Figure 6.
Figure 6. MBD2 is a critical component of the MeCPC in primary mouse splenocytes and MEL-ρ cells. (A) Graphic depiction of the ρ-globin mini-locus introduced into MEL cells. The locus contains (1) a 4.5-kb ρ-globin genomic sequence, (2) a 4-kb chicken LCR enhancer element (HSS2 and HSS3), and (3) 5′ and 3′ cHS4 insulator elements that surround the gene and enhancer. A 2.5-kb fragment of the ρ-globin genomic sequence extending from 248 bp upstream to 2.2 kb downstream of the cap site was excised, in vitro methylated, and religated prior to transfection into MEL cells. In this way the ρ-globin gene is methylated at the same sites as the endogenous gene in chicken adult erythroid cells. (B) EMSA performed with 20 μg nuclear extract from primary mouse splenocytes. Extracts derived from spleens of MBD2+/+ mice form the MeCPC and can be supershifted by the addition of anti-mMBD2 IgG but not control IgG. In contrast, extracts derived from the spleens of MBD2-/- mice do not form a complex on the M-ρ248 probe. (C) RNase protection assay analyzing expression of ρ-globin, mMBD2, and 18S RNAs in MEL-ρ cells treated with shRNAs targeting mMBD2. Significant knock-down of mMBD2 expression is seen in MEL-ρ cells containing shRNA-expressing plasmids that target mMBD2, as compared with control cells (lanes 3 and 4 as compared with lanes 1 and 2). No ρ-globin expression is seen in MEL-ρ cells with wild-type MBD2 expression (lane 2). In contrast, robust ρ-globin expression is seen in MEL-ρ cells in which mMBD2 expression has been knocked down by shRNA (lane 4). Loading of RNA for all MEL samples was equal, as shown by equal amounts of 18S RNA present in the samples. This datum indicates that MBD2 is functionally required for full transcriptional silencing of the methylated ρ-globin gene. (D) Slot blots performed with genomic DNA from wild-type MEL or MEL-ρ cells that contain a stably integrated ρ-globin mini-locus. Genomic DNA from MEL-ρ cells contains ρ-globin gene sequences, whereas wild-type MEL cells display only background hybridization. A 2-fold difference in copy number is observed between mMBD2+ MEL-ρ and mMBD2- knock-down MEL-ρ cells. In contrast, a similar amount of endogenous mouse GAPDH gene sequences is seen in all 3 samples, demonstrating equal DNA loading. Chicken genomic DNA was used as a positive control for the ρ-globin probe.

MBD2 is a critical component of the MeCPC in primary mouse splenocytes and MEL-ρ cells. (A) Graphic depiction of the ρ-globin mini-locus introduced into MEL cells. The locus contains (1) a 4.5-kb ρ-globin genomic sequence, (2) a 4-kb chicken LCR enhancer element (HSS2 and HSS3), and (3) 5′ and 3′ cHS4 insulator elements that surround the gene and enhancer. A 2.5-kb fragment of the ρ-globin genomic sequence extending from 248 bp upstream to 2.2 kb downstream of the cap site was excised, in vitro methylated, and religated prior to transfection into MEL cells. In this way the ρ-globin gene is methylated at the same sites as the endogenous gene in chicken adult erythroid cells. (B) EMSA performed with 20 μg nuclear extract from primary mouse splenocytes. Extracts derived from spleens of MBD2+/+ mice form the MeCPC and can be supershifted by the addition of anti-mMBD2 IgG but not control IgG. In contrast, extracts derived from the spleens of MBD2-/- mice do not form a complex on the M-ρ248 probe. (C) RNase protection assay analyzing expression of ρ-globin, mMBD2, and 18S RNAs in MEL-ρ cells treated with shRNAs targeting mMBD2. Significant knock-down of mMBD2 expression is seen in MEL-ρ cells containing shRNA-expressing plasmids that target mMBD2, as compared with control cells (lanes 3 and 4 as compared with lanes 1 and 2). No ρ-globin expression is seen in MEL-ρ cells with wild-type MBD2 expression (lane 2). In contrast, robust ρ-globin expression is seen in MEL-ρ cells in which mMBD2 expression has been knocked down by shRNA (lane 4). Loading of RNA for all MEL samples was equal, as shown by equal amounts of 18S RNA present in the samples. This datum indicates that MBD2 is functionally required for full transcriptional silencing of the methylated ρ-globin gene. (D) Slot blots performed with genomic DNA from wild-type MEL or MEL-ρ cells that contain a stably integrated ρ-globin mini-locus. Genomic DNA from MEL-ρ cells contains ρ-globin gene sequences, whereas wild-type MEL cells display only background hybridization. A 2-fold difference in copy number is observed between mMBD2+ MEL-ρ and mMBD2- knock-down MEL-ρ cells. In contrast, a similar amount of endogenous mouse GAPDH gene sequences is seen in all 3 samples, demonstrating equal DNA loading. Chicken genomic DNA was used as a positive control for the ρ-globin probe.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal