Minor Satellite Research Articles

Determinants of minor satellite RNA function in chromosome segregation in mouse embryonic stem cells.

The centromere is a fundamental higher-order structure in chromosomes ensuring their faithful segregation upon cell division. Centromeric transcripts have been described in several species and suggested to participate in centromere function. However, low sequence conservation of centromeric repeats appears inconsistent with a role in recruiting highly conserved centromeric proteins. Here, we hypothesized that centromeric transcripts may function through a secondary structure rather than sequence conservation. Using mouse embryonic stem cells (ESCs), we show that an imbalance in the levels of forward or reverse minor satellite (MinSat) transcripts leads to severe chromosome segregation defects. We further show that MinSat RNA adopts a stem-loop secondary structure, which is conserved in human α-satellite transcripts. We identify an RNA binding region in CENPC and demonstrate that MinSat transcripts function through the structured region of the RNA. Importantly, mutants that disrupt MinSat secondary structure do not cause segregation defects. We propose that the conserved role of centromeric transcripts relies on their secondary RNA structure.

CFDP1 regulates the stability of pericentric heterochromatin thereby affecting RAN GTPase activity and mitotic spindle formation.

The densely packed centromeric heterochromatin at minor and major satellites is comprised of H3K9me2/3 histones, the heterochromatin protein HP1α, and histone variants. In the present study, we sought to determine the mechanisms by which condensed heterochromatin at major and minor satellites stabilized by the chromatin factor CFDP1 affects the activity of the small GTPase Ran as a requirement for spindle formation. CFDP1 colocalized with heterochromatin at major and minor satellites and was essential for the structural stability of centromeric heterochromatin. Loss of CENPA, HP1α, and H2A.Z heterochromatin components resulted in decreased binding of the spindle nucleation facilitator RCC1 to minor and major satellite repeats. Decreased RanGTP levels as a result of diminished RCC1 binding interfered with chromatin-mediated microtubule nucleation at the onset of mitotic spindle formation. Rescuing chromatin H2A.Z levels in cells and mice lacking CFDP1 through knock-down of the histone chaperone ANP32E not only partially restored RCC1-dependent RanGTP levels but also alleviated CFDP1-knockout-related craniofacial defects and increased microtubule nucleation in CFDP1/ANP32E co-silenced cells. Together, these studies provide evidence for a direct link between condensed heterochromatin at major and minor satellites and microtubule nucleation through the chromatin protein CFDP1.

DNA satellite and chromatin organization at mouse centromeres and pericentromeres

BackgroundCentromeres are essential for faithful chromosome segregation during mitosis and meiosis. However, the organization of satellite DNA and chromatin at mouse centromeres and pericentromeres is poorly understood due to the challenges of assembling repetitive genomic regions.ResultsUsing recently available PacBio long-read sequencing data from the C57BL/6 strain, we find that contrary to the previous reports of their homogeneous nature, both centromeric minor satellites and pericentromeric major satellites exhibit a high degree of variation in sequence and organization within and between arrays. While most arrays are continuous, a significant fraction is interspersed with non-satellite sequences, including transposable elements. Using chromatin immunoprecipitation sequencing (ChIP-seq), we find that the occupancy of CENP-A and H3K9me3 chromatin at centromeric and pericentric regions, respectively, is associated with increased sequence enrichment and homogeneity at these regions. The transposable elements at centromeric regions are not part of functional centromeres as they lack significant CENP-A enrichment. Furthermore, both CENP-A and H3K9me3 nucleosomes occupy minor and major satellites spanning centromeric-pericentric junctions and a low yet significant amount of CENP-A spreads locally at centromere junctions on both pericentric and telocentric sides. Finally, while H3K9me3 nucleosomes display a well-phased organization on major satellite arrays, CENP-A nucleosomes on minor satellite arrays are poorly phased. Interestingly, the homogeneous class of major satellites also phase CENP-A and H3K27me3 nucleosomes, indicating that the nucleosome phasing is an inherent property of homogeneous major satellites.ConclusionsOur findings reveal that mouse centromeres and pericentromeres display a high diversity in satellite sequence, organization, and chromatin structure.

