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. 2015 Feb 26;125(9):1477-87.
doi: 10.1182/blood-2014-10-605022. Epub 2015 Jan 5.

Compound loss of function of nuclear receptors Tr2 and Tr4 leads to induction of murine embryonic β-type globin genes

Shuaiying Cui  1 , Osamu Tanabe  2 , Michael Sierant  1 , Lihong Shi  3 , Andrew Campbell  4 , Kim-Chew Lim  1 , James Douglas Engel  1
Affiliations

Compound loss of function of nuclear receptors Tr2 and Tr4 leads to induction of murine embryonic β-type globin genes

Shuaiying Cui et al. Blood. .

Abstract

The orphan nuclear receptors TR2 and TR4 have been shown to play key roles in repressing the embryonic and fetal globin genes in erythroid cells. However, combined germline inactivation of Tr2 and Tr4 leads to periimplantation lethal demise in inbred mice. Hence, we have previously been unable to examine the consequences of their dual loss of function in adult definitive erythroid cells. To circumvent this issue, we generated conditional null mutants in both genes and performed gene inactivation in vitro in adult bone marrow cells. Compound Tr2/Tr4 loss of function led to induced expression of the embryonic εy and βh1 globins (murine counterparts of the human ε- and γ-globin genes). Additionally, TR2/TR4 function is required for terminal erythroid cell maturation. Loss of TR2/TR4 abolished their occupancy on the εy and βh1 gene promoters, and concurrently impaired co-occupancy by interacting corepressors. These data strongly support the hypothesis that the TR2/TR4 core complex is an adult stage-specific, gene-selective repressor of the embryonic globin genes. Detailed mechanistic understanding of the roles of TR2/TR4 and their cofactors in embryonic and fetal globin gene repression may ultimately enhance the discovery of novel therapeutic agents that can effectively inhibit their transcriptional activity and be safely applied to the treatment of β-globinopathies.

