Defective erythropoiesis caused by mutations of the thyroid hormone receptor α gene

Patients with mutations of the THRA gene exhibit classical features of hypothyroidism, including erythroid disorders. We previously created a mutant mouse expressing a mutated TRα1 (denoted as PV; Thra1PV/+ mouse) that faithfully reproduces the classical hypothyroidism seen in patients. Using Thra1PV/+ mice, we explored how the TRα1PV mutant acted to cause abnormalities in erythropoiesis. Thra1PV/+ mice exhibited abnormal red blood cell indices similarly as reported for patients. The total bone marrow cells and erythrocytic progenitors were markedly reduced in the bone marrow of Thra1PV/+ mice. In vitro terminal differentiation assays showed a significant reduction of mature erythrocytes in Thra1PV/+ mice. In wild-type mice, the clonogenic potential of progenitors in the erythrocytic lineage was stimulated by thyroid hormone (T3), suggesting that T3 could directly accelerate the differentiation of progenitors to mature erythrocytes. Analysis of gene expression profiles showed that the key regulator of erythropoiesis, the Gata-1 gene, and its regulated genes, such as the Klf1, β-globin, dematin genes, CAII, band3 and eALAS genes, involved in the maturation of erythrocytes, was decreased in the bone marrow cells of Thra1PV/+ mice. We further elucidated that the Gata-1 gene was a T3-directly regulated gene and that TRα1PV could impair erythropoiesis via repression of the Gata-1 gene and its regulated genes. These results provide new insights into how TRα1 mutants acted to cause erythroid abnormalities in patients with mutations of the THRA gene. Importantly, the Thra1PV/+ mouse could serve as a preclinical mouse model to identify novel molecular targets for treatment of erythroid disorders.

Introduction Thyroid hormones have long been known to play an important role in erythropoiesis. Early in vitro studies demonstrated that L-thyroxine (T4) stimulates the biosynthesis of hemoglobin [1]. Experimental animal models also showed that T4 enhances red blood cell formation and stimulates hemoglobin synthesis [2]. Studies in humans have shown a causal association between hypothyroidism and anemia [3]. Persons with subclinical hypothyroidism had lower hemoglobin levels [4,5] and a higher prevalence of anemia than euthyroid persons. Treatment of patients with subclinical hypothyroidism with thyroid hormone resulted in a significant increase in hemoglobin content [6] or erythropoietin levels [7]. However, how hypothyroidism results in erythroid disorders at the molecular level remains largely unknown.
Recently, patients with mutations of the thyroid hormone receptor α gene (THRA) have been reported to exhibit some of the classical symptoms and signs of hypothyroidism with impaired growth and delayed bone development [8][9][10][11]. These patients also display anemia. These findings suggested that erythropoietic disorders in humans are mediated by TRα1 mutants and support the critical role of TRα1 in erythropoiesis. The availability of a mouse model (Thra1 PV/+ mice), harboring a mutated TRα1 (designated as TRα1PV) [12], has made it possible for us to elucidate the role of TRα1 mutants in erythroid disorders. TRα1PV has a Cterminal mutated sequence (398-PPFVLGSVRGLD-409) [12], similar to the truncated C-terminal sequence in two patients (398-PPTLPRGL -405) [9]. The PV mutation was first identified from a patient with severe resistance to thyroid hormone (RTHβ), characterized by elevated thyroid hormone levels accompanied by normal TSH, short stature, goiter, and tachycardia [13]. The PV mutated sequence was targeted to the Thra gene at the corresponding position as in THRB gene to assess the functional consequence of TRα1 mutations at the time when no patients with the mutations of the THRA gene was discovered. Thra1 PV/+ mice exhibit displayed phenotype of hypothyroidism as in the patients with severe growth retardation [12] and delayed bone development [12,14,15]. The Thra1 PV/+ mouse has been used as a preclinical model to test the effectiveness of T4 treatment for the correction of impaired bone development due the actions of mutated TRα1 [16]. These studies further validate the usefulness of the Thra1 PV/+ mouse to understand how mutations of the THRA gene result in deleterious abnormalities in patients.
In the present study, we first characterized the erythroid phenotypes in Thra1 PV/+ mice and showed that Thra1 PV/+ mice exhibit anemia with decreased red blood cells and reduced hemoglobin content similar to patients with mutations of the THRA gene [11]. We further identified the genes that were abnormally regulated by TRα1PV, resulting in defective erythropoiesis. Thus, our study has provided direct molecular evidence to show that mutations of the THRA gene could impair erythropoiesis and has uncovered novel molecular actions of TRα1 in the erythroid differentiation and development.

Alterations of peripheral blood composition in Thra1 PV/+ mice
We first analyzed peripheral blood composition to characterize erythropoietic phenotypes in Thra1 PV/+ mice. Complete blood count revealed that major indices for erythrocytes, such as red blood cell count (RBC), hemoglobin levels (Hb) and hematocrit (HCT), were significantly lower in Thra1 PV/+ mice than in wild-type (WT) mice. As shown in Fig 1, RBC, Hb, and HCT were decreased by 14% (panel A), 13% (panel B), and 10% (panel C), respectively. Other erythrocyte peripheral indices, namely, the mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), and platelets (PLT), were 3% (panel D), 3% (panel E), and 30% (panel F) lower in Thra1 PV/+ mice than in WT mice. These results indicated that mutations of the Thra gene could affect different lineages leading to defects.
