Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin.

Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct territories. Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin

Emerging Roles of Repetitive and Repeat-Containing RNA in Nuclear and Chromatin Organization and Gene Expression

Genomic repeats have been intensely studied as regulatory elements controlling gene transcription, splicing and genome architecture. Our understanding of the role of the repetitive RNA such as the RNA coming from genomic repeats, or repetitive sequences embedded in mRNA/lncRNAs, in nuclear and cellular functions is instead still limited. In this review we discuss evidence supporting the multifaceted roles of repetitive RNA and RNA binding proteins in nuclear organization, gene regulation, and in the formation of dynamic membrane-less aggregates. We hope that our review will further stimulate research in the consolidating field of repetitive RNA biology

DNA as a programmable viscoelastic nanoelement

The two strands of a DNA molecule with a repetitive sequence can pair into many different basepairing patterns. For perfectly periodic sequences, early bulk experiments of Poerschke indicate the existence of a sliding process, permitting the rapid transition between different relative strand positions [Biophys. Chem. 2 (1974) 83]. Here, we use a detailed theoretical model to study the basepairing dynamics of periodic and nearly periodic DNA. As suggested by Poerschke, DNA sliding is mediated by basepairing defects (bulge loops), which can diffuse along the DNA. Moreover, a shear force f on opposite ends of the two strands yields a characteristic dynamic response: An outward average sliding velocity v~1/N is induced in a double strand of length N, provided f is larger than a threshold f_c. Conversely, if the strands are initially misaligned, they realign even against an external force less than f_c. These dynamics effectively result in a viscoelastic behavior of DNA under shear forces, with properties that are programmable through the choice of the DNA sequence. We find that a small number of mutations in periodic sequences does not prevent DNA sliding, but introduces a time delay in the dynamic response. We clarify the mechanism for the time delay and describe it quantitatively within a phenomenological model. Based on our findings, we suggest new dynamical roles for DNA in artificial nanoscale devices. The basepairing dynamics described here is also relevant for the extension of repetitive sequences inside genomic DNA.Comment: 10 pages, 7 figures; final version to appear in Biophysical Journa

Sequence Expression of Supernumerary B Chromosomes: Function or Fluff?

B chromosomes are enigmatic heritable elements found in the genomes of numerous plant and animal species. Contrary to their broad distribution, most B chromosomes are non-essential. For this reason, they are regarded as genome parasites. In order to be stably transmitted through generations, many B chromosomes exhibit the ability to "drive", i.e., they transmit themselves at super-Mendelian frequencies to progeny through directed interactions with the cell division apparatus. To date, very little is understood mechanistically about how B chromosomes drive, although a likely scenario is that expression of B chromosome sequences plays a role. Here, we highlight a handful of previously identified B chromosome sequences, many of which are repetitive and non-coding in nature, that have been shown to be expressed at the transcriptional level. We speculate on how each type of expressed sequence could participate in B chromosome drive based on known functions of RNA in general chromatin- and chromosome-related processes. We also raise some challenges to functionally testing these possible roles, a goal that will be required to more fully understand whether and how B chromosomes interact with components of the cell for drive and transmission

Imaging-based analysis of 5-methylcytosine at low-repetitive genomic loci using transcription activator-like effector probes

5-Methylcytosine (5mC) is the main epigenetic modification of mammalian genomes. It plays significant roles during cell development and differentiation and is involved in the regulation of essential cellular processes such as the control of gene expression. Dysregulation of methylation can lead to aberrant epigenetic patterns associated with a variety of diseases. To analyze cellular 5mC in situ, fluorescently labeled transcription-activator-like effector (TALE) proteins can be used as 5mC-sensitive probes in imaging studies. TALEs are DNA-binding proteins that provide sequence and 5mC selectivity via a domain of modular repeats, each recognizing a specific nucleobase. This enables the design of TALE probes for sequence-specific analysis of 5mC in user-defined target sequences. In imaging studies, 5mC-sensitive and 5mC-insensitive TALE pairs are used in co-stainings to allow the analysis of 5mC independently of changes in target accessibility. However, until now this has been limited to highly repetitive genomic DNA sequences. To extend this approach for the analysis of 5mC in low-repetitive coding gene loci, this work develops a straightforward signal amplification strategy to increase the imaging sensitivity with TALEs. This is achieved by additional immunostaining of the employed TALE probes, enabling the imaging of only 32 theoretical repeat sequences in the low repetitive MUC4 gene locus. In co-staining experiments, this allows the detection of 5mC changes in this locus between cell types with different methylation levels, introduced by DNA methyltransferase knockouts or overexpression. The ability to detect 5mC differences in this small number of target sequences opens up new perspectives for the analysis of 5mC in non-repetitive genomic loci, providing new insights into the regulation of gene expression

