Characterization of a Helicase-Like Transcription Factor Involved in the Expression of the Human Plasminogen Activator Inhibitor-1 Gene

Abstract

A 5.4-kb cDNA encoding the protein that binds to the B Box of the plasminogen activator inhibitor-1 (PAI-1) gene was isolated and sequenced. The protein, named helicase-like transcription factor (HLTF), contains a DNA-binding domain, a RING finger domain, and seven helicase domains and is homologous to SWI/SNF proteins. Two HLTF mRNAs of 5.5 and 4.5 kb were detected in most human tissues, a single gene was located on chromosome 3q24-25, and the protein was located in the nucleoplasm. Two HLTF proteins differing in translation start site (Met-1 or Met-123) were obtained by in vitro translation in reticulocyte lysate or by immunoprecipitation from HeLa cell nuclear extracts. In vitro transcription from the PAI-1 promoter in HeLa cell extracts was inhibited by HLTF antibodies and by the HLTF DNA binding domain. Over-expression of HLTF or HLTFMet123 produced a three-fold induction of PAI-1-LUC transient expression in HeLa cells. Mutation of the PAI-1 B Box led to an eight-fold reduction of basal PAI-1-LUC expression in these cell lines, but did not affect the four- to six-fold induction by phorbol esters.

Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination

“…For example, HLTF activates the PA 1-1 promoter via specific binding to a promoter element (16), and in other studies, HLTF has been shown to bind to the simian virus 40 (SV40) enhancer, the myosin light chain locus enhancer, and HIV long terminal repeats (17)(18)(19)(20). Interestingly, humans have at least two different forms of HLTF protein that differ in their translation initiation site, and it is the shorter form that lacks the Ϸ120 N-terminal residues that exhibit the sequencespecific DNA binding activity and is transcriptionally active (16). Because the sequence-specific DNA binding region in HLTF has been mapped to lie between residues Ϸ125 and 220, it would seem that the HIRAN domain common to both HLTF and Rad5 is not involved in the binding of the gene regulatory regions.…”

“…The cDNA of HLTF in pZL2 plasmid was a gift from Alexandra Belayew (University of Leuven, Belgium) (16). The HLTF cDNA was cloned into N-terminal fusion with the GST gene in the yeast GAL-PGK expression vector pBJ842, resulting in plasmid pIL1000.…”

HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages

“…By ensuring the completion of S-phase following DNA damage, Rad5/HLTF/SHPRH contribute to genome integrity. HLTF is a DNA-binding protein (39-41) first described as a transcription factor (42,43), but later authenticated as a protein involved in DNA repair, in tumor suppression, and in the early stages of carcinogenesis (44)(45)(46)(47). Although both HLTF and SHPRH are able to polyubiquitinate PCNA, they do not act redundantly (48).…”

Functional Interactions between Sp1 or Sp3 and the Helicase-like Transcription Factor Mediate Basal Expression from the Human Plasminogen Activator Inhibitor-1 Gene

“…We have previously cloned and characterized a novel transcription factor involved in basal expression of the human PAI-1 gene in HeLa cells (17). The protein, named helicase-like transcription factor (HLTF) has a specific DNA-binding domain, a RING finger domain, and the seven conserved DNA helicase domains; it is homologous to proteins of the SNF/SWI family that play a role in chromatin remodeling and facilitate trans-factor interaction with nucleosomes (reviewed in Refs.…”

“…The same protein was independently isolated by other groups because it interacted with other DNA targets (the human immunodeficiency virus long terminal repeat and the simian virus enhancer, the myosin light chain locus enhancer, the rabbit uteroglobin promoter, a tumor necrosis factor response element), and shown to display DNA-dependent ATPase activity (22)(23)(24)(25). HLTF activates the PAI-1 promoter via specific interaction with the B box that was initially identified as a phorbol ester-responsive element (13), but was later shown only to be involved in basal expression (17). The B box is highly similar to the GT box (also called CACCC motif), which has been shown to bind Sp1 as well as other recently identified members of the expanding Sp family of transcription factors (26 -28).…”