1. The function of the bcl-x promoter in erythroid progenitor cells
by Cuixia Tian, Paul Gregoli, and Maurice Bondurant
Blood
Volume 101(6):
March 15, 2003
©2003 by American Society of Hematology

2. The mouse bcl-x promoter and illustration of probes used
The mouse bcl-x promoter and illustration of probes used.(A) Features of the mouse bcl-x gene upstream of the protein coding sequence.
The mouse bcl-x promoter and illustration of probes used.(A) Features of the mouse bcl-x gene upstream of the protein coding sequence. The sequences shown are taken from reference 16 and from the GenBank (accession number AF088904). The indicated splice donor and acceptor sites have been previously published16 17 and were verified here. The indicated cluster of transcription start sites was determined by us to be the predominant site in erythroid cells and corresponds to one of several sites published by others Position −1804 is the farthest upstream point of conservation between sequences of the mouse and human genomes in the region of the bcl-x genes (comparison of Genbank sequences AF and AL160175). (B) An illustration of 5 of the most useful probes used in nuclease protection mapping of the transcription start site. Probes 3 and 4 were generated by RT-PCR and thus lack the intron sequence, −396 to −112.
Cuixia Tian et al. Blood 2003;101:
©2003 by American Society of Hematology

3. S1 nuclease protection analyses of the mousebcl-x transcription start sites.(A) Labeled, single-stranded (ss) DNA probe was the noncoding strand of the region shown in Figure 1B as Probe 1 (−754 through −412).
S1 nuclease protection analyses of the mousebcl-x transcription start sites.(A) Labeled, single-stranded (ss) DNA probe was the noncoding strand of the region shown in Figure 1B as Probe 1 (−754 through −412). The probe was hybridized to total RNA from mouse erythroblasts (FVA cells) that had been cultured for 36 hours with EPO. Lane 1: the DNA fragments that were protected from S1 nuclease. Lane 2: purified probe, not subjected to digestion (about 7000 cpm). Products of a set of sequencing reactions for a portion of the human coagulation Factor XI gene of known sequence are shown as precise size markers in the first 4 lanes (not numbered). The inset photograph to the right of lane 2 shows the triplet bands of lane 1 at higher resolution. (B) Labeled, ssDNA probe was from the clone shown in Figure 1B as Probe 3 (−433 through +26 lacking the intron). The full length of the bcl-xsequence in Probe 3 is 176 bp. Lane 1: probe hybridized with erythroblast cell RNA (36 hours of culture) and digested with S1 nuclease. Lane M: 5′ end–labeled ΦX174 HinfI fragments. Lane 2: undigested probe (1000 cpm). (C) Labeled, ssDNA probe represented the sequence shown in Figure 1B as Probe 4 (−498 through +49 lacking the intron). Sequence in Probe 4 complementary tobcl-x mRNA is 264 bp. Analyses as for (A) and (B) using S1 nuclease. Lane 1: undigested probe (1000 cpm). Lane 2: probe hybridized with yeast RNA. Lanes 3-6: probe hybridized with total RNAs from thymus, brain, mouse erythroblasts (FVA cells), mouse fetal liver (day 15 gestation). Lane M: as described previously. (D) Labeled, ssDNA was from the clone shown in Figure 1B as Probe 5 (−431 through −34 including the intron). Probe 5 contains 397 bp of sequence of the gene. Lane M: as described in the legend to panel B. Lane 1: probe hybridized to erythroblast RNA (36 hours of culture) and digested with S1 nuclease. Numbers with asterisks are fragments protected from nucleases. Numbers without asterisks are sizes of ΦX174 marker fragments.
Cuixia Tian et al. Blood 2003;101:
©2003 by American Society of Hematology

