Minor Satellite Research Articles

Non-Random Occurrence of Robertsonian Translocations in the House Mouse (Mus musculus domesticus): Is It Related to Quantitative Variation in the Minor Satellite?

The house mouse, Mus musculus domesticus, shows extraordinary chromosomal diversity driven by fixation of Robertsonian (Rb) translocations. The high frequency of this rearrangement, which involves the centromeric regions, has been ascribed to the architecture of the satellite sequence (high quantity and homogeneity). This promotes centromere-related translocations through unequal recombination and gene conversion. A characteristic feature of Rb variation in this subspecies is the non-random contribution of different chromosomes to the translocation frequency, which, in turn, depends on the chromosome size. Here, the association between satellite quantity and Rb frequency was tested by PRINS of the minor satellite which is the sequence involved in the translocation breakpoints. Five chromosomes with different translocation frequencies were selected and analyzed among wild house mice from 8 European localities. Using a relative quantitative measurement per chromosome, the analysis detected a large variability in signal size most of which was observed between individuals and/or localities. The chromosomes differed significantly in the quantity of the minor satellite, but these differences were not correlated with their translocation frequency. However, the data uncovered a marginally significant correlation between the quantity of the minor satellite and chromosome size. The implications of these results on the evolution of the chromosomal architecture in the house mouse are discussed.

Carbonate orientational order and superlattice structure in vaterite

Vaterite is considered to play an important role as a precursor phase in the formation of calcium carbonate phases, including those related to biomineralization. An accurate description of vaterite׳s structure associated with the order of carbonate groups is essential to understanding the formation, stabilization, and functionality of vaterite in organisms. Molecular dynamics simulations, synchrotron X-ray diffraction, and transmission electron microscopy have been combined in order to investigate the structure of vaterite. The electrostatic interactions between Ca and neighboring CO3 groups promote local and long-range ordering of CO3 groups, which may result in a superstructure of vaterite. Molecular dynamics simulations show that the superstructure (P6522) with ordered carbonate ions has a relatively lower energy than the disordered structure. The kinetics of the disorder-to-order transition suggests that the transition is rapid and that the superstructure is expected to form. X-ray diffraction data confirm the presence of the P6522 superstructure. The measured diffraction peaks are consistent with the calculated diffraction peaks, especially those weak peaks predicted as a result of the superstructure. Transmission electron microscopy also reveals minor satellite electron diffraction peaks with the more intense peaks of the primary pattern, suggesting a superlattice structure resulted from ordering in both crystallographic ab plan and c direction, which is consistent with the proposed superstructure.

The maternal nucleolus plays a key role in centromere satellite maintenance during the oocyte to embryo transition

The oocyte (maternal) nucleolus is essential for early embryonic development and embryos originating from enucleolated oocytes arrest at the 2-cell stage. The reason for this is unclear. Surprisingly, RNA polymerase I activity in nucleolus-less mouse embryos, as manifested by pre-rRNA synthesis, and pre-rRNA processing are not affected, indicating an unusual role of the nucleolus. We report here that the maternal nucleolus is indispensable for the regulation of major and minor satellite repeats soon after fertilisation. During the first embryonic cell cycle, absence of the nucleolus causes a significant reduction in major and minor satellite DNA by 12% and 18%, respectively. The expression of satellite transcripts is also affected, being reduced by more than half. Moreover, extensive chromosome bridging of the major and minor satellite sequences was observed during the first mitosis. Finally, we show that the absence of the maternal nucleolus alters S-phase dynamics and causes abnormal deposition of the H3.3 histone chaperone DAXX in pronuclei of nucleolus-less zygotes.

Sequence-specific microscopic visualization of DNA methylation status at satellite repeats in individual cell nuclei and chromosomes

Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis.

