Regulatory elements and DNA-binding proteins mediating transcription from the chicken very-low-density apolipoprotein II gene

Abstract

The chicken Very-Low-Density Apolipoprotein II (apoVLDL II) gene is specifically expressed in liver in response to estrogen. In this study, we performed a functional analysis of the 300-base pair region immediately 5' to the gene by gene transfer of chloramphenicol acetyl transferase (CAT) constructs into chicken embryonic hepatocytes (CEH). Two estrogen response elements (EREs) could be distinguished which together form a potent estrogen response unit. Stimulation of transient expression by co-transfection with a plasmid expressing rat C/EBP confirmed that a similar protein in chicken liver may be involved in apoVLDL II transcription. In vitro DNaseI footprinting and band-shift analysis with liver, oviduct and spleen nuclear extract revealed the tissue distribution of the proteins binding to the promoter region. A liver-specific protein bound to multiple sites of which some resembled the recognition sequence of the CCAAT/Enhancer binding protein, C/EBP. Of the other proteins binding to the apoVLDL II promoter, one was identified as the liver-specific LF-A1 by mobility shift analysis, using purified bovine LF-A1, and another as the general COUP-transcription factor, using an antiserum against the human COUP-TF.

Reactivation of apolipoprotein II gene transcription by cycloheximide reveals two steps in the deactivation of estrogen receptor-mediated transcription.

“…These changes may result from binding of specific transcription factors, including the receptor (4,67), but may also reflect changes in nucleosomal organization on the apoll promoter. A structural change in the chromatin organization of the mouse mammary tumor virus promoter occurs upon glucocorticoid treatment as a result of an altered interaction of promoter DNA with a specifically positioned nucleosome (52).…”

“…Studies of gene activation and estrogen-dependent alterations in chromatin structure and DNA methylation patterns have been restricted to animal studies or tissue homogenates (2,3,14,15,38,42,67,68). Similarly, transcriptional analysis with gene transfer techniques has been limited to transient assays in primary liver cultures or heterologous cell lines (4,13,56). The LMH cell line is a chicken hepatoma that expresses many hepatocyte properties (41) and may provide a homologous cell line for studies of avian liver gene expression.…”

Cis‐acting elements reinforcing the activity of the estrogen‐response element in the very‐low‐density apolipoprotein II gene promoter

“…However, our observation that mutation of E l abolished expression suggests that the half-ERE motifs within the apoVLDLII promoter are not capable of mediating the estrogen response independently of E l . Additional experiments have shown that a single element E2 is not sufficient to confer estrogen-responsiveness to the minimal promoter, but that two copies of element E2 act synergistically as an estrogen response unit [6] (and our unpublished data). Obviously, element E2 is a weak estrogen receptor target site.…”

“…+ 34)CAT, using the exonuclease 111 method, was described previously [6]. For the construction of TATA-CAT, previously called P,,,,CAT, the apoVLDLI1 promoter fragment spanning the -40 to +34 region was cloned into the XhoI site of the otherwise promoterless pUCl8-based plasmid pBLCAT3 [21] as described [6].…”

“…For the construction of TATA-CAT, previously called P,,,,CAT, the apoVLDLI1 promoter fragment spanning the -40 to +34 region was cloned into the XhoI site of the otherwise promoterless pUCl8-based plasmid pBLCAT3 [21] as described [6]. Double-stranded oligonucleotides containing individual protein binding sites were cloned in the multiple cloning site upstream of the minimal apoVLDLI1 promoter using standard procedures [6, 191. Firstly, oligonucleotides encompassing E l or E l D were cloned in the BamHI site.…”