Comprehensive analysis of the palindromic motif TCTCGCGAGA: a regulatory element of the HNRNPK promoter.
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.
Mikula
Michal
M
Department of Gastroenterology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Roentgena, Warsaw, Poland.
Gaj
Pawel
P
Dzwonek
Karolina
K
Rubel
Tymon
T
Karczmarski
Jakub
J
Paziewska
Agnieszka
A
Dzwonek
Artur
A
Bragoszewski
Piotr
P
Dadlez
Michal
M
Ostrowski
Jerzy
J
D047369:Chromatin Immunoprecipitation / Q000379:methods; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D006367:HeLa Cells; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012261:Ribonucleoproteins / Q000235:genetics*
D004268:DNA-Binding Proteins; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D012261:Ribonucleoproteins; C072074:HNRNPK protein, human; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases