A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron

Abstract

Genomic imprinting is an epigenetic regulation that leads to gene expression in a parent-of-origin specific manner. AFF3, the central component of the Super Elongation Complex-like 3 (SEC-L3), is enriched at both the intergenic-differentially methylated region (IG-DMR) and the Meg3 enhancer within the imprinted Dlk1-Dio3 locus to regulate the allele-specific gene expression in this locus. The localization of AFF3 to IG-DMR requires ZFP57. However, how AFF3 functions at the Meg3 enhancer in maintaining allele-specific gene expression remains unclear. Here, we demonstrate that AFF3 is associated with the Krüppel-like zinc finger protein ZFP281 in mouse embryonic stem (ES) cells. ZFP281 recruits AFF3 to the Meg3 enhancer within the imprinted Dlk1-Dio3 locus, thus regulating the allele-specific expression of the Meg3 polycistron. Our genome-wide analyses further identify ZFP281 as a critical factor generally associating with AFF3 at enhancers and functioning together with AFF3 in regulating the expression of a subset of genes. Our study suggests that different zinc finger proteins can recruit AFF3 to different regulatory elements and differentially regulate the function of AFF3 in a context-dependent manner.

Figure 1.

ZFP281 binding motif is highly enriched at AFF3 occupied enhancer regions in mouse ES cells. (A) AFF3 and TRIM28 co-occupy 104 genomic loci, including the imprinting control regions (ICRs) of known imprinted loci. The enrichment of the enhancer mark p300 is relatively low at the 104 loci. An average occupancy plot for AFF3, TRIM28 and p300 is shown. (B) ZFP57 DNA-binding motif is the most significantly overrepresented motif among AFF3 and TRIM28 co-bound sites. Statistical significance (P-value) of the over-representation of the ZFP57 binding motif is shown. (C) p300 is relatively high-enriched at the rest of AFF3 binding sites, where the level of TRIM28 is low. An average occupancy plot for AFF3, TRIM28 and p300 is shown. (D) ZFP281 DNA-binding motif is the most significantly overrepresented motif among AFF3 and TRIM28 non-overlapped sites. Statistical significance (P-value) of the over-representation of the ZFP281 binding motif is shown. (E) ZFP281 is enriched at AFF3 bound regions. ChIP of ZFP281 at five randomly selected AFF3 bound regions. The Hba2 gene serves as a negative control for ChIP-qPCR. Error bars represent the standard deviation of two independent measurements.

Figure 2.

ZFP281 is required for the localization of AFF3 to the Meg3 upstream enhancer within the imprinted Dlk1-Dio3 locus and the transcriptional elongation of the maternally expressed Meg3 polycistron. (A) AFF3 and ZFP281 are co-localized at the Meg3 upstream enhancer within the Dlk1-Dio3 locus. Shown are ChIP-seq binding profiles of AFF3, ZFP281, enhancer mark p300 and ICR marks TRIM28 and ZFP57 at the genomic region upstream of the Meg3 promoter. The intergenic-differentially methylated region (IG-DMR) is highlighted with a blue bar, and Meg3 upstream enhancer region is highlighted with a pink bar. (B) ZFP281 is required for the recruitment of AFF3 to the Meg3 upstream enhancer. Shown is ChIP-seq binding profile of AFF3 at the genomic region upstream of the Meg3 promoter in both control and ZFP281 knockdown mouse ES cells. (C) The occupancy of ZFP281 at the Meg3 upstream enhancer is increased about 2-fold in ZFP57-null cells, where the loss of heterochromatin occurs at the nearby IG-DMR. Shown is ChIP-seq binding profile of ZFP281 at the genomic region upstream of the Meg3 promoter in both ZFP57 wild-type and knockout ES cells. (D) The expression of the maternally expressed Meg3 polycistron is regulated by ZFP281. Shown are RNA-seq track files of the Meg3 polycistron in mouse ES cells bearing control or either of the two independent ZFP281 shRNAs. (E) The Meg3 polycistron is regulated by ZFP281 at the transcriptional elongation stage. ZFP281 knockdown results in reduced occupancy of Pol II in the gene body of the Meg3 polycistron in mouse ES cells. Shown are Pol II ChIP-seq track files at the Meg3 polycistron region in mouse ES cells bearing either control or ZFP281 shRNA.

Figure 3.

ZFP281 co-localizes with AFF3 in mouse ES cells. (A) Heat maps of binding profiles in mouse ES cell for AFF3, ZFP57, ZFP281 and p300 are shown within 5 kb of the center of AFF3 peaks. (B) Endogenous immunoprecipitations (IP) followed by Western blotting show the interaction between AFF3 and ZFP281. (C–E) Genome browser track examples for the binding profiles of ZFP281, p300 and AFF3, and histone markers H3K4me1, H3K4me3 and H3K27ac. ZFP281 and AFF3 co-bind at the enhancers of the (C) Meg3, (D) Cdkn1c and (E) Lin28a genes. The red bar indicated the primer pair amplified regions in Figure 1E.

Figure 4.

ZFP281 is a major cofactor of AFF3 in regulating gene expression in mouse ES cells. (A) Heat maps of binding profiles in mouse ES cell are shown, within 5 kb of the center of AFF3 peaks, to compare the occupancy of AFF3 in control and ZFP281 knockdown ES cells. The binding profile of ZFP281 is also shown. The loss of AFF3 occupancy occurs at the regions where ZFP281 and AFF3 are co-localized. (B) Scatter plot demonstrating differentially expressed genes, assessed by RNA-seq, in ZFP281 knockdown versus AFF3 knockdown. Correlation coefficient is shown. (C) ZFP281 is required for the recruitment of AFF3 to region upstream of the Vrtn gene. Shown is ChIP-seq binding profile of AFF3 at the Vrtn gene locus in both control and ZFP281 knockdown mouse ES cells. (D) Pol II occupancies at the Vrtn gene locus is reduced after either ZFP281 or AFF3 depletion. Shown are Pol II ChIP-seq track files at the Vrtn gene locus in mouse ES cells bearing control, ZFP281 or AFF3 shRNA. (E) The expression of the Vrtn gene is regulated by both ZFP281 and AFF3. Shown are RNA-seq track files of the Vrtn gene in mouse ES cells bearing control, ZFP281 or AFF3 shRNA. RNA-seq and Pol II ChIP-seq after AFF3 knockdown were downloaded from Luo 2016.

Figure 5.

Cartoon model illustrates the functional mode of ZFP281 and AFF3 in regulating imprinted gene expression. AFF3 is localized to the methylated IG-DMR on paternal allele and the unmethylated Meg3 upstream enhancer on maternal allele, respectively. The recruitment of AFF3 to methylated IG-DMR at the paternal allele is mediated by ZFP57 and/or the heterochromatic environment that ZFP57 maintains. ZFP281 is required for the loading of AFF3 to the Meg3 upstream enhancer of the maternal allele. When ZFP281 is depleted by shRNA mediated RNAi, the recruitment of AFF3 to the Meg3 upstream enhancer is affected and the expression of the Meg3 polycistron is down-regulated. In the condition of ZFP57 knockout, where heterochromatin is lost at the IG-DMR on the paternal allele, ZFP281 is able to bind to corresponding Meg3 enhancer on the paternal allele and recruit AFF3 to activate the expression of the downstream Meg3 polycistron, leading the bi-allelic expression of the Meg3 polycistron.