Defective cellularity in the bone marrow of Thra1 PV/+ mice After birth and throughout adult life, the bone marrow remains the major hematopoietic organ in mice [17]. We therefore examined the histology of H & E stained femur sections to assess the cellularity of the bone marrow. As shown in Fig 2A, fewer bone marrow cells with a higher fat deposit were apparent in Thra1 PV/+ mice than wild-type mice (Fig 2A, compare panel b with panel a). By counting cell numbers, we found that total bone marrow cells were decreased 57% in Thra1 PV/+ mice (Fig 2A-c). We have measured the fat areas in the bone marrow of wild-type mice (n = 3) and Thra1 PV/+ mice (n = 3). The quantitative data is now shown as Fig 2A-d, indicating that the fat (% of total area) was 6.2-fold higher in the bone marrow of Thra1 PV/+ mice than in wild-type mice.
We next assessed which abnormal population of progenitors in the bone marrow cells of Thra1 PV/+ mice contributed to anemia. It is known that a small population of hematopoietic stem cells (HSC) gives rise to multipotent progenitors (MPP), which subsequently differentiate into common myeloid population cells (CMP population cells) and common lymphoid progenitors (CLP). CMP is differentiated into megakaryocytic/erythroid (MEP) and granulocytemyeloid (GMP) progenitors. Because patients with mutations of the THRA gene exhibit anemia, we focused on the analysis of the lineage derived from MEP, and its subsequent downstream progenitors: burst-forming unit-erythroid (BFU-E) to colony-forming unit-erythroid  The distribution of respective erythroid and megakaryocytic populations in the bone marrow of wild type and Thra1 PV/+ mice. Erythroid and megakaryocytic progenitors and mature cells were analyzed by FACS using cell surface markers. CFU-E erythrocyte progenitors identified in the bone marrow cells that (CFU-E), then to erythroblasts, and ultimately to mature erythrocytes [18][19][20]. To assess the effect of TRα1PV mutation on the number of progenitors in the MEP lineage, we analyzed the progenitors by specific cell surface markers in the bone marrow using flow cytometry. Erythroid cells at different developmental stages can be identified by different cell surface markers: Ter119 (erythroid specific glycophorin) and CD71 (the transferrin receptor). Ter119 expression increases as maturation of erythrocyte progresses [21,22]. CD71 is the transferrin receptor that expresses at high levels in early erythroid precursors, but its levels decrease toward erythroid maturation [21,22]. cKit (CD117) is a cytokine receptor expressed on the surface of hematopoietic stem cells, MPP, and CMP. Stem cells antigen 1 (Sca1) is expressed in HSC [19,23]. Using these specific cell markers, we identified which sub-populations were altered in the bone marrow cells of Thra1 PV/+ mice.
CFU-E progenitors reside in the lineage negative (Lin-)Ter119-CD71+cKit+ populations of the bone marrow ( Fig 2B, panel a). CFU-E progenitors were decreased 24% in Thra1 PV/+ mice compared with wild-type mice (Fig 2B, panel d). Mature red blood cells and reticulocyte precursors exhibited characteristic Ter119+CD71-expression on the cell surface ( Fig 2B, panel b). We found that erythrocytes were decreased 56% in the bone marrow of Thra1 PV/+ mice ( Fig  2B, panel e). CFU-Mk progenitors and megakaryocytes reside in Ter119-Sca1-CD41+CD61+ population (Fig 2B panel c). The only difference between CFU-Mk and megakaryocyte is that CFU-Mk expresses cKit as a marker in the progenitors. We found that CFU-Mk progenitors and megakaryocytes were decreased 19% and 52%, respectively, in the bone marrow of Thra1 PV/+ mice (Fig 2B, panels f and g, respectively). Notably, the extent of differences between WT and Thra1 PV/+ mice was greater in the CFU-E and erythrocytes than in CFU-Mk and megakaryocytes.
We further carried out in vitro colony forming unit assays. BFU-E progenitors were defined as cells that colonize after 14 days under defined-in vitro culture conditions ( Fig 2C, panel a). CFU-E progenitors were colonized cells after 2 days in defined culture conditions ( Fig 2C,  panel b). CFU-Mk progenitors were counted after 7 days in defined culture conditions ( Fig  2C, panel c). BFU-E, CFU-E, and CFU-Mk (expressed as relative colonies versus total bone marrow cells) were decreased 68% (Fig 2C-d), 62% (Fig 2C-e), and 68% (Fig 2C-f), respectively, in Thra1 PV/+ mice as compared with WT mice. We further carried out colony assays for CFU-GEMM and CFU-GM. The number of CFU-GEMM and CFU-GM colonies as shown in Fig 2D-a and 2D-c, respectively, was 70% and 60% lower, respectively, in Thra1 PV/+ mice than in WT mice (Fig 2D-b and 2D-d). These results imply that the capacity of progenitor cells to differentiate from MEP to erythroblasts as well as to megakaryocytes was impaired, leading to erythroid disorders in Thra1 PV/+ mice.

