RPL11 Haploinsufficient Mice Have a CFU-E/Proerythroblast Block, Elevated Erythroblast Heme, Reduced Gata1, and Increased Ribosomal Protein Gene Expression

Abstract Diamond-Blackfan anemia (DBA) is associated with hypomorphic mutations in at least 16 ribosomal proteins. Additionally, mutations in GATA1 that result in the preferential expression of variants lacking the transactivation domain cause a similar but not identical disease. Ribosomal haploinsufficiency can lead to imbalanced ribosome protein production resulting in the activation of p53 and additionally slows protein translation, including globin, resulting in excess heme during the early stages of erythropoiesis when heme synthesis (an enzymatic process) is brisk but globin levels are insufficient. Mice lacking the heme export protein FLVCR1 develop a severe macrocytic anemia similar to DBA resulting from a block in differentiation at the CFU-E/proerythroblast stage which is caused by excess heme and ROS (J Clin Invest 125:4681, 2015). This macrocytic anemia occurs independent of ribosomal haploinsufficiency or p53 activation, suggesting that elevated heme is a key factor in the pathophysiology of DBA. Indeed, erythroid cultures of marrow from individuals with DBA demonstrate delayed globin synthesis, excess heme, elevated ROS, and increased cell death of CFU-E/proerythroblasts (Sci Transl Med 8:338RA67, 2016). Rescue studies showed in vitro erythroid differentiation improved when heme synthesis was decreased. To further understand the role of heme and ribosomal haploinsufficiency in DBA, we are characterizing RPL11 haploinsufficient mice. RPL11 heterozygous mice develop a cell intrinsic macrocytic anemia with increased susceptibility to radiation-induced lymphomagenesis (Cell Reports 13:712, 2015). Our studies confirm RPL11 heterozygous mice have a chronic macrocytic anemia (HGB 12.0±1.7 vs 14.3±0.4 g/dL; MCV 58.0±2.2 vs 46.8±1.4 fL) concurrent with a block at the CFU-E/proerythroblasts stage (63% reduction in basophilic erythroblasts). Erythroblast heme content is 2-fold higher than control by the polychromatic erythroblast stage while ROS is elevated (15-75%) throughout terminal differentiation. To understand the pathophysiology leading to ineffective erythropoiesis in DBA we performed single cell RNA sequencing and cell surface protein quantification on erythroid precursors from control and Flvcr1 -deleted mice and are comparing these data to data from RPL11 haploinsufficient mice. Principal component analysis identified 4 transcriptionally unique clusters with negative, low, intermediate, and high Ter119 levels respectively. α- and β-globin transcription were highly correlated (r=0.975) and increased as Ter119 expression increased. Gene set enrichment analysis comparing control cells to Flvcr1 -deleted cells revealed significant upregulation of the ribosome pathway genes and downregulation of the hallmark heme metabolism pathway genes including GATA1 and GATA1-target genes. Quantitative PCR analysis of RPL11 haploinsufficient erythroid cells show 2-fold increases in Rps19, Rps14, Rpl4, and Rpl35 transcript levels during terminal erythroid differentiation, however, Rpl11 transcript levels are reduced 50% in precursor cells and fail to increase to comparable levels with other ribosomal protein genes. Cdkn1a was increased, consistent with activation of the p53 pathway. Comparable studies with Flvcr1 -deleted mice do not show any activation of the p53 pathway, indicating that p53 pathway activation is unique to ribosomal haploinsufficiency and not a result of excess heme. Both Flvcr1 -deleted and RPL11 haploinsufficient erythroid cells have reduced Gata1 expression. To resolve the role of heme from other driving factors, we tested the effect of ALA and iron treatment to induce endogenous heme synthesis in sorted human marrow cells. Within 15 minutes of treatment the early erythroid cells (Lin-CD36+GlyA-) upregulated ribosomal gene transcript levels while later erythroid cells (Lin-CD36+GlyA+) did not. Additionally, GATA1 protein levels were rapidly decreased by ALA and iron, but not by exogenous heme or iron treatment alone. Thus poor translation of globin in ribosomal haploinsufficiencies leads to excess heme. It is this excess heme which leads to premature termination of erythroid differentiation by reducing GATA1 levels, additionally, it exacerbates ribosomal protein imbalance, increasing p53 activation and cell death. Thus the key pathologies in DBA are a direct result of excess heme, suggesting new approaches for treatment. Disclosures No relevant conflicts of interest to declare.