Centromere Innovations with a Mouse Species

Mammalian centromeres direct faithful genetic inheritance at cell division and are typically found in regions of highly repetitive DNA, such as a‐satellite in humans and minor satellite in many mouse species. In the house mouse M. musculus, the 120 bp DNA monomers are nearly identical (~95%) to each other, and CENP‐A nucleosomes containing the centromeric histone H3 variant, CENP‐A, are positioned with their center position, the nucleosomal dyad, within the DNA recognition element for the sequence‐specific DNA‐binding protein, CENP‐B. We find that CENP‐B‐/‐ mice have precisely the same positioning of CENP‐A nucleosomes as CENP‐B+/+ mice, indicating a potential role for DNA sequence in the positioning of CENP‐A nucleosomes. M. pahari diverged from M. musculus ~ 4 M years ago and has been reported to have a different general DNA sequence organization at centromeres, so we sought to determine the features of centromeric chromatin that were conserved between these species. We found that, on most M. pahari centromeres, CENP‐B is of very low abundance but is extremely enriched on a single chromosome. Using multiple genomic strategies, we identified the most abundant centromere repeat in M. pahari that we term pi‐satellite, which is ~189 bp long and organized into tandem repeat arrays. Chromosomes harboring pi‐satellite lack CENP‐B boxes entirely. Additionally, we employed a more sophisticated strategy to identify a less abundant centromere satellite that contains the CENP‐B box. This variant exhibits greater homogenization, is 188 bp long, and is found exclusively on the single centromere containing extremely high levels of CENP‐B. This centromere also recruits more constitutive heterochromatin, marked by H3K9me3, consistent with a role for CENP‐B in driving pericentromeric heterochromatin. Together, these findings reveal that a mouse species is undergoing dramatic molecular differences directed by evolutionary innovations in centromere DNA sequence.

Loss of centromeric RNA activates the spindle assembly checkpoint in mammalian female meiosis I.

The repetitive sequences of DNA centromeric regions form the structural basis for kinetochore assembly. Recently they were found to be transcriptionally active in mitosis, with their RNAs providing noncoding functions. Here we explore the role, in mouse oocytes, of transcripts generated from within the minor satellite repeats. Depletion of minor satellite transcripts delayed progression through meiosis I by activation of the spindle assembly checkpoint. Arrested oocytes had poorly congressed chromosomes, and centromeres were frequently split by microtubules. Thus, we have demonstrated that the centromeric RNA plays a specific role in female meiosis I compared with mitosis and is required for maintaining the structural integrity of centromeres. This may contribute to the high aneuploidy rates observed in female meiosis.

Population and subspecies diversity at mouse centromere satellites

BackgroundMammalian centromeres are satellite-rich chromatin domains that execute conserved roles in kinetochore assembly and chromosome segregation. Centromere satellites evolve rapidly between species, but little is known about population-level diversity across these loci.ResultsWe developed a k-mer based method to quantify centromere copy number and sequence variation from whole genome sequencing data. We applied this method to diverse inbred and wild house mouse (Mus musculus) genomes to profile diversity across the core centromere (minor) satellite and the pericentromeric (major) satellite repeat. We show that minor satellite copy number varies more than 10-fold among inbred mouse strains, whereas major satellite copy numbers span a 3-fold range. In contrast to widely held assumptions about the homogeneity of mouse centromere repeats, we uncover marked satellite sequence heterogeneity within single genomes, with diversity levels across the minor satellite exceeding those at the major satellite. Analyses in wild-caught mice implicate subspecies and population origin as significant determinants of variation in satellite copy number and satellite heterogeneity. Intriguingly, we also find that wild-caught mice harbor dramatically reduced minor satellite copy number and elevated satellite sequence heterogeneity compared to inbred strains, suggesting that inbreeding may reshape centromere architecture in pronounced ways.ConclusionTaken together, our results highlight the power of k-mer based approaches for probing variation across repetitive regions, provide an initial portrait of centromere variation across Mus musculus, and lay the groundwork for future functional studies on the consequences of natural genetic variation at these essential chromatin domains.

Distinct Regulation of the Expression of Satellite DNAs in the Beetle Tribolium castaneum.

