Heterochromatic Regions Research Articles

Abstract 4726: KMT5C-dependent regulation of mesenchymal-related genes and identification of KMT5C interactome using BioID

Abstract Epigenetic dysregulation has recently been recognized as a new hallmark of cancer. Dysregulation of chromatin remodelers is estimated to affect 10-20% of cancer. Thus, understanding how epigenetic-based mechanisms contribute to cancer initiation and progression is critical. KMT5C is a histone methyltransferase that catalyzes histone H4 lysine 20 trimethylation (H4K20me3), which has historically been associated with formation and maintenance of constitutive heterochromatin regions. Albeit recent evidence from our lab also suggests regulation of gene-rich euchromatin regions. In this previous body of work we discovered that loss of KMT5C promotes development of resistance to tyrosine kinase inhibitors (TKI) in EGFR-mutant non-small cell lung cancer (NSCLC) cells (PC9 and HCC827). Patient data further supports that the KMT5C transcript level is downregulated following resistance to osimertinib, a third-generation TKI. Our data suggests that KMT5C is involved in non-canonical regulation of genes outside of heterochromatic regions through its methyltransferase activity. Analysis of data deposited into the Cell Miner database and publicly available RNA-sequencing and ChIP-sequencing data revealed that the KMT5C-H4K20me3 axis has significant negative correlations with several genes involved in epithelial-to-mesenchymal transition (EMT). Our validation studies support this observation. However the mechanisms involved in KMT5C facilitating this change at EMT-related genes is unknown. KMT5C is well understood to be recruited to heterochromatin regions through its interaction with HP1, yet the mechanism for recruitment of KMT5C to gene-rich euchromatin regions remains to be elucidated. We hypothesize that KMT5C is recruited to its target genes in euchromatin in an HP1-independent mechanism. In this study, we aim to identify the interactome of KMT5C using an unbiased proximity labeling BioID approach. This study is expected to uncover a novel epigenetic mechanism that contributes to development of resistance to TKIs and may inform of strategies to target KMT5C-downregulated TKI-resistant cancers. Citation Format: Jihye Son, Alejandra Agredo, Ally Glaws, Andrea L. Kasinski. KMT5C-dependent regulation of mesenchymal-related genes and identification of KMT5C interactome using BioID. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4726.

Analysis in Proceratophrys boiei genome illuminates the satellite DNA content in a frog from the Brazilian Atlantic forest.

Satellite DNAs (satDNAs) are one of the most abundant elements in genomes. Characterized as tandemly organized sequences that can be amplified into multiple copies, mainly in heterochromatic regions. The frog P. boiei (2n = 22, ZZ♂/ZW♀) is found in the Brazilian Atlantic forest and has an atypical pattern of heterochromatin distribution when compared to other anuran amphibians, with large pericentromeric blocks on all chromosomes. In addition, females of Proceratophrys boiei have a metacentric sex chromosome W showing heterochromatin in all chromosomal extension. In this work, we performed high-throughput genomic, bioinformatic, and cytogenetic analyses to characterize the satellite DNA content (satellitome) in P. boiei, mainly due to high amount of C-positive heterochromatin and the highly heterochromatic W sex chromosome. After all the analyses, it is remarkable that the satellitome of P. boiei is composed of a high number of satDNA families (226), making P. boiei the frog species with the highest number of satellites described so far. Consistent with the observation of large centromeric C-positive heterochromatin blocks, the genome of P. boiei is enriched with high copy number of repetitive DNAs, with total satDNA abundance comprising 16.87% of the genome. We successfully mapped via Fluorescence in situ hybridization the two most abundant repeats in the genome, PboSat01-176 and PboSat02-192, highlighting the presence of certain satDNAs sequences in strategic chromosomal regions (e.g., centromere and pericentromeric region), which leads to their participation in crucial processes for genomic organization and maintenance. Our study reveals a great diversity of satellite repeats that are driving genomic organization in this frog species. The characterization and approaches regarding satDNAs in this species of frog allowed the confirmation of some insights from satellite biology and a possible relationship with the evolution of sex chromosomes, especially in anuran amphibians, including P. boiei, for which data were not available.

