (chromatin insulator) and H3K36me3 histone modification (RNA elongation) are detected only in the localization of housekeeping genes (interbands and gray bands of polytene chromosomes) and never in the regions of developmental genes (black bands and large puffs arising from them). Antibodies to H3S10P histone modification, which is associated with the initial elongation of the RNA strand during transcription, are found exclusively in small puffs, but not in housekeeping gene localization sites or large ecdysone-induced puffs, where housekeeping genes are localized. Antibodies to H4R3me2 histone modification (a co-repressor of the ecdysone receptor) are detected only in large ecdysone-induced puffs.

Otu and Rif1 Double Mutant Enables Analysis of Satellite DNA in Polytene Chromosomes of Ovarian Germ Cells in Drosophila melanogaster.

Polytene chromosomes in Drosophila serve as a classical model for cytogenetic studies. However, heterochromatic regions of chromosomes are typically under-replicated, hindering their analysis. Mutations in the Rif1 gene lead to additional replication of heterochromatic sequences, including satellite DNA, in salivary gland cells. Here, we investigated the impact of the Rif1 mutation on heterochromatin in polytene chromosomes formed in ovarian germ cells due to the otu gene mutation. By the analysis of otu11; Rif11 double mutants, we found that, in the presence of the Rif1 mutation, ovarian cells undergo additional polytenization of pericentromeric regions. This includes the formation of large chromatin blocks composed of satellite DNA. Thus, the effects of the Rif1 mutation are similar in salivary gland and germ cells. The otu11; Rif11 system opens new possibilities for studying factors associated with heterochromatin during oogenesis.

Incomplete replication generates somatic DNA alterations within Drosophila polytene salivary gland cells.

DNA replication remains unfinished in many Drosophila polyploid cells, which harbor disproportionately fewer copies of late-replicating chromosomal regions. By analyzing paired-end high-throughput sequence data from polytene larval salivary gland cells, we define 112 underreplicated (UR) euchromatic regions 60-480 kb in size. To determine the effects of underreplication on genome integrity, we analyzed anomalous read pairs and breakpoint reads throughout the euchromatic genome. Each UR euchromatic region contains many different deletions 10-500 kb in size, while very few deletions are present in fully replicated chromosome regions or UR zones from embryo DNA. Thus, during endocycles, stalled forks within UR regions break and undergo local repair instead of remaining stable and generating nested forks. As a result, each salivary gland cell contains hundreds of unique deletions that account for their copy number reductions. Similar UR regions and deletions were observed in ovarian DNA, suggesting that incomplete replication, fork breakage, and repair occur widely in polytene cells. UR regions are enriched in genes encoding immunoglobulin superfamily proteins and contain many neurally expressed and homeotic genes. We suggest that the extensive somatic DNA instability described here underlies position effect variegation, molds the structure of polytene chromosomes, and should be investigated for possible functions.

Faint gray bands in Drosophila melanogaster polytene chromosomes are formed by coding sequences of housekeeping genes.

In Drosophila melanogaster, the chromatin of interphase polytene chromosomes appears as alternating decondensed interbands and dense black or thin gray bands. Recently, we uncovered four principle chromatin states (4НММ model) in the fruit fly, and these were matched to the structures observed in polytene chromosomes. Ruby/malachite chromatin states form black bands containing developmental genes, whereas aquamarine chromatin corresponds to interbands enriched with 5' regions of ubiquitously expressed genes. Lazurite chromatin supposedly forms faint gray bands and encompasses the bodies of housekeeping genes. In this report, we test this idea using the X chromosome as the model and MSL1 as a protein marker of the lazurite chromatin. Our bioinformatic analysis indicates that in the X chromosome, it is only the lazurite chromatin that is simultaneously enriched for the proteins and histone marks associated with exons, transcription elongation, and dosage compensation. As a result of FISH and EM mapping of a dosage compensation complex subunit, MSL1, we for the first time provide direct evidence that lazurite chromatin forms faint gray bands. Our analysis proves that overall most of housekeeping genes typically span from the interbands (5' region of the gene) to the gray band (gene body). More rarely, active lazurite chromatin and inactive malachite/ruby chromatin may be found within a common band, where both the housekeeping and the developmental genes reside together.

Chromatin Structure and "DNA Sequence View": The Role of Satellite DNA in Ectopic Pairing of the Drosophila X Polytene Chromosome.