4. Quantification of transcripts initiated at the main start site during erythroblast differentiation.Left panel: probe for RNase protection analysis was labeled RNA of complementary sequence to bcl-x mRNA from −930 through −412 (Probe 2, Figure 1B).
Quantification of transcripts initiated at the main start site during erythroblast differentiation.Left panel: probe for RNase protection analysis was labeled RNA of complementary sequence to bcl-x mRNA from −930 through −412 (Probe 2, Figure 1B). Lanes labeled C contain samples of probe hybridized with yeast RNA and digested with RNase A and T1. Lanes labeled 0 hours to 48 hours indicate that the RNA used for hybridization was from mouse erythroblast cells cultured for the indicated period in the presence of EPO. Samples were digested with RNases. Lane M is marker, as described in the legend to Figure 2B. Lanes Pr2 contain undigested probe 2 samples (10 000 cpm). Right panel: probe for RNase protection analysis was labeled RNA complementary to bcl-x mRNA sequence from −754 through −412 (Probe 1, Figure 1B). Probe was hybridized with total RNA from mouse erythroblasts cultured for 36 hours and 48 hours in the presence of EPO. Hybridized samples were treated with RNase A and T1. Lane M is ΦX174 marker. Lanes Pr1 contain undigested probe 1.
Cuixia Tian et al. Blood 2003;101:
©2003 by American Society of Hematology

5. Major transcription start site for bcl-x in human erythroblasts
Major transcription start site for bcl-x in human erythroblasts.Left panel: labeled, ssDNA consisting of the noncoding strand of the sequence from −858 through −389 of the human bcl-xsequence (GenBank accession number AL160175) was hybridized to 25 μg total...
Major transcription start site for bcl-x in human erythroblasts.Left panel: labeled, ssDNA consisting of the noncoding strand of the sequence from −858 through −389 of the human bcl-xsequence (GenBank accession number AL160175) was hybridized to 25 μg total RNA from human erythroblasts derived by 10 days of culture of partially purified erythroid progenitors.22 Lanes 1 and 2 are duplicates showing the major protected fragments after S1 nuclease digestion. Full-length probe (not shown) was 519 nucleotides long. The leftmost lanes of the panel contain labeled sequencing reactions from a portion of the human coagulation Factor XI gene, as in Figure 2A. Right panel is a reproduction of Figure 2A.
Cuixia Tian et al. Blood 2003;101:
©2003 by American Society of Hematology

6. Expression of reporter luciferase directed by portions of the bcl-x promoter in transient transfection assays.Portions of the bcl-x promoter bearing the complete sequence from −1 (nucleotide preceding A of the ATG codon) to the indicated nucleotide on the h...
Expression of reporter luciferase directed by portions of the bcl-x promoter in transient transfection assays.Portions of the bcl-x promoter bearing the complete sequence from −1 (nucleotide preceding A of the ATG codon) to the indicated nucleotide on the horizontal axis were placed in the luciferase reporter vector pGL3 Basic Vector (Promega). The constructs were transfected into HCD57 cells, and the firefly luciferase was measured after 24 hours. A control expression plasmid, pRL-TK, was cotransfected, as described in “Materials and methods.” The ratio of firefly luciferase to Renilla luciferase was determined in the transfected cells. That ratio for the pGL3 luciferase vector without any inserted promoter sequence was adopted as the baseline expression value. Each transfection assay was performed 3 to 5 times, and representative values are shown. For all test plasmids, the range of values obtained in repetitive experiments was less than 15% of the values shown.
Cuixia Tian et al. Blood 2003;101:
©2003 by American Society of Hematology

7. Function of bcl-x promoter sequences in stable transfection assays (integrated constructs).Several of the bcl-x/reporter constructs depicted in Figure5 were further modified to contain a neomycin resistance gene.
Function of bcl-x promoter sequences in stable transfection assays (integrated constructs).Several of the bcl-x/reporter constructs depicted in Figure5 were further modified to contain a neomycin resistance gene. These were transfected into HCD57 cells, and clones of cells bearing integrated plasmids were selected by culturing in G418. Approximately 50 luciferase-positive cell clones were isolated for each plasmid construct. Luciferase expression was measured for each cell clone; each “x” on the graph represents a cell clone. Numbers on the x-axis indicate group numbers. Group 1 clones bear a plasmid withbcl-x promoter sequence from −1 through −197; Group 2 have promoter sequence from −1 through −1217; Group 3 have sequence from −1 through −1804; and Group 4 have sequence from −1 through −2331. Relative luciferase units are standardized on a per 0.5 × 106 cells basis.
Cuixia Tian et al. Blood 2003;101:
©2003 by American Society of Hematology

