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.

The regulatory repertoire of PLZF and SALL4 in undifferentiated spermatogonia

“…This sequence was previously observed in human heterogeneous nuclear ribonucleoprotein K (HNRNPK) and ADP-ribosylation factor 3 (ARF3) gene promoters and is known to be bound by KAISO (Haun et al, 1993;Mikula et al, 2010). We found this same motif in a shared PLZF/SALL4 binding site in THY1 + spermatogonia in intron 1 of the mouse Hnrnpk gene and in a PLZF binding site in the promoter region of the mouse Arf3 gene (Table S2).…”

“…2A, Table S3). Among these, the top PLZF motif (5′-TCTCGCGAGA-3′) was similar to a motif identified previously for another ZBTB family member (Mikula et al, 2010) and was more significantly represented among PLZF unique and shared sites (P≤2.64×10…”

Nucleotide composition-linked divergence of vertebrate core promoter architecture

“…It can function in transcriptional regulation by recruiting YY1, but if present in the plus-orientation downstream from the TSS, it can act either as a Kozak consensus site in the transcribed mRNA for translation or as a binding site for YY1 (Xi et al 2007). The Clus1 element, xt7, identified previously (FitzGerald et al 2004;Wyrwicz et al 2007;Yamashita et al 2007;Guo et al 2008;Mikula et al 2010) is one of the most well-positioned motifs identified in the Xenopus promoters. The exact identity of this motif remains unclear, but the highly specific positioning relative to the TBP binding peak could indicate that it is bound by an important element in the core transcriptional machinery, warranting further investigation.…”

“…The relatively uncharacterized xt7 motif enriched in the Xenopus promoters was previously identified and called Clus1, because it clusters in human promoters (FitzGerald et al 2004). It is a conserved motif, present in the promoters of many housekeeping genes (Wyrwicz et al 2007), and shown to be important for the human HNRNPK and FBN1 promoters (Guo et al 2008;Mikula et al 2010). The identity of the protein that binds this element is unknown, although ZBED1 (zinc finger, BED-type containing 1) can bind to this element in the promoters of ribosomal genes (Yamashita et al 2007).…”

Changes in urine proteome accompanying diabetic nephropathy progression

“…A database search using the MASCOT search engine was carried out in a 3-step procedure described elsewhere [7] to calculate MS and MS/MS measurement errors and to recalibrate the data for the repeated MASCOT search. The initial search parameters were set as follows: enzyme, semitrypsin; fixed modification, cysteine modification by MMTS and iTRAQ labeling of the N-terminus of peptides and lysine side chains; variable modifications, oxidation (M); max missed cleavages, 1.…”