Mammalian Interspersed Repeats Research Articles

Intronic Architecture Links DNA Methylation to Gene Expression and Helps Drive Subtype-Specific Transcriptional Landscapes in DNMT3A- and IDH1/2-Mutant Acute Myeloid Leukemias (AML)

Mutations in DNMT3A and IDH1/2 are each found in ~20% of AML patients. 10-15% of AMLs carry mutations in both genes (herein, double mutants), resulting in a unique methylation landscape and upregulation of a signaling signature. In murine models, the presence of both mutations results in greater leukemogenic potential. However, the specific mechanism through which DNA methylation (DNAme) drives gene expression programs in double mutants remains unclear. We hypothesized that the link between DNAme and gene expression would be explained by more than simple proximity, and that the genomic architecture of the affected genes would play a key role.To test this, we first performed an unbiased correlation analysis of gene expression with DNAme at all CpG sites (mCs) located within the same topologically associated domain (TAD). We identified 406 genes with significant (FDR> 5% and absolute rho > 0.5) expression-methylation correlations with mCs proximal to the respective genes (herein the E-M gene set). In addition, another 2,088 genes (the L E-M set) were identified with long-range correlations (>2Kb from the gene body) with mCs in the respective TAD (median distance = 451 Kb). As a set, the E-M genes significantly overlapped (P < 10 -2) with genes identified as either differentially expressed (DE; n=890) or differentially methylated (DM; n= 4,006) between IDH1/2 and DNMT3A mutant AMLs. Notably, a simple overlap analysis of DE and DM genes showed no significant overlap between them, thus demonstrating that correlation analysis performed better in bridging the epigenome with the transcriptome. DAVID and Gene Set Enrichment Analysis on the genes ranked by correlation strength revealed that signaling, fructose and lipid metabolism pathways are enriched in the E-M gene set (FDR < 5%) but not in the L E-M set. Analysis of transcription factor (TF) binding profiles did not reveal a common set of TF(s) binding to the mCs proximal to the genes of the identified pathways. Thus, we hypothesized that the E-M genes have other structural characteristics in common that drive regulation through DNAme, for which we focused on their genomic architecture. This analysis revealed that introns of genes in both the E-M and L E-M sets are significantly denser in Mammalian Interspersed Repeats (MIR) than expected by random chance (P < 10 -2). Additionally, E-M genes were significantly sparser in endogenous retroviruses (ERVL) and primate-specific Alu elements. mCs with significant correlations were also enriched at MIR and depleted from Alu elements (P < 10 -2), thus creating a regulatory network between mCs and genes with MIR sequences as the common denominator. Genome-wide, CpGs within retrotransposons that were differentially methylated among the three AML subtypes were enriched at enhancer regions or coding genes, particularly the E-M genes. Furthermore, the Dnmt3a knock-out (KO) or Idh2 R140Q knock-in mouse models display the same architectural biases at genes correlated with DNAme as the E-M genes identified in the human samples. Next, we sought to put our findings in the context of normal hematopoiesis and found that genes upregulated during normal hematopoietic differentiation are significantly denser in MIR elements and sparser of Alu elements than expected (P < 10 -2). Alignment of the leukemic samples within normal differentiation trajectories revealed that double mutants resembled differentiated cell types more closely, while DNMT3A and IDH1/2 single mutants resembled hematopoietic stem cells. The E-M and L E-M sets significantly overlapped (P < 10 -2) with those genes upregulated during myeloid but not erythroid or lymphoid differentiation, demonstrating that genes regulated by DNAme are at the core of the biology of these AMLs.In summary, our integrative work sheds light on a novel mechanism in which epigenetic modifications can regulate gene expression through MIR sequences within introns of hematopoietic-relevant genes and we posit that overlapping CpG dinucleotides may act as recruiters or substrates of DNMT3A and/or TET proteins. This mechanism seems to also be active in normal hematopoiesis and thus, is hijacked by leukemic cells. Therefore, our findings identify retrotransposons as a missing link in the understanding of epigenetic regulation of gene expression, reveal a previously uncharacterized role for these elements in leukemogenesis, and point to different cells of origin for each AML subtype. DisclosuresNo relevant conflicts of interest to declare.