Roles of Mis18α in Epigenetic Regulation of Centromeric Chromatin and CENP-A Loading

The Mis18 complex has been identified as a critical factor for the centromeric localization of a histone H3 variant, centromeric protein A (CENP-A), which is responsible for the specification of centromere identity in the chromosome. However, the functional role of Mis18 complex is largely unknown. Here, we generated Mis18α conditional knockout mice and found that Mis18α deficiency resulted in lethality at early embryonic stage with severe defects in chromosome segregation caused by mislocalization of CENP-A. Further, we demonstrate Mis18α's crucial role for epigenetic regulation of centromeric chromatin by reinforcing centromeric localization of DNMT3A/3B. Mis18α interacts with DNMT3A/3B, and this interaction is critical for maintaining DNA methylation and hence regulating epigenetic states of centromeric chromatin. Mis18α deficiency led to reduced DNA methylation, altered histone modifications, and uncontrolled noncoding transcripts in centromere region by decreased DNMT3A/3B enrichment. Together, our findings uncover the functional mechanism of Mis18α and its pivotal role in mammalian cell cycle.

Tandemly repeated DNA families in the mouse genome

BackgroundFunctional and morphological studies of tandem DNA repeats, that combine high portion of most genomes, are mostly limited due to the incomplete characterization of these genome elements. We report here a genome wide analysis of the large tandem repeats (TR) found in the mouse genome assemblies.ResultsUsing a bioinformatics approach, we identified large TR with array size more than 3 kb in two mouse whole genome shotgun (WGS) assemblies. Large TR were classified based on sequence similarity, chromosome position, monomer length, array variability, and GC content; we identified four superfamilies, eight families, and 62 subfamilies - including 60 not previously described. 1) The superfamily of centromeric minor satellite is only found in the unassembled part of the reference genome. 2) The pericentromeric major satellite is the most abundant superfamily and reveals high order repeat structure. 3) Transposable elements related superfamily contains two families. 4) The superfamily of heterogeneous tandem repeats includes four families. One family is found only in the WGS, while two families represent tandem repeats with either single or multi locus location. Despite multi locus location, TRPC-21A-MM is placed into a separated family due to its abundance, strictly pericentromeric location, and resemblance to big human satellites.To confirm our data, we next performed in situ hybridization with three repeats from distinct families. TRPC-21A-MM probe hybridized to chromosomes 3 and 17, multi locus TR-22A-MM probe hybridized to ten chromosomes, and single locus TR-54B-MM probe hybridized with the long loops that emerge from chromosome ends. In addition to in silico predicted several extra-chromosomes were positive for TR by in situ analysis, potentially indicating inaccurate genome assembly of the heterochromatic genome regions.ConclusionsChromosome-specific TR had been predicted for mouse but no reliable cytogenetic probes were available before. We report new analysis that identified in silico and confirmed in situ 3/17 chromosome-specific probe TRPC-21-MM. Thus, the new classification had proven to be useful tool for continuation of genome study, while annotated TR can be the valuable source of cytogenetic probes for chromosome recognition.

Comparison of the genetic and ecological diversity of the native to the introduced tilapiines (Pisces: Cichlidae), and their population structures in the Lake Victoria region, East Africa

The impact of introduced tilapiines in Lake Victoria Region (LVR) was studied using experimental fishing survey methods and microsatellite DNA marker analysis. The experiments were carried out between 1993 and 2000. Oreochromis esculentus and O. variabilis, the two native forms of the LVR and original principals of the fisheries of these waters, were found to be completely displaced from the major lakes and were only occasionally found in a few minor satellite lakes surrounding Lakes Kyoga and Victoria. Nile tilapia, O. niloticus, is now the dominant tilapiine, and one of the three key fisheries species of LVR along with Nile perch, Lates niloticus, and the minnow-like Rastrineobola argentea. Oreochromis leucostictus, also introduced to the LVR from the nilotic system, was largely restricted to swampy fringes of LVR water bodies. Tilapia zillii, also introduced from nilotic waters and earlier recorded to have been the most prevalent introduced tilapiine in LVR, occurred only sparsely and was very restricted in distribution. Genetic studies showed Nile tilapia having the highest genetic diversity, and being the least subdivided among LVR tilapiines. O. leucostictus was next in variability, but had the greatest gene flow, probably due to its ability to traverse low oxygen waters such as swamps which separate most LVR water bodies. The native forms were genetically the least variable, except where they coexist with introduced congeners. ‘Uncontaminated’ native forms were observed only in satellite Lakes of Kayanja, Kayugi, Manywa, Kawi and Kanyaboli. The population structure of native forms of LVR suggest they are being genetically swamped, especially for O. variabilis by Nile tilapia alleles, due to genetic exchange with introduced forms, resulting in highly localized differentiation of native species. T. zillii was equally highly subdivided, but has higher genetic variation within subpopulations, a situation linked to monogamous breeding behaviour which allows most individuals of its population to participate in breeding. Hybridization with T. rendalli, which was only occasionally encountered and previously mistaken for dwarf Nile tilapia, may also contribute to high within population variation in small T. zillii populations. On a macro-evolutionary level, the sister relationship between O. niloticus and O. esculentus is questionable and in need of further phylogenetic analysis. Overall the study showed a changed fishery, and the need to manage satellite lakes in a manner that will preserve the previous premier elements of the region's fishery (O. esculentus and O. variabilis) within natural refugia.