Chromatin 3D structure plays a crucial role in regulation of gene activity. Previous studies have envisioned spatial contact formations between chromatin domains with different epigenetic properties, protein compositions and transcription activity. This leaves specific DNA sequences that affect chromosome interactions. The Drosophila melanogaster polytene chromosomes are involved in non-allelic ectopic pairing. The mutant strain agnts3, a Drosophila model for Williams-Beuren syndrome, has an increased frequency of ectopic contacts (FEC) compared to the wild-type strain Canton-S (CS). Ectopic pairing can be mediated by some specific DNA sequences. In this study, using our Homology Segment Analysis software, we estimated the correlation between FEC and frequency of short matching DNA fragments (FMF) for all sections of the X chromosome of Drosophila CS and agnts3 strains. With fragment lengths of 50 nucleotides (nt), CS showed a specific FEC-FMF correlation for 20% of the sections involved in ectopic contacts. The correlation was unspecific in agnts3, which may indicate the alternative epigenetic mechanisms affecting FEC in the mutant strain. Most of the fragments that specifically contributed to FMF were related to 1.688 or 372-bp middle repeats. Thus, middle repetitive DNA may serve as an organizer of ectopic pairing.

In Vivo Silencing of Genes Coding for dTip60 Chromatin Remodeling Complex Subunits Affects Polytene Chromosome Organization and Proper Development in Drosophila melanogaster.

Chromatin organization is developmentally regulated by epigenetic changes mediated by histone-modifying enzymes and chromatin remodeling complexes. In Drosophila melanogaster, the Tip60 chromatin remodeling complex (dTip60) play roles in chromatin regulation, which are shared by evolutionarily-related complexes identified in animal and plants. Recently, it was found that most subunits previously assigned to the dTip60 complex are shared by two related complexes, DOM-A.C and DOM-B.C, defined by DOM-A and DOM-B isoforms, respectively. In this work, we combined classical genetics, cell biology, and reverse genetics approaches to further investigate the biological roles played during Drosophila melanogaster development by a number of subunits originally assigned to the dTip60 complex.

Molecular and genetic organization of bands and interbands in the dot chromosome of Drosophila melanogaster.

The fourth chromosome smallest in the genome of Drosophila melanogaster differs from other chromosomes in many ways. It has high repeat density in conditions of a large number of active genes. Gray bands represent a significant part of this polytene chromosome. Specific proteins including HP1a, POF, and dSETDB1 establish the epigenetic state of this unique chromatin domain. In order to compare maps of localization of genes, bands, and chromatin types of the fourth chromosome, we performed FISH analysis of 38 probes chosen according to the model of four chromatin types. It allowed clarifying the dot chromosome cytological map consisting of 16 loose gray bands, 11 dense black bands, and 26 interbands. We described the relation between chromatin states and bands. Open aquamarine chromatin mostly corresponds to interbands and it contains 5'UTRs of housekeeping genes. Their coding parts are embedded in gray bands substantially composed of lazurite chromatin of intermediate compaction. Polygenic black bands contain most of dense ruby chromatin, and also some malachite and lazurite. Having an accurate map of the fourth chromosome bands and its correspondence to physical map, we found that DNase I hypersensitivity sites, ORC2 protein, and P-elements are mainly located in open aquamarine chromatin, while element 1360, characteristic of the fourth chromosome, occupies band chromatin types. POF and HP1a proteins providing special organization of this chromosome are mostly located in aquamarine and lazurite chromatin. In general, band organization of the fourth chromosome shares the features of the whole Drosophila genome.

Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains.

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

Combgap Promotes Ovarian Niche Development and Chromatin Association of EcR-Binding Regions in BR-C.

The development of niches for tissue-specific stem cells is an important aspect of stem cell biology. Determination of niche size and niche numbers during organogenesis involves precise control of gene expression. How this is achieved in the context of a complex chromatin landscape is largely unknown. Here we show that the nuclear protein Combgap (Cg) supports correct ovarian niche formation in Drosophila by controlling ecdysone-Receptor (EcR)- mediated transcription and long-range chromatin contacts in the broad locus (BR-C). Both cg and BR-C promote ovarian growth and the development of niches for germ line stem cells. BR-C levels were lower when Combgap was either reduced or over-expressed, indicating an intricate regulation of the BR-C locus by Combgap. Polytene chromosome stains showed that Cg co-localizes with EcR, the major regulator of BR-C, at the BR-C locus and that EcR binding to chromatin was sensitive to changes in Cg levels. Proximity ligation assay indicated that the two proteins could reside in the same complex. Finally, chromatin conformation analysis revealed that EcR-bound regions within BR-C, which span ~30 KBs, contacted each other. Significantly, these contacts were stabilized in an ecdysone- and Combgap-dependent manner. Together, these results highlight Combgap as a novel regulator of chromatin structure that promotes transcription of ecdysone target genes and ovarian niche formation.

Gene dunce Localization in the Polytene Chromosome of Drosophila melanogaster Long Span Batch of Adjacent Chromosomal Structures.

The molecular and chromosomal localization of the dunce gene was studied. This gene (167.3 kb) consists almost entirely of introns, in which a cluster of seven short tissue-specific genes is located. On the basis of the results of FISH analysis of the gene fragments, we established that the dunce gene is located within nine chromosomal structures (four bands and five interbands), which contradicts the common idea that genes are located in only one structure (band or interband) or at the boundary of these structures. Our results are quite unexpected and original and greatly expand the current understanding of the genetic organization of interphase chromosomes.

