Distinct Ldb1/NLI complexes orchestrate γ-globin repression and reactivation through ETO2 in human adult erythroid cells

Abstract

The Ldb1/GATA-1/TAL1/LMO2 complex mediates long-range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated NLI (Ldb1 homolog) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we found robust NLI complex occupancy at a site downstream of the (A)γ-globin gene within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members, together with corepressor ETO2 and by γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is reactivated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. We conclude that alternative NLI complexes mediate γ-globin transcription or silencing through long-range LCR interactions involving an intergenic site of noncoding RNA transcription and that ETO2 is critical to this process.

Figure 1

Substantial co-occupancy of genomic sites by NLI and GATA-1 in human cells. Localization of NLI, GATA-1, and RNA pol II in K562 cells was examined by ChIP-chip. (A) Venn diagram showing intersection of genomic binding sites of GATA-1, NLI, and pol II within the region probed by tiling array. Total sites are indicated in parentheses; some pol II sites overlap more than one GATA-1 or NLI site, preventing the sum of interactions in the pol II circle from being equal to the total sites observed. (B) Bar plot of fraction of sites for each factor within promoters, exons, introns, and intergenic space based on RefSeq annotations. Promoter regions are defined as TSS plus 1 kb upstream. Exon and intron categories are not mutually exclusive because of multiple isoforms and overlapping genes. Intergenic space is defined as a lack of annotated transcripts and excludes the 1-kb promoter regions. (C). Tiling array signal (log2 ratios) across the globin locus on chr11. Regulatory elements and globin gene promoters are indicated by dotted lines. Peaks were called by ACME (using the parameters described in “NL1/GATA-1/Pol II ChIP-chip”) and are indicated below each track as dark gray boxes. Results for NLI, GATA-1, and RNA pol II ChIP-chip are shown. Data have been deposited in GEO under accession number GSE30047.

Figure 2

BGL3 is preferentially expressed in cells containing high HbF. (A) Map of the human β-globin locus showing the positions of primer pairs used to determine RNA transcript levels and factor occupancy by ChIP and real-time quantitative PCR (vertical lines). The globin genes and LCR DNase I–hypersensitive sites are represented by blue and red rectangles, respectively. The BGL3 region is depicted by a green rectangle. (B) Globin gene and intergenic transcription in K562 cells was evaluated using reverse-transcribed total RNA and quantitative RT-PCR with primers specific to the regions indicated on the x-axis. White bars indicate no reverse transcriptase. Results were normalized to GAPDH and error bars indicate SD. (C) Globin gene and intergenic transcription was evaluated for low-HbF (black bars) and high-HbF cells (white bars) as in panel B. Dark and light gray bars indicate values for reactions with no reverse transcriptase for low- and high-HbF cells, respectively. (D) ChIP was performed using an RNA Polymerase II Ab for low- and high-HbF cells. The locations of primers for PCR are indicated on the x-axis. Results were calculated against an input sample and normalized to GAPDH. Error bars indicate SD; black bars, low-HbF cells; white bars, high-HbF cells. Dark and light gray bars indicate values for an isotype matched control Ab in low- and high-HbF cells, respectively. The dotted line indicates the average of IgG control precipitations across all amplicons. (E) ChIP was performed using an H3K4me3 Ab to determine occupancy in low- and high-HbF cells. All data were calculated and plotted as described in panel D. *P < .05.

Figure 3

BCL11A occupancy in BGL3 changes when γ-globin is reactivated. ChIP was performed using a BCL11A Ab to determine occupancy in low- and high-HbF cells. Solid black bars indicate low-HbF cells; white bars, high-HbF cells. Dark and light gray bars indicate values for an isotype control Ab in low- and high-HbF cells, respectively. The dotted line indicates the average of IgG control precipitations across all amplicons. Results were calculated and plotted as described in Figure 2D. *P < .05.

Figure 4

NLI and complex members TAL1, GATA-1, and ETO2 differentially bind to the LCR and globin genes in low- and high-HbF cells. ChIP was performed using Abs specific for NLI (A), GATA-1 (B), TAL1 (C), and ETO2 (D) to determine occupancy in low- and high-HbF cells. Solid black bars indicate low-HbF cells; white bars, high-HbF cells. Dark and light gray bars indicate values for an isotype-matched control Ab in low- and high-HbF cells, respectively. The shaded area shows the 900-bp region encompassed by the 3 primer pairs within BGL3. The dotted line in all panels indicates the average of IgG control precipitations across all amplicons. All data were calculated and plotted as described in Figure 2D. *P < .05.

Figure 5

Increasing the NLI/ETO2 ratio in K562 cells induces γ-globin transcription. (A) The ratio of abundance of NLI/ETO2 at locations across the locus is plotted using the values obtained by ChIP assays shown in Figure 4. (B) The expression of γ-globin was monitored by quantitative RT-PCR after manipulating the NLI/ETO2 ratio in K562 cells. Cells were collected 48 hours after transfection with a control RNAi vector or with an ETO2 RNAi vector alone or together with an NLI-expressing vector (+, 2 μg; ++, 4 μg). Results were normalized to GAPDH and error bars indicate SD. *P < .05; **P < .001. The expression of BGL3 (C) and ϵ-globin (D) were monitored by quantitative RT-PCR after manipulating the NLI/ETO2 ratio in K562 cells exactly as described for panel B. (E) Western blot analysis was performed using Abs to ETO2 and NLI to confirm ETO2 knockdown and NLI overexpression. Actin served as the loading control.

Figure 6

Long-range interactions in the β-globin locus include the BGL3 region. 3C analysis was performed for cells expressing high- and low-HbF. Each EcoRI cleavage site is represented on the genome browser by a yellow triangle, and the interaction frequency with the designated anchor fragment is plotted in the middle of each fragment (dotted vertical lines). The globin genes and BGL3 sequences are shown in the middle track as gray and black rectangles, respectively. Colored line graphs represent data from low-HbF cells (green) and high-HbF cells (red). (A) Relative interaction frequency between the LCR2-4 fragment as anchor and other regions of the locus. (B) Relative interaction frequency between the BGL3 region and other parts of the locus. Note that the interaction frequency reported for the EcoRI fragment containing the ^Gγ-globin gene represents the value for both γ-globin gene interactions with the LCR (see “Chromosome conformation capture”). Error bars indicate SD.