Validation of select GABP-activated direct target genes in HSCs
| Gene symbol . | Gene name . | Fold enrichment on ChIP-Seq of human HPCs . | Validation of GABP binding in Lin− BM cells (fold enrichment) . | Validation of GABP binding in c-Kit+Lin− BM cells (fold enrichment) . | Validation of gene-expression changes in GABPα-null Flt3−LSKs (ratio of KO/WT) . |
|---|---|---|---|---|---|
| Apoptosis regulation | |||||
| Bcl2 | Bcl-2 (B-cell leukemia/lymphoma 2) | 14* | 2.6 ± 1.3† | 3.4 ± 1.8† | 0.06 ± 0.09‡ |
| Bcl2l1 | Bcl-XL (Bcl-2–like 1) | 18 | 35.5 ± 15.1‡ | 33.7 ± 5.9‡ | 0.24 ± 0.25‡ |
| Mcl1 | Mcl-1 (myeloid cell leukemia sequence 1) | 6 | 6.9 ± 3.5‡ | 5.8 ± 1.8‡ | 0.26 ± 0.09‡ |
| Transcription factors | |||||
| Zfx | Zfx (zinc finger protein X-linked) | 14 | 2.7 ± 1.0‡ | 2.1 ± 0.2‡ | 0.03 ± 0.04‡ |
| Etv6 | Etv6/Tel (ets variant gene 6) | 6 | 2.1 ± 1.0† | 1.6 ± 1.0§ | 0.24 ± 0.11‡ |
| Smad4 | Smad4 (MAD homolog 4) | 14 | 1.9 ± 0.7† | 2.2 ± 0.8† | 0.29 ± 0.26‡ |
| Foxo3 | Foxo3a (forkhead box O3) | 4 | 1.8 ± 0.5‡ | 2.9 ± 1.7† | 0.27 ± 0.13‡ |
| Gabpa | GABPα | 90 | 8.2 ± 3.2‡ | 39.5 ± 7.9‡ | 0.09 ± 0.15‡ |
| Gabpb1 | GABPβ1 | 32 | 2.1 ± 0.6‡ | 2.1 ± 1.0† | 0.28 ± 0.25‡ |
| DNA repair | |||||
| Atm | ATM (ataxia telangiectasia mutated homolog) | 43 | 5.1 ± 3.1† | 3.5 ± 0.8‡ | The Atm transcript was not consistently detected in KO cells. |
| Telomere maintenance | |||||
| Terf2 | TRF2 (telomeric repeat binding factor 2) | 20 | 8.0 ± 2.8‡ | 5.2 ± 1.6‡ | 0.42 ± 0.32† |
| Signaling molecules | |||||
| Pten | PTEN (phosphatase and tensin homolog) | 17 | 1.9 ± 0.6† | 1.5 ± 0.5† | 0.11 ± 0.11‡ |
| DNA methylation | |||||
| Dnmt1 | DNMT-1 (DNA methyltransferase 1) | 19 | 7.4 ± 1.9‡ | 4.7 ± 1.4‡ | 0.54 ± 0.17‡ |
| Histone acetylation | |||||
| Ep300 | p300 (E1A binding protein p300) | 11 | 2.3 ± 0.6‡ | 1.8 ± 0.2‡ | 0.38 ± 0.16‡ |
| Myst4 | MYST-4 (MYST histone acetyltransferase 4) | 13 | 9.5 ± 3.6‡ | 10.9 ± 4.4‡ | 0.2 ± 0.25‡ |
| Chromatin remodeling | |||||
| Smarca2 | Brm/BAF190B | 12 | 2.3 ± 1.6§ | 1.9 ± 0.8† | 0.05 ± 0.05‡ |
| Smarca4 | Brg1/BAF190A | 15 | 3.5 ± 1.9† | 2.9 ± 1.1‡ | 0.38 ± 0.11‡ |
| Gene symbol . | Gene name . | Fold enrichment on ChIP-Seq of human HPCs . | Validation of GABP binding in Lin− BM cells (fold enrichment) . | Validation of GABP binding in c-Kit+Lin− BM cells (fold enrichment) . | Validation of gene-expression changes in GABPα-null Flt3−LSKs (ratio of KO/WT) . |
|---|---|---|---|---|---|
| Apoptosis regulation | |||||
| Bcl2 | Bcl-2 (B-cell leukemia/lymphoma 2) | 14* | 2.6 ± 1.3† | 3.4 ± 1.8† | 0.06 ± 0.09‡ |
| Bcl2l1 | Bcl-XL (Bcl-2–like 1) | 18 | 35.5 ± 15.1‡ | 33.7 ± 5.9‡ | 0.24 ± 0.25‡ |
| Mcl1 | Mcl-1 (myeloid cell leukemia sequence 1) | 6 | 6.9 ± 3.5‡ | 5.8 ± 1.8‡ | 0.26 ± 0.09‡ |
| Transcription factors | |||||
| Zfx | Zfx (zinc finger protein X-linked) | 14 | 2.7 ± 1.0‡ | 2.1 ± 0.2‡ | 0.03 ± 0.04‡ |