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Figures

Figure 1
Figure 1
Expression of NRs in differentiating human CD34+ cells. (A-C) RNA-seq analyses were performed on primary human CD34+ cells induced to differentiate ex vivo under serum-free culture conditions., The relative abundances (FKPM) of all 48 known NRs on differentiation days 4, 8, 11, and 14 are graphed in order of decreasing abundance. Note that the ordinate axis decreases by10-fold consecutively from the top panel down. (D-E) TR2/TR4, but not COUP-TFII, mRNAs and proteins were readily detected by qRT-PCR and western blotting assays in human CD34+ cells subjected to 2 different erythroid differentiation conditions., Similarly, TR4, but not COUP-TFII, proteins were detected in MELs and flow-sorted Ter119+ (erythroid) cells from adult mouse spleen. Abundant COUP-TFII protein, on the other hand, were detected in Ter119− (nonerythroid) adult mouse splenocytes. OAZ1 mRNA and anti-β-actin immunoreactivity were used as normalization controls in qRT-PCR and immunoblotting assays.
Figure 2
Figure 2
Generation of Tr2 and Tr4 null and conditionally mutant mice. Schematic illustrations of the wild-type, targeted, null, floxed, and conditional null alleles of Tr2 (A) and Tr4 (B). The null and floxed alleles of Tr2 or Tr4 were generated by intercrossing the respective gene targeted mice with Cre- or Flp-expressing transgenic lines, respectively. To generate the Tr2 null allele, exons 4 and 5 encoding the DNA binding domain were excised, whereas the Tr4 null allele was deleted for exon 5 encoding part of the DNA binding domain. In both floxed alleles, a DNA fragment containing the rest of the coding exons for each gene and the SV40 polyadenylation sequence was fused in-frame to the respective exons 5. The Tr2 or Tr4 conditional null allele was generated by Cre-mediated excision of the respective floxed allele, leaving behind a fluorescent reporter gene (CFP or YFP) flanked by 1 loxP and 1 Frt site. CFP, cyan fluorescent protein; YFP, yellow fluorescent protein.
Figure 3
Figure 3
Combinatorial genetic loss of Tr2/Tr4 leads to elevated expression of the murine embryonic β-type globin genes. (A) Breeding strategy to generate conditional tandem loss of Tr2 and Tr4 function and schematic showing the location of the primers used in qPCR for detection of the floxed (unrecombined) and total DNA. (B-C) Time-course analyses of Cre-mediated Tr4f/f deletion and TR4 mRNA change in Tr2−/−:Tr4f/f Lin− BM cells, either mock-infected (Cre−) or infected with Ad-Cre (Cre+), on days 0 and 2 of culture. Tr4f/f genomic DNA and TR4 mRNA quantification were performed using SBYR Green-based qPCR. (D-I) Combined loss of TR2/TR4 leads to elevated embryonic β-type globin gene expression. Values of tested genes are first expressed relative to the expression of housekeeping gene Oaz1, and then relative to the transcript of interest in Cre− cells (arbitrarily set at 100% or onefold).
Figure 4
Figure 4
Compound loss of TR2/TR4 impairs erythropoiesis. (A) WT and Tr2−/−:Tr4f/f Lin− BM cells were mock infected (Cre−) or infected with Ad-Cre (Cre+) on days 0 and 2 of culture. An aliquot of cells was stained daily with anti-CD71 and anti-Ter119 antibodies prior to flow cytometric analyses. As erythroid differentiation progressed, CD71+Ter119− immature erythroid cells (quadrant II) became CD71+Ter119+ (quadrant III) before becoming CD71−Ter119+ (quadrant IV). Numbers represent the fractional percentages of cells in each quadrant. Only Tr2−/−:Tr4f/f Lin− BM cells, which had been exposed to Ad-Cre infection, had far fewer circulation mature CD71−Ter119+ cells and an increased number of immature CD71+Ter119+ cells. (B) Data from erythroid cells at each maturational state (quadrants I-IV) from 4 to 6 mice of either genotype are summarized in the histogram. WT, wild type.
Figure 5
Figure 5
Genetic loss of Tr2/Tr4 abolishes the chromatin occupancy of TR2/TR4 and their cofactors at the DR-binding sites in the embryonic globin gene promoters. The binding of TR2/TR4 and other corepressors to select murine β-like globin gene promoters: εy (A), βh1 (B), and βmaj (C) as well as to an irrelevant control sequence located 5.9 kbp 5′ to the βmaj promoter (D) in infected Lin− cells were analyzed in ChIP experiments. Statistically significant enrichment of the orphan receptors and/or the cofactors at the promoters and control sequence is indicated in comparison with the control IgG values (indicated with asterisks; *P < .05).
Figure 6
Figure 6
The effects of Lsd1 conditional inactivation on erythropoiesis and chromatin occupancy by TR2 and TR4 and corepressors. (A) Lsd1f/f Lin− BM cells were mock-infected (Cre−) or infected with Ad-Cre (Cre+) on days 0 and 2 of culture. An aliquot of cells was stained daily with anti-CD71 and anti-Ter119 antibodies prior to flow cytometric analyses. Conditional loss of Lsd1 in Lin− BM cells, which had been exposed to Ad-Cre infection, led to far fewer mature CD71−Ter119+ cells and an increased number of immature CD71+Ter119+ cells. (B-E) LSD1 inactivation obliterated its chromatin occupancy, as expected, and of its partner CoREST. The binding of TR2/TR4 and other corepressor components to the β-type globin gene promoters were either unchanged or quite modestly reduced in infected Lsd1f/f Lin− cells.
Figure 7
Figure 7
Conditional Dnmt1 ablation alters erythroid differentiation and cofactor chromatin occupancy in globin gene promoters. (A) Flow cytometry following the differentiation of Ad-Cre–infected Lin− cells from Dnmt1f/f mice (see legend to Figure 6A). (B-E) Loss of DNMT1 activity led to enhanced LSD1 chromatin occupancy as detected by ChIP assays in Cre-infected Dnmt1f/f Lin− cells.
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