Classification Problems of Repetitive DNA Sequences

Repetitive DNA sequences, satellite DNAs (satDNAs) and transposable elements (TEs) are essential components of the genome landscape, with many different roles in genome function and evolution. Despite significant advances in sequencing technologies and bioinformatics tools, detection and classification of repetitive sequences can still be an obstacle to the analysis of genomic repeats. Here, we summarize how specificities in repetitive DNA organizational patterns can lead to an inability to classify (and study) a significant fraction of bivalve mollusk repetitive sequences. We suggest that the main reasons for this inability are: the predominant association of satDNA arrays with Helitron/Helentron TEs ; the existence of many complex loci ; and the unusual, highly scattered organization of short satDNA arrays or single monomers across the whole genome. The specificities of bivalve genomes confirm the need for introducing diverse organisms as models in order to understand all aspects of repetitive DNA biology. It is expected that further development of sequencing techniques and synergy among different bioinformatics tools and databases will enable quick and unambiguous characterization and classification of repetitive DNA sequences in assembled genomes

Complexities associated with expression of Epstein-Barr virus (EBV) lytic origins of DNA replication.

EBV has two lytic origins (oriLyt) of DNA replication lying at divergent sites on the viral genome within a duplicated sequence (DS). The latter contains potential hairpin loops, ‘hinge’ elements and the promoters for transcripts from viral genes BHLF1 and LF3. These genes themselves consist largely of 125 and 102 bp repetitive sequences, respectively, and encode basic proteins. We have examined these genomic regions in detail in attempts to understand why lytic replication—necessary for virus survival—is so inefficient, and to identify controlling elements. Our studies uncovered a diverse family of promoters (P) for BHLF1 and LF3, only one pair of which (P1) proved sensitive to chemical inducing agents. The others (P2–P3/4), abutting the replication ‘core’ origin elements in DS and extending into 50-unique sequences, may play roles in the maintenance of viral latency. We further identified a family of overlapping small complementary-strand RNAs that transverse the replication ‘core’ origin elements in a manner suggesting a role for them as ‘antisense’ species and/or DNA replication primers. Our data are discussed in terms of alternative lytic replication models. We suggest our findings might prove useful in seeking better control over viral lytic replication and devising strategies for therapy

Distinct forms of the ß subunit of GTP-binding regulatory proteins identified by molecular cloning

Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 2 subunits. The bovine transducin β subunit (β 1) has been cloned previously. We have now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue Mr 37,329 β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expreβed at lower levels than β 1 subunit mRNA in all tiβues examined. The β 1 and β 2 meβages are expreβed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

Two genetic codes: Repetitive syntax for active non-coding RNAs; non-repetitive syntax for the DNA archives

Current knowledge of the RNA world indicates 2 different genetic codes being present throughout the living world. In contrast to non-coding RNAs that are built of repetitive nucleotide syntax, the sequences that serve as templates for proteins share—as main characteristics—a non-repetitive syntax. Whereas non-coding RNAs build groups that serve as regulatory tools in nearly all genetic processes, the coding sections represent the evolutionarily successful function of the genetic information storage medium. This indicates that the differences in their syntax structure are coherent with the differences of the functions they represent. Interestingly, these 2 genetic codes resemble the function of all natural languages, i.e., the repetitive non-coding sequences serve as appropriate tool for organization, coordination and regulation of group behavior, and the nonrepetitive coding sequences are for conservation of instrumental constructions, plans, blueprints for complex protein-body architecture. This differentiation may help to better understand RNA group behavioral motifs

DNA sequences classification and computation scheme based on the symmetry principle

The DNA sequences containing multifarious novel symmetrical structure frequently play crucial role in how genomes work. Here we present a new scheme for understanding the structural features and potential mathematical rules of symmetrical DNA sequences using a method containing stepwise classification and recursive computation. By defining the symmetry of DNA sequences, we classify all sequences and conclude a series of recursive equations for computing the quantity of all classes of sequences existing theoretically; moreover, the symmetries of the typical sequences at different levels are analyzed. The classification and quantitative relation demonstrate that DNA sequences have recursive and nested properties. The scheme may help us better discuss the formation and the growth mechanism of DNA sequences because it has a capability of educing the information about structure and quantity of longer sequences according to that of shorter sequences by some recursive rules. Our scheme may provide a new stepping stone to the theoretical characterization, as well as structural analysis, of DNA sequences