In the flour beetle, Tribolium castaneum (peri)centromeric heterochromatin is mainly composed of a major satellite DNA TCAST1 interspersed with minor satellites. With the exception of heterochromatin, clustered satellite repeats are found dispersed within euchromatin. In order to uncover a possible satellite DNA function within the beetle genome, we analysed the expression of the major TCAST1 and a minor TCAST2 satellite during the development and upon heat stress. The results reveal that TCAST1 transcription was strongly induced at specific embryonic stages and upon heat stress, while TCAST2 transcription is stable during both processes. TCAST1 transcripts are processed preferentially into piRNAs during embryogenesis and into siRNAs during later development, contrary to TCAST2 transcripts, which are processed exclusively into piRNAs. In addition, increased TCAST1 expression upon heat stress is accompanied by the enrichment of the silent histone mark H3K9me3 on the major satellite, while the H3K9me3 level at TCAST2 remains unchanged. The transcription of the two satellites is proposed to be affected by the chromatin state: heterochromatin and euchromatin, which are assumed to be the prevalent sources of TCAST1 and TCAST2 transcripts, respectively. In addition, distinct regulation of the expression might be related to diverse roles that major and minor satellite RNAs play during the development and stress response.

MIWI prevents aneuploidy during meiosis by cleaving excess satellite RNA.

MIWI, a murine member of PIWI proteins mostly expressed during male meiosis, is crucial for piRNA biogenesis, post-transcriptional regulation, and spermiogenesis. However, its meiotic function remains unknown. Here, we report that MIWI deficiency alters meiotic kinetochore assembly, significantly increases chromosome misalignment at the meiosis metaphase I plate, and causes chromosome mis-segregation. Consequently, Miwi-deficient mice show elevated aneuploidy in metaphase II and spermatid death. Furthermore, in Miwi-null and Miwi slicer-deficient mutants, major and minor satellite RNAs from centromeric and pericentromeric satellite repeats accumulate in excess. Over-expression of satellite repeats in wild-type spermatocytes also causes elevated chromosome misalignment, whereas reduction of both strands of major or minor satellite RNAs results in lower frequencies of chromosome misalignment. We show that MIWI, guided by piRNA, cleaves major satellite RNAs, generating RNA fragments that may form substrates for subsequent Dicer cleavage. Furthermore, Dicer cleaves all satellite RNAs in conjunction with MIWI. These findings reveal a novel mechanism in which MIWI- and Dicer-mediated cleavage of the satellite RNAs prevents the over-expression of satellite RNAs, thus ensuring proper kinetochore assembly and faithful chromosome segregation during meiosis.

The acute myeloid leukemia variant DNMT3A Arg882His is a DNMT3B-like enzyme.

We have previously shown that the highly prevalent acute myeloid leukemia (AML) mutation, Arg882His, in DNMT3A disrupts its cooperative mechanism and leads to reduced enzymatic activity, thus explaining the genomic hypomethylation in AML cells. However, the underlying cause of the oncogenic effect of Arg882His in DNMT3A is not fully understood. Here, we discovered that DNMT3A WT enzyme under conditions that favor non-cooperative kinetic mechanism as well as DNMT3A Arg882His variant acquire CpG flanking sequence preference akin to that of DNMT3B, which is non-cooperative. We tested if DNMT3A Arg882His could preferably methylate DNMT3B-specific target sites in vivo. Rescue experiments in Dnmt3a/3b double knockout mouse embryonic stem cells show that the corresponding Arg878His mutation in mouse DNMT3A severely impairs its ability to methylate major satellite DNA, a DNMT3A-preferred target, but has no overt effect on the ability to methylate minor satellite DNA, a DNMT3B-preferred target. We also observed a previously unappreciated CpG flanking sequence bias in major and minor satellite repeats that is consistent with DNMT3A and DNMT3B specificity suggesting that DNA methylation patterns are guided by the sequence preference of these enzymes. We speculate that aberrant methylation of DNMT3B target sites could contribute to the oncogenic potential of DNMT3A AML variant.

Locus-Specific Chromatin Proteome Revealed by Mass Spectrometry-Based CasID.