Mouse centromeric tandem repeats in silico and in situ

The search for all sequences containing centromeric (CEN) minor satellite (MiSat) or pericentromeric (peri-CEN) mouse major satellite (MaSat) was conducted in the whole genome shotgun (WGS) database. The sequences were checked for the presence of the known dispersed repeats using the Censor software. The presence of tandem repeats was tested using Tandem Repeat Finder (TRF). Monotonous MiSat and MaSat arrays and MaSat to MiSat array transitions were detected. Moreover, two other types of contacts were revealed: (1) MiSat transition to fragments of retroelements LINE and IAP (ERV family, intracisternal A-type particles), mainly to ORF2 and 5'-LTR containing elements; (2) MaSat transition to two tandem repeats with monomers 21 bp and 31 bp in size. The presence of the MiSat/IAP transition could be checked experimentally. The common DNA motif among the IAP fragments close to MiSat was isolated. IAP-specific primers were constructed and the fragments obtained in PCR with LAP and MiSat primers compiled the plasmid vector library. Clone n51 with the maximum length of the possible insertion (approximately no. 800 bp) was selected from the library. FISH on extended chromatin fibers (fiberFISH) carried out on the n51 clone demonstrated that the main signal definitely belonged to CEN. However, the signals on the chromosome arms were also detected that could be due to the partial homology of n51 to the dispersed repeats. The duplicated fiberFISH with MiSat and n51 allowed to measure the distances between the fragments. The previously obtained MS3 sequence has some homology to IAP and CEN localization. Accordingly, the regular associations of MiSat with IAP retroelements were shown in silico and in situ. Together with the published data, the present findings suggest that retroelements or their fragments may be essential components of the normal centromere of higher eukaryotes.

Determination of distinctive carbohydrate signatures obtained from the Aeromonas hydrophila (chemotype II) core oligosaccharide pinpointing the presence of the 4‐O‐phosphorylated 5‐O‐linked Kdo reducing end group using electrospray ionization quadrupole orthogonal time‐of‐flight mass spectrometry and tandem mass spectrometry

The electrospray quadrupole orthogonal time-of-flight mass spectrometric (ESI-QqTOF-MS) structural elucidation of the core oligosaccharide of Aeromonas hydrophila (chemotype II) lipopolysaccharide has been investigated and it was demonstrated that it contained an 4-O-phosphorylated Kdo reducing end group, which was glycosylated by the remaining outer core oligosaccharide through its O-5 position. After releasing the core oligosaccharide from the native LPS with acid, the phosphorylated Kdo residue eliminated phosphoric acid, to produce a core oligosaccharide containing a mixture of diastereomeric 4,8- and 4,7-anhydro-alpha-keto acids and an open-chain olefinic Kdo residue. The characteristic glycone sequence was elucidated by collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of the protonated molecule of the native core oligosaccharide. In addition, the analysis of the Hakomori permethylated core oligosaccharide was carried out by electrospray ionization quadrupole orthogonal time-of-flight mass spectrometry (ESI-QqTOF-MS) and matrix-assisted laser desorption/ionization (MALDI)-QqTOF-MS analyses. The presence of more than nine isobaric isomers of this core was detected. The CID-MS/MS analysis of the various protonated permethylated core oligosaccharide molecules showed a similar and diagnostic fragmentation pattern. The over-methylation of the permethylated core oligosaccharide containing either the 4,7- or the 4,8-anhydro-alpha-keto acid unit and the open-chain olefinic Kdo unit was reported. It was realized that the extra minor satellite signals obtained in the ESI-QqTOF-MS and MALDI-TOF-MS analyses were dimethyl sulfoxide (DMSO) stable covalent addition products, which have occurred by a Michael addition on the 4,8-Kdo exocyclic double bond. The occurrence of this series of covalent addition products during the MS analysis of a permethylated core oligosaccharide should be considered as 'carbohydrate-distinctive signatures' establishing and confirming the presence of a 4-O-phosphorylated-5-O-linked Kdo reducing end group.