Cytogenetic Analysis of the Fungus-Farming Ant Cyphomyrmex rimosus (Spinola, 1851) (Formicidae: Myrmicinae: Attini) Highlights Karyotypic Variation

The fungus-farming ant genus Cyphomyrmex (subtribe Attina, clade Neoattina) comprises 23 described species that are widely distributed throughout the Neotropics. Species within Cyphomyrmex have taxonomic issues such as Cyphomyrmex rimosus (Spinola, 1851) which is likely a species complex. Cytogenetics is a useful tool for evolutionary studies and understanding species with dubious taxonomy. In this study, we characterized the karyotype of C. rimosus from Viçosa, Minas Gerais State, southeastern Brazil using classical and molecular cytogenetic techniques to enrich the chromosomal information about Cyphomyrmex. The karyotype of C. rimosus from the rainforest of southeastern Brazil (2n = 22, 18m + 4sm) notably contrasts with that previously described for this species in Panama (2n = 32). This intraspecific chromosomal variation suggests the existence of a species complex within this taxon according to the previous hypothesis derived from morphological analysis. We detected GC-rich heterochromatic regions in C. rimosus and, using repetitive DNA probes, showed that this heterochromatin shares repetitive sequences with other Neoattina species already studied, enhancing the importance of this specific genome region in the understanding of Attina evolution. Mapping of microsatellite (GA)₁₅ on C. rimosus was restricted to the euchromatic regions of all chromosomes. The single intrachromosomal rDNA sites observed in C. rimosus follow the general genomic organization trend of ribosomal genes in Formicidae. Our study extends the data of chromosome mapping on Cyphomyrmex and reinforces the importance of cytogenetic studies in different localities to better understand taxonomic issues in widely distributed taxa such as C. rimosus.

Supernumerary Marker Chromosome Identified in Asian Elephant (Elephas maximus).

We identified a small, supernumerary marker chromosome (sSMC) in two phenotypically normal Asian elephants (Elephas maximus): a female (2n = 57,XX,+mar) and her male offspring (2n = 57,XY,+mar). sSMCs are defined as structurally abnormal chromosomes that cannot be identified by conventional banding analysis since they are usually small and often lack distinct banding patterns. Although current molecular techniques can reveal their origin, the mechanism of their formation is not yet fully understood. We determined the origin of the marker using a suite of conventional and molecular cytogenetic approaches that included (a) G- and C-banding, (b) AgNOR staining, (c) preparation of a DNA clone using laser microdissection of the marker chromosome, (d) FISH with commercially available human painting and telomeric probes, and (e) FISH with centromeric DNA derived from the centromeric regions of a marker-free Asian elephant. Moreover, we present new information on the location and number of NORs in Asian and savanna elephants. We show that the metacentric marker was composed of heterochromatin with NORs at the terminal ends, originating most likely from the heterochromatic region of chromosome 27. In this context, we discuss the possible mechanism of marker formation. We also discuss the similarities between sSMCs and B chromosomes and whether the marker chromosome presented here could evolve into a B chromosome in the future.

Chromosome diversity in Buthidae and Chactidae scorpions from Brazilian fauna: Diploid number and distribution of repetitive DNA sequences.

In this work, we analyzed cytogenetically eight Chactidae and Buthidae, including the localization of repetitive DNA sequences. The chactids possess monocentric chromosomes and the highest diploid numbers (2n=50 in Brotheas amazonicus, 2n=36 in Chactopsis amazonica, 2n=30 in Neochactas sp.) when compared with buthids (2n=10 in Tityus bahiensis, 2n=14 in Tityus apiacas and Tityus metuendus, 2n=18 in Tityus aba, 2n=26 in Ischnotelson peruassu). The localization of rDNA genes and (TTAGG)n sequences exhibited a conserved pattern of two terminal/subterminal ribosomal cistrons and terminal telomere signals. However, the comparison between the data of C-banding, DAPI after FISH and Cot-DNA fraction indicated a variable quantity and distribution of these regions, as follow: (i) positive heterochromatin and Cot-DNA signals (B. amazonicus and I. peruassu), (ii) small blocks of heterochromatin with large Cot-DNA signals (T. metuendus), (iii) positive heterochromatic regions and absence of Cot-DNA signals (T. aba and T. apiacas), and (iv) negative heterochromatin and Cot-DNA signals (T. bahiensis). Therefore, our results revealed that there still is not a clear relation between quantity of heterochromatin and presence of monocentric or holocentric chromosomes and occurrence of chromosomal rearrangements, indicating that repetitive regions in scorpions must be analyzed using different cytogenetic approaches.