Biotin proximity labeling has largely extended the toolbox of mass spectrometry-based interactomics. To date, BirA, engineered BirA variants, or other biotinylating enzymes have been widely applied to characterize protein interactions. By implementing chromatin purification-based methods the genome-wide interactome of proteins can be defined. However, acquiring a high-resolution interactome of a single genomic locus preferably by multiplexed measurements of several distinct genomic loci in parallel remains challenging. We recently developed CasID, a novel approach where the catalytically inactive Cas9 (dCas9) is coupled to the promiscuous biotin ligase BirA (BirA∗). With CasID, first the local proteome at repetitive telomeric, major satellite, and minor satellite regions was determined. With more efficient biotin ligases and sensitive mass spectrometry, others have successfully identified the chromatin composition at even smaller genomic, non-repetitive regions of a few hundred base pairs in length. Here, we summarize the most recent developments towards interactomics at a single genomic locus and provide a step-by-step protocol based on the CasID approach.

PML modulates H3.3 targeting to telomeric and centromeric repeats in mouse fibroblasts

Targeted deposition of histone variant H3.3 into chromatin is paramount for proper regulation of chromatin integrity, particularly in heterochromatic regions including repeats. We have recently shown that the promyelocytic leukemia (PML) protein prevents H3.3 from being deposited in large heterochromatic PML-associated domains (PADs). However, to what extent PML modulates H3.3 loading on chromatin in other areas of the genome remains unexplored. Here, we examined the impact of PML on targeting of H3.3 to genes and repeat regions that reside outside PADs. We show that loss of PML increases H3.3 deposition in subtelomeric, telomeric, pericentric and centromeric repeats in mouse embryonic fibroblasts, while other repeat classes are not affected. Expression of major satellite, minor satellite and telomeric non-coding transcripts is altered in Pml-null cells. In particular, telomeric Terra transcripts are strongly upregulated, in concordance with a marked reduction in H4K20me3 at these sites. Lastly, for most genes H3.3 enrichment or gene expression outcomes are independent of PML. Our data argue towards the importance of a PML-H3.3 axis in preserving a heterochromatin state at centromeres and telomeres.

Repli-FISH (Fluorescence in Situ Hybridization): Application of 3D-(Immuno)-FISH for the Study of DNA Replication Timing of Genetic Repeat Elements

Background: Genetic repeat elements (interspersed or tandem repeats) have diverse functions within cells and at different phases of the cell cycle. Yet, their investigation at a genome-wide scale has been difficult ever since due to their repetitive nature. Here, we describe a method to study the DNA replication kinetics of different repeat elements in single cells throughout the S-phase of the cell cycle. Methods: Mouse major satellite, minor satellite and telomere repeat elements as well as human LINE-1 and Alu repeats are detected in Fluorescence In Situ Hybridization (FISH) assays by specific hybridization probes containing biotinylated nucleotides. We combine their visualization via fluorophore-tagged streptavidin with the detection of cellular DNA replication signals at the single-cell level. Results: All probes hybridized specifically to their intended target sequence as verified by microscopical analysis and with regard to their location on individual chromosomes. We detected DNA replication by labeling replisome components or nascent DNA synthesis by detecting incorporated thymidine analogues. The different spatial distribution of replication signals allowed us to classify the cells in different S-phase substages. We additionally measured genomic DNA content to time the progression throughout S-phase. Then, we performed colocalization analysis of the degree of signal overlap between DNA replication and DNA probe signal to determine the kinetics of DNA replication of repeat DNA in individual cells and by high-throughput/high-content microscopical analysis. Conclusions: Application of the probes mentioned above in 3-dimensionally preserved interphase nuclei with the simultaneous detection of newly synthesized DNA or components of the replication machinery allows to determine the replication kinetics of all elements during the synthesis phase of the cell cycle in human as well as embryonic and somatic mouse cells.

The New Horizons and Hubble Space Telescope search for rings, dust, and debris in the Pluto-Charon system