The NoRC complex mediates the heterochromatin formation and stability of silent rRNA genes and centromeric repeats

Maintenance of specific heterochromatic domains is crucial for genome stability. In eukaryotic cells, a fraction of the tandem-repeated ribosomal RNA (rRNA) genes is organized in the heterochromatic structures. The principal determinant of rDNA silencing is the nucleolar remodelling complex, NoRC, that consists of TIP5 (TTF-1-interacting protein-5) and the ATPase SNF2h. Here we showed that TIP5 not only mediates the establishment of rDNA silencing but also the formation of perinucleolar heterochromatin that contains centric and pericentric repeats. Our data indicated that the TIP5-mediated heterochromatin is indispensable for stability of silent rRNA genes and of major and minor satellite repeats. Moreover, depletion of TIP5 impairs rDNA silencing, upregulates rDNA transcription levels and induces cell transformation. These findings point to a role of TIP5 in protecting genome stability and suggest that it can play a role in the cellular transformation process.

Resistance to 5-aza-2'-deoxycytidine in Genic Regions Compared to Non-genic Repetitive Sequences

The DNA methyltransferase (Dnmt) inhibitor and demethylating agent 5-aza-2'-deoxycytidine (5azadC) has been used to induce cellular differentiation and gene activation. It has been approved for treating several kinds of malignancies due to its ability to reactivate silenced tumor suppressor genes. Considering the potential effect of 5azadC on non-targeted genomic regions in normal cells, we investigated its effect on repetitive sequences and selected gene loci, Oct-4, Sall3, Per1, Clu, Dpep1 and Igf2r, including tissue-dependent and differentially methylated regions, by treating mouse NIH/3T3 fibroblast cells with concentrations of 5azadC ranging from 0.001 to 5 microM. Demethylation of minor satellite repeats and endogenous viruses was concentration dependent, and the demethylation was strong at 1 and 5 microM. In genic regions, the methylation level decreased only at 0.1 microM, but was minimally altered at concentrations lower or higher, regardless of the abundance of CpG sites. Thus, repeats are strongly demethylated, but genic regions are only demethylated at effective doses. Genes were activated by 5azadC treatment and were accompanied by a unique combination of histone modifications in genic regions, including an increased level of H3K9me3 and a decreased level of AcH3. Increase of H3K9me3 in genic regions was not observed in Dnmt knock out cells. We identified differential effects of 5azadC on repetitive sequences and genic regions and revealed the importance of choosing appropriate 5azadC doses to achieve targeted gene recovery.

Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase

Non-coding RNAs are emerging as key players in many fundamental biological processes, including specification of higher-order chromatin structure. We examined the implication of RNA transcribed from mouse centromeric minor satellite repeats in the formation and function of centromere-associated complexes. Here we show that the levels of minor satellite RNA vary during cell-cycle progression, peaking in G2/M phase, concomitant with accumulation of proteins of the chromosomal passenger complex near the centromere. Consistent with this, we describe that murine minor satellite RNA are components of CENP-A-associated centromeric fractions and associate with proteins of the chromosomal passenger complex Aurora B and Survivin at the onset of mitosis. Interactions of endogenous Aurora B with CENP-A and Survivin are sensitive to RNaseA. Likewise, the kinase activity of Aurora B requires an RNA component. More importantly, Aurora B kinase activity can be potentiated by minor satellite RNA. In addition, decreased Aurora B activity after RNA depletion can be specifically rescued by restitution of these transcripts. Together, our data provide new functional evidence for minor satellite transcripts as key partners and regulators of the mitotic kinase Aurora B.

Use of centromeric and telomeric DNA probes in in situ hybridization for differentiation of micronuclei induced by lomefloxacin

Classification of micronuclei induced by lomefloxacin, a difluorinated quinolone bactericidal agent, in mouse bone marrow was performed by fluorescence in situ hybridization using DNA probes for the centromere repeated minor satellite DNA and the telomeric hexamer repeat (5'-TTAGGG-3'). Colchicine and mitomycin C were used as a positive control aneugen and clastogen, respectively, and these compounds produced the expected responses. Single doses of 40, 80, 160, or 320 mg/kg lomefloxacin were given via oral intubations and bone marrow was sampled at 24 and 48 hr after treatment. The micronuclei showed significant increases in both sampling times after doses of 320 mg/kg. A statistically significant increase of micronuclei frequency was also detected for 160 mg/kg lomefloxacin at 48 hr after treatment. The responses were directly correlated with bone-marrow cytotoxicity. Following treatment with 160 and 320 mg/kg lomefloxacin, 48.2 and 50.0% of the induced micronuclei, respectively, showed double labeling with centromeric signals and several telomeric signals, indicating that the induced micronuclei were composed of whole chromosomes. Similarly, 51.8 and 50.0% of the induced micronuclei, respectively, were centromere-negative, demonstrating that lomefloxacin not only induces chromosome loss but also chromosome breakage. The results also showed that chromosomes can be enclosed in a micronucleus before and after centromere separation. Overall, this study provides the first evidence of the potential of lomefloxacin to induce aneugenic effect in mice. However, given the high doses used in this study, the clinical significance of this finding is uncertain.

Localization of satellite DNA and associated proteins in respect to nucleolar precursor bodies in one- and two-cell mouse embryos

Nucleolar precursor bodies (NPB) are characteristic structures in the nuclei of one- and two cell mouse embryos. The alignment of centromeric (CEN) and pericentromeric (periCEN) chromosome regions to the chromatin layer surrounding NPB is known. Mus musculus 4 satellite DNA (satDNA) types are known to be located in CEN region--mouse minor satellite (MiSat) and mouse satellite 3 (MS3); and periCEN region--mouse major satellite (MaSat) and mouse satellite (MS4). We determined the localization of 4 types of mouse satDNA CEN and periCEN regions and associated proteins: RNA-helicase p68, SMC3, Rad21 subunits of the cohesin complex and SYCP3 subunit of the synaptonemal complex (SC). Partially flattened nuclei of the one- and two-cell embryos and embryos treated with ocadaic acids (OA) were used. Different satDNA fragments revealed distinct domains at the surface of NPB: periCEN MaSat was always localized in NPB more internally covering almost entire surface of NPB while CEN MiSat, MS3 and periCEN MS4 showed more peripheral localization. All 4 satDNA did not cover the entire areas of the NPB, indicating the presence of other DNA sequence involved in its formation. RNA-helicase p68 and components of multiprotein cohesin and synaptonemal complexes are the necessary components of NPB. Our results support the opinion that NPB serve as a precursor of chromocenters.

Abnormal DNA Methyltransferase Expression in Mouse Germline Stem Cells Results in Spermatogenic Defects1

Although spermatogonial stem cells (SSCs) are committed to spermatogenesis, they may also convert to an embryonic stem cell-like pluripotent state at a low frequency. Because changes in DNA methylation patterns are associated with this conversion, we examined the effect of manipulating DNA methyltransferase (Dnmt) expression on the fate of cultured SSCs, germline stem (GS) cells. Dnmt1 knockdown induced apoptosis in GS cells, which was attenuated by the loss of Trp53. In contrast, GS cells proliferated normally in vitro after Dnmt3a/Dnmt3b ablation or during Dnmt3l overexpression. However, Dnmt3a/Dnmt3b double-mutant cells showed hypomethylation in the SineB1 repetitive sequence, and Dnmt3l-overexpressing cells showed hypermethylation in major and minor satellite sequences; neither cell type formed teratomas and completed spermatogenesis following transplantation into the seminiferous tubules. Although genetic manipulation did not increase the conversion of GS cells to a pluripotent state, these results underscore the important role of DNMTs in survival and spermatogenic differentiation in SSCs.