Generating PARP Knockout D. melanogaster with CRISPR/Cas9 System.

Long-branched negatively charged poly(ADP-ribose) (pADPr) is a posttranslation modification of nuclear proteins that play a key role in many chromatin remodeling events. While several enzymes of PARP family could synthesize it across all multicellular organisms, Drosophila melanogaster is very suitable model to study pADPr-regulated processes because only one PARP gene is present. Despite the fact that PARP is an intensively studied protein with multiple important functions, no total knockout PARP flies were obtained in mobile element mutagenesis-based projects, mainly because PARP gene localizes in heterochromatic region. Here, we describe all steps of generating PARP mutated D. melanogaster with CRISPR/Cas9 system from the gRNA design, plasmid cloning to fly crosses and mutation detection. Provided gRNAs sequences target the region with high efficiency and results in more than 90% mutant stocks. This method could also be modified to generate PARP mutations in other gene locus, knockins with donor sequences for homology recombination or to be adjusted for other pADPr turnover-regulating enzymes.

How enzymatic activity is involved in chromatin organization.

Spatial organization of chromatin plays a critical role in genome regulation. Previously, various types of affinity mediators and enzymes have been attributed to regulate spatial organization of chromatin from a thermodynamics perspective. However, at the mechanistic level, enzymes act in their unique ways and perturb the chromatin. Here, we construct a polymer physics model following the mechanistic scheme of Topoisomerase-II, an enzyme resolving topological constraints of chromatin, and investigate how it affects interphase chromatin organization. Our computer simulations demonstrate Topoisomerase-II's ability to phase separate chromatin into eu- and heterochromatic regions with a characteristic wall-like organization of the euchromatic regions. We realized that the ability of the euchromatic regions to cross each other due to enzymatic activity of Topoisomerase-II induces this phase separation. This realization is based on the physical fact that partial absence of self-avoiding interaction can induce phase separation of a system into its self-avoiding and non-self-avoiding parts, which we reveal using a mean-field argument. Furthermore, motivated from recent experimental observations, we extend our model to a bidisperse setting and show that the characteristic features of the enzymatic activity-driven phase separation survive there. The existence of these robust characteristic features, even under the non-localized action of the enzyme, highlights the critical role of enzymatic activity in chromatin organization.

Heterochromatin in the fungal plant pathogen, Zymoseptoria tritici: Control of transposable elements, genome plasticity and virulence.

Heterochromatin is a repressive chromatin state that plays key roles in the functional organisation of eukaryotic genomes. In fungal plant pathogens, effector genes that are required for host colonization tend to be associated with heterochromatic regions of the genome that are enriched with transposable elements. It has been proposed that the heterochromatin environment silences effector genes in the absence of host and dynamic chromatin remodelling facilitates their expression during infection. Here we discuss this model in the context of the key wheat pathogen, Zymoseptoria tritici. We cover progress in understanding the deposition and recognition of heterochromatic histone post translational modifications in Z. tritici and the role that heterochromatin plays in control of genome plasticity and virulence.

TGM2-mediated histone transglutamination is dictated by steric accessibility

Recent studies have identified serotonylation of glutamine-5 on histone H3 (H3Q5ser) as a novel posttranslational modification (PTM) associated with active transcription. While H3Q5ser is known to be installed by tissue transglutaminase 2 (TGM2), the substrate characteristics affecting deposition of the mark, at the level of both chromatin and individual nucleosomes, remain poorly understood. Here, we show that histone serotonylation is excluded from constitutive heterochromatic regions in mammalian cells. Biochemical studies reveal that the formation of higher-order chromatin structures associated with heterochromatin impose a steric barrier that is refractory to TGM2-mediated histone monoaminylation. A series of structure-activity relationship studies, including the use of DNA-barcoded nucleosome libraries, shows that steric hindrance also steers TGM2 activity at the nucleosome level, restricting monoaminylation to accessible sites within histone tails. Collectively, our data indicate that the activity of TGM2 on chromatin is dictated by substrate accessibility rather than by primary sequence determinants or by the existence of preexisting PTMs, as is the case for many other histone-modifying enzymes.

