Genome-wide evidence for an essential role of the human Staf/ZNF143 transcription factor in bidirectional transcription

Abstract

In the human genome, ∼ 10% of the genes are arranged head to head so that their transcription start sites reside within <1 kbp on opposite strands. In this configuration, a bidirectional promoter generally drives expression of the two genes. How bidirectional expression is performed from these particular promoters constitutes a puzzling question. Here, by a combination of in silico and biochemical approaches, we demonstrate that hStaf/ZNF143 is involved in controlling expression from a subset of divergent gene pairs. The binding sites for hStaf/ZNF143 (SBS) are overrepresented in bidirectional versus unidirectional promoters. Chromatin immunoprecipitation assays with a significant set of bidirectional promoters containing putative SBS revealed that 93% of them are associated with hStaf/ZNF143. Expression of dual reporter genes directed by bidirectional promoters are dependent on the SBS integrity and requires hStaf/ZNF143. Furthermore, in some cases, functional SBS are located in bidirectional promoters of gene pairs encoding a noncoding RNA and a protein gene. Remarkably, hStaf/ZNF143 per se exhibits an inherently bidirectional transcription activity, and together our data provide the demonstration that hStaf/ZNF143 is indeed a transcription factor controlling the expression of divergent protein-protein and protein-non-coding RNA gene pairs.

Figure 1.

Features of bidirectional promoter sizes in the human genome. The length of the bidirectional promoter in base pair is plotted versus the frequency of its occurrence (promoter number for a 10 bp interval). Dotted line indicates the position of the promoters pools.

Figure 2.

Characterization of bidirectional promoters that are target for hStaf/ZNF143 and detection of promoter occupancy in vivo by chromatin immunoprecipitation (ChiP). (A) Outline showing identification of the target promoters. The results of the ChIP and PCR assays performed on 100 promoters (87 protein coding pairs, 11 protein coding–nc RNA pairs and two noncoding–noncoding RNA pairs) are indicated. (B) SBS motif analysis with sequence logos depicting nucleotide distribution in the SBS (left panel) and in the SBS with the 5′-associated submotif (right panel) (C–F). Binding of the endogenous hStaf/ZNF143 to genomic sites in HeLa cells was analyzed by ChIP. Genomic DNA fragments, recovered from input material or immunoprecipitated with hStaf/ZNF143 antibody or no-antibody, were subjected to semi-quantitative PCR amplification in the presence of (α³²P)-dCTP with specific primer pairs. Lanes 1,2 and 3,4: serial dilutions of DNA immunoprecipitated with anti-hStaf/ZNF143 or no-antibody (no Ab), respectively. Lanes 5–7: serial dilutions of input material. Lane 8 (NC, negative control): PCR lacking the chromatin DNA template. Positive (C) and negative (D) controls for ChIP assays. Promoters used are indicated on the right of panel C. PCR products generated with the PP1–PP4 primer pairs originate from unique regions 2.4, 2.1, 6.5 and 2.5 kbp upstream of the tRNA^Sec, U4 ATAC, GAPDH and BUB1B genes, respectively. Typical positive and three negative results obtained from bidirectional promoter regions are depicted in E and F, respectively. Bidirectional promoters are indicated by gene names.

Figure 3.

The human TMEM186–PMM2 and C19orf6–snRNAU6.2 intergenic regions act as bidirectional promoters. Mutational analysis of the promoter identified the functional importance of the SBS in bidirectional transcription. (A) Characteristics of the extended intergenes EI4 and EI12 (length and name of genes in pair), intergenes (name and length) and SBS (position, orientation and score) are indicated. (B) Sequences of the EI4 (TMEM186–PMM2) and EI12 (C19orf6–snRNAU6.2) regions cloned into the dual and firefly luciferase reporters, respectively. The sequence of the intergene is underlined, the SBS are highlighted in gray and the U6 gene is in italics. (C) Schematic diagrams of the reporter genes used in the transfection assays. Mutations (−) are indicated; the relative transcription activities of the different constructs are indicated, representing either the luciferase activity/empty vector ratio or the relative activity in the reverse transcription assay for the U6 snRNA expression. The yield of extended maxiU6 was normalized to that of the extended maxi5S added as the internal standard. Values are mean ± SE (error bars), n = 3 independent experiments with two replicates/group.

Figure 4.

The staf binding site (SBS) bears the capacity to direct bidirectional transcription. Schematic diagrams of the reporter genes used in transfection assays and dual luciferase activities. The BUB1B promoter fragment −464/−194 (relative to the translation initiation codon) containing two SBS (SBS1 and SBS2) and two RRACTACAN motifs was inserted into the dual luciferase vector. Boxed are RRACTACAN (light gray) and SBS (solid box) elements. mut RRACTACAN (open box) and mut SBS (open box) constructs contain ACTAC to TATGG and CCCA to TTTC substitutions, respectively. The relative transcription activities of the different constructs are indicated, representing the firefly and Renilla luciferase activity/empty vector ratios. Values are mean ± SE (error bars), n = 3 independent experiments with two replicates/group.

Figure 5.

Effects of the hStaf/ZNF143 knock-down and overexpression on the mRNA level produced by divergent gene pairs. (A) hStaf/ZNF143 protein was knocked-down in HeLa cells by RNAi. Cells, either treated with RNAi control or treated with hStaf/ZNF143 siRNAs were harvested 72 h after transfection. hStaf/ZNF143 protein overexpression was obtained by transfecting HeLa cells with a DNA construct containing the hStaf/ZNF143 cDNA under the control of the CMV promoter. Total cellular protein were subjected to western blotting with polyclonal antibodies against a C-terminal epitope of hStaf/ZNF143 or anti-AspRS as the internal control. (B) Total RNA was extracted from 72 h treated and control cells, and relative quantification of hStaf/ZNF143 mRNA was carried out by quantitative RT–PCR. The bar indicated the relative content of mRNA normalized to U3 snoRNA (endogenous control) in treated with respect to control cells, fixed as 1. Values are expressed as a ratio and results are mean ± SD (n = 8). Statistical analysis was performed using paired two-tailed Student’s t-test. (C) Relative mRNA quantification by quantitative RT-PCR in 10 divergent gene pairs. The measures were performed for one of the partners only in the two gene pairs C14orf133–AHSA1 and DOCK4–ZNF277. The undetermined partner is marked as nd, not determined. Values are expressed as in (B).