Stimulation of the erythroid lineage differentiation by T3
TRα1PV is a dominantly negative mutant and cannot bind T3 [12]. That the capacity of the progenitors to form BFU-E, CFU-E, and CFU-Mk was impaired in the bone marrow of Thra1 PV/+ mice prompted us to ascertain whether the differentiation from MEP to downstream BFU and CFU was regulated by thyroid hormones. We therefore rendered Thra1 PV/+ mice hypothyroid by treating them with PTU and rendered the PTU-treated mice hyperthyroid by T3 treatment. As shown in Fig 3A, PTU treatment was effective in lowering serum total T3 and T4 levels (bar 2 in panels a and b, respectively) in WT mice. In line with the TRα1 mutations disrupt erythropoiesis lowering of T3 and T4, TSH was highly elevated (bar 2 in Fig 3A-c). T3 treatment of PTUtreated WT mice led to highly elevated T3 (Fig 3A-a, bar 3) and so suppressed TSH levels ( Fig 3A-c, bar 3). Treatment of Thra1 PV/+ mice with PTU followed by T3 injection led to similar changes as shown for WT mice (Fig 3A-a, 3A-b and 3A-c, bars 4-6). These findings are consistent with the earlier reports that the feedback loop in the pituitary-thyroid axis was not affected by expressing TRα1PV mutant in Thra1 PV/+ mice [12].
We next isolated bone marrow cells from hypothyroid (PTU-treated) and hyperthyroid (T3-treated) mice and carried out colony forming assays. No significant differences in BFU-E (Fig 3B- by 42%, 77% and 25%, respectively from hypothyroid to hyperthyroid mice. In contrast, in Thra1 PV/+ mice, compared to WT mice, there was markedly decreased BFU-E (Fig 3B-a), CFU-E (Fig 3B-b), and CFU-Mk (Fig 3B-c) (compare bar 4-6 to bars 1-3). Moreover, the extent of decreases in Thra1 PV/+ mice were not affected by T3 treatment (bars 4-6). These results indicated that colony forming units derived from MEP were stimulated by T3 in WT mice, but were not affected by T3 in Thra1 PV/+ mice due to the actions of dominant negative TRα1PV mutant.
To further confirm that TRα1PV mutant acted to inhibit the differentiation in the erythroid lineage, we isolated lineage depleted bone marrow cells (Lin-BM) by eliminating mature lineage cells including T cells, B cells, macrophages, granulocytes, and erythrocytes. Using Lin-BM, we compared the maturation of erythrocytes in WT and Thra1 PV/+ mice using an in vitro terminal erythropoiesis system [24]. Using an equal number of total bone marrow cells from WT (Fig 4A-a) and Thra1 PV/+ mice (Fig 4B-a), we found 40% and 31%, respectively, of Ter119+ with low FSC population (boxed in red). As shown by May-GrÜ nwald-Giemsa staining ( Fig 4A and 4B). The gated Ter119-positive cells associated with low FSC fraction (boxed in red) in panel c were quantified: 30% and 17% of mature erythrocytes were detected for WT and Thra1 PV/+ mice, respectively. These results represent a 47% reduction in the capacity of Lin-MB cells from Thra1 PV/+ mice to mature into erythrocytes ( Fig 4C).
The expression of the Gata1 and its regulated genes is repressed in the bone marrow of Thra1 PV/+ mice To investigate the molecular mechanisms by which TRα1PV mutant induced erythropoietic disorders, we first analyzed the expression of a major regulator of erythropoiesis, the Gata1 gene (erythroid transcription factor; GATA-binding factor 1). GATA1 is a member of the GATA transcription factor family and is essential for erythroid development by regulating a large ensemble of genes that mediate both the development and function of red blood cells [25,26]. The loss of Gata1 expression leads to erythroid maturation arrest and embryonic lethality due to anemia [27][28][29][30][31]. We found the expression of the Gata1 gene was~50% lower in the bone marrow of Thra1 PV/+ mice than in WT mice ( Fig 5A-a, compare bar 2 with bar 1). Direct western blot analysis shows that the GATA1 protein level in the bone marrow was lower than that in the spleen of WT mice (compare lane 2 with lane 1, Fig 5A-b). However, when the bone marrow cell lysates of WT mice were first enriched by immunoprecipitation followed by western blot analysis (Co-IP), GATA1 proteins were detected (lane 3, Fig 5A-b). In contrast, under identical experimental conditions, GATA1 proteins were not detected (lane 4), indicating that the GATA1 protein level in the bone marrow of Thra1 PV/+ mice was lower than that of WT mice. Lanes 5 and 6 were the corresponding negative controls in that an irrelevant IgG was used in the immunoprecipitation step. That no signals were detected in lanes 5 and 6 indicated that the protein detected by Co-IP was specific. GATA1 regulates the expression of the Klf1 gene, which drives erythropoiesis by affecting its downstream target genes critical for maturation of erythrocytes [25,32,33]. Consistent with decreased expression of the Gata1 gene, the expression of the Klf1 gene was 53% lower in the bone marrow of Thra1 PV/+ mice ( Fig 5B, bar 2 versus bar 1). We also analyzed the expression of KLF1 and GATA1 target genes, such as the β-globin (beta major globin) and the dematin genes (erythroid membrane and cytoskeleton related gene). The β-globin protein along with α-globin makes up the most common form of hemoglobin in adult humans. Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. Dematin binds to spectrin and dynamically regulates red cell membrane mechanical function [34]. Consistent with the reduced expression of the Klf1 gene, the expressions of the β-globin and dematin genes were also repressed by 70% and 50%, respectively, in the bone marrow of Thra1 PV/+ mice (Fig 5C and 5D). We further analyzed the expression of CAII, band 3 and eALAS genes, which are regulated by GATA-1 during erythropoiesis. Moreover, these TRα1 mutations disrupt erythropoiesis three genes are known to be directly regulated by TRα1 and T3 in birds [35][36][37]. The expression of these three genes was inhibited 93%, 90% and 86%, respectively, in the bone marrow of Thra1 PV/+ mice (Fig 5E, 5F and 5G, respectively). Taken together, these data indicate that suppression of Gata-1 gene and the known T3-target genes led to impaired erythropoiesis.