Su(var)2-10- and Su(var)205-dependent upregulation of the heterochromatic gene neverland is required for developmental transition in Drosophila.

Animals develop from juveniles to sexually mature adults through the action of steroid hormones. In insect metamorphosis, a surge of the steroid hormone ecdysone prompts the transition from the larval to the adult stage. Ecdysone is synthesized by a series of biosynthetic enzymes that are specifically expressed in an endocrine organ, the prothoracic gland. At the late larval stage, the expression levels of ecdysone biosynthetic enzymes are upregulated through the action of numerous transcription factors, thus initiating metamorphosis. In contrast, the mechanism by which chromatin regulators support the expression of ecdysone biosynthetic genes is largely unknown. Here, we demonstrate that Su(var)2-10 and Su(var)205, suppressor of variegation [Su(var)] genes encoding a chromatin regulator Su(var)2-10 and nonhistone heterochromatic protein 1a, respectively, regulate the transcription of one of the heterochromatic ecdysone biosynthetic genes, neverland, in Drosophila melanogaster. Knockdown of Su(var)2-10 and Su(var)205 in the prothoracic gland caused a decrease in neverland expression, resulting in a defect in larval-to-prepupal transition. Furthermore, overexpression of neverland and administration of 7-dehydrocholesterol, a biosynthetic precursor of ecdysone produced by Neverland, rescued developmental defects in Su(var)2-10 and Su(var)205 knockdown animals. These results indicate that Su(var)2-10- and Su(var)205-mediated proper expression of neverland is required for the initiation of metamorphosis. Given that Su(var)2-10-positive puncta are juxtaposed with the pericentromeric heterochromatic region, we propose that Su(var)2-10- and Su(var)205-dependent regulation of inherent heterochromatin structure at the neverland gene locus is essential for its transcriptional activation.

Chromatin structure can introduce systematic biases in genome-wide analyses of Plasmodium falciparum.

Background: The maintenance, regulation, and dynamics of heterochromatin in the human malaria parasite, Plasmodium falciparum, has drawn increasing attention due to its regulatory role in mutually exclusive virulence gene expression and the silencing of key developmental regulators. The advent of genome-wide analyses such as chromatin-immunoprecipitation followed by sequencing (ChIP-seq) has been instrumental in understanding chromatin composition; however, even in model organisms, ChIP-seq experiments are susceptible to intrinsic experimental biases arising from underlying chromatin structure. Methods: We performed a control ChIP-seq experiment, re-analyzed previously published ChIP-seq datasets and compared different analysis approaches to characterize biases of genome-wide analyses in P. falciparum. Results: We found that heterochromatic regions in input control samples used for ChIP-seq normalization are systematically underrepresented in regard to sequencing coverage across the P. falciparum genome. This underrepresentation, in combination with a non-specific or inefficient immunoprecipitation, can lead to the identification of false enrichment and peaks across these regions. We observed that such biases can also be seen at background levels in specific and efficient ChIP-seq experiments. We further report on how different read mapping approaches can also skew sequencing coverage within highly similar subtelomeric regions and virulence gene families. To ameliorate these issues, we discuss orthogonal methods that can be used to characterize bona fide chromatin-associated proteins. Conclusions: Our results highlight the impact of chromatin structure on genome-wide analyses in the parasite and the need for caution when characterizing chromatin-associated proteins and features.

Chromosomal-level assembly of Bactericera cockerelli reveals rampant gene family expansions impacting genome structure, function and insect-microbe-plant-interactions.