Identification of an Epigenetic Profile Classifier That Is Associated with Survival in Head and Neck Cancer

Panels of prognostic biomarkers selected using candidate approaches often do not validate in independent populations, so additional strategies are needed to identify reliable classifiers. In this study, we used an array-based approach to measure DNA methylation and applied a novel method for grouping CpG dinucleotides according to well-characterized genomic sequence features. A hypermethylation profile among 13 CpG loci, characterized by polycomb group target genes, mammalian interspersed repeats, and transcription factor-binding sites (PcG/MIR/TFBS), was associated with reduced survival (HR, 3.98; P = 0.001) in patients with head and neck squamous cell carcinoma. This association was driven by CpGs associated with the TAP1 and ALDH3A1 genes, findings that were validated in an independent patient group (HR, 2.86; P = 0.04). Together, the data not only elucidate new potential targets for therapeutic intervention in head and neck cancer but also may aid in the identification of poor prognosis patients who may require more aggressive treatment regimens.

The role of transposable elements in shaping the combinatorial interaction of transcription factors

Motivations. In the last few years several studies showed that transposable elements (TEs) in the human genome are significantly associated with transcription factor binding sites (TFBSs) and that in several cases their expansion within the genome led to a substantial rewiring of the regulatory network. Here we suggest another possible role played by TEs in the evolution of the regulatory networks. We discuss a set of evidences supporting the idea that the evolution of particular patterns on combinatorial interactions among Transcription Factors (TFs) was mediated and supported by the expansion of specific classes of TEs in the human genome. Methods. We tested our conjecture looking, as a case study, at the binding of Estrogen Receptor alpha (ERalpha) to DNA using two chromatin immunoprecipitation sequencing (ChIP-seq) public datasets on MCF7 cell lines corresponding to different modalities of exposure to estrogen. On these datasets we performed a genome-wide analysis of Transposable Elements overlapping ChIP-seq binding peaks and for each of these sequences we performed a genome-wide scan for putative TFBSs employing canonical Positional Weight Matrices (PWMs) Results. We found a remarkable enrichment of a few well defined types and classes of transposable elements overlapping the ChiP-seq peaks in our two datasets. Among these enriched TEs a prominent role was played by MIR (Mammalian Interspersed Repeats) transposons. These TEs underwent a dramatic expansion at the beginning of the mammalian radiation and then stabilized. We conjecture that the special affinity of ERalpha for the MIR class of TEs could be at the origin of the important role which ERalpha assumed in mammalians. Then, looking at the results of the scan for TFBSs, we found strong enrichment and correlated presence of a few specific TFs. In several cases these TFs were known cofactors of ERalpha, thus supporting the idea of a co-regulatory role of Transcription Factors located within the sane TE. Most of these correlations turned out to be strictly associated to specific classes of TEs thus suggesting the presence of a well defined transposon code within the regulatory network. Altogether our results support the idea that transposition events, besides rewiring the network, also played a central role in the emergence and success of combinatorial gene regulation in complex eukaryotes and that the evolution of specific combinations of TFs interactions was actually mediated and driven by the expansion of a few specific classes of Transposable Elements.

The role of Transposable Elements in shaping the combinatorial interaction of Transcription Factors

BackgroundIn the last few years several studies have shown that Transposable Elements (TEs) in the human genome are significantly associated with Transcription Factor Binding Sites (TFBSs) and that in several cases their expansion within the genome led to a substantial rewiring of the regulatory network. Another important feature of the regulatory network which has been thoroughly studied is the combinatorial organization of transcriptional regulation. In this paper we combine these two observations and suggest that TEs, besides rewiring the network, also played a central role in the evolution of particular patterns of combinatorial gene regulation.ResultsTo address this issue we searched for TEs overlapping Estrogen Receptor α (ERα) binding peaks in two publicly available ChIP-seq datasets from the MCF7 cell line corresponding to different modalities of exposure to estrogen. We found a remarkable enrichment of a few specific classes of Transposons. Among these a prominent role was played by MIR (Mammalian Interspersed Repeats) transposons. These TEs underwent a dramatic expansion at the beginning of the mammalian radiation and then stabilized. We conjecture that the special affinity of ERα for the MIR class of TEs could be at the origin of the important role assumed by ERα in Mammalians. We then searched for TFBSs within the TEs overlapping ChIP-seq peaks. We found a strong enrichment of a few precise combinations of TFBS. In several cases the corresponding Transcription Factors (TFs) were known cofactors of ERα, thus supporting the idea of a co-regulatory role of TFBS within the same TE. Moreover, most of these correlations turned out to be strictly associated to specific classes of TEs thus suggesting the presence of a well-defined "transposon code" within the regulatory network.ConclusionsIn this work we tried to shed light into the role of Transposable Elements (TEs) in shaping the regulatory network of higher eukaryotes. To test this idea we focused on a particular transcription factor: the Estrogen Receptor α (ERα) and we found that ERα preferentially targets a well defined set of TEs and that these TEs host combinations of transcriptional regulators involving several of known co-regulators of ERα. Moreover, a significant number of these TEs turned out to be conserved between human and mouse and located in the vicinity (and thus candidate to be regulators) of important estrogen-related genes.

