RNA Pol II Promotes Transcription of Centromeric Satellite DNA in Beetles

Transcripts of centromeric satellite DNAs are known to play a role in heterochromatin formation as well as in establishment of the kinetochore. However, little is known about basic mechanisms of satellite DNA expression within constitutive heterochromatin and its regulation. Here we present comprehensive analysis of transcription of abundant centromeric satellite DNA, PRAT from beetle Palorus ratzeburgii (Coleoptera). This satellite is characterized by preservation and extreme sequence conservation among evolutionarily distant insect species. PRAT is expressed in all three developmental stages: larvae, pupae and adults at similar level. Transcripts are abundant comprising 0.033% of total RNA and are heterogeneous in size ranging from 0.5 kb up to more than 5 kb. Transcription proceeds from both strands but with 10 fold different expression intensity and transcripts are not processed into siRNAs. Most of the transcripts (80%) are not polyadenylated and remain in the nucleus while a small portion is exported to the cytoplasm. Multiple, irregularly distributed transcription initiation sites as well as termination sites have been mapped within the PRAT sequence using primer extension and RLM-RACE. The presence of cap structure as well as poly(A) tails in a portion of the transcripts indicate RNA polymerase II–dependent transcription and a putative polymerase II promoter site overlaps the most conserved part of the PRAT sequence. The treatment of larvae with alpha-amanitin decreases the level of PRAT transcripts at concentrations that selectively inhibit pol II activity. In conclusion, stable, RNA polymerase II dependant transcripts of abundant centromeric satellite DNA, not regulated by RNAi, have been identified and characterized. This study offers a basic understanding of expression of highly abundant heterochromatic DNA which in beetle species constitutes up to 50% of the genome

DNA demethylation and heterochromatin remodelling upon heat stress

Conversion of environmental signals into epigenetic information is thought to occur widely but has been poorly studied as yet. It is proposed that changes in the expression of molecules involved in chromatin remodelling or their modification might play a role in this process. Here we study remodelling of heterochromatin upon heat stress in the red flour beetle Tribolium castaneum (Tenebrionidae, Coleoptera), one of the most important stored products pest. We show that the expression of abundant satellite DNA TCAST which is located within the constitutive pericentromeric heterochromatin, is strongly induced following heat shock and is accompanied by increase in repressive epigenetic modifications of histones at TCAST regions. Upon recovery from heat stress, the expression of satellite DNA-associated siRNAs as well as histone modifications is quickly restored. Bisulphite sequencing of TCAST satellite DNA reveals cytosine methylation not only restricted to CpG sites but also found at CpA, CpT and CpC sites, and preserved within heterochromatin during all developmental phases. The level of methylation decreases after heat shock depending on the duration and results in a complete demethylation after prolonged treatment. Our results indicate that heat shock induced satellite DNA demethylation and expression affect epigenetic state of constitutive heterochromatin in insects. It can be hypothesized that transient remodelling of heterochromatin is part of a physiological gene expression program activated under stress conditions. We propose the existence of a negative-feedback mechanism in which satellite DNA-associated siRNAs induced by heat stress, repress their own transcription by providing additional H3K9me3 anchorage sites for the chromodomain protein such as HP1, and in this way restore heterochromatic state

Dispersed satellite DNA elements and their effect on gene expression

Different from dispersed transposable elements whose role in evolution of gene regulation was investigated in diverse model organism, influence of satellites DNA on gene regulation was not investigated till now. To perform potential regulatory function, satellite DNA elements are predicted not only to be present within heterochromatin, but to be distributed in euchromatic portion of the genome, in the vicinity of genes. Within insect species Tribolium castaneum satellite DNAs make a substantial portion of the genome and are major constituents of pericentromeric heterochromatin. The expression of a major heterochromatic T. castaneum satellite DNA TCAST1 proceeds in the form of long double-strand transcripts which are rapidly processed into small interfering RNAs (siRNAs). Satellite DNA expression is strongly induced by heat shock, and increased level of satellite-derived siRNAs is accompanied by increase of repressive epigenetic modifications of histones, H3K9me2-3 at satellite DNA regions. Single repeats or short stretches of the same TCAST1 satellite DNA are also dispersed in the close vicinity of protein-coding genes within euchromatin of T. castaneum. To explore the potential gene-regulatory role of TCAST1 satellite elements, we examined variation of TCAST1 elements among 10 T. castaneum wild-type strains originating from diverse geographic locations, and followed expression of genes that either contain or have in the vicinity polymorphic TCAST1 elements. Gene expression was explored at normal as well as under heat stress conditions. Expression analysis of genes that contain polymorphic TCAST1 elements within introns indicates influence of TCAST1 elements on gene expression under heat stress conditions. The gene expression is effected by the presence of TCAST1 element as well as by the number of repeats within dispersed TCAST1 satellite element. In all cases, TCAST1 elements partially repressed activity of genes after heat shock treatment. It is proposed that TCAST1-associated siRNAs, significantly induced after heat stress, affect epigenetic state of euchromatic regions containing dispersed TCAST1 satellite elements by increasing repressive epigenetic modifications of histones. This is the first demonstration of satellite DNA involvement in the modulation of protein-gene expression and indication for the role of satellite DNA in the evolution of gene regulation