Lineage specific expansions and gene duplications are some of the most important sources of evolutionary novelty in eukaryotes. Although not as prevalent in eukaryotes compared to bacteria, horizontal gene transfer events can also result in key adaptations for insects, especially for those involved in insect-microbe interactions. In this study we assemble the first chromosomal assembly of the psyllid Bactericera cockerelli and reveal that the B.cockerelli genome has experienced significantly more gene expansion events compared to other Hemipteran representatives with fully sequenced genomes. We also reveal that B.cockerelli's genome is the largest psyllid genome (567 Mb) sequenced to date and is ~15% larger than the other two psyllid species genomes sequenced (Pachypsylla venusta and Diaphorina citri). Structurally, B.cockerelli appears to have an additional chromosome compared to the distantly related psyllid species P.venusta due to a previous chromosomal fission or fusion event. The increase in genome size and dynamic nature of the B.cockerelli genome may largely be contributed to the widespread expansion of type I and II repeat elements that are rampant across all of B.cockerelli's. chromosomes. These repeat elements are distributed near equally in both euchromatic and heterochromatic regions. Furthermore, significant gene family expansions and gene duplications were uncovered for genes that are expected to be important in its adaptation to insect-plant and microbe interactions, which include transcription factors, proteases, odorant receptors, and horizontally transferred genes that are involved in the nutritional symbioses with their long-term nutritional endosymbiont Carsonella.

Cytological heterogeneity of heterochromatin among 10 sequenced Drosophila species.

In Drosophila chromosomal rearrangements can be maintained and are associated with karyotypic variability among populations from different geographic localities. The abundance of variability in gene arrangements among chromosomal arms is even greater when comparing more distantly related species and the study of these chromosomal changes has provided insights into the evolutionary history of species in the genus. In addition, the sequencing of genomes of several Drosophila species has offered the opportunity to establish the global pattern of genomic evolution, at both genetic and chromosomal level. The combined approaches of comparative analysis of syntenic blocks and direct physical maps on polytene chromosomes have elucidated changes in the orientation of genomic sequences and the difference between heterochromatic and euchromatic regions. Unfortunately, the centromeric heterochromatic regions cannot be studied using the cytological maps of polytene chromosomes because they are underreplicated and therefore reside in the chromocenter. In Drosophila melanogaster, a cytological map of the heterochromatin has been elaborated using mitotic chromosomes from larval neuroblasts. In the current work, we have expanded on that mapping by producing cytological maps of the mitotic heterochromatin in an additional 10 sequenced Drosophila species. These maps highlight 2 apparently different paths, for the evolution of the pericentric heterochromatin between the subgenera Sophophora and Drosophila. One path leads toward a progressive complexity of the pericentric heterochromatin (Sophophora) and the other toward a progressive simplification (Drosophila). These maps are also useful for a better understanding how karyotypes have been altered by chromosome arm reshuffling during evolution.

A Perspective of Molecular Cytogenomics, Toxicology, and Epigenetics for the Increase of Heterochromatic Regions and Retrotransposable Elements in Tambaqui (Colossoma macropomum) Exposed to the Parasiticide Trichlorfon.

Simple SummaryThe aim of the present study was to evaluate the Trichlorfon effects on the retrotransposable elements in tambaqui (Colossoma macropomum) genome, which is a highly popular and well-known fish in the Amazon with a large reproduction number mediated by pisciculture. Thereby, tambaqui specimens were submitted to two different Trichlorfon concentrations (30% and 50% of LC50–96 h) under experimental conditions. The retrotransposons were analyzed using the FISH technique and the heterochromatin standard with the C-band technique. The retrotransposons studied presented a dispersed distribution profile in the tambaqui karyotype with Rex3 being more prominent than the others, showing the greatest increase in markings. Furthermore, the heterochromatin profile showed that these retrotransposons can be found in the heterochromatic portions of the chromosomes. Thus, it was observed that Trichlorfon has an activation mechanism for these retroelements, especially Rex3.Rex retroelements are the best-known transposable elements class and are broadly distributed through fish and also individual genomes, playing an important role in their evolutionary dynamics. Several agents can stress these elements; among them, there are some parasitic compounds such as the organochlorophosphate Trichlorfon. Consequently, knowing that the organochlorophosphate Trichlorfon is indiscriminately used as an antiparasitic in aquaculture, the current study aimed to analyze the effects of this compound on the activation of the Transposable Elements (TEs) Rex1, Rex3, and Rex6 and the structure of heterochromatin in the mitotic chromosomes of the tambaqui (Colossoma macropomum). For this, two concentrations of the pesticide were used: 30% (0.261 mg/L) and 50% (0.435 mg/L) of the recommended LC50–96 h concentration (0.87 mg/L) for this fish species. The results revealed a dispersed distribution for Rex1 and Rex6 retroelements. Rex3 showed an increase in both marking intensity and distribution, as well as enhanced chromosomal heterochromatinization. This probably happened by the mediation of epigenetic adaptive mechanisms, causing the retroelement mobilization to be repressed. However, this behavior was most evident when Trichlorfon concentrations and exposure times were the greatest, reflecting the genetic flexibility necessary for this species to successfully adapt to environmental changes.

Duplex sequencing identifies genomic features that determine susceptibility to benzo(a)pyrene-induced in vivo mutations

Exposure to environmental mutagens increases the risk of cancer and genetic disorders. We used Duplex Sequencing (DS), a high-accuracy error-corrected sequencing technology, to analyze mutation induction across twenty 2.4 kb intergenic and genic targets in the bone marrow of MutaMouse males exposed to benzo(a)pyrene (BaP), a widespread environmental pollutant. DS revealed a linear dose-related induction of mutations across all targets with low intra-group variability. Heterochromatic and intergenic regions exhibited the highest mutation frequencies (MF). C:G > A:T transversions at CCA, CCC and GCC trinucleotides were enriched in BaP-exposed mice consistent with the known etiology of BaP mutagenesis. However, GC-content had no effect on mutation susceptibility. A positive correlation was observed between DS and the “gold-standard” transgenic rodent gene mutation assay. Overall, we demonstrate that DS is a promising approach to study in vivo mutagenesis and yields critical insight into the genomic features governing mutation susceptibility, spectrum, and variability across the genome.

A Chromosome-Length Reference Genome for the Endangered Pacific Pocket Mouse Reveals Recent Inbreeding in a Historically Large Population.

High-quality reference genomes are fundamental tools for understanding population history, and can provide estimates of genetic and demographic parameters relevant to the conservation of biodiversity. The federally endangered Pacific pocket mouse (PPM), which persists in three small, isolated populations in southern California, is a promising model for studying how demographic history shapes genetic diversity, and how diversity in turn may influence extinction risk. To facilitate these studies in PPM, we combined PacBio HiFi long reads with Omni-C and Hi-C data to generate a de novo genome assembly, and annotated the genome using RNAseq. The assembly comprised 28 chromosome-length scaffolds (N50 = 72.6 MB) and the complete mitochondrial genome, and included a long heterochromatic region on chromosome 18 not represented in the previously available short-read assembly. Heterozygosity was highly variable across the genome of the reference individual, with 18% of windows falling in runs of homozygosity (ROH) >1 MB, and nearly 9% in tracts spanning >5 MB. Yet outside of ROH, heterozygosity was relatively high (0.0027), and historical Ne estimates were large. These patterns of genetic variation suggest recent inbreeding in a formerly large population. Currently the most contiguous assembly for a heteromyid rodent, this reference genome provides insight into the past and recent demographic history of the population, and will be a critical tool for management and future studies of outbreeding depression, inbreeding depression, and genetic load.

Different transcriptional responses by the CRISPRa system in distinct types of heterochromatin in Drosophila melanogaster

