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.

Fig. 1

Chromosomal association of newly expressed HA-MeCP2. Newly made HA-MeCP2 in stage IV–V Xenopus oocytes targets the chromomeric regions of LBCs. DAPI staining (in red) shows that the chromosomal association of MeCP2 is proportional to the DNA density. While many lateral loops are present (they are readily visible by DIC), they appear to be devoid of HA-MeCP2. However, very short HA-MeCP2 positive lateral projections are readily visible and are likely to correspond to the bases of many loops. HA-MeCP2 also associates with nucleoli, but at a low concentration and primarily with the granular region

Fig. 2

The MBD is necessary and sufficient for chromosomal association of MeCP2. a Schematic representation of three deletions of MeCP2. All truncated forms received a HA tag at the N-terminus. In addition, both ∆C203-486 and MBD were fused to a SV40 type NLS to promote efficient nuclear targeting (∆N1-161 already contains a NLS). Deletions were expressed in stage IV–V Xenopus oocytes and a summary of their respective subcellular distributions is provided. b DIC and corresponding fluorescent micrographs of the HA-MBD deletion. Just like wild-type HA-MeCP2, the newly made protein HA-MBD primarily targets the chromomeric regions of LBCs

Fig. 3

Expression of Human MeCP2 and three single substitution mutants. HA-tagged human MeCP2 was expressed in stage IV–V Xenopus oocytes. All proteins were expressed to comparable levels and recruited to the nucleus. Wild-type MeCP2 and, surprisingly, F155S mutant were the only two proteins able to associate with the chromomeric regions of LBCs

Fig. 4

HA-MeCP2 targets the bases of loops transcribed by RNA pol II. a DIC and corresponding fluorescent micrographs of nuclear spreads where HA-MeCP2 (green) and NF7 (red) were co-localized. At a lower magnification, NF7 and HA-MeCP2 appear segregated into two distinct chromosomal domains: NF7 associates with the RNP matrix of most lateral loops, while HA-MeCP2 is primarily associated with chromomeres. Magnified views, however, reveal that HA-MeCP2 is also found at the base of some loops (arrows). b These short lateral projections of HA-MeCP2 disappear when loops retract upon treatment with AMD but the chromomeric distribution persists

Fig. 5

Immunostaining of Xenopus laevis LBC with 5-methylcytosine mAb. At lower magnifications a general chromomeric staining for 5mC is evident that is proportional to the DNA concentration indicated by DAPI staining. The region shown at higher magnification in the insets is indicated by the red box in the DIC image. Arrowheads in the insets indicate two of the 5mC-stained fibrils that project laterally from the chromomeric axis and that presumably correspond to the bases of some lateral loops. Note that the amplified rDNA that can be specifically detected in the fibrillar centres of extrachromosomal nucleoli by DAPI staining (arrow) appears unstained for 5mC, consistent with the lack of methylation in amplified rDNA determined by biochemical analyses (Dawid et al. 1970)

Fig. 6

Overview of 5mC-immunostaining of axolotl LBCs. a In the portion of a single LBC bivalent shown here a predominantly chromomeric staining pattern that parallels the DAPI staining intensity is apparent under low magnification. The bulk of the loop chromatin, as indicated by pol II (mAb H5) immunostaining appears not to show general 5mC immunostaining. b Regions of centromeric heterochromatin are reflected by the appearance of loopless chromatin bars (brackets)—the examples here are close to the HLB loci (arrowheads) on LBC6. The intensity of 5mC in the heterochromatin appears similar to that of the DAPI staining, as in flanking chromomeres, suggesting that 5mC is not enriched in lampbrush heterochromatin. Note some laterally projecting fibrils of 5mC staining are apparent in neighbouring regions even at this low magnification (two examples indicated by arrows)

Fig. 7

Detail of 5mC-immunostaining of axolotl LBC loops. a Laterally projecting 5mC-stained fibrils can be seen emerging from the brightly stained chromomeres of the chromosome axis. The fibrils often appear to be contiguous with, but not to overlap, the transcribed regions of lateral loops that are indicated by pol II staining (mAb H5). One example is indicated by the arrow. b Detail of interstitial 5mC immunostaining of lateral loops in axolotl LBCs. Short-stained regions of methylated DNA (green in the merge) again appear contiguous with, not to overlap, the pol II-transcribed regions (red). Correspondingly, the loop pol II staining pattern exhibits gaps, with the gaps also matching discontinuities in the nascent RNP matrix (phase contrast image). These features are indicated by arrows for one such region in one particular loop, which interestingly appears to comprise a single matrix unit/transcription unit

Fig. 8

Distribution of 5-hydroxymethylcytosine in axolotl LBCs. a Widely distributed immunostaining of chromomeres obtained with a monoclonal antibody against 5hmC in contrast to fewer, more localized foci of immunostaining obtained with a 5hmC polyclonal antibody. Low magnification image of part of LBC3. b Central region of LBC13 showing the contrasting patterns of immunolocalization produced by 5hmC monoclonal and polyclonal antibodies. This region of LBC13 contains two loci at which histone locus bodies (HLBs) are always attached, and in this example the HLBs attached at homologous loci are fused to each other (arrows in DIC image). A localized region of intense immunostaining with the 5hmC polyclonal antibody occurs about midway between the HLB loci

Fig. 9

Co-localization of 5hmC and pol III in axolotl LBCs. a Central region of axolotl LBC13 immunostained for pol III with α-RPC15 antibody, showing the homologous pol III sites located between the two HLB loci that mark this bivalent. The HLBs at the homologous loci on the left are separate in this example, whereas the HLBs of the right-hand loci are fused (arrows in merge). b Central region of LBC13 immunostained first for pol III using mAb No34 and then with the α5hmC rabbit polyclonal antibody. The same single region of immunostaining is seen on both homologues with both antibodies. As in (a), the HLBs at the left-hand loci are separate while the right-hand HLBs are fused (arrows in merge). c Two homologous loci from a different LBC in the same preparation as (b) also showing co-localization of pol III and 5hmC