Analysis of Copy Number Variation in the Abp Gene Regions of Two House Mouse Subspecies Suggests Divergence during the Gene Family Expansions

The Androgen-binding protein (Abp) gene region of the mouse genome contains 64 genes, some encoding pheromones that influence assortative mating between mice from different subspecies. Using CNVnator and quantitative PCR, we explored copy number variation in this gene family in natural populations of Mus musculus domesticus (Mmd) and Mus musculus musculus (Mmm), two subspecies of house mice that form a narrow hybrid zone in Central Europe. We found that copy number variation in the center of the Abp gene region is very common in wild Mmd, primarily representing the presence/absence of the final duplications described for the mouse genome. Clustering of Mmd individuals based on this variation did not reflect their geographical origin, suggesting no population divergence in the Abp gene cluster. However, copy number variation patterns differ substantially between Mmd and other mouse taxa. Large blocks of Abp genes are absent in Mmm, Mus musculus castaneus and an outgroup, Mus spretus, although with differences in variation and breakpoint locations. Our analysis calls into question the reliance on a reference genome for interpreting the detailed organization of genes in taxa more distant from the Mmd reference genome. The polymorphic nature of the gene family expansion in all four taxa suggests that the number of Abp genes, especially in the central gene region, is not critical to the survival and reproduction of the mouse. However, Abp haplotypes of variable length may serve as a source of raw genetic material for new signals influencing reproductive communication and thus speciation of mice

Differential enrichment of H3K9me3 at annotated satellite DNA repeats in human cell lines and during fetal development in mouse

Background: Trimethylation of histone H3 on lysine 9 (H3K9me3) at satellite DNA sequences has been primarily studied at (peri)centromeric regions, where its level shows differences associated with various processes such as development and malignant transformation. However, the dynamics of H3K9me3 at distal satellite DNA repeats has not been thoroughly investigated. Results: We exploit the sets of publicly available data derived from chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-Seq), produced by the The Encyclopedia of DNA Elements (ENCODE) project, to analyze H3K9me3 at assembled satellite DNA repeats in genomes of human cell lines and during mouse fetal development. We show that annotated satellite elements are generally enriched for H3K9me3, but its level in cancer cell lines is on average lower than in normal cell lines. We find 407 satellite DNA instances with differential H3K9me3 enrichment between cancer and normal cells including a large 115-kb cluster of GSATII elements on chromosome 12. Differentially enriched regions are not limited to satellite DNA instances, but instead encompass a wider region of flanking sequences. We found no correlation between the levels of H3K9me3 and noncoding RNA at corresponding satellite DNA loci. The analysis of data derived from multiple tissues identified 864 instances of satellite DNA sequences in the mouse reference genome that are differentially enriched between fetal developmental stages. Conclusions: Our study reveals significant differences in H3K9me3 level at a subset of satellite repeats between biological states and as such contributes to understanding of the role of satellite DNA repeats in epigenetic regulation during development and carcinogenesis

Dispersion Profiles and Gene Associations of Repetitive DNAs in the Euchromatin of the Beetle Tribolium castaneum

ABSTRACT Satellite DNAs are tandemly repeated sequences clustered within heterochromatin. However, in some cases, such as the major TCAST1 satellite DNA from the beetle Tribolium castaneum, they are found partially dispersed within euchromatin. Such organization together with transcriptional activity enables TCAST1 to modulate the activity of neighboring genes. In order to explore if other T. castaneum repetitive families have features that could provide them with a possible gene-modulatory role, we compare here the structure, organization, dispersion profiles, and transcription activity of 10 distinct TCAST repetitive families including TCAST1. The genome organization of TCAST families exhibit either satellite-like or transposon-like characteristics. In addition to heterochromatin localization, bioinformatic searches of the assembled genome have revealed dispersion of all families within euchromatin, preferentially in the form of single repeats. Dispersed TCAST repeats are mutually correlated in distribution and are grouped in distinct regions of euchromatin. The repeats are associated with genes, are enriched in introns relative to intergenic regions, and very rarely overlap exons. In spite of the different mechanisms of repeat proliferation, such as transposition and homologous recombination, all TCAST families share a similar frequency of spreading as well as dispersion and gene association profiles. Additionally, TCAST families are transcribed and their transcription is significantly activated by heat stress. A possibility that such common features of TCAST families might be related to their potential gene-modulatory role is discussed