We conducted an extensive search for dust or debris rings in the Pluto-Charon system before, during, and after the New Horizons encounter in July 2015. Methodologies included attempting to detect features by back-scattered light during the approach to Pluto (phase angle α ∼ 15°), in situ detection of impacting particles, a search for stellar occultations near the time of closest approach, and by forward-scattered light imaging during departure (α ∼ 165°). An extensive search using the Hubble Space Telescope (HST) prior to the encounter also contributed to the final ring limits. No rings, debris, or dust features were observed, but our new detection limits provide a substantially improved picture of the environment throughout the Pluto-Charon system. Searches for rings in back-scattered light covered the range 35,000–250,000 km from the system barycenter, a zone that starts interior to the orbit of Styx, the innermost minor satellite, and extends out to four times the orbital radius of Hydra, the outermost known satellite. We obtained our firmest limits using data from the New Horizons LORRI camera in the inner half of this region. Our limits on the normal I/F of an unseen ring depends on the radial scale of the rings: 2×10−8 (3σ) for 1500 km wide rings, 1×10−8 for 6000 km rings, and 7×10−9 for 12,000 km rings. Beyond ∼ 100, 000 km from Pluto, HST observations limit normal I/F to ∼8×10−8. Searches for dust features from forward-scattered light extended from the surface of Pluto to the Pluto-Charon Hill sphere (rHill=6.4×106 km). No evidence for rings or dust clouds was detected to normal I/F limits of ∼8.9×10−7 on ∼ 104 km scales. Four stellar occulation observations also probed the space interior to Hydra, but again no dust or debris was detected. The Student Dust Counter detected one particle impact 3.6 × 106 km from Pluto, but this is consistent with the interplanetary space environment established during the cruise of New Horizons. Elsewhere in the solar system, small moons commonly share their orbits with faint dust rings. Our results support recent dynamical studies suggesting that small grains are quickly lost from the Pluto-Charon system due to solar radiation pressure, whereas larger particles are orbitally unstable due to ongoing perturbations by the known moons.

Geomorfología y petrografía de la colada Ángeles y del cono Monte de la Cruz, volcán Barva, Costa Rica

En la falda sureste del volcán Barva se encuentra el cono piroclástico compuesto Monte de la Cruz, a partir del cual fue emitida la colada de lava Ángeles (7,3 km, área 12,8 km2). Por medio de estudios geomorfológicos, petrográficos y trabajo de campo se dividió en dos unidades, Ángeles Inferior y Superior. Petrográficamente, Ángeles Inferior corresponde con una lava andesítico basáltica vesicular de textura hipocristalina porfirítica a levemente glomeroporfirítica, con fenocristales de plagioclasa, clinopiroxenos, ortopiroxenos, olivinos y opacos. Ángeles Superior, por su parte, es andesítica vesicular a andesítica con textura hipocristalina, glomeroporfirítica a glomeroporfirítica seriada, con fenocristales de plagioclasa, clinopiroxenos, ortopiroxenos, olivinos y opacos. Morfológicamente, fueron observadas kipukas y lévees. Regionalmente, se observa que el cono Monte de la Cruz, junto con otros conos satélites menores, están alineados N19°W a lo largo de 8,5 km, evidenciando un origen asociado a una fractura volcano-tectónica.

Automated Improvement of Geolocation Accuracy in AVHRR Data Using a Two-Step Chip Matching Approach—A Part of the TIMELINE Preprocessor

The geolocation of Advanced Very High Resolution Radiometer (AVHRR) data is known to be imprecise due to minor satellite position and orbit uncertainties. These uncertainties lead to distortions once the data are projected based on the provided orbit parameters. This can cause geolocation errors of up to 10 km per pixel which is an obstacle for applications such as time series analysis, compositing/mosaicking of images, or the combination with other satellite data. Therefore, a fusion of two techniques to match the data in orbit projection has been developed to overcome this limitation, even without the precise knowledge of the orbit parameters. Both techniques attempt to find the best match between small image chips taken from a reference water mask in the first, and from a median Normalized Difference Vegetation Index (NDVI) mask in the second round. This match is determined shifting around the small image chips until the best correlation between reference and satellite data source is found for each respective image part. Only if both attempts result in the same shift in any direction, the position in the orbit is included in a third order polynomial warping process that will ultimately improve the geolocation accuracy of the AVHRR data. The warping updates the latitude and longitude layers and the contents of the data layers itself remain untouched. As such, original sensor measurements are preserved. An included automated quality assessment generates a quality layer that informs about the reliability of the matching.

Noncoding Centromeric RNA Expression Impairs Chromosome Stability in Human and Murine Stem Cells.

