Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity

Abstract

Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer.

Keywords: elongation control; enhancers; transcription.

Figure 1.

AFF3 binds to both gDMRs and enhancers of imprinted genes in mESCs. (A) AFF3 occupies an intergenic region within the Dlk1-Dio3 locus in mESCs. ChIP-seq binding profiles of AFF3 at the Dlk1-Dio3 loci in v6.5, KH2, and TC1 mESCs are shown. Two different antibodies were generated and used to chromatin immunoprecipitate AFF3 in v6.5 cells (indicated by Ab1 and Ab2). The two intergenic AFF3 peaks within the Dlk1-Dio3 locus are highlighted with an orange bar. (B) The distal intergenic peak to the Meg3 transcription start site (TSS) within the Dlk1-Dio3 locus is associated with the gDMR marks TRIM28 (Rowe et al. 2013), SETDB1 (Bilodeau et al. 2009), and H3K9me3 (Karimi et al. 2011), while the proximal peak is associated with the enhancer marks ELL3 (Lin et al. 2013), p300 (official protein symbol EP300) (Creyghton et al. 2010), H3K27ac, and H3K4me1 (Creyghton et al. 2010). Shown are ChIP-seq binding profiles of AFF3, gDMR marks (TRIM28, SETDB1, and H3K9me3), and enhancer marks (ELL3, p300, H3K27ac, and H3K4me1) at the genomic region upstream of the Meg3 TSS. AFF3 ChIP-seq in v6.5 cells using Ab1 is shown. The gDMR region is highlighted with a blue bar, and the enhancer region upstream of the Meg3 TSS is highlighted with a pink bar. Schematic illustrations of the Dlk1-Dio3 locus show the maternally and paternally expressed genes indicated by red and black, respectively. (C) AFF3 co-occupies the gDMRs of imprinted loci with TRIM28. An average occupancy plot of AFF3 and TRIM28 is shown for the known imprinted loci in Supplemental Figure S1C.

Figure 2.

AFF3 is recruited to methylated gDMRs. (A) AFF3 binds to the methylated H19, Snrpn, and Peg1 gDMRs in mESCs. Bisulfite sequencing of whole-cell extract (WCE; input for ChIP) and AFF3 ChIP at the H19, Snrpn, and Peg1 gDMRs. Methylated and unmethylated cytosines are designated by filled and unfilled circles, respectively. Each line indicates a unique DNA clone. In contrast to whole-cell extract, the AFF3 ChIP DNA is predominantly methylated at all tested gDMRs. (B) AFF3 binds to the methylated gDMRs in AG and PG MEF cell lines, shown by ChIP-qPCR. (C) Differential DNA methylation at gDMRs in AG and PG MEF cells is shown by MeDIP-qPCR. The Herc3 gene served as a positive control for the ChIP-qPCR and MeDIP-qPCR experiments. (D) DNA methylation is required for the recruitment of AFF3 to the methylated gDMRs. The association of AFF3 in the DNMT triple knockouts with gDMRs was compared with its association with gDMRs in wild-type ESCs by ChIP-qPCR. The Prkcsh gene served as a positive control for the ChIP-qPCR. The Hba gene served as a negative control for ChIP-qPCR. Error bars represent the standard deviation of two independent measurements of a representative ChIP experiment. (E) ZFP57 is required for the recruitment of AFF3 to gDMRs. MA plot showing changes in AFF3 occupancy in the ZFP57 knockout ESCs. The X-axis shows the log₂ geometric average of AFF3 occupancy in the two conditions. The Y-axis shows the log₂ fold change between conditions. Each dot represents one of the 973 high-confidence AFF3 peaks. gDMRs are highlighted by black circles. Colored dots indicate a significant change was measured (adjusted P-value < 0.05).

Figure 3.

The activity of the Meg3 upstream enhancer is regulated by allele-specific binding of AFF3. (A) Schematic illustration of the amplified genomic regions of the Meg3-distal gDMR (B) and the Meg3-proximal enhancer (C). AFF3 occupies the Meg3-distal gDMR on the paternal allele (B), while binding to the active Meg3-proximal enhancer on the maternal allele (C). Methylated and unmethylated cytosines are designated by filled and unfilled circles, respectively. Each line indicates a unique DNA clone. (D) AFF3 is required for the activity of the Meg3-proximal enhancer. Shown are ChIP-seq binding profiles of H3K27ac at the genomic regions upstream of the Meg3 gene in mESCs bearing either NonT or AFF3 shRNA. H3K27ac is significantly reduced at the Meg3 upstream enhancer upon AFF3 RNAi. (E) AFF3 is required for the expression of the maternally expressed genes within the Dlk1-Dio3 locus. Shown are RNA sequencing (RNA-seq) track files in the Dlk1-Dio3 locus in mESCs bearing either NonT or AFF3 shRNA. Both AFF3 (F) and H3K27ac (G) levels are increased about twofold in ZFP57-null cells, reflecting the loss of heterochromatin. Shown are ChIP-seq binding profiles of AFF3 (F) and H3K27ac (G) at the genomic region upstream of the Meg3 TSS in both ZFP57 wild-type and knockout ESCs.

Figure 4.

The maternally expressed Meg3 gene is transcribed as a polycistron that is regulated by AFF3 at the transcriptional elongation stage. (A) Pol II is continuously transcribing from Meg3 to Mirg. Depletion of AFF3 leads to the reduction of elongating Pol II in the body of the Meg3 polycistron. (B) AFF3 knockdown leads to reduced nascent RNA at the Meg3 polycistron. GRO-seq (Min et al. 2011) (C) and the ChIP-seq binding profiles (D) of the elongation marks (SPT5 [Rahl et al. 2010], AFF4 [Lin et al. 2011], and H3K36me3 [Marson et al. 2008]) demonstrate continuous transcription of the Meg3 polycistron. (E) The ChIP-seq binding profiles of the TSS marks (TBP [Liu et al. 2011], NELFA [Rahl et al. 2010], and H3K4me3 [Mikkelsen et al. 2007]) indicate that the TSS of the Meg3 polycistron overlaps with the annotated Meg3 TSS. The paternally expressed Rtl1 gene is not transcribed in ESCs. A schematic illustration of the Dlk1-Dio3 locus (with maternally and paternally expressed genes indicated by red and black, respectively) is shown below.

Figure 5.

Cartoon model illustrating the locations of IG-DMR-bound AFF3 and Meg3 upstream enhancer-bound AFF3 at the Dlk1-Dio3 locus. Methylation of the IG-DMR inhibits the genesis of an active enhancer on the paternal allele through sequestering AFF3, while AFF3 on the active allele is free to bind the active enhancer on the maternal allele to regulate transcription of the Meg3 polycistron at the elongation stage.