Molecular cytogenetic evaluation of the mechanism of micronuclei formation induced by camptothecin, topotecan, and irinotecan

We used the conventional bone marrow micronucleus test complemented with the fluorescent in situ hybridization with the minor satellite DNA probe to investigate the mechanisms of induction of micronuclei in mice treated with camptothecin and its clinical antineoplastic analogues topotecan and irinotecan. All experiments were performed with male Swiss albino mice. Single doses of 1 mg/kg camptothecin or 0.6 mg/kg topotecan were injected intraperitoneally and bone marrow was sampled at 30 hr (camptothecin) or 24 hr (topotecan) after treatment. A dose of 60 mg/kg irinotecan was injected intravenously, once every fourth day for 13 days and bone marrow was sampled 24 hr after the last treatment. In animals treated with camptothecin, a total of 1.07% micronuclei were found and 70% of them were centromere-negative, indicating their formation by DNA strand breaks and reflecting the predominant clastogenic activity of camptothecin. Exposure to topotecan and irinotecan yielded 1.71 and 0.83% micronuclei, respectively. About 52.7 and 48.8% of the induced micronuclei, respectively, were centromere-positive, indicating their formation by whole chromosomes and reflecting the aneugenic activity of both compounds. Correspondingly, about 47.3 and 51.2% of the induced micronuclei, respectively were centromere-negative, demonstrating that topotecan and irinotecan not only induce chromosome loss but also DNA strand breaks. Both the clastogenic and aneugenic potential of these drugs can lead to the development of secondary tumors and abnormal reproductive outcomes. Therefore, the clinical use of these agents must be weighed against the risks of secondary malignancies in cured patients and persistent genetic damage of their potential offspring.

Dynamic Nuclear Organization of Constitutive Heterochromatin During Fetal Male Germ Cell Development in Mice1

In mice, male germ cells enter mitotic arrest beginning at 13.5 days postcoitum (dpc), and remain suspended in the G(0)/G(1) cell cycle stage until after birth. During this period, male germ cells undergo extensive epigenetic reprogramming, which is essential for their subsequent function as male gametes. A global reorganization and spatial clustering of constitutive heterochromatin has been implicated in epigenetic plasticity during cellular differentiation. Here, we have studied the dynamics of heterochromatin in fetal (12.5-19.5 dpc) and neonatal (4 days postpartum) male germ cells. We monitored constitutive heterochromatin-specific markers, and observed changes in the association of histone H3 trimethylation of lysine 9 (H3K9me3), binding of heterochromatin protein 1, and patterns of 4',6-diamino-2-phenylindole staining in pericentric regions of chromosomes, along with a coincident loss of chromocenters in fetal prospermatogonia during mitotic arrest. We also observed a transient loss of H3K9me3 associated with major and minor satellite repeat sequences, plus inactivation of histone methyltransferases (Suv39h1 and Suv39h2), and transient activation of histone demethylase (Jmjd2b) in these same cells. These epigenetic changes were correlated with relocation of centromeric regions toward the nuclear periphery in prospermatogonia during mitotic arrest. Taken together, these results show that constitutive heterochromatin undergoes dramatic reorganization during prespermatogenesis. We suggest that these dynamic changes in heterochromatin contribute to normal epigenetic reprogramming of the paternal genome in fetal prospermatogonia suspended in the G(0)/G(1) stage, and that this also represents an epigenomic state that is particularly amenable to reprogramming.