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The Interplay of GATA1 with Heme Regulates an Erythroid Cell's Differentiation

Blood ◽

10.1182/blood.v128.22.541.541 ◽

2016 ◽

Vol 128 (22) ◽

pp. 541-541

Author(s):

Raymond T Doty ◽

Xiaowei Yan ◽

Christopher Lausted ◽

Zhantao Yang ◽

Li Liu ◽

...

Keyword(s):

Ribosomal Protein ◽

Aminolevulinic Acid ◽

Macrocytic Anemia ◽

Erythroid Differentiation ◽

Gene Set Enrichment Analysis ◽

Erythroid Cells ◽

Ineffective Erythropoiesis ◽

P53 Pathway ◽

Heme Synthesis ◽

Marrow Cells

Abstract GATA1 promotes the transcription of ALAS2, the first and rate limiting step of heme synthesis, and the transcription of many other erythroid-specific genes. It also increases its own transcription while silencing proliferation genes active in early progenitors and thus assures that erythroid differentiation correctly initiates. Heme then transcriptionally and translationally upregulates globin to guarantee adequate hemoglobin production in each cell as it matures. In mice lacking the heme exporter, FLVCR1, excess heme and ROS accumulate and erythropoiesis fails at the CFU-E/proerythroblast stage, resulting in a severe macrocytic anemia (HGB 4.4±0.97 vs 14.8±0.57 g/dL; MCV 66.9±6.2 vs 48.4±0.65 fL). To determine how excess heme causes ineffective erythropoiesis and whether heme is key to terminating differentiation in normal erythroid cells, we performed RNA sequencing of single early erythroid cells (BFU-E to basophilic erythroblasts) from wildtype control and Flvcr1-deleted mice and linked this transcription data to the total quantity of Ter119 on that cell. Principal component analysis (PCA) identified 4 transcriptionally unique clusters A, B, C, & D, which contained cells with negative, low, intermediate, and high Ter119 levels respectively. α- and β-globin transcription were highly correlated (r=0.975), occurred in all cells, increased as Ter119 expression increased, and upregulated in Flvcr1-deleted cells. Gene set enrichment analysis (GSEA) comparing control cells to Flvcr1-deleted cells revealed excess heme results in significant downregulation of the hallmark heme metabolism pathway genes (heme biosynthesis and erythroid differentiation genes), upregulation of the ribosome pathway genes, and no alteration of the P53 pathway genes. All eight heme biosynthetic enzyme genes were expressed equivalently in cluster A cells from control and Flvcr1-deleted mice; however expression in Flvcr1-deleted cells was significantly reduced in clusters B-D. Of the 181 erythroid differentiation genes in the hallmark heme pathway, Gata1 had the greatest reduction (67%) in Flvcr1-deleted cells. Coupled two-way clustering analysis (CTWC) identified 150 genes co-regulated with Gata1 including 106 known GATA1 target genes which were all poorly upregulated in Flvcr1-deleted cells in clusters B-D. Independent microarray analysis of mRNA from control and Flvcr1-deleted CD71+ erythroid cells confirmed low Gata1 mRNA and low GATA1-dependent gene expression in the Flvcr1-deleted cells. To determine if excess heme was directly responsible for Gata1 downregulation, we treated K562, HEL-R, and primary human erythroid marrow cells with aminolevulinic acid (ALA) and iron to increase endogenous heme synthesis. In the primary cells, GATA1 protein decreased by 30-43% (p=0.03) within 15 minutes and 66% by 90 minutes (similar decreases observed in cell lines), suggesting that heme disrupts GATA1 protein function resulting in the loss of autoregulation and reduced GATA1 mRNA. Of 88 genes in the ribosome pathway, 73 were significantly upregulated in Flvcr1-deleted cells, including 16 of the 17 ribosomal protein genes linked to Diamond-Blackfan anemia (DBA) or del(5q) myelodysplastic syndrome (MDS). When heme synthesis was induced in primary human erythroid marrow cells with ALA and iron, the transcription of ribosome protein genes such as Rps19, Rps14, and Rpl35 increased, further supporting the concept that heme assures sufficient ribosome production for globin protein synthesis. While P53 activation is a key factor in ineffective erythropoiesis caused by ribosomal protein imbalance (i.e., DBA and del(5q) MDS), GSEA did not reveal any increased activation of the P53 pathway in Flvcr1-deleted cells. To confirm that P53 was not involved in the ineffective erythropoiesis caused by excess heme, we generated mice lacking both P53 and FLVCR1. These double mutant mice had severe macrocytic anemia (HGB 2.4±0.70 g/dL; MCV 56.5±4.3 fL) comparable to mice lacking just FLVCR1. Thus, GATA1 turns on heme synthesis and initiates the erythroid differentiation program. GATA1 with heme assure each cell's appropriate progression. Then heme turns off GATA1 to end differentiation. By linking excess heme to prematurely low GATA1, our data may also explain the ineffective (early termination of) erythropoiesis in DBA and reconcile the observations of Sci Transl Med 8:338ra67, 2016 and Nat Med 20:748, 2014. Disclosures No relevant conflicts of interest to declare.