Transposable elements in disease-associated cryptic exons

Transposable elements (TEs) make up a half of the human genome, but the extent of their contribution to cryptic exon activation that results in genetic disease is unknown. Here, a comprehensive survey of 78 mutation-induced cryptic exons previously identified in 51 disease genes revealed the presence of TEs in 40 cases (51%). Most TE-containing exons were derived from short interspersed nuclear elements (SINEs), with Alus and mammalian interspersed repeats (MIRs) covering >18 and >16% of the exonized sequences, respectively. The majority of SINE-derived cryptic exons had splice sites at the same positions of the Alu/MIR consensus as existing SINE exons and their inclusion in the mRNA was facilitated by phylogenetically conserved changes that improved both traditional and auxiliary splicing signals, thus marking intronic TEs amenable for pathogenic exonization. The overrepresentation of MIRs among TE exons is likely to result from their high average exon inclusion levels, which reflect their strong splice sites, a lack of splicing silencers and a high density of enhancers, particularly (G)AA(G) motifs. These elements were markedly depleted in antisense Alu exons, had the most prominent position on the exon-intron gradient scale and are proposed to promote exon definition through enhanced tertiary RNA interactions involving unpaired (di)adenosines. The identification of common mechanisms by which the most dynamic parts of the genome contribute both to new exon creation and genetic disease will facilitate detection of intronic mutations and the development of computational tools that predict TE hot-spots of cryptic exon activation.

Tuatara (Sphenodon) Genomics: BAC Library Construction, Sequence Survey, and Application to the DMRT Gene Family

The tuatara (Sphenodon punctatus) is of "extraordinary biological interest" as the most distinctive surviving reptilian lineage (Rhyncocephalia) in the world. To provide a genomic resource for an understanding of genome evolution in reptiles, and as part of a larger project to produce genomic resources for various reptiles (evogen.jgi.doe.gov/second_levels/BACs/our_libraries.html), a large-insert bacterial artificial chromosome (BAC) library from a male tuatara was constructed. The library consists of 215 424 individual clones whose average insert size was empirically determined to be 145 kb, yielding a genomic coverage of approximately 6.3x. A BAC-end sequencing analysis of 121 420 bp of sequence revealed a genomic GC content of 46.8%, among the highest observed thus far for vertebrates, and identified several short interspersed repetitive elements (mammalian interspersed repeat-type repeats) and long interspersed repetitive elements, including chicken repeat 1 element. Finally, as a quality control measure the arrayed library was screened with probes corresponding to 2 conserved noncoding regions of the candidate sex-determining gene DMRT1 and the DM domain of the related DMRT2 gene. A deep coverage contig spanning nearly 300 kb was generated, supporting the deep coverage and utility of the library for exploring tuatara genomics.

Evaluation of Mammalian Interspersed Repeats to investigate the goat genome

RiassuntoValutazione di sequenze intersperse ripetute per l’analisi del genoma caprino. Tra le classi di sequenze intersperse ripetute, le Mammalian Interspersed Reapeats (MIR) sono utilizzate per la definizione di relazioni filogenetiche tra diversi gruppi tassonomici. Analizzando un ristretto numero di individui non imparentati, appartenenti a 7 diverse razze caprine italiane, é stata valutata la presenza di polimorfismi interspecie per questa classe di sequenze. I risultati ottenuti indicano che il 66% dei marcatori, prodotti amplificando sequenze inter-MIR, sono polimorfici tra le razze analizzate. Il calcolo dei valori di eterozigosi attesa entro e tra razze ed il valore di Fst indica che, nel campione da noi considerato, la variabilità genetica osservata é legata a differenze tra razze e corrisponde al 35,5% della variabilità genetica totale. Questi dati suggeriscono il possibile utilizzo delle MIR in studi di diversità genetica, completando il quadro delle informazioni già disponibili per geni, mtDNA e marcatori neutri.