8. Analysis of regulatory sequences of the upstream element
Analysis of regulatory sequences of the upstream element.(A) The sequence of the upstream element of the bcl-xpromoter that has strong enhancerlike activity.
Analysis of regulatory sequences of the upstream element.(A) The sequence of the upstream element of the bcl-xpromoter that has strong enhancerlike activity. Also depicted are the structures of several mutations created in that sequence that were analyzed for functional activity. Mutants 1 through 6 are indicated by brackets encompassing a sequence deleted from the normal sequence. Bases shown within a bracket indicate that those bases were substituted for the deleted sequence. The transcription factors indicated above the sequence were retrieved by the Transcription Element Search Software ( based on consensus sequence motifs found by that program. The motifs of the factors shown are present in homologous sequences of the mouse and human bcl-x genes. GAGA factor has great importance for transcription in Drosophila, but no homolog of it has been identified in mammalian cells. (B) Results of transient transfection assays in HCD57 cells using constructs of the enhancer test vector, PGL3-Promoter Vector (Promega). This vector contains the SV40 promoter. The plasmids represented by the histogram columns contain, from left to right: vector alone; vector containing the SV40 enhancer; vector with bcl-x promoter sequence from −1804 through −1734 (upele70), forward orientation; vector withbcl-x promoter −1801 through −1213 (upele589), forward orientation; vector with upele589 in reverse orientation; vector with upele 589 placed downstream of luciferase gene. Other columns labeled as “del” represent experiments with upele70, mutated as indicated in (A), inserted upstream of the luciferase gene and in the normal orientation. Test plasmids were cotransfected with the control plasmid, pRL-TK, as an internal control for experimental variation. The activity of the unmodified PGL3-Promoter Vector was chosen as 1. Transfection experiments for each test plasmid were repeated 3 to 5 times. In all cases, the range of the determined values was equal to or less than 13% of the values shown.
Cuixia Tian et al. Blood 2003;101:
©2003 by American Society of Hematology

9. Binding of nuclear proteins to the upstream element
Binding of nuclear proteins to the upstream element.(A) The end-labeled, double-stranded oligonucleotide had a sense strand sequence 5′-GCTCGCCAGGGGTCGCTGCTCTCCCATG-3′, which includes the most upstream 24 bp of the upstream element (Figure 7A).
Binding of nuclear proteins to the upstream element.(A) The end-labeled, double-stranded oligonucleotide had a sense strand sequence 5′-GCTCGCCAGGGGTCGCTGCTCTCCCATG-3′, which includes the most upstream 24 bp of the upstream element (Figure 7A). Lanes 1-3 and lanes 4-6 represent complexes formed by extracts from mouse splenic erythroblasts (FVA cells) cultured for 0 hours (freshly isolated cells) or 24 hours, respectively. Lanes 1 and 4, no unlabeled competitor oligonucleotide; lanes 2 and 5, a 100-fold excess of a heterologous competitor oligonucleotide bearing a binding sequence for the transcription factor TAL1 (5′-CTCCCAGCAGCTGGCCTAGGAGATAGCAGCAG-3′); lanes 3 and 6, a 100-fold excess of unlabeled homologous competitor. (B) The end-labeled, double-stranded oligonucleotide had a sense strand sequence 5′-GTCCCTTTCTGGCGCGCACTCCTTTTGC-3′, which includes the sequence of the upstream element deleted in mutant 6 depicted in Figure 7A-B. Lanes 1-3 represent complexes formed with nuclear extracts of 0-hour splenic erythroblasts (FVA cells), and lanes 4-6 represent complexes formed with extracts from FVA cells cultured for 24 hours. Lanes 1 and 4, no unlabeled competitor oligonucleotide; lanes 2 and 5, a 100-fold excess of unlabeled homologous competitor oligonucleotide; lanes 3 and 6, a 100-fold excess of unlabeled heterologous competitor (TAL1 binding sequence as in Figure 8A).
Cuixia Tian et al. Blood 2003;101:
©2003 by American Society of Hematology