A novel human polycomb binding site acts as a functional polycomb response element in Drosophila

Abstract

Polycomb group (PcG) proteins are key chromatin regulators implicated in multiple processes including embryonic development, tissue homeostasis, genomic imprinting, X-chromosome inactivation, and germ cell differentiation. The PcG proteins recognize target genomic loci through cis DNA sequences known as Polycomb Response Elements (PREs), which are well characterized in Drosophila. However, mammalian PREs have been elusive until two groups reported putative mammalian PREs recently. Consistent with the existence of mammalian PREs, here we report the identification and characterization of a potential PRE from human T cells. The putative human PRE has enriched binding of PcG proteins, and such binding is dependent on a key PcG component SUZ12. We demonstrate that the putative human PRE carries both genetic and molecular features of Drosophila PRE in transgenic flies, implying that not only the trans PcG proteins but also certain features of the cis PREs are conserved between mammals and Drosophila.

Figure 1. PcG proteins bind to the potential PREs in human cells.

(A) Assessment of H3K27me3 levels at SLC, A3, and A13 regions using PCR. H3K27me3 ChIP DNA samples from resting T cells and their input controls were analyzed using ³²P-labeled specific primers. Actin was used as control. Band intensities were quantified using Phospho Imager and indicated below the panel. (B, C, D) PcG proteins SUZ12, BMI1, and RING1B are enriched at the SLC and A3 regions compared to the A13 region in resting T cells (B), HeLa cells (C), and SW-13 cells (D), respectively. ChIP assays were performed using antibodies specific for SUZ12, BMI1, and RING1B with chromatin prepared from CD4⁺ T cells, HeLa cells and SW-13 cells. The ChIP DNA was analyzed by qPCR using primers specific for the SLC, A3 and A13 regions (primer sequences in Table 1).

Figure 2. Normal PcG protein activities are required for the PRE-mediated transcriptional repression.

Knocking down SUZ12 decreased the binding of PRC1 proteins at the endogenous SLC (A) and endogenous A3 (B) regions. ChIP assays were performed using the indicated antibodies, with chromatin from HeLa cells transfected with pREP4-Puro-siSUZ12 or the control vector. ChIP DNA was analyzed by qPCR using primers specific for the SLC-PRE and A3-PRE regions (Table 1). The specificity of ChIP experiment was confirmed by evaluating PcG binding at a region upstream of the BRG1 gene locus, which showed very low level of PcG proteins in both the control and SUZ12 knockdown cells. (C) Knocking down SUZ12 in HeLa cells increased the expression of the endogenous SLC17A7 (SLC locus) and HoxA3 (A3 locus) genes but not the HoxA13 (A13 locus) gene. Total RNAs were isolated from HeLa cells transfected with pREP4-Puro-siSUZ12 or a control vector and selected with puromycin. The expression level of the genes was determined by qRT-PCR analysis.

Figure 3. The putative human PREs repress reporter gene expression in Drosophila.

Quantification of the white gene expression controlled by putative human PREs. mRNA of white was quantified by qRT-PCR and normalized to a constitutively expressed gene rp32L transcript level, followed by multiplying with a factor of 100. For each transgenic line, the qRT-PCR (white/rp32L) data is obtained from 2–3 qPCR reactions and averaged. And for each human DNA element, the data is the average of 5–6 independent lines and the error bars indicate standard error from all independent lines tested.

Figure 4. The putative human PREs have characteristics resembling Drosophila PREs in transgenic flies.

(A) Pairing-sensitive silencing of human PREs: The same transgene in heterozygous (upper panels) or homozygous (lower panels) flies. (B) Quantification of results shown in (A) by qRT-PCR analyses. 2–3 PCR reactions were performed for each genotype. (C) Derepression of miniwhite transcription by a mutation in the ph gene. Quantification of white gene transcript from the same miniwhite transgene at either a wild-type background or the ph (ph⁴⁰¹) mutant background by qRT-PCR analyses. 2–3 PCR reactions were performed for each genotype. (D) Repression of miniwhite transcription by a mutation in the trx gene. Quantification of white gene transcript from the same miniwhite transgene in a temperature-sensitive trx (trx¹) background at either the permissive temperature or the restrictive temperature by qRT-PCR analyses. 2–3 PCR reactions were performed for each genotype.

Figure 5. The putative human PRE SLC element has enriched Drosophila PcG protein binding in transgenic flies.

The H3K27me3 modification and Drosophila PcG proteins are enriched at the SLC region compared to the A13 region. The H3K27me3 modification is also enriched at the A3 region compared to the A13 region, but no enrichment of Drosophila PcG proteins has been detected at the A3 region. ChIP assays were performed using antibodies specific for H3K27me3, E(z), and Pc with chromatin prepared from fly heads. The ChIPed DNA was analyzed by qPCR using primers specific for either the SLC or the A3 region and normalized to the A13 region in the same ChIP experiment. 2–3 independent ChIP experiments were performed for each antibody and three qPCR reactions were performed for each region in every ChIP experiment.