Repression of the Gata1 gene by TRα1PV mediates erythroid disorders in Thra1 PV/+ mice That the expression of the Gata1 gene was suppressed by TRα1PV prompted us to ascertain whether the Gata1 gene was directly regulated by TR/T3. We searched for putative thyroid hormone response elements (TREs) in the GATA1 hematopoietic regulatory domain [38], located upstream of the transcription starting site (TSS) of the Gata1 gene ( Fig 6A). We also searched for putative TREs in the +4691 intronic sequence between exon I and exon II ( Fig  6A). In this -4096 to +4691 region, we found putative TREs-containing regions with the core consensus sequences of the hexa-nucleotide "half-site" (A/G)GGT(C/A/G)A (T#1 -T#8, Fig  6B-I). To identify relevant TREs, we used chromatin immunoprecipitation (ChIP) to ascertain the binding of TRα1 to these eight putative TREs in the bone marrow of WT and Thra1 PV/+ mice. When anti-TRα1 antibodies were used, a significantly higher binding of TRα1 to TRE1 (AGTGGGGTCCATT in the region +1410 to +1549 bp) were found in the bone marrow of WT mice than when anti-IgG antibodies (negative controls) were used (bar 3 versus bar 1, Fig  6B-II-a). In the bone marrow of Thra1 PV/+ mice, when specific anti-TRα1PV antibodies (rabbit polyclonal antibody T1) were used, a significantly higher binding of TRα1PV to TRE1 than when anti-IgG antibodies (negative controls) were used (bar 6 versus bar 2, Fig 6B-II-a). Similar significantly higher binding of TRα1 (bar 3 versus bar 1, Fig 6B-II-b) and TRα1PV (bar 6 versus bar 2, Fig 6B-II-b) to TRE2 (CCCACGGAGATTCCTGT, in the region -3699 to -3325 bp) were also detected. However, we did not find specific binding of TRα1 and TRα1PV to other regions containing putative TREs (i.e., T#1, T#2, T#3, T#5, T#7, and T#8, Fig 6B-I).
These ChIP results suggested that TRα1 and TRα1PV could bind directly to these TRE-containing regions in TRE1 (region T#4) and TRE2 (region T#6) on the intronic region and the proximal promoter, respectively, of the Gata1 gene.
We next used electrophoretic mobility shift assay (EMSA) to demonstrate directly the binding of TRα1 and TRα1PV to TREs in TRE1 and TRE2 regions (Fig 6C-I). As shown in Fig  6C-II-a, TRα1 prepared by in vitro transcription/translation system, bound to TRE1 (the TRE sequence is shown in Fig 6C-I) as heterodimers with the retinoid X receptor (RXR) (lanes 5 and 6 show the increasing concentrations of TRα1). The TRE1-bound TRα1 was supershifted by anti-TRα1 antibodies (lane 7), but not by control anti-IgG antibodies (lane 8). In the presence of unlabeled TRE, no binding to 32 P-labeled TRE1 was detected (lane 9). These data indicate that TRE1 bound specifically to TRα1 as heterodimers with RXR. Similarly, TRα1PV also bound to TRE1 as heterodimers with RXR (lanes 11-12), which was supershifted by anti-TRα1PV (lane 13). No specific binding to labeled TRE in the presence of unlabeled TRE was observed (lane 15). Lanes 16-19 were positive controls using a labeled F2 (TRE with inverted two half-sites). Moreover, when TRE1 from AGTGGGGTCCATT was mutated to AGACGG GTCTGTT, no binding was detected by EMSA (Fig 6C-II-b; compare lanes 11-16 with lanes 3-8 in which wild-type TRE was used in EMSA). These EMSA results indicate that TRE1 bound directly and specifically to TRα1 and TRα1PV. Using similar EMSA, we also found specific binding of TRα1 (Fig 6C-III-a, lanes 5-9) and TRα1PV (Fig 6C-III-a, lanes 11-15) to TRE2. The binding was further confirmed by mutational analysis in which, when TRE2 was mutated from CCCACGGAGATTCCTGT to TTTGAGGAGATCTTCTC, no binding was detected by EMSA (Fig 6C-III-b; compare lanes 11-16 with lanes 3-8 in which wild-type TRE was used in EMSA). Taken together, these results indicate that we have uncovered two specific TRα1 and TRα1PV binding TREs in the promoter of the Gata1 gene.