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Single-cell analyses demonstrate that a heme–GATA1 feedback loop regulates red cell differentiation

Blood ◽

10.1182/blood-2018-05-850412 ◽

2019 ◽

Vol 133 (5) ◽

pp. 457-469 ◽

Cited By ~ 8

Author(s):

Raymond T. Doty ◽

Xiaowei Yan ◽

Christopher Lausted ◽

Adam D. Munday ◽

Zhantao Yang ◽

...

Keyword(s):

Ribosomal Protein ◽

Single Cell ◽

Aminolevulinic Acid ◽

Developmental Trajectories ◽

Erythroid Differentiation ◽

Surface Proteins ◽

Red Cell ◽

Erythroid Cells ◽

Heme Synthesis ◽

Globin Synthesis

Abstract Erythropoiesis is the complex, dynamic, and tightly regulated process that generates all mature red blood cells. To understand this process, we mapped the developmental trajectories of progenitors from wild-type, erythropoietin-treated, and Flvcr1-deleted mice at single-cell resolution. Importantly, we linked the quantity of each cell’s surface proteins to its total transcriptome, which is a novel method. Deletion of Flvcr1 results in high levels of intracellular heme, allowing us to identify heme-regulated circuitry. Our studies demonstrate that in early erythroid cells (CD71+Ter119neg-lo), heme increases ribosomal protein transcripts, suggesting that heme, in addition to upregulating globin transcription and translation, guarantees ample ribosomes for globin synthesis. In later erythroid cells (CD71+Ter119lo-hi), heme decreases GATA1, GATA1-target gene, and mitotic spindle gene expression. These changes occur quickly. For example, in confirmatory studies using human marrow erythroid cells, ribosomal protein transcripts and proteins increase, and GATA1 transcript and protein decrease, within 15 to 30 minutes of amplifying endogenous heme synthesis with aminolevulinic acid. Because GATA1 initiates heme synthesis, GATA1 and heme together direct red cell maturation, and heme stops GATA1 synthesis, our observations reveal a GATA1–heme autoregulatory loop and implicate GATA1 and heme as the comaster regulators of the normal erythroid differentiation program. In addition, as excessive heme could amplify ribosomal protein imbalance, prematurely lower GATA1, and impede mitosis, these data may help explain the ineffective (early termination of) erythropoiesis in Diamond Blackfan anemia and del(5q) myelodysplasia, disorders with excessive heme in colony-forming unit-erythroid/proerythroblasts, explain why these anemias are macrocytic, and show why children with GATA1 mutations have DBA-like clinical phenotypes.

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Delayed Globin Synthesis Leads to Excessive Heme and the Macrocytic Anemia of Diamond Blackfan Anemia and del(5q) Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v130.suppl_1.sci-18.sci-18 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. SCI-18-SCI-18

Author(s):

Janis L. Abkowitz ◽

Raymond T Doty ◽

Zhantao Yang ◽

Xiaowei Yan ◽

Christopher Lausted ◽

...

Keyword(s):

