Association of modified cytosines and the methylated DNA-binding protein MeCP2 with distinctive structural domains of lampbrush chromatin

Abstract

We have investigated the association of DNA methylation and proteins interpreting methylation state with the distinctive closed and open chromatin structural domains that are directly observable in the lampbrush chromosomes (LBCs) of amphibian oocytes. To establish the distribution in LBCs of MeCP2, one of the key proteins binding 5-methylcytosine-modified DNA (5mC), we expressed HA-tagged MeCP2 constructs in Xenopus laevis oocytes. Full-length MeCP2 was predominantly targeted to the closed, transcriptionally inactive chromomere domains in a pattern proportional to chromomeric DNA density and consistent with a global role in determining chromatin state. A minor fraction of HA-MeCP2 was also found to associate with a distinctive structural domain, namely a short region at the bases of some of the extended lateral loops. Expression in oocytes of deleted constructs and of point mutants derived from Rett syndrome patients demonstrated that the association of MeCP2 with LBCs was determined by its 5mC-binding domain. We also examined more directly the distribution of 5mC by immunostaining Xenopus and axolotl LBCs and confirmed the pattern suggested by MeCP2 targeting of intense staining of the chromomeres and of some loop bases. In addition, we found in the longer loops of axolotl LBCs that short interstitial regions could also be clearly stained for 5mC. These 5mC regions corresponded precisely to unusual segments of active transcription units from which RNA polymerase II (pol II) and nascent transcripts were simultaneously absent. We also examined by immunostaining the distribution in lampbrush chromatin of the oxidized 5mC derivative, 5-hydroxymethylcytosine (5hmC). Although in general, the pattern resembled that obtained for 5mC, one antibody against 5hmC produced intense staining of restricted chromosomal foci. These foci corresponded to a third type of lampbrush chromatin domain, the transcriptionally active but less extended structures formed by clusters of genes transcribed by pol III. This raises the possibility that 5hmC may play a role in establishing the distinctive patterns of gene repression and activation that characterize specific pol III-transcribed gene families in amphibian genomes.

Low-voltage scanning electron microscopy study of lampbrush chromosomes and nuclear bodies in avian and amphibian oocytes

“…2). These regions of lateral loops apparently correspond to the regions of lampbrush chromatin enriched with the 5-methylcytosine72 (and our unpublished data).…”

Effects of Tributyltin Chloride on Developing Mouse Oocytes and Preimplantation Embryos

“…DNA methyltransferases (DNMTs) catalyze the methylation of cytosines. Subsequent studies reveal that 5-hmC is further oxidized by TET enzymes to form 5-formylcytosine and 5-carboxylcytosine (Morgan et al, 2012). The DNMTs 3A and 3B are responsible for DNA de novo methylation in cooperation with the cofactor DNMT3L during embryogenesis, which establishes the somatic methylation pattern for the organism (Okano et al, 1998).…”

“…Several publications have demonstrated that the ten-eleven-translocation (TET) proteins catalyze oxidation of 5-mC into 5-hydroxymethylcytosine (5-hmC). Subsequent studies reveal that 5-hmC is further oxidized by TET enzymes to form 5-formylcytosine and 5-carboxylcytosine (Morgan et al, 2012). Imbalanced active demethylation or aberrant methylation could disturb the expression of key developmental genes, which in turn would induce cancer or tumors.…”

FABP7 Regulates Acetyl-CoA Metabolism Through the Interaction with ACLY in the Nucleus of Astrocytes

“…The coding region of mouse Fabp7 was amplified by PCR and amplified cDNA was subcloned into the pcDNA ™3.1 (+) mammalian expression vector (Thermo Fisher Scientific Inc.). The sequence of nuclear localization signal peptide (NLS, CCA AAG AAG AAG CGA AAG ATG) and nuclear export signal peptide (NES, AGT CTG GCA GCT GAG TTC CGA CAC CTG CAA CTG AAG GAA) were reported previously [33,34]. Primer list to obtain amplified cDNA for Fabp7 with NLS at N or C terminus was shown in Supplemental Table 1.…”