Upstream stimulatory factors are involved in the P1 promoter directed transcription of the AbetaH-J-J locus

Abstract

Background: Alternative splicing of the locus AβH-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) β-hydroxylase (AAH), truncated homologs of AAH with a role in calcium homeostasis humbug and junctate and a structural protein of the sarcoplasmic reticulum membranes junctin. AAH and humbug are over expressed in a broad range of malignant neoplasms. We have previously reported that this locus contains two promoters, P1 and P2. While AAH and humbug are expressed in most tissues under the regulation of the P1 promoter, AAH, junctin and junctate are predominantly expressed in excitable tissues under the control of the P2 promoter. We previously demonstrated that Sp transcription factors positively regulate the P1 promoter.

Identification and functional characterization of the human EXT1 promoter region

“…When adding standard nuclear protein extract from untransfected HEK293 cells, no signal could be observed. For positive control "PI/USFmer", an aspecific signal as well as a second specific signal observed, which are in accordance with the literature (Finotti et al, 2008) No binding was observed for "rs34016643[C]" for any of both protein extracts (Fig. 6).…”

“…Specific signals are indicated by a black arrow. A white arrow indicates an aspecific signal, observed in all binding reactions and in accordance with a previous report (Finotti et al, 2008). The same accounts for the double specific signal observed for positive control "PI/USFmer" when adding standard nuclear protein extract representing USF2-binding (lane B), which is lost in the presence of an USF1 excess (lane A).…”

“…Sense oligos were ordered with a 5′-biotin linker as indicated by "5′-BiosG". (Finotti et al, 2008) Positive control 20 5′-BiosG/GGCGGCCCACGTGTCCACCT-3′ Sp1mer_BIO (Finotti et al, 2008) Negative control 29 5′-BiosG/CCCTTGGTGGGGGCGGGGCCTAAGCTGCG-3′ Table 4 Promoter predictions. Overview of the in silico predicted EXT1 promoter regions for the respective prediction programs.…”

“…For both rs34016643-alleles (G/C), 25 bp oligos were purchased (Integrated DNA Technologies, Coralville, IA, USA) with the polymorphic nucleotide centered and with a biotin label at the 5′-end of the sense oligos. Oligo "PI/USFmer" (Finotti et al, 2008) was used as a positive control for USF1 binding, while the SpI-binding "SpImer" (Finotti et al, 2008) was used as a negative control. Sequences of all oligos can be found in Table 3.…”

“…For both rs34016643-alleles (G/C), 25 bp oligodeoxyribonucleotides (oligos) were purchased (Integrated DNA Technologies, Coralville, IA, USA) with the polymorphic location centered and with 5′-GATC sticky ends to provide a substrate for radiolabeling ("rs34016643[G]"; "rs34016643[C]"). Oligo "PI/USFmer" (Finotti et al, 2008) was included as a positive control for USF1 binding. Oligo sequences can be found in Table 2.…”