Comprehensive analysis of the palindromic motif TCTCGCGAGA: a regulatory element of the HNRNPK promoter

Abstract

Definitive identification of promoters, their cis-regulatory motifs, and their trans-acting proteins requires experimental analysis. To define the HNRNPK promoter and its cognate DNA-protein interactions, we performed a comprehensive study combining experimental approaches, including luciferase reporter gene assays, chromatin immunoprecipitations (ChIP), electrophoretic mobility shift assays (EMSA), and mass spectrometry (MS). We discovered that out of the four potential HNRNPK promoters tested, the one containing the palindromic motif TCTCGCGAGA exhibited the highest activity in a reporter system assay. Although further EMSA and MS analyses, performed to uncover the identity of the palindrome-binding transcription factor, did identify a complex of DNA-binding proteins, neither method unambiguously identified the pertinent direct trans-acting protein(s). ChIP revealed similar chromatin states at the promoters with the palindromic motif and at housekeeping gene promoters. A ChIP survey showed significantly higher recruitment of PARP1, a protein identified by MS as ubiquitously attached to DNA probes, within heterochromatin sites. Computational analyses indicated that this palindrome displays features that mark nucleosome boundaries, causing the surrounding DNA landscape to be constitutively open. Our strategy of diverse approaches facilitated the direct characterization of various molecular properties of HNRNPK promoter bearing the palindromic motif TCTCGCGAGA, despite the obstacles that accompany in vitro methods.

Figure 1

Graphical localization of HNRNPK promoters and their activity in HeLa cells. (A) Information from the annotated sequence databases GenBank and dbEST was analysed by computational tools, permitting the identification of four potential HNRNPK promoters. Localization of potential promoters sequences (vertical bars) are superimposed on three HNRNPK mRNA transcripts derived from the UCSC browser (hg18 genome assembly) http://genome.ucsc.edu (chr9:85772500-85786000). (B) HeLa cells were transiently transfected with constructs of four human HNRNPK promoters (P1, Promoter 1, containing the TCTCGCGAGA motif; P2, Promoter 2; P3, Promoter 3; P4, Promoter 4) fused to a luciferase reporter gene (pGL4.10) and cotransfected with the phrl-CMV plasmid encoding the Renilla luciferase gene as an internal control. (C) HeLa cells were transfected with mutated forms of the HNRNPK promoter 1 (P1M1, single mutation; P1M2, double mutation; P1M3, triple mutation; pGL4.10, null plasmid). Luciferase activities were measured 24 h after transfection. Mean firefly luciferase activities were normalized to Renilla luciferase activities measured for different promoter reporter plasmids. Data are expressed as a percentage of promoter fragment 1 activity, and represent the mean ± standard deviation of three independent experiments.

Figure 2

Electrophoretic mobility shift assay of in vitro binding of TCTCGCGAGA motif probes. Intact or Ku80-depleted NEs, prepared from proliferating HeLa cells, were incubated with a ³²P-labelled (A) ds 16 bp probe, (B) ds 30 bp probe, (C) ss 16 nt probe, or (D) ss 30 nt probe and resolved on the gel. WT, wild-type probe, containing proper palindrome motif; MT, mutated probe.

Figure 3

Principal component analysis of the relative abundances of 132 proteins selected for MS-based quantitative analysis. Experiments are colour-coded as follows: red, 16 nt single-stranded (ss) DNA probe, starving cells; blue, 16 bp ds DNA probe; green, 30 nt ss DNA probe; black, 30 bp ds DNA probe.

Figure 4

Cellular proliferation state increases the affinity of non-specific DNA binders. (A) Electrophoretic mobility shift assay of in vitro binding of 16 bp ds DNA (P1-16), and a corresponding mutated probe (P1M-16), to HeLa NEs after 0, 1, 6, and 24 h of serum stimulation. (B) NEs (5 µg) as in (A) were resolved by SDS–PAGE and electrotransferred to a PVDF membrane. Blotted proteins were immunostained with antibodies against Ku80, PARP1, hnRNP K, and Histone H3.

Figure 5

Promoter levels of RNA polymerase II and histone modifications. HeLa resting (0 h) and serum-treated cells (1, 6, and 24 h) were used in ChIP assays with antibodies against RNAPII, H3, H3Ac(K9/K18), H3K4me3, and H3K27me3. Isolated DNA was used in real-time PCR with primers to the promoters of HBB, HNRNPK, TUBB6, GAPDH, HNRNPH, RPS7, HSP70, and EGR1. Data are expressed as percent of input chromatin for RNAPII, or as ratios of modified histone marks to total H3 ChIP signals. Data represent the mean ± sd of four independent experiments.

Figure 6

Distribution of PARP1 at heterochromatic and euchromatic sites. HeLa quiescent (0 h) and serum-treated cells (1, 6, and 24 h) were used in ChIP assays with antibodies against PARP1. Isolated DNA was used in real-time PCR with primers to the intergenic site, two sites within the RHO gene, and the promoter of HBB (A), as well as the promoters of HNRNPK, TUBB6, GAPDH, HNRNPH, RPS7, HSP70, and EGR1 (B). Data are expressed as percent of input chromatin and represent the mean ± sd of four independent experiments. (C) The comparison of PARP1 abundance at heterochromatic and euchromatic sites. The data represent an averaged % of input for all heterochromatin and euchromatin genes tested separately for each time point. The differences between heterochromatin and euchromatin in PARP1 occupancy were statistically significant (*P < 0.005; unpaired t-test) for all time points tested.

Figure 7

An open chromatin profile at the promoters with the TCTCGCGAGA motif. (A) Averaged profile of DNase-seq tracks surrounding the HNRNPK promoter (chr9:85785291-85785515) acquired for HepG2, HeLa, k562, and GM12878 cell lines, sd as whiskers. Data were obtained from the ENCODE Open Chromatin, Duke DNase-seq Base Overlap Signal datasets. The mammal conservation data were taken from Placental Mammal Basewise Conservation track deposited in the UCSC browser (hg18 genome assembly) http://genome.ucsc.edu. (B) Averaged HeLa cell profile of DNase-seq tracks surrounding 50 promoters bearing the TCTCGCGAGA motif. The CisRED (http://www.cisred.org) Human 9 database was queried with TCTCGCGAGA, and the resulting coordinates of 50 promoters were used to extract the DNase-seq tracks from the ENCODE Open Chromatin, Duke DNase-seq Base Overlap Signal dataset. The open chromatin profiles were superimposed over the TCTCGCGAGA sequence present in each of the 50 promoters and extended over a 150 bp window. The plot represents an averaged value of DNase-seq signal for 50 promoters.