To assess whether these two TREs mediated the TRα1-dependent regulation of the transcription of the Gata1 gene, we constructed reporters in which the expression of luciferase is mediated by TRE1 or TRE2. We cloned the 0.140 kb fragment containing the TRE1 (Fig 7A-Ia and 7A-I-b) and 1.553 kb fragment containing the TRE2 into the pGL4.23 luciferase plasmid (Fig 7A-I-b and 7A-I-c, respectively). It is of interest to note that in the 1.553 kb fragment, one GATA box and two E-boxes, enhancers to regulate the transcription of the Gata1 gene, were also present [39]. These two Luc-reporters were transfected into human erythroleukemia 562 (K562) cells, which express endogenous TRα1 [40]. Indeed, using a Luc-reporter containing palindromic TRE (Pal-Luc), the reporter activity mediated by the endogenous TRα1 was detected in the presence of T3 (Fig 7A-II-a, bar 2 versus bar 1). When the TRα1PV expression vector was transfected into K562 cells, the Pal-Luc reporter activity was suppressed by dominant negative activity of TRα1PV (bar 4 versus bar 2). Transfection of the TRα1 expression plasmid led to an additional 15-fold activation of Pal-Luc reporter activity (bar 6 versus bar 2). This TRα1/T3-mediated activation was repressed by the transfection of the TRα1PV expression plasmid (bar 8 versus bar 6). These results indicate that K562 cells are a good model cell line to evaluate the activity of TRE1 and TRE2 in the regulation of the Gata1 gene by TRα1.
In contrast to Pal-Luc, in the presence of T3, a small, but significant repression of TRE1-Luc activity was observed (Fig 7A-II-b, bar 2 versus 1). Interestingly, transfection of TRα1PV led to a 1.3-fold activation of TRE1-Luc activity (bar 4 versus 2). When TRα1 expression plasmid was transfected into K562 cells, more suppression of TRE1-Luc activity than by endogenous TRα1 was observed (bar 6 versus 2). Remarkably, co-transfection of the TRα1PV expression plasmid with TRα1 expression plasmid into K562 cells resulted in a 2.5-fold activation of TRE1-Luc activity (bar 8 versus bar 6). These results demonstrated that TRE1 mediated the negative regulation activity of TRα1. These findings are reminiscent of the regulation of the Tshα common subunit (α-SU) gene by TR/T3 in the pituitary of Thra1 PV/+ mice [12].
In contrast to TRE1-Luc activity, the luciferase activity mediated by TRE2 was activated two-fold by T3 in K562 cells (Fig 7A-II-c, bar 2 versus bar 1). This T3-stimuated reporter activity was totally abolished by transfection of the TRα1PV expression plasmid (bar 4 versus bar 2). Transfection of the TRα1 expression plasmid led to an additional 1.9-fold activation of the reporter activity (bar 6 versus bar 2). This T3-activated reporter activity mediated by TRα1 was totally abolished by co-transfection of the TRα1PV expression plasmid (bar 8 versus bar 6). These results indicate that TRE2, via T3-TRα1, mediates the positive regulation of the Gata1 gene transcription.
The identification of one positive TRE and one negative TRE in the promoter prompted us to assess the overall regulation by TRα1of the Gata1 gene in the bone marrow. We rendered WT and Thra1 PV/+ mice hypothyroid by treating them with PTU and then some PTU-treated mice with T3 to make them hyperthyroid (see also Fig 3). The expression of the Gata1 mRNA was~50% lower in hypothyroid WT mice than in euthyroid WT mice (Fig 7B-a, bar 3 versus bar 1). The expression of the Gata1 mRNA was~3-fold higher in hyperthyroid WT mice than in hypothyroid WT mice (Fig 7B-a, bar 5 versus bar 3). These results indicate that the Gata1 gene was positively regulated by T3. In Thra1 PV/+ mice, Fig 7B-a also shows that the expression of the Gata1 mRNA in the bone marrow of euthyroid Thra1 PV/+ mice was~50% lower than TRα1 mutations disrupt erythropoiesis that in euthyroid WT mice (bar 2 versus 1). The Gata1 mRNA expression in hypothyroid Thra1 PV/+ mice was also lower than that in hypothyroid WT mice (bar 4 versus 3). However, the expression of the Gata1 mRNA in the hyperthyroid in Thra1 PV/+ mice was not significantly increased as compared with that in hypothyroid mice (Fig 7B-a, bar 6 versus bar 4), indicating that TRα1PV expressed in the bone marrow of Thra1 PV/+ mice has lost transcription capacity due to its inability to bind T3.