Ribosomal Protein ◽

Macrocytic Anemia ◽

Protein Translation ◽

Cell Surface Expression ◽

Red Cell ◽

Erythroid Cells ◽

P53 Expression ◽

Diamond Blackfan Anemia ◽

Heme Synthesis ◽

Globin Synthesis

Abstract Thirty percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of ribosomal protein S19 and ~40% from haploinsufficiencies of 15 other ribosomal proteins. The macrocytic anemia of myelodysplasia with deletion of chromosome 5q (del(5q)MDS), which results from the acquisition of RPS14 haploinsufficiency, has a similar clinical phenotype. Although these mutations disrupt ribosome assembly and impair protein translation, how this causes macrocytic anemia remains uncertain and controversial. Since 95% of the protein content of red cells is globin, we hypothesized that any germline or somatic mutation that slows protein synthesis would impair globin production relative to heme production. This is because the synthesis of heme, a chemical chelate, depends on only small amounts of protein (enzymes) and the rate limiting enzyme, ALAS2, is an early GATA1 target. Studies of marrow cells from patients with DBA and del(5q) MDS show that heme synthesis indeed progresses normally, while globin synthesis is delayed. This results in excess heme in CFU-E/proerythroblasts, excessive ROS and cell death (Sci Transl Med 8:338RA67, 2016). Similar results are seen in a murine model of heme excess (Flvcr1 -deletion) (J Clin Invest 125:4681, 2015) and murine models of DBA. As slowing heme synthesis improves the coordination of heme and globin and improves red cell production, a phase 2 study is underway (PI Bart Scott) to determine the efficacy of aggressive iron chelation to slow heme synthesis in patients with very low to intermediate risk MDS and anemia. More recently, we have quantitated the cell surface expression of CD71, CD44 and Ter119 on individual murine erythroid cells from normal, Flvcr1 -deleted mice with macrocytic anemia, and erythropoietin-treated mice. We then barcoded and assessed the cell's total transcriptome. By linking these datasets, we uncovered a GATA1-heme autoregulatory loop which regulates normal erythropoiesis and contributes to the failed erythropoiesis of ribosomal protein haploinsufficiency. We show that heme normally upregulates ribosome protein transcription in early erythroid cells. Thus, in addition to increasing globin transcription and translation (via Bach1 and HRI), heme assures adequate ribosomes for globin synthesis. In later erythroid cells, heme decreases GATA1, GATA1 target genes and mitotic spindle gene expression, assuring that red cell differentiation appropriately terminates and cell division ceases. In human marrow CD36+GlyA- or CD36+GlyA+ cells, these changes occur within 15 minutes of inducing endogenous heme synthesis with ALA (bypasses ALAS2) and iron. As excess heme would increase ROS, increase ribosomal protein imbalance to intensify P53 expression, prematurely lower GATA1, and impede mitosis, our data explain the ineffective (early termination of) erythropoiesis in DBA and del(5q) MDS, help explain why these anemias are macrocytic, and reconcile the disparate observations of others. Disclosures No relevant conflicts of interest to declare.

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Trafficking Kinesin Binding Protein Is Essential for Human Erythropoiesis

Blood ◽

10.1182/blood.v118.21.683.683 ◽

2011 ◽

Vol 118 (21) ◽

pp. 683-683

Author(s):

Seung-Jae Noh ◽

Y.Terry Lee ◽

Colleen Byrnes ◽

Antoinette Rabel ◽

Jeffery L. Miller

Keyword(s):

Conflicts Of Interest ◽

Ex Vivo ◽

Annexin V ◽

K562 Cells ◽

Erythroid Differentiation ◽

Mitochondrial Outer Membrane ◽

Slight Reduction ◽

Erythroid Cells ◽

Heme Synthesis

Abstract Abstract 683 Mitochondrial specialization in erythroblasts is important for efficient heme synthesis, with defects or reduced expression of several mitochondrial proteins causing anemia. Trafficking kinesin binding 2 (TRAK2) is known to participate in mitochondrial movement along microtubule by interacting with kinesin motor protein and making a complex with Miro that is localized on the mitochondrial outer membrane. Transcriptome data suggest that TRAK2 is highly and specifically expressed in early erythroid cells. Here the role of TRAK2 was studied among human CD34+ cells that were grown in ex vivo serum-free cultures supplemented with erythropoietin (EPO, total culture period 21 days). Quantitative PCR studies indicated that TRAK2 expression is highly regulated during erythropoiesis. Its expression pattern was nearly identical to aminolevulinate synthase 2, the erythroid specific enzyme for the committed step of the heme biosynthetic pathway, and mitoferrin 1, the erythroid specific mitochondrial iron transporter. Western analyses revealed that TRAK2 protein is detected as a doublet band with molecular weights of 130kD and 105kD. Mitochondrial co-localization of TRAK2 was verified by confocal microscopy in TRAK2-overexpressing K562 cells. To study a potential role of TRAK2 in erythropoiesis, TRAK2 expression was reduced in cultured human erythroid cells using lentiviral shRNA transduction. TRAK2 knockdown (TRAK2-KD) was confirmed by Western analysis in K562 cells. In primary erythroblasts, TRAK2-KD caused slight reduction of CD36+ immature erythroblasts at culture day 7 prior to the addition of EPO (CD36+ population 58% in control vs 40% in TRAK2-KD). After the addition of erythropoietin to the culture medium, TRAK2-KD severely restricted erythroblast proliferation (5.0 million cells/ml in control vs 0.25 million cells/ml in TRAK2-KD on culture day 18). Flow cytometric analyses showed that <1% of the CD36+ progenitors cells differentiated into glycophorin A erythroblasts compared with >90% in control cultures. Annexin-V staining indicated that more than 90% of cells had undergone apoptosis by day 14. These data suggest that TRAK2 expression is required for erythroid differentiation. As such, defects in TRAK2 expression should be considered in cases of unexplained anemia. The data also support the notion that mitochondrial location or mobility within erythroblasts may be important for iron trafficking or heme synthesis. Disclosures: No relevant conflicts of interest to declare.

