DNA damage-binding proteins and heterogeneous nuclear ribonucleoprotein A1 function as constitutive KCS element components of the interferon-inducible RNA-dependent protein kinase promoter.
Protein kinase regulated by RNA (PKR) plays important roles in many cellular processes including virus multiplication and cell growth, differentiation, and apoptosis. The promoter of the PKR gene possesses a novel 15-bp element designated KCS, positioned upstream of a consensus interferon (IFN)-stimulated response element, that is required for both basal and interferon-inducible transcription. Protein binding to the KCS element is not dependent upon IFN treatment and correlates with transcriptional activity of the PKR promoter. The identity of KCS-binding proteins (KBP) that selectively bind at the KCS element is largely unknown, except for the transcription factor Sp1. We now have purified KBP from HeLa cell nuclear extracts by ion-exchange and DNA-affinity chromatography steps and then identified four constituent proteins of the KBP complex by mass spectrometry and immunochemistry: KBP120 and KBP45 are the damaged DNA-binding protein subunits, p127 DDB1 and p48 DDB2, respectively; KBP100 is the transcription factor Sp1; and KBP35 is the heterogeneous nuclear ribonucleoprotein A1. The steady-state levels of these four KCS-binding proteins in human cells are not altered by IFN treatment. Components of the KBP complex bind selectively and constitutively to the KCS element in the absence of IFN treatment, both in vitro as measured by competition electrophoretic mobility shift assay (EMSA) and DNA pull-down assays and in vivo as measured by chromatin immunoprecipitation assays. Depletion of DDB2 by antisense strategy reduces KBP complex formation by EMSA. These results provide new insight into the biochemical identity and activity of proteins involved in PKR promoter function.
Das
Sonali
S
Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106, USA.
Ward
Simone Visosky
SV
Markle
Danielle
D
Samuel
Charles E
CE
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007372:Interferons / Q000378:metabolism*; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000737:chemistry; D019892:eIF-2 Kinase / Q000235:genetics* / Q000378:metabolism
D002843:Chromatin; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D016376:Oligonucleotides, Antisense; D016329:Sp1 Transcription Factor; C000619656:hnRNPA1 protein, human; D004247:DNA; D007372:Interferons; D019892:eIF-2 Kinase