Kruppel-like factor 2 Transcriptional Regulation Involves Heterogeneous Nuclear Ribonucleoproteins and Acetyltransferases

Abstract

Kruppel-like factor 2 (KLF2) is expressed in several cell types, and knockout animals have shown that KLF2 gene regulation is involved in multiple biological processes. These include maintaining T-cells in the quiescent state, preventing preadipocytes from differentiating into mature adipocytes, stabilizing blood vessel walls through endothelial cell function, and advancing the later stages of lung development. Defining the regulation of KLF2 expression is important to understand these diverse functions. Promoter analysis of KLF2 has revealed that a region between -138 and -111 base pairs is required for its transcription, and this nucleotide sequence occurs in a region that is highly conserved in evolution. The present study was carried out to identify transcription factors that bind to this region of the KLF2 promoter. Nuclear factors were enriched by DNA affinity chromatography using the conserved nucleotide sequence of the KLF2 promoter. Mass spectrometry analysis of the proteins eluted from the affinity matrix identified several proteins, including glucose regulated protein-78 kDa (GRP-78), heterogeneous nuclear ribonucleoprotein (hnRNP)-U, hnRNP-D, CArG binding factor (CBF), P300/CBP associated factor (PCAF), cAMP response element binding protein (CREB) and SWI/SNF. The binding of these proteins to the highly conserved region of the KLF2 promoter element was tested by electrophoretic mobility supershift assays and chromatin immunoprecipitation analysis. These procedures confirmed that hnRNP-U, hnRNP-D, PCAF, and P-300 bind to the KLF2 promoter. Transactivation experiments demonstrated that these proteins are important for regulating KLF2 transcription. Of special interest is the role of hnRNPs in the transcription of the KLF2 gene.

Regulation of Arterial-Venous Differences in Tumor Necrosis Factor Responsiveness of Endothelial Cells by Anatomic Context

“…It has been proposed that KLF2 can bind p300 and compete with phosphorylated NF-B p65 antagonizing NF-B transcriptional activity. 8,10,32 Our ChIP data are not fully consistent with this proposal, because a putative competition for histone acetylases does not explain why NF-B binding to the E-selectin enhancer is also reduced in arterial versus venous ECs in organ culture or in KLF2 transductants. However, the difference between HUAECs and HUVECs in situ in the TNF-induced binding of p300 does appear more profound than the difference in NF-B binding to the E-selectin enhancer.…”

Up-regulation of the KLF2 Transcription Factor by Fluid Shear Stress Requires Nucleolin

“…Myocyte enhancement factor 2 has also been found to bind the Ϫ138/Ϫ111 promoter region in T cells (25). We have also demonstrated that this same region binds several other factors to induce KLF2 expression in macrophages, including hnRNP-D, hnRNP-U, PCAF, and p300 (26). The full complement of trans-acting factors is not known, however; nor is the complete mechanism of activation fully understood.…”

“…DNA Affinity Chromatography and Mass Spectrometry-DNA affinity chromatography and mass spectrometry were performed as described previously (26) with the exception that complimentary strands of the 62-bp region of the KLF2 promoter (nucleotides Ϫ157 to Ϫ95) were annealed and cross-linked to Sepharose beads. Following washing and incubation in DNA binding buffer, 10 mg of nuclear extract was incubated with the beads for 30 min on ice.…”

Endothelial cell activation by antiphospholipid antibodies is modulated by Krüppel-like transcription factors

“…24,[33][34][35] This consideration, taken together with the (A) Endothelial cells were transfected with expression vectors containing scrambled DNA (Scr DNA, control) or KLF2, and/or CBP/p300. All cells were also transfected with an NF-B-luciferase reporter and Renilla luciferase.…”