Transcription factors (TFs) activate gene expression by binding to elements close to promoters or enhancers. Some TFs can bind to heterochromatic regions to initiate gene activation, suggesting that if a TF is able to bind to any type of heterochromatin, it can activate transcription. To investigate this possibility, we used the CRISPRa system based on dCas9-VPR as an artificial TF in Drosophila. dCas9-VPR was targeted to the TAHRE telomeric element, an example of constitutive heterochromatin, and to promoters and enhancers of the HOX Ultrabithorax (Ubx) and Sex Combs Reduced (Scr) genes in the context of facultative heterochromatin. dCas9-VPR robustly activated TAHRE transcription, showing that although this element is heterochromatic, dCas9-VPR was sufficient to activate its expression. In the case of HOX gene promoters, although Polycomb complexes epigenetically silence these genes, both were ectopically activated. When the artificial TF was directed to enhancers, we found that the expression pattern was different compared to the effect on the promoters. In the case of the Scr upstream enhancer, dCas9-VPR activated the gene ectopically but with less expressivity; however, ectopic activation also occurred in different cells. In the case of the bxI enhancer located in the third intron of Ubx, the presence of dCas9-VPR is capable of increasing transcription initiation while simultaneously blocking transcription elongation, generating a lack of functional phenotype. Our results show that CRISPRa system is able to activate transcription in any type of heterochromatin; nevertheless, its effect on transcription is subject to the intrinsic characteristics of each gene or regulatory element.

Genome organization of major tandem repeats and their specificity for heterochromatin of macro- and microchromosomes in Japanese quail.

Tandemly repeated DNAs form heterochromatic regions of chromosomes, including the vital centromeric chromatin. Despite the progress in new genomic technologies, tandem repeats remain poorly deciphered and need targeted analysis in the species of interest. The Japanese quail is one of the highest-producing poultry species as well as a model organism. Its genome differs by a noticeable accumulation of heterochromatin, which led to an increase by 1/7 compared to the chicken genome size. Prominent heterochromatin blocks occupy the short arms of acrocentric macrochromosomes and of microchromosomes. We have applied de novo repeat finder approach to unassembled raw reads of the Japanese quail genome. We identified the 20 most common tandem repeats with the abundance >1Mb, which represent about 4.8% of the genome. We found that tandem repeat CjapSAT primarily contributes to the centromeric regions of the macrochromosomes CJA1-8. Cjap31B together with previously characterized BglII makes up centromere regions of microchromosomes and W chromosome. Other repeats populate heterochromatin of microchromosomal short arms in unequal proportions, as revealed by fluorescence in situ hybridization. The Cjap84A, Cjap408A, and CjapSAT repeat sequences show similarities to retrotransposon motifs. This suggests that retroelements may have played a crucial role in the distribution of repeats throughout the Japanese quail genome.

OCT4 activates a Suv39h1-repressive antisense lncRNA to couple histone H3 Lysine 9 methylation to pluripotency.

Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. Accordingly, in undifferentiated and pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. How global H3K9 methylation levels are coupled with the loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of Suv39h1as, an antisense long non-coding RNA to Suv39h1. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, affecting all heterochromatic regions, particularly peri-centromeric major satellites and retrotransposons. This increase in heterochromatinization efficiency leads to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the genetic control of pluripotency with the global efficiency of H3K9 methylation associated with a major cell fate restriction, the irreversible loss of pluripotency.

Topoisomerase VI participates in an insulator-like function that prevents H3K9me2 spreading

The organization of the genome into transcriptionally active and inactive chromatin domains requires well-delineated chromatin boundaries and insulator functions in order to maintain the identity of adjacent genomic loci with antagonistic chromatin marks and functionality. In plants that lack known chromatin insulators, the mechanisms that prevent heterochromatin spreading into euchromatin remain to be identified. Here, we show that DNA Topoisomerase VI participates in a chromatin boundary function that safeguards the expression of genes in euchromatin islands within silenced heterochromatin regions. While some transposable elements are reactivated in mutants of the Topoisomerase VI complex, genes insulated in euchromatin islands within heterochromatic regions of the Arabidopsis thaliana genome are specifically down-regulated. H3K9me2 levels consistently increase at euchromatin island loci and decrease at some transposable element loci. We further show that Topoisomerase VI physically interacts with S-adenosylmethionine synthase methionine adenosyl transferase 3 (MAT3), which is required for H3K9me2. A Topoisomerase VI defect affects MAT3 occupancy on heterochromatic elements and its exclusion from euchromatic islands, thereby providing a possible mechanistic explanation to the essential role of Topoisomerase VI in the delimitation of chromatin domains.