1. Multiple functions of Ldb1 required for β-globin activation during erythroid differentiation
by Sang-Hyun Song, AeRi Kim, Tobias Ragoczy, M. A. Bender, Mark Groudine, and Ann Dean
Blood
Volume 116(13):
September 30, 2010
©2010 by American Society of Hematology

2. Recruitment of elongation competent Ser2P RNA pol II to the β-globin locus during mouse MEL cell differentiation.
Recruitment of elongation competent Ser2P RNA pol II to the β-globin locus during mouse MEL cell differentiation. (A) The mouse β-globin locus is diagrammed, and the positions of TaqMan probes used for real-time PCR are indicated below and named on the graphs. (B) MEL cells were treated with 2% DMSO for 4 days (IMEL) or without DMSO (UMEL), and globin mRNA was analyzed by quantitative real-time RT-PCR. Data are mean ± SEM, and 18S ribosomal RNA transcripts served as a control. (C-D) ChIP was performed MEL cell chromatin and antibodies to (C) RNA pol II or (D) Ser2P pol II. Three different chromatin preparations were analyzed by quantitative PCR. Error bars represent the SEM. Necdin, not transcribed in these cells, served as a negative control.
Sang-Hyun Song et al. Blood 2010;116:
©2010 by American Society of Hematology

3. Enrichment of P-TEFb kinase at the β-globin LCR and promoter region in differentiating MEL cells and alterations in H3ac and H3K4me3.
Enrichment of P-TEFb kinase at the β-globin LCR and promoter region in differentiating MEL cells and alterations in H3ac and H3K4me3. (A-C) Chromatin was prepared from MEL cells before and after 4 days of 2% DMSO treatment. ChIP was performed with antibodies to (A) Cdk9 or (B) CycT1. Necdin served as a negative control. Data are mean ± SEM. (C) Antibodies to acH3 or H3K4me3 were used in ChIP and graphed together by setting the highest value obtained with each antibody equal to 1. Lines represent AcH3; and bars, H3K4me3.
Sang-Hyun Song et al. Blood 2010;116:
©2010 by American Society of Hematology

4. Ldb1 knockdown inhibits enrichment of the Ldb1 complex and Ser2P RNA pol II at the β-globin promoter and LCR in MEL cells.
Ldb1 knockdown inhibits enrichment of the Ldb1 complex and Ser2P RNA pol II at the β-globin promoter and LCR in MEL cells. (A) MEL cells were stably transduced with Ldb1 shRNA or with an empty virus (Ctrl) and individual clones were isolated. Reduced Ldb1 expression in 2 stable Ldb1 knockdown clones (KD1 and KD2) was confirmed by Western blot analysis before (−) and after 4 days of 2% DMSO treatment (+). GATA-1 and α-tubulin served as positive and internal controls, respectively. (B) Stable clones were treated with 2% DMSO. Total RNA was isolated at day 4, and β-globin expression was analyzed by quantitative real-time RT-PCR. Each value was normalized with 18S ribosomal RNA. Three RNA preparations were analyzed. Error bars represent the SEM. (C) ChIP was carried out using KD1 cells after 4 days of 2% DMSO induction using antibodies to acH3 and H3K4me3, and the data were graphed together by setting the highest level for each antibody equal to 1. (D) The ChIP assay was performed with KD1 cells after 4 days of 2% DMSO induction using antibodies to Ldb1, GATA-1, LMO2, and SCL. Error bars represent the SEM for multiple independent chromatin preparations. (E-F) ChIP was carried out as for panel D using antibodies to Ser2P pol II (E) or Cdk9 (F). Necdin served as a negative control. Error bars represent the SEM for multiple independent chromatin preparations. ChIP assays with KD2 cells gave similar results (not shown) to those in panels C through F.
Sang-Hyun Song et al. Blood 2010;116:
©2010 by American Society of Hematology

5. The Ldb1 complex and Ser2P RNA pol II are enriched at the β-globin promoter in fetal liver erythroid cells of mice homozygous for a deletion of the LCR. (A) The mouse β-globin locus is diagrammed, and the positions of TaqMan probes used for real-time PCR ar...
The Ldb1 complex and Ser2P RNA pol II are enriched at the β-globin promoter in fetal liver erythroid cells of mice homozygous for a deletion of the LCR. (A) The mouse β-globin locus is diagrammed, and the positions of TaqMan probes used for real-time PCR are indicated below and named on the graphs. Endogenous (WT) and LCR-deleted (ΔLCR) murine β-globin loci are depicted. (B) Total RNA was isolated from E14.5 fetal liver of WT or ΔLCR mice, and globin expression was analyzed by quantitative real-time RT-PCR. α-Globin was used as a positive control of RNA expression. Each value was normalized with 18S ribosomal RNA. Error bars represent the SEM for several independent RNA preparations. (C-F) Chromatin was prepared from E14.5 fetal liver of WT (+) or ΔLCR (−) mice, and then ChIP and quantitative PCR were performed with (C) Ldb1, GATA-1, LMO2, and SCL, (D) Ser2P pol II, and (E) Cdk9, or (F) DSIF and FACT components as indicated on the graphs. Necdin served as a negative control. Error bars represent the SEM among independent chromatin preparations.
Sang-Hyun Song et al. Blood 2010;116:
©2010 by American Society of Hematology

6. Ldb1 is required for localization of the β-globin locus away from the nuclear periphery during differentiation.
Ldb1 is required for localization of the β-globin locus away from the nuclear periphery during differentiation. (A) Three-dimensional immuno-FISH analysis of untreated cells and cells treated with DMSO. WT indicates MEL cells; Ctrl KD, MEL cells with control shRNA; KD, MEL cells with shRNA directed to Ldb1; U, uninduced; and 4D, treated with DMSO for 4 days. Red represents nuclear lamin immunofluorescence; and green, probe detecting the β-globin locus. Bar represents 5 μm. For details see “Image acquisition and analysis.” (B) Quantitation of association of globin loci with the nuclear lamina before and after DMSO treatment for 3 days (3d) or 4 days (4d). More than 100 cells were scored for each determination. Error bars represent the SE.
Sang-Hyun Song et al. Blood 2010;116:
©2010 by American Society of Hematology

7. Importance of Ldb1 to embryonic and adult β-globin gene expression during erythroid differentiation.
Importance of Ldb1 to embryonic and adult β-globin gene expression during erythroid differentiation. Wild-type (WT) or Ldb1-null mutant (Ldb1−/−) ES cells were differentiated along erythroid lines into EBs with erythropoietin for 8 days as described under “EB differentiation.” (A) Morphology of WT or Ldb1−/− EB after 8 days of differentiation. (B-C) Total RNA was isolated at the indicated day of differentiation to determine expression of (B) Ldb1, or (C) ϵγ, βH1, and βmaj-globin. Each value was normalized with 18S ribosomal RNA. Error bars represent the SEM from independent RNA preparations.
Sang-Hyun Song et al. Blood 2010;116:
©2010 by American Society of Hematology

8. A model of dynamic long-range communication between the β-globin LCR and gene.
A model of dynamic long-range communication between the β-globin LCR and gene. Association of the Ldb1 complex (GATA-1/SCL/LMO2) and other activation components (shaded ovals), whose recruitment might be dependent on Ldb1, occupying the LCR and β-globin gene. Previous work comparing induced and uninduced MEL cells suggests that the Ldb1 complex binds first to the LCR.11 In the absence of the LCR, these components can co-occupy the β-globin gene, implying that promoter occupancy may normally occur independently; however, such occupancy is insufficient for high-level transcription. Full occupancy may be sufficient to establish LCR/β-globin proximity, before nuclear migration, via unknown mechanisms, and TF occupancy. Alternatively, the locus might migrate, again by unknown mechanisms, and establish LCR/β-globin proximity as a result of positioning within a TF. Proximity would then be stabilized by protein-protein interactions, possibly dependent on Ldb1.
Sang-Hyun Song et al. Blood 2010;116:
©2010 by American Society of Hematology