Ldb1 regulates carbonic anhydrase 1 during erythroid differentiation

Abstract

Carbonic anhydrase 1 (Car1), an early specific marker of the erythroid differentiation, has been used to distinguish fetal and adult erythroid cells since its production closely follows the γ- to β-globin transition, but the molecular mechanism underlying transcriptional regulation of Car1 is unclear. Here, we show that Car1 mRNA decreases significantly when erythroid differentiation is induced in MEL cells. The Ldb1 protein complex including GATA1/SCL/LMO2 binds to the Car1 promoter in uninduced cells and reduced enrichment of the complex during differentiation correlates with loss of Car1 expression. Knockdown of Ldb1 results in a reduction of Ser2 phosphorylated RNA Pol II and Cdk9 at the Car1 promoter region, suggesting that Ldb1 is required for recruitment of Pol II as well as the transcription regulator P-TEFb to enhance elongation of Car1 transcripts. Taken together, these data show that Ldb1 forms a regulatory complex to maintain Car1 expression in erythroid cells.

Fig. 1. Changes in erythroid-specific gene transcription during differentiation of MEL cells

MEL cells were treated with 2% DMSO, and then total RNA was prepared at the indicated times (days). (A) α-globin, (B) Gypa, (C) Epb4.2, (D) Slc4a1, (E) Car1, and (F) Car2 mRNA expression was analyzed by qRT-PCR. Each value was normalized with 18S ribosomal RNA. Error bars represent the SEM for three independent RNA preparations.

Fig. 2. Rank plots of induction or Ldb1 reduction in MEL cells

For each contrast in the microarray experiment (effect of induction in WT cells, effect of knockdown in uninduced cells and effect of knockdown in induced cells), probe sets in the array were sorted by fold change. For each gene highlighted in Table 1, the probe set with the best adjusted p-value is indicated. Those probes with an adjusted p-value > 0.01 are indicated in gray.

Fig. 3. Enrichment of the Ldb1/GATA1/LMO2/SCL complex at the Car1 promoter during MEL cell differentiation

(A) The Car1-3 locus is illustrated to scale showing the location of primer pairs used for qPCR with names below. ChIP was carried out using MEL cells before (UMEL) and after 4 days of 2% DMSO induction (IMEL) using antibodies to (B) Ldb1, (C) GATA1, (D) LMO2, and (E) SCL. HS26 (hypersensitive site 26) of mouse α-globin locus served as a positive control and necdin as a negative control for Ldb1 complex binding [19]. Error bars represent the SEM among four independent chromatin preparations.

Fig. 4. Dissociation of transcription complex components from the Car1 promoter during MEL cell differentiation

(A) The mouse Car1 gene is illustrated to scale. Exons are indicated by vertical lines. The positions of amplicons used for qPCR are indicated below and are named. Car1 pro/Ex1 amplifies an E box/GATA motif at position −203 to −198 of the erythoid-specific distal promoter/exon1 region [24]. Ex2 denotes an E box/GATA motif over the intron1/exon2 region (proximal promoter for adult colon epithelia). −1.9K and +29K are putative E box/GATA motifs upstream and downstream of the erythoid-specific promoter, respectively. Pr/Ex1 denotes a positive control sequence at the promoter of the mouse α-globin gene [19]. Inactive necdin served as a negative control [9]. Chromatin was prepared from MEL cells before (UMEL) and after 4 days of 2% DMSO treatment (IMEL). ChIP was performed with antibodies to (B) RNA Pol II, (C) Ser2P Pol II, or (D) Cdk9. The results of four independent experiments ± SEM are graphed.

Fig. 5. Knockdown of Ldb1 by shRNA results in failure of Ldb1/GATA1/LMO2/SCL complex formation at the Car1 promoter

MEL cells were stably transfected with control or Ldb1-shRNA [9]. Ldb1-KD1 and Ldb1-KD2 represent two stable Ldb1- shRNA expressing MEL cell clones. (A) Western blot analysis was performed with the indicated antibodies. α-tubulin served as an internal control. (B) Car1 mRNA expression was analyzed by qRT-PCR from control or stable Ldb1-shRNA clones. Each value was normalized with 18S rRNA. Error bars represent the SEM of three independent experiments. (C-F) Chromatin was prepared from control or stable Ldb1-shRNA clones. ChIP assay was carried out with antibodies to (C) Ldb1, (D) GATA1, (E) Ser2P Pol II, and (F) Cdk9. Data are mean ± SEM of four independent experiments.