Phylogenetic relationships between Afrotropical and Palaearctic Crocidura species inferred from Inter-SINE-PCR

The relationships between Afrotropical and Palaearctic Crocidurinae species have been evaluated on the basis of inter-SINE-PCR (IS-PCR). Two SINE families have been studied: mammalian interspersed repeats (MIR) and a short retroposon SOR, specific to the family Soricidae. Use of neighbor-joining and maximum parsimony analyses allowed us to recognize three major groupings: (i) 50-chromosome African species ( C. parvipes and C. olivieri) and 28-chromosome European C. leucodon; (ii) species endemic to Ethiopia (36-chromosome C. thalia, C. glassi, Crocidura sp. B, united with C. macmillani and C. baileyi); (iii) Palaearctic 40-chromosome species group. Therefore, the major groupings of Crocidura shrews in the phylogenetic analysis of IS-PCR results cannot be distinguished as Palaearctic and Afrotropical groups of species: European C. leucodon unites with African 50-chromosome species with a high bootstrap support. The affiliation of Suncus murinus with Eurasian grouping is only supported by low bootstrap values.

Presence of MIR-elements in the complete nucleotide sequence of human chromosome 22

The location of mammalian interspersed repeats (MIRs) and their density have been determined in the complete nucleotide sequence of human chromosome 22. The approach developed by us has allowed detection of 9675 MIRs at a statistically significant level, which by 15% exceeds the MIR number revealed by all previous approaches. It has been demonstrated that a considerable amount of MIRs missed by the algorithms applied earlier occurs in known DNA sequences of the human genome. The study of the MIR density revealed substantial irregularity of their distribution along the chromosome. The data on the MIRs thus found and the computer program searching for diverged sequences are available by E-mail: katrin2@mail.ru or katrin22@mtu-net.ru.

Evolution of the MIR Elements Located in Coding Regions of Human Genome

Mammalian interspersed repeats (MIRs) are classical tRNA-like SINEs of 260 bp in length, whose sequences are substantially diverged. The evolution of mammalian MIRs is now considered as a part of a common evolution of a CORE-SINE repeat family bearing a generic CORE-SINE that has been spread by borrowing reverse transcriptase from different LINE families by addition of the 3’ end containing the binding site for this enzyme [2]. The age of MIRs has been suggested of about 550 million years [2, 5]. In addition to the origination and spreading of MIRs, there is another aspect of the repeat evolution, their influence on a host genome. Unlike the Alu elements, whose role in a genome has been well studied, very little is known about the consequences of the MIR insertion. Here we have tried to focus on the MIRs evolution in the translated regions of human genes, using our own high powerful program for a search MIRs in the GenBank.

MIR: Family of repeats common to vertebrate genomes

We studied the occurrence of mammalian interspersed repeats (MIRs) in DNA and RNA of vertebrates, invertebrates, and bacteria using the data from GenBank. A special algorithm based on a weight position matrix with optimal alignment using dynamic programming was developed to search for the traces of MIR dissemination. This allowed us to search for highly divergent MIRs carrying deletions and insertions. MIRs were detected in genomes of various fishes, includingLatimeria. This suggests that the origin of MIRs dates back more than 400 million years. The method to search for similarity between highly divergent sequences may be used to find the genome fragments from various ancient repeat families and from various gene families.

MIRs are present in coding regions of human genes.

By using a weighted function and the method of enlarged similarity a search has been performed to identify mammalian interspersed repeats (MIRs) in DNA sequences from the EMBL data bank. The existence of MIRs is shown in coding regions of human genes and also in chicken and duck genomes. It is possible to conclude from the results obtained that MIRs were established in the coding regions of some genes and may have taken part in gene evolution. Furthermore, MIRs may have been amplified in vertebrate genomes before the origin of mammals.

DNA Scoring Matrices: Application to Detect Divergent Repeats

The log-odd scoring matrices for proteins (PAM series) were first published nearly 20 years ago. We describe scoring matrices for DNA that are based on the frequencies of nucleotide substitutions and insertions/deletions observed in the evolution of repeats. Series of scoring matrices with different relative entropies for several repeat families were calculated. These matrices are asymmetric; for example, c→t has a different score from t→c; insertions occur much less frequently (and have lower scores) than deletions. We calculated separate scores for c and g when they form a eg dinucleotide. The scoring matrices with different relative entropies describe nearly neutral DNA evolution at various evolutionary distances. We used scoring matrices derived from mammalian interspersed repeats (MIRs) to search for copies of this repeat in GenBank. The sensitivity of both Smith-Waterman and BLAST searches improves dramatically when we use scoring matrices instead of default match/mismatch scores. These matrices can...