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

SATELLITE DNA - UKE ELEMENTS DISPERSED WlTHlN EUCHROMATIN OF BEETTE Tribolium castoneum

Satellite DNA - like elements dispersed in the within euchromatin of Tribolium castaneu

Satellite DNAs in Tenebrionid Species: Structure, Organization and Evolution

The heterochromatic region of chromosomes contains highly repeated satellite DNAs with no or very few genes and is usually transcriptionally inactive. Its repetitive character has led to the suggestion that it is »junk DNA« with no utility, although some important inheritance functions have been mapped to it. Beetles from the family Tenebrionidae (Insecta, Coleoptera) contain a substantial amount of heterochromatin and satellite DNA in their genome and represent a suitable system for structural and organizational studies. Here, the data on the primary and higher order structure and organization of satellite DNAs from ten different tenebrionid species are presented, as well as on mutational processes effecting their evolution. Using these data, we have tried to determine conserved structural elements within satellites which can be important for maintaining the heterochromatin structure and compactness. Such analyses could help to define the structural components of the chromosome responsible for some heterochromatic functions, like chromosome pairing, centromere function and sister chromatid adhesion

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

Satellite DNA Modulates Gene Expression in the Beetle Tribolium castaneum after Heat Stress

Non-coding repetitive DNAs have been proposed to perform a gene regulatory role, however for tandemly repeated satellite DNA no such role was defined until now. Here we provide the first evidence for a role of satellite DNA in the modulation of gene expression under specific environmental conditions. The major satellite DNA TCAST1 in the beetle Tribolium castaneum is preferentially located within pericentromeric heterochromatin but is also dispersed as single repeats or short arrays in the vicinity of protein-coding genes within euchromatin. Our results show enhanced suppression of activity of TCAST1-associated genes and slower recovery of their activity after long-term heat stress relative to the same genes without associated TCAST1 satellite DNA elements. The level of gene suppression is not influenced by the distance of TCAST1 elements from the associated genes up to 40 kb from the genes’ transcription start sites, but it does depend on the copy number of TCAST1 repeats within an element, being stronger for the higher number of copies. The enhanced gene suppression correlates with the enrichment of the repressive histone marks H3K9me2/3 at dispersed TCAST1 elements and their flanking regions as well as with increased expression of TCAST1 satellite DNA. The results reveal transient, RNAi based heterochromatin formation at dispersed TCAST1 repeats and their proximal regions as a mechanism responsible for enhanced silencing of TCAST1-associated genes. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum strains after long-term heat stress and might have an impact on adaptation to different environmental conditions