Mammalian genomes employ heritable cytosine methylation in the long-term silencing of retrotransposons and genes subject to genomic imprinting and X chromosome inactivation. Little is known of the mechanisms that direct cytosine methylation to specific sequences. Here we show that DNA methyltransferase 3-like (Dnmt3L (ref. 1)) is expressed in testes during a brief perinatal period in the non-dividing precursors of spermatogonial stem cells at a stage where retrotransposons undergo de novo methylation. Deletion of the Dnmt3L gene prevented the de novo methylation of both long-terminal-repeat (LTR) and non-LTR retrotransposons, which were transcribed at high levels in spermatogonia and spermatocytes. Loss of Dnmt3L from early germ cells also caused meiotic failure in spermatocytes, which do not express Dnmt3L. Whereas dispersed repeated sequences were demethylated in mutant germ cells, tandem repeats in pericentric regions were methylated normally. This result indicates that the Dnmt3L protein might have a function in the de novo methylation of dispersed repeated sequences in a premeiotic genome scanning process that occurs in male germ cells at about the time of birth.

Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L.

https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/33666/Cell2001.pdf

The Shortest Telomere, Not Average Telomere Length, Is Critical for Cell Viability and Chromosome Stability

Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold

Nuclear Architecture of Rod Photoreceptor Cells Adapts to Vision in Mammalian Evolution

https://www.cell.com/cell/pdf/0092-8674(91)90318-S.pdf

A position-effect assay for boundaries of higher order chromosomal domains.

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species.

/pdf/construction-of-a-small-mus-musculus-repetitive-dna-library-175u69vuwx.pdf

Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

Annealing studies were performed on DNA fragments associated with rat and mouse liver interphase nuclear matrix and the metaphase scaffold of Chinese hamster DON cells. Matrix and scaffold bound DNA fragments, reassociated with an excess of total genomic DNA, displayed kinetics virtually identical with total nuclear DNA probes. Moreover, both the extent and kinetics of these hybridizations were independent of the matrix DNA fragment size (less than 350--5000 base pairs) and the method of nuclease digestion used in their preparation (DNase I, micrococcal nuclease or endogenous digestion). The repetitive DNA component of the matrix DNA was examined by reacting discrete sizes of matrix DNA fragments (less than 350--5000 base pairs) from mouse liver with a library of cloned repetitive sequence DNA fragments which included mouse major satellite sequences. Our results demonstrate that short DNA fragments anchored to the nuclear matrix contain these cloned sequences is similar proportion of total nuclear DNA and, when viewed in light of the annealing results, indicate that matrix DNA is not enriched in either repetitive or unique sequences. Furthermore, the matrix DNA fragments appear to contain the entire sequence complexity of the genome. Finally, we hybridized both matrix and total nuclear DNA fragments with cDNA to total nuclear polyadenylated RNA. The kinetics and extent of hybridization indicate that most, if not all, of the actively transcribed DNA sequences are present in similar concentrations. We conclude that in the overall organization of eukaryotic DNA within the nucleus, the repeating domains or loops which have been demonstrated by a number of investigators are not anchored at specific attachment sequences in interphase cells or during mitosis. These findings are discussed with regard to current concepts of eukaryotic DNA loop organization.

Dennis Pietras

Papers

Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

Hybridization of nuclear matrix attached deoxyribonucleic acid fragments

Hybridization of nuclear matrix attached deoxyribonucleic acid fragments