| Etv6 | Etv6/Tel (ets variant gene 6) | 6 | 2.1 ± 1.0† | 1.6 ± 1.0§ | 0.24 ± 0.11‡ |
| Smad4 | Smad4 (MAD homolog 4) | 14 | 1.9 ± 0.7† | 2.2 ± 0.8† | 0.29 ± 0.26‡ |
| Foxo3 | Foxo3a (forkhead box O3) | 4 | 1.8 ± 0.5‡ | 2.9 ± 1.7† | 0.27 ± 0.13‡ |
| Gabpa | GABPα | 90 | 8.2 ± 3.2‡ | 39.5 ± 7.9‡ | 0.09 ± 0.15‡ |
| Gabpb1 | GABPβ1 | 32 | 2.1 ± 0.6‡ | 2.1 ± 1.0† | 0.28 ± 0.25‡ |
| DNA repair | |||||
| Atm | ATM (ataxia telangiectasia mutated homolog) | 43 | 5.1 ± 3.1† | 3.5 ± 0.8‡ | The Atm transcript was not consistently detected in KO cells. |
| Telomere maintenance | |||||
| Terf2 | TRF2 (telomeric repeat binding factor 2) | 20 | 8.0 ± 2.8‡ | 5.2 ± 1.6‡ | 0.42 ± 0.32† |
| Signaling molecules | |||||
| Pten | PTEN (phosphatase and tensin homolog) | 17 | 1.9 ± 0.6† | 1.5 ± 0.5† | 0.11 ± 0.11‡ |
| DNA methylation | |||||
| Dnmt1 | DNMT-1 (DNA methyltransferase 1) | 19 | 7.4 ± 1.9‡ | 4.7 ± 1.4‡ | 0.54 ± 0.17‡ |
| Histone acetylation | |||||
| Ep300 | p300 (E1A binding protein p300) | 11 | 2.3 ± 0.6‡ | 1.8 ± 0.2‡ | 0.38 ± 0.16‡ |
| Myst4 | MYST-4 (MYST histone acetyltransferase 4) | 13 | 9.5 ± 3.6‡ | 10.9 ± 4.4‡ | 0.2 ± 0.25‡ |
| Chromatin remodeling | |||||
| Smarca2 | Brm/BAF190B | 12 | 2.3 ± 1.6§ | 1.9 ± 0.8† | 0.05 ± 0.05‡ |
| Smarca4 | Brg1/BAF190A | 15 | 3.5 ± 1.9† | 2.9 ± 1.1‡ | 0.38 ± 0.11‡ |
KO indicates Mx1Cre-GABPαFL/−; and WT, Mx1Cre-GABPαFL/+.
Select genes under different functional categories were validated for GABP binding and GABP-dependent expression. The gene symbols, commonly gene names, and fold enrichment of GABP binding in human HPCs were summarized in first 3 columns. Lin− and c-Kit+Lin− murine BM cells from WT C57BL/6 mice were used for validation of GABP binding using quantitative PCR. Data are means ± SD of 2 independent experiments with each sample measured in duplicates or triplicates. For validation of gene-expression changes, Flt3−LSK cells were sorted from pIpC-treated Mx1Cre-GABPαFL/+ and Mx1Cre-GABPαFL/− mice, cDNA amplified and quantitatively determined. Data are means ± SD of 3 independent experiments with each sample measured in duplicates. Statistical significance was determined using the Student t test.
Note that in terms of fold enrichment, the binding of GABPα to the Etv6, Pten, Ep300, and Smarca2 gene loci was relatively weak especially in c-Kit+Lin− murine BM cells. One possible explanation might be that the affinity of GABPα binding to these loci was low and the detection was limited by low numbers of c-Kit+Lin− BM cells that can be obtained. Alternatively, enriched binding of GABPα might be more specific in primitive HSCs, and the binding signal was diluted by other non-HSC cells included in the c-Kit+Lin− BM cells.
Binding of GABP in the BCL2 locus was in intron 1, more than 2 kb from its TIS (see Figure 4A).
P < .05.
P < .01.
Not significant.