Consistent with the regulation of the Gata1 gene by T3, similar T3-regulatory patterns in the expression of the Klf1 gene was found in the WT mice (Fig 7B-b). The Klf1 mRNA level was lower in the hypothyroid mice than in euthyroid mice (bar 3 versus bar 1), but was higher in hyperthyroid mice than in hypothyroid mice (Fig 7B-b, bar 5 versus bar 3). The Klf1 mRNA level was lower in euthyroid Thra1 PV/+ mice than in euthyroid WT mice (bar 2 versus 1). No apparent increase in the expression of the Klf1 mRNA level was found in hyperthyroid Thra1 PV/+ mice as compared with hypothyroid Thra1 PV/+ mice (Fig 7B-b, bar 6 versus bar 4) due to the loss of T3 binding activity and transcriptional activity of TRα1PV. Taken together, these results indicate that the Gata1 gene is directly positively regulated by T3/TRα1. Mutations of TRα1, such as TRα1PV, suppressed the expression of the Gata1 gene to impair erythropoiesis in Thra1 PV/+ mice. To further support this notion, we carried out rescue experiments. We exogenously expressed the GATA1 gene in the Lin-bone marrow cells followed by terminal differentiation assay. As shown in Fig 8A, GATA1-tagged with V5 protein was detected by anti-V5 antibodies (lane 2), whereas it was not detected in the bone marrow cells transfected with on the control plasmid (lane 1). FACS analysis shows that exogenously expressed GATA1 did not further increase mature erythrocytes in WT mice, as indicated by Ter119+ with low FSC population (Fig 8B-a versus 8B-b, areas boxed in red). In contrast, exogenously expressed GATA1 led to increase in mature erythrocytes (39% in the Fig 8C-b versus 27% in the Fig 8C-a, areas boxed in red) in Thra1 PV/+ mice. Quantitation graph represent a 43% increase in mature erythrocytes from Thra1 PV/+ mice (Fig 8C, panel c). These data demonstrated that the defective terminal erythropoiesis from Lin-BM in Thra1 PV/+ mice was partially corrected by exogenous expression of GATA1 protein.
We further evaluated whether the exogenously transfected GATA1 as described above could affect the erythroid genes critical for erythropoiesis.  3 in all panels). These results indicated that the elevated expression of these erythroid genes contributed to the partial rescue of the defective erythropoiesis and further support the essential role of GATA1 in mediating the impaired erythropoiesis in Thra1 PV/+ mice.

Discussion
In 2002, we created the Thra1 PV/+ mouse that displayed aspects of the phenotype of hypothyroidism with retarded growth, delayed bone development, and marginally abnormal thyroid function tests [12]. These phenotypic manifestations are distinct from those of the Thrb PV/+ mice that faithfully reproduce resistance to thyroid hormone (RTH) in patients caused by mutations of the THRB gene [41]. The distinct phenotypic manifestations by Thra1 PV/+ mice and Thrb PV/+ mice suggested TR isoform-dependent actions of TR mutants in vivo. However, because of the lack of patients with mutations of the THRA gene at the time, it was not clear whether this postulate held true. The discovery of patients with mutations of the THRA gene in 2012 [8], exhibiting similar hypothyroidism as in the Thra1 PV/+ mouse, has validated that the Thra1 PV/+ mouse is a valuable model to elucidate the molecular basis of hypothyroidism caused by mutated TRα1. Indeed, Thra1 PV/+ mice have been used to predict the outcome of prolonged treatment with a supraphysiologic dose of T4 aimed at ameliorating the skeletal abnormalities in patients [16]. The studies provided valuable information showing that patients with different THRA mutations would display responses to T4 treatment that vary depending on the severity of the causative mutation.
In the present studies, we used Thra1 PV/+ mice to understand the molecular actions of TRα1 mutants that lead to erythroid disorders in patients. We found that Thra1 PV/+ mice exhibited abnormal red blood cell indices, decreased erythroid lineage progenitors in the bone TRα1 mutations disrupt erythropoiesis marrow, and reduction in the terminal differentiation of progenitors in the erythroid lineage. Moreover, for the first time, we have identified a key erythropoietic gene, the Gata1 gene, as a direct TR/T3 target gene by the discovery of thyroid hormone response elements in the promoter region of that gene. Thus, using the Thra1 PV/+ mouse, we have provided direct evidence to indicate that mutations of TRα1 could lead to erythroid disorders and that the disorders are mediated, at least in part, by the suppressed expression of a key erythropoietic regulator, the Gata1 gene, by TRα1 mutants. The present studies have shed new light on the molecular basis of the erythroid disorders found in patients with mutations of the THRA gene.
The critical role of TRα1 was also demonstrated in another mouse model, the Thra -/mouse [42]. Deficiency of TRα1 led to defective fetal and adult erythropoiesis in that erythroid progenitor numbers were decreased in fetal livers; moreover, terminal maturation of erythrocytes was impaired [42]. While the erythroid defects observed in Thra -/mice were similar to those of Thra1 PV/+ mice, the underlying mechanisms would be different. The defective TRα1 mutations disrupt erythropoiesis erythropoiesis in Thra -/mice would be mediated by the lack of TRα1 in regulating the transcription of erythroid-related TRα1 target genes. In contrast, in Thra1 PV/+ mice, the impaired erythropoiesis was due to the dominant negative action of TRα1 mutants on erythroid-related TRα1 target genes, such as the suppression of the T3-positively regulated Gata1 gene demonstrated in the present studies. At present, in Thra -/mice, the erythroid-related TRα1 target genes affected by TRα1 deficiency have not been elucidated. It is difficult to compare the extent and the scope of erythroid abnormalities from the TRα1 deficiency or mutated TRα1 until a comprehensive analysis of global gene expression profiles becomes available. Still, the similar erythroid defects observed in Thra -/mice and Thra1 PV/+ mice clearly highlight the important role of TRα1 in erythropoiesis.
The loss of normal functions of TRα1 due to mutations as in Thra1 PV/+ mice or due to TRα1 deficiency as in Thra -/mice could lead to erythroid disorders as shown in the present studies and in Kendrick et al. [42], respectively. However, that wild-type TRα1 plays a critical role in erythropoiesis was also demonstrated by the findings that thyroid hormones promoted the clonogenic forming ability of BFU-E, CFU-E, and CFU-Mk in hyperthyroid mice shown in the present studies ( Fig 3B). These findings are consistent with the observations from clinical studies, indicating that thyroid hormones play a critical role in erythropoiesis. Qualitative and quantitative studies of erythropoiesis in patients with hyperthyroidism exhibit mild erythrocytosis with erythyroid hyperplasia and increased erythropoietic activity in the bone marrow [43]. In addition, erythrocyte counts, serum erythropoietin, and hypoxia-inducible factor 1α levels patients with untreated Graves' hyperthyroidism were significantly higher than those in the age-and sex-matched healthy controls. Methimazole or subsequent radioiodine therapy of patients with hyperthyroidism reduced erythrocytosis and thyroid function returned to normal, suggesting that thyroid hormone promotes erythrocytosis [44]. Moreover, recent population-based studies on euthyroid subjects revealed a significant positive association between thyroid hormones and erythrocyte indices, such as erythrocyte counts, and hemoglobin levels [45]; [46]. These association studies support the notion that thyroid hormones stimulate erythropoiesis. Still, the molecular mechanisms by which thyroid hormones promote erythropoiesis in hyperthyroidism are not clear. The models of Thra1 PV/+ and Thra -/mice would provide valuable tools for such studies.
Early studies have shown that v-erbA is one of the two oncogenes of the avian erythroblastosis virus (AEV) [47], an acute chicken retrovirus that induces lethal erythroleukemia and sarcoma in vivo. V-erbA is a mutated TRα1, which acts in neoplasia by blocking erythroid differentiation and by altering the growth properties of fibroblasts [47]. Similar to TRα1PV, v-erbA functions as a transcription repressor by dominant negative interference with the transcription activity of its normal cellular homolog, c-erbA (TRα1). While the v-erbB locus alone is sufficient to induce erythroleukemia and sarcoma independent of the v-erbA gene, the v-erbA by itself is not capable of independently causing transformation in either erythroid cells or fibroblasts [47,48], The expression of the v-erbA gene in erythroid cells blocks terminal differentiation and keeps the cells in very immature and highly proliferative stages [47]. Similar to v-erbA, TRα1PV is a potent dominant negative mutant of TRα1. These observations on the functional characteristics of v-erbA raised an important question about whether TRα1PV could act as an oncogene in Thra1 PV/+ mice. Up to now, we have not observed any transformed phenotypes in erythroid cells of Thra1 PV/+ mice. It is likely that another oncogene such as v-erbB (a mutated version of epidermal growth factor receptor; EGFR) would be needed, as in AEV-induced erythroleukemia and sarcoma, to collaborate with TRα1PV to bring out the transformed phenotypes in erythroid cells of Thra1 PV/+ mice. This possibility is testable and awaits future studies.
The present studies have shown that Thra1 PV/+ mice exhibited erythroid disorders with abnormal red blood cell indices, decreased total bone marrow cells, and reduced clonogenic potential of erythroid progenitors. The defective erythropoiesis was mediated by TRα1PVmediated suppression of a key erythropoietic gene (the Gata1 gene), resulting in concurrent repression of other genes involved in the maturation of erythrocytes. Two TREs were identified on the Gata1 gene that responded to T3 differently in the reporter assay. TRα1/T3 via interacting with TRE1 mediated suppression of the transcription whereas TRα1/T3 via interacting with TRE2 activated transcription. It is of interest to point out that located upstream of TRE2 are two E-boxes and one GATA box (GATA1 gene hematopoietic enhancers) critical to the GATA1 gene transcription (see Fig 7A-I). In view of the findings that in vivo, T3 activated the overall transcription of the Gata1 gene as shown in the hyperthyroid WT mice (Fig 7B-a, makes it tempting to speculate that TRE1, though shown to be negatively regulated by T3 in the reporter assay (Fig 7A-II-b), could conceivably be affected by the GATA1 gene hematopoietic enhancers via a long-range looping mechanism, functionally acting as a positive TRE. However, interestingly, in the chicken GATA1 promoter, a negative TRE was identified [49]. TRα1 binds to this TRE as heterodimers with the chicken ovalbumin upstream promoter transcription factor [49], suggesting that the regulatory role of TRα1 in the GATA1 gene transcription is conserved between chicken and mouse.
The identification of the Gata1 gene as a key regulator would suggest that the Gata1 gene or the genes it regulates could potentially be targets for treatment. Moreover, novel TR isoformspecific thyroid hormone analogs are being developed. The Thra1 PV/+ mouse would be a valuable model to test the effectiveness of these potential targets to correct the erythroid disorders.

