DNA Environment of Centromeres and Non-homologous Chromosomes Interactions in Mouse

Pericentromeric regions of chromosomes enriched in tandemly repeated satellite DNA although representing a significant part of eukaryotic genomes are still understudied mainly due to interdisciplinary knowledge gaps. Recent studies suggest their important role in genome regulation, karyotype stability and evolution. Thus, the idea of satellite DNA as a junk part of the genome was refuted. Integration of data about molecular composition, chromosome behaviour and details of in situ organization of pericentromeric regions is of great interest. The objective of this work was a cytogenetic analysis of the interactions of pericentromeric regions non-homologous chromosomes in mouse spermatocytes using immuno-FISH. We analysed two events: the associations between cerntomeric regions of X chromosome and autosomes, and associations between centromeric regions of autosomal bivalents forming chromocenters. We conclude that X chromosome form temporary synaptic associations with different autosomes in early meiotic prophase I which normally can be found at pachytene-diplotene without signs of pachytene arrest. These associations are formed between the satellite DNA-enriched centomeric regions of X chromosome and different autosomes but not involve the satellite-poor centromeric region of Y-chromosome. We suggest the mechanism of X chromosome competitive replacement from such associations during synaptic correction. We showed that centromeric region of the X chromosome remains free of γH2Ax-dependent chromatin inactivation, while Y chromosome is completely inactivated. This findings highlights the predominant role of associations between satellite DNA-enriched regions of different chromosomes including X. We assume that X-autosome temporary associations is a manifestation of an additional synaptic disorders checkpoint. These associations are normally corrected before the late diplotene. We revealed that the intense spreading conditions applied to the spermatocytes I nuclei did not lead to destruction of stretched chromatin fibers i.e. elongated chromocenters enriched in satellite DNA. Revealed by us tight associations between pericentromeric regions of different autosomal bivalents and X chromosome may represent the basis for repeat stability maintenance in autosomes an X chromosome. The consequences of our findings are discussed. We obtained the preparations of mouse spermatocytes nuclei in the meiotic prophase I using two approaches: standard and extremely intense surface spread techniques. Using immuno-FISH we visualized tandemly repeated mouse Major and Minor satellite DNA located in the pericentromeric regions of chromosomes and performed a morphological comparison of the standard- and intensely spreaded meiotic nuclei. Based on our results, we assume the remarkable strength of the chromocenter-mediated associations, “chromatin “bridges”, between different bivalents at the pachytene and diplotene stages. We have demonstrated that the chromocenter “bridges” between the centromeric ends of meiotic bivalents are enriched in both tandemly repeated Major and Minor satellite DNA. Association of centromeric regions of autosomal bivalents and X-chromosome but not with Y-chromosome correlates with the absence of Major and Minor satellites on Y-chromosome. We suggest that revealed tight associations between pericentromeric regions of bivalents may represent the network-like system providing dynamic stability of chromosomal territories, as well as add new data for the hypothesis of ectopic recombination in these regions which supports sequence homogeneity between non-homologous chromosomes and does not contradict the meiotic restrictions imposed by the crossing-over interference near centromeres. We conclude that nuclear architecture in meio-sis may play an essential role in contacts between the non-homologous chromosomes providing the specific characteristics of pericentromeric DNA.

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

Background Mammalian 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. Results We 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. Conclusion Taken 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.

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.

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.

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.