CCCTC-binding Factor Activates PARP-1 Affecting DNA Methylation Machinery

Our previous data have shown that in L929 mouse fibroblasts the control of methylation pattern depends in part on poly(ADP-ribosyl)ation and that ADP-ribose polymers (PARs), both present on poly(ADP-ribosyl)ated PARP-1 and/or protein-free, have an inhibitory effect on Dnmt1 activity. Here we show that transient ectopic overexpression of CCCTC-binding factor (CTCF) induces PAR accumulation, PARP-1, and CTCF poly(ADP-ribosyl)ation in the same mouse fibroblasts. The persistence in time of a high PAR level affects the DNA methylation machinery; the DNA methyltransferase activity is inhibited with consequences for the methylation state of genome, which becomes diffusely hypomethylated affecting centromeric minor satellite and B1 DNA repeats. In vitro data show that CTCF is able to activate PARP-1 automodification even in the absence of nicked DNA. Our new finding that CTCF is able per se to activate PARP-1 automodification in vitro is of great interest as so far a burst of poly(ADP-ribosyl)ated PARP-1 has generally been found following introduction of DNA strand breaks. CTCF is unable to inhibit DNMT1 activity, whereas poly(ADP-ribosyl)ated PARP-1 plays this inhibitory role. These data suggest that CTCF is involved in the cross-talk between poly(ADP-ribosyl)ation and DNA methylation and underscore the importance of a rapid reversal of PARP activity, as DNA methylation pattern is responsible for an important epigenetic code.

Vezf1 regulates genomic DNA methylation through its effects on expression of DNA methyltransferase Dnmt3b

The zinc finger protein vascular endothelial zinc finger 1 (Vezf1) has been implicated in the development of the blood vascular and lymphatic system in mice, and has been characterized as a transcriptional activator in some systems. The chicken homolog, BGP1, has binding sites in the beta-globin locus, including the upstream insulator element. We report that in a mouse embryonic stem cell line deletion of both copies of Vezf1 results in loss of DNA methylation at widespread sites in the genome, including Line1 elements and minor satellite repeats, some imprinted genes, and several CpG islands. Loss of methylation appears to arise from a substantial decrease in the abundance of the de novo DNA methyltransferase, Dnmt3b. These results suggest that naturally occurring mutations in Vezf1/BGP1 might have widespread effects on DNA methylation patterns and therefore on epigenetic regulation of gene expression.

Abstract LB-330: A novel role of tumor suppressor BRCA1 in regulating integrity of heterochromatin structure.

Abstract Introduction: BRCA1, as well as BRCA2, were identified as the hereditary breast and ovarian cancer susceptibility genes that can account for almost all of inherited cases of breast cancers. BRCA1 is a multifunctional nuclear protein with a RING domain at the N-terminus. Heterochromatin is chromatin containing inactive genes. Pericentromeric heterochromatin is composed of highly repetitive tandem DNA repeats whose transcription is constitutively silenced. In mice, the repeats are classified as major and minor satellite repeats. Heterochromatin protein 1 (HP1) plays a critical role in maintaining heterochromatin structure. Methods: To study the biological function of the tumor suppressor BRCA1, we deleted BRCA1 in nestin positive neuronal stem cells in the brain and showed that the animals displayed severe neuro-developmental deficits. In the course of this work we discovered a novel molecular defect in BRCA1 deficient cells, i.e., a failure to maintain integrity of the heterochromatic structure. Results: In the absence of BRCA1, the number of heterochromatic centers is severely reduced in mouse neuronal cells. HP1 proteins are destabilized, and more importantly, the transcription of satellite DNA is derepressed in mouse neural progenitor cells, mouse fibroblasts, and human cancer cells, including a BRCA1 deficient breast cancer cell line. The defect in silencing invariably appears after the loss of the BRCA1 allele in our paradigm prior to exhibiting any neoplastic phenotype. What is the mechanism of BRCA1 in regulating the heterochromatin integrity? ChIP analysis showed that BRCA1 physically associates with both major satellite DNA and minor satellite DNA, whereas the association is diminished in the BRCA1 knockout animals. More intriguingly, the absence of BRCA1 binding to satellite DNA correlates with a drastic reduction of ubiquitinated histone H2A at the constitutive heterochromatin region. Because BRCA1 contains an E3 ubiquitin ligase activity which is enhanced by BARD1 and the complex ubiquitinate histone H2A in vitro, we examined the heterochromatin silencing in cells with different BRCA1 mutants. These experiments showed that the E3 ligase activity of BRCA1 is required for repression of satellite DNA transcription. Conclusions: Our working model is that mutations of BRCA1 may destabilize the protein complex and lead to defects in the heterochromatin integrity, thereby affecting the stability of the genome which is a hallmark of cancer. Given that mutants in the RING domains are the same as in women who have pathological diseases, we hypothesize that deregulation of satellite DNA transcription can be explored as an early detection marker for breast cancer progression in BRCA1 patients.