Mice and treatment
All animal studies were performed according to the approved protocols of the National Cancer Institute Animal Care and Use Committee. The animal study protocol is NCI LMB-036. Mice harboring the mutated Thra1 PV gene (Thra1 PV mice) were prepared and genotyped by PCR as described earlier [12]. Wild-type (Thra1 +/+ ) and Thra1 PV/+ female siblings were used in this study. To induce hypothyroidism, mice were fed a low-iodine diet supplemented with 0.15% propylthiouracil (LoI/PTU) (Cat# TD 95125, Harlan Teklad, Madison, WI) for 10 days. To induce hyperthyroidism, T3 (5 μg; Cat# T2752, Sigma-Aldrich, St. Louis, MO) was injected intraperitoneally to each mouse for 6 days while they were being fed with LoI/PTU diet. The same volume of vehicle (phosphate-buffered saline) was injected in the control group.

Cells
Erythroleukemia K562 cell line was maintained in RPMI1650 (Thermo Fisher Scientific, Waltham, MA) with 10% fetal bovine serum (FBS; GE Healthcare life science, Marlborough, MA) with 50 units/ml penicillin G and 50 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA). Bone marrow cells were isolated from femurs and tibiae of wild-type and Thra1 PV mice (age: 3-5 months). Single cell suspensions were prepared by passing bone marrow through a 70 μM cell strainer.

Peripheral blood profile analysis
For analysis of complete blood counts, peripheral blood was collected in a heparinized microtube and analyzed by hematology analyzer (HEMAVET HV950FS, Drew Scientific, Miami Lakes, FL).

RNA extraction and quantitative RT-PCR
Total RNA was isolated from bone marrow cells using Trizol (Thermo Fisher Scientific, Waltham, MA). RT-qPCR was performed with one step SYBR Green RT-qPCR Master Mix (Qiagen, Valencia, CA). The mRNA level of each gene was normalized to the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA level. The primer sequences are listed in S1 Table. Chromatin immunoprecipitation assays (ChIP) ChIP assay with bone marrow cells was performed as described previously [51]. Quantitative PCR was performed to detect the upstream fragment in Gata1 genes (primer sequences are listed in S1 Table). The fold of changes in binding was relative to the control of IgG level as 1.

Plasmid constructs, and transient transfection
The Gata1 TRE2 and Gata1 TRE1 luciferase constructs were generated by cloning upstream Gata1 promoter fragments into the pGL4.23 luciferase plasmid. The Gata1-TRE2 luciferase construct was made by insertion of a 1.553 kb XhoI-HindIII fragment representing the sequences between -3.741 kb and -2.189 kb. The Gata1-TRE1 luciferase construct was made by insertion of a 140 bp XhoI-HindIII fragment representing the sequences between +1.385 kb and +1.524 kb. Insert sequence validated by DNA sequencing. K562 cells were transfected with the TRE-luc reporters with Genepulse X cell electrophorators (Biorad). Bone marrow cells (2X10 6 cells) were transfected with the GATA1-pLenti6/V5 plasmid (3 μg) provided by Dr. GP Rodgers (NHLBI; [53]) using 4D nucleofector (Lonza) in accordance with the manufacturer's instructions. The Gata1 reporter plasmids cloned in pGL3 basic (5 μg), and the expressing plasmid for TRα1 (pcDNA3.1-TRα1; 10 μg) with or without the expression plasmid for TRα1PV (pcDNA3.1-TRα1PV; 80 μg) were transfected into K562 cells according to Guigon et al [54]. Luciferase activity was measured using Victor 3 (PerkinElmer Life and Analytical Sciences, Waltham, MA). Luciferase values were standardized to the ratio of β-galactosidase activity and protein concentration. The fold of changes in activity was based on using the values of negative control (no plasmid transfected cell without T3) as 1.

In vitro terminal erythropoiesis assay
For lineage depleted bone marrow cell preparation, linage marker positive cells were depleted using the biotin based selection kit (cat# 19856, STEMCELL Technologies, Vancouver, BC) according to the manufacturer's instructions. Lin-BM cells were seeded in fibronectin-coated wells (Corning Inc, Corning, NY). To induce erythropoiesis, Lin-BM cells were cultured as described [24].

Flow cytometry analysis
All antibodies used in flow cytometry were from eBiosciences (Thermo Fisher Scientific, Waltham, MA). The sources of antibodies and fluorophore-labeled antibodies used in FACS analyses are listed in S2 Table. The flow cytometry analyses were performed on a BD LSR II flow cytometer (BD bioscience, San Jose, CA) and analyzed with FloJo, LLC (Tree Star Inc, Ashland, OR).

Western blot analysis and co-immunoprecipitation
The western blot analysis of bone marrow lysates was carried as described previously [50]. To determine the GATA1 tagged with V5 (The V5 tag is derived from a small epitope found on the P and V proteins of the paramyxovirus of simian virus 5 (SV5). after transfection with GATA1-V5 expression plasmid, anti-V5 antibodies (1:2000 dilution; Thermo Fisher Scientific) was used to detect the expressed GATA1-V5. GAPDH (1:4000 dilution; Cell Signaling Technology (Danvers, MA) was used as a loading control.

Statistical analysis
All statistical analyses and the graphs were performed and generated using GraphPad Prism version 6.0 (GraphPad Software, La Jolla, CA). P < 0.05 is considered statistically significant. All data are expressed as mean ± SEM.
Supporting information S1 Table. The primer sequences used for the determination of expression levels of the gene listed in the Table. (DOCX) S2 Table. The sources of antibodies used in FACS analysis. (DOCX)