Architecture of Promoters of House-Keeping Genes in Polytene Chromosome Interbands of Drosophila melanogaster.

This is the first study to investigate the molecular-genetic organization of polytene chromosome interbands located on both molecular and cytological maps of Drosophila genome. The majority of the studied interbands contained one gene with a single transcription initiation site; the remaining interbands contained one gene with several alternative promoters, two or more unidirectional genes, and "head-to-head" arranged genes. In addition, intricately arranged interbands containing three or more genes in both unidirectional and bidirectional orientation were found. Insulator proteins, ORC, P-insertions, DNase I hypersensitive sites, and other open chromatin structures were situated in the promoter region of the genes located in the interbands. This area is critical for the formation of the interband, an open chromatin region in which gene transcription and replication are combined.

Non-farnesylated B-type lamin can tether chromatin inside the nucleus and its chromatin interaction requires the Ig-fold region.

Lamins are thought to direct heterochromatin to the nuclear lamina (NL); however, this function of lamin has not been clearly demonstrated in vivo. To address this, we analyzed polytene chromosome morphology when artificial lamin variants were expressed in Drosophila endoreplicating cells. We found that the CaaX-motif-deleted B-type lamin Dm0, but not A-type lamin C, was able to form a nuclear envelope-independent layer that was closely associated with chromatin. Other nuclear envelope proteins were not detected in this "ectopic lamina," and the associated chromatin showed a repressive histone modification maker but not a permissive histone modification marker nor RNA polymerase II proteins. Furthermore, deletion of the C-terminal lamin-Ig-fold domain prevents chromatin association with this ectopic lamina. Thus, non-farnesylated B-type lamin Dm0 can form an ectopic lamina and induce changes to chromatin structure and status inside the interphase nucleus.

Widespread colocalization of the Drosophila histone acetyltransferase homolog MYST5 with DREF and insulator proteins at active genes.

MYST family histone acetyltransferases play important roles in gene regulation. Here, we have characterized the Drosophila MYST histone acetyltransferase (HAT) encoded by cg1894, whose closest homolog is Drosophila MOF, and which we have termed MYST5. We found it localized to a large number of interbands as well as to the telomeres of polytene chromosomes, and it showed strong colocalization with the interband protein Z4/Putzig and RNA polymerase II. Accordingly, genome-wide location analysis by ChIP-seq showed co-occurrence of MYST5 with the Z4-interacting partner Chriz/Chromator. Interestingly, MYST5 bound to the promoter of actively transcribed genes, and about half of MYST5 sites colocalized with the transcription factor DNA replication-related element-binding factor (DREF), indicating a role for MYST5 in gene expression. Moreover, we observed substantial overlap of MYST5 binding with that of the insulator proteins CP190, dCTCF, and BEAF-32, which mediate the organization of the genome into functionally distinct topological domains. Altogether, our data suggest a broad role for MYST5 both in gene-specific transcriptional regulation and in the organization of the genome into chromatin domains, with the two roles possibly being functionally interconnected.

Polyteny: still a giant player in chromosome research.

In this era of high-resolution mapping of chromosome territories, topological interactions, and chromatin states, it is increasingly appreciated that the positioning of chromosomes and their interactions within the nucleus is critical for cellular function. Due to their large size and distinctive structure, polytene chromosomes have contributed a wealth of knowledge regarding chromosome regulation. In this review, we discuss the diversity of polytene chromosomes in nature and in disease, examine the recurring structural features of polytene chromosomes in terms of what they reveal about chromosome biology, and discuss recent advances regarding how polytene chromosomes are assembled and disassembled. After over 130 years of study, these giant chromosomes are still powerful tools to understand chromosome biology.

Banding Pattern of Polytene Chromosomes as a Representation of Universal Principles of Chromatin Organization into Topological Domains.

Drosophila polytene chromosomes are widely used as a model of eukaryotic interphase chromosomes. The most noticeable feature of polytene chromosome is transverse banding associated with alternation of dense stripes (dark or black bands) and light diffuse areas that encompass alternating less compact gray bands and interbands visible with an electron microscope. In recent years, several approaches have been developed to predict location of morphological structures of polytene chromosomes based on the distribution of proteins on the molecular map of Drosophila genome. Comparison of these structures with the results of analysis of the three-dimensional chromatin organization by the Hi-C method indicates that the morphology of polytene chromosomes represents direct visualization of the interphase nucleus spatial organization into topological domains. Compact black bands correspond to the extended topological domains of inactive chromatin, while interbands are the barriers between the adjacent domains. Here, we discuss the prospects of using polytene chromosomes to study mechanisms of spatial organization of interphase chromosomes, as well as their dynamics and evolution.