Evolutionary history of alpha satellite DNA repeats dispersed within human genome euchromatin

Major human alpha satellite DNA repeats are preferentially assembled within (peri)centromeric regions but are also dispersed within euchromatin in the form of clustered or short single repeat arrays. To study the evolutionary history of single euchromatic human alpha satellite repeats (ARs), we analyzed their orthologous loci across the primate genomes. The continuous insertion of euchromatic ARs throughout the evolutionary history of primates starting with the ancestors of Simiformes (45-60 Ma) and continuing up to the ancestors of Homo is revealed. Once inserted, the euchromatic ARs were stably transmitted to the descendant species, some exhibiting copy number variation, whereas their sequence divergence followed the species phylogeny. Many euchromatic ARs have sequence characteristics of (peri)centromeric alpha repeats suggesting heterochromatin as a source of dispersed euchromatic ARs. The majority of euchromatic ARs are inserted in the vicinity of other repetitive elements such as L1, Alu, and ERV or are embedded within them. Irrespective of the insertion context, each AR insertion seems to be unique and once inserted, ARs do not seem to be subsequently spread to new genomic locations. In spite of association with (retro)transposable elements, there is no indication that such elements play a role in ARs proliferation. The presence of short duplications at most of ARs insertion sites suggests site-directed recombination between homologous motifs in ARs and in the target genomic sequence, probably mediated by extrachromosomal circular DNA, as a mechanism of spreading within euchromatin

Androgen-Binding Protein (Abp) Evolutionary History: Has Positive Selection Caused Fixation of Different Paralogs in Different Taxa of the Genus Mus?

Comparison of the androgen-binding protein (Abp) gene regions of six Mus genomes provides insights into the evolutionary history of this large murid rodent gene family. We identified 206 unique Abp sequences and mapped their physical relationships. At least 48 are duplicated and thus present in more than two identical copies. All six taxa have substantially elevated LINE1 densities in Abp regions compared with flanking regions, similar to levels in mouse and rat genomes, although nonallelic homologous recombination seems to have only occurred in Mus musculus domesticus. Phylogenetic and structural relationships support the hypothesis that the extensive Abp expansion began in an ancestor of the genus Mus. We also found duplicated Abpa27's in two taxa, suggesting that previously reported selection on a27 alleles may have actually detected selection on haplotypes wherein different paralogs were lost in each. Other studies reported that a27 gene and species trees were incongruent, likely because of homoplasy. However, L1MC3 phylogenies, supposed to be homoplasy-free compared with coding regions, support our paralog hypothesis because the L1MC3 phylogeny was congruent with the a27 topology. This paralog hypothesis provides an alternative explanation for the origin of the a27 gene that is suggested to be fixed in the three different subspecies of Mus musculus and to mediate sexual selection and incipient reinforcement between at least two of them. Finally, we ask why there are so many Abp genes, especially given the high frequency of pseudogenes and suggest that relaxed selection operates over a large part of the gene clusters

Subcellular localization and polyadenylation of transcripts.

<p><i>(A)</i> Agarose gel electrophoresis of cytoplasmic (cy), nuclear (nu), polyadenylated (a+) and non-polyadenylated (a-) RNA and hybridization profile obtained with RNA probe for forward strand. 13 µg of cy and 1.3 µg of nu RNA extracted in parallel from the same sample of adults are loaded as well as 1 µg of a+ and 17 µg of a-. Cytoplasmic (13 µg) and non-polyadenylated RNAs (17 µg) treated with RNase A (+Rc and +Ra, respectively) serve as controls for genomic DNA contamination. <i>S. cerevisiae</i> RNA (c) represents negative hybridization control. <i>(B)</i> Dot blot analysis of total RNA (45 µg), polyadenylated RNA (a+, 6 µg), non-polyadenylated RNA (a-, 40 µg) using PRAT RNA probes for forward and reverse strand, as well as of cytoplasmic (cy) and nuclear (nu) RNA, in amounts indicated, using RNA probe for reverse strand. <i>S. cerevisiae</i> RNA is used as a negative control in amounts exactly matching those of above mentioned samples (−control). For the purpose of PRAT transcripts quantification in different RNA fractions, signal intensity was reduced for the signal detected in the corresponding negative control. Due to the low signal intensity in the a+ fraction of reverse strand precise quantification was not possible. Complementary PRAT transcripts, in amounts indicated, are used as a positive control (+control) and for calibration. Cytoplasmic and nuclear RNA digested with RNase A represent control for genomic DNA contamination (+RNase).</p