We analyzed the effect of transcribed noncoding RNA centromeric satellites on chromosome segregation in normal human and murine stem and fibrosarcoma cells. The overexpression of different centromeric alphoid DNAs in all cell lines induced a marked increase in chromosome mis-segregation in anaphase. Overexpression of centromeric mouse minor satellite also increased chromosome instability in the murine stem but not in human cells. Analysis of chromosome segregation in vivo showed disturbances in the mitotic progression, which was frequently unresolved. Live cell imaging revealed that overexpression of centromeric satellites resulted in several different chromosomal morphological errors in the cell nuclei. Our findings correlated with other reports that several centromeric noncoding RNAs are detected in different carcinoma cells and their expression resulted in segregation errors. Our study furnishes further insights into a novel source of genomic instability in human and murine cells. It has recently been shown that noncoding centromeric RNAs are present in some form of cancer, and thus, overexpression of several types of centromeric noncoding RNAs may be useful as a specific maker for neoplastic cells.

Determination of local chromatin composition by CasID

ABSTRACTChromatin structure and function are determined by a plethora of proteins whose genome-wide distribution is typically assessed by immunoprecipitation (ChIP). Here, we developed a novel tool to investigate the local chromatin environment at specific DNA sequences. We combined the programmable DNA binding of dCas9 with the promiscuous biotin ligase BirA* (CasID) to biotinylate proteins in the direct vicinity of specific loci. Subsequent streptavidin-mediated precipitation and mass spectrometry identified both known and previously unknown chromatin factors associated with repetitive telomeric, major satellite and minor satellite DNA. With super-resolution microscopy, we confirmed the localization of the putative transcription factor ZNF512 at chromocenters. The versatility of CasID facilitates the systematic elucidation of functional protein complexes and locus-specific chromatin composition.

Aurora Kinase B Regulates Telomerase Activity via a Centromeric RNA in Stem Cells.

Non-coding RNAs can modulate histone modifications that, at the same time, affect transcript expression levels. Here, we dissect such a network in mouse embryonic stem cells (ESCs). It regulates the activity of the reverse transcriptase telomerase, which synthesizes telomeric repeats at the chromosome ends. We find that histone H3 serine 10 phosphorylation set by Aurora kinase B (AURKB) in ESCs during the S phase of the cell cycle at centromeric and (sub)telomeric loci promotes the expression of non-coding minor satellite RNA (cenRNA). Inhibition of AURKB induces silencing of cenRNA transcription and establishment of a repressive chromatin state with histone H3 lysine 9 trimethylation and heterochromatin protein 1 accumulation. This process results in a continuous shortening of telomeres. We further show that AURKB interacts with both telomerase and cenRNA and activates telomerase in trans. Thus, in mouse ESCs, telomere maintenance is regulated via expression of cenRNA in a cell-cycle-dependent manner.

ATRX tolerates activity-dependent histone H3 methyl/phos switching to maintain repetitive element silencing in neurons

ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of “repressive” histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX’s ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this “methyl/phos” switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction.

Satellite DNA as a driver of population divergence in the red flour beetle Tribolium castaneum.

Tandemly repeated satellite DNAs are among most rapidly evolving sequences in eukaryotic genome, usually differing significantly among closely related species. By inducing changes in heterochromatin and/or centromere, satellite DNAs are expected to drive population and species divergence. However, despite high evolutionary dynamics, divergence of satellite DNA profiles at the level of natural population which precedes and possibly triggers speciation process is not readily detected. Here, we characterize minor TCAST2 satellite DNA of the red flour beetle Tribolium castaneum and follow its dynamics among wild-type strains originating from diverse geographic locations. The investigation revealed presence of three distinct subfamilies of TCAST2 satellite DNA which differ in monomer size, genome organization, and subfamily specific mutations. Subfamilies Tcast2a and Tcast2b are tandemly arranged within pericentromeric heterochromatin whereas Tcast2c is preferentially dispersed within euchromatin of all chromosomes. Among strains, TCAST2 subfamilies are conserved in sequence but exhibit a significant content variability. This results in overrepresentation or almost complete absence of particular subfamily in some strains and enables discrimination between strains. It is proposed that homologous recombination, probably stimulated by environmental stress, is responsible for the emergence of TCAST2 satellite subfamilies, their copy number variation and dispersion within genome. The results represent the first evidence for the existence of population-specific satellite DNA profiles. Partial organization of TCAST2 satellite DNA in the form of single repeats dispersed within euchromatin additionally contributes to the genome divergence at the population level.