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Regulation of hemoglobin synthesis and proliferation of differentiating erythroid cells by heme-regulated eIF-2α kinase

Blood ◽

10.1182/blood.v96.9.3241 ◽

2000 ◽

Vol 96 (9) ◽

pp. 3241-3248 ◽

Cited By ~ 30

Author(s):

John S. Crosby ◽

Peter J. Chefalo ◽

Irene Yeh ◽

Shong Ying ◽

Irving M. London ◽

...

Keyword(s):

Protein Synthesis ◽

Erythroid Differentiation ◽

Dominant Negative ◽

Erythroid Cells ◽

3T3 Cells ◽

Binding Domains ◽

Inhibition Of Protein Synthesis ◽

Nih 3T3 ◽

Hemoglobin Production

Abstract Protein synthesis in reticulocytes depends on the availability of heme. In heme deficiency, inhibition of protein synthesis correlates with the activation of heme-regulated eIF-2α kinase (HRI), which blocks the initiation of protein synthesis by phosphorylating eIF-2α. HRI is a hemoprotein with 2 distinct heme-binding domains. Heme negatively regulates HRI activity by binding directly to HRI. To further study the physiological function of HRI, the wild-type (Wt) HRI and dominant-negative inactive mutants of HRI were expressed by retrovirus-mediated transfer in both non-erythroid NIH 3T3 and mouse erythroleukemic (MEL) cells. Expression of Wt HRI in 3T3 cells resulted in the inhibition of protein synthesis, a loss of proliferation, and eventually cell death. Expression of the inactive HRI mutants had no apparent effect on the growth characteristics or morphology of NIH 3T3 cells. In contrast, expression of 3 dominant-negative inactive mutants of HRI in MEL cells resulted in increased hemoglobin production and increased proliferative capacity of these cells upon dimethyl-sulfoxide induction of erythroid differentiation. These results directly demonstrate the importance of HRI in the regulation of protein synthesis in immature erythroid cells and suggest a role of HRI in the regulation of the numbers of matured erythroid cells.

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Hemin Augments Growth and Hemoglobinization of Erythroid Precursors Derived from Patients with Diamond-Blackfan Anemia (DBA).

Blood ◽

10.1182/blood.v112.11.2037.2037 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2037-2037

Author(s):

Eitan Fibach ◽

Memet Aker

Keyword(s):

De Novo ◽

Therapeutic Potential ◽

Early Stage ◽

Macrocytic Anemia ◽

Erythroid Cells ◽

Primary Defect ◽

Two Phase ◽

Differentiation Potential ◽

Heme Synthesis ◽

Erythroid Precursors

Abstract DBA is a congenital form of pure red cell anemia characterized by a macrocytic anemia, reticulocytopenia, and a block in erythroid differentiation at the proerythroblast stage, often in association with physical anomalies and growth retardation. About 25% of the patients carry mutations in genes that encode for proteins (RPS19, RPS24 and RPS17) that bind to the 40S subunit of the ribosome. The resultant defect in ribosomal biogenesis has been proposed to impair the initiation of globin translation, leading to mismatch between intracellular levels of heme and globin chains. It has been hypothesized that the transient excess of intracellular free heme resulting from the delay in globin synthesis exerts direct toxicity to erythroid precursors and plays a major role in pathogenesis of DBA through apoptosis of proerythroblasts (Keel et al., Science319;825,2008). Free hemin, however, is not necessarily toxic to developing erythroid precursors. Exogenously supplied hemin is readily taken up by erythroid cells in culture and its iron is incorporated into hemoglobin or stored in ferritin (Fibach et al., J Cell Physiol130;460,1987). Following addition of succinylacetone, a potent inhibitor of heme synthesis, exogenously supplied hemin can replace intracellularly synthesized heme and be incorporated into de novo formed hemoglobin (Fibach et al., Blood85;2967,1995). Hemin supplementation to semi-solid cultures promotes the growth of normal erythroid precursors (e.g., Lu and Broxmeyer, Exp Hematol11;721,1983). We showed in a two-phase liquid culture that exogenous hemin promotes normal erythropoiesis by accelerating the proliferation and hemoglobinization of erythroid precursors in the presence or absence of transferrin (Fibach et al., Blood85;2967,1995). This effect was particularly prominent during the early stages of hemoglobinization, when iron-uptake and heme synthesis are rate-limiting. In the present study we show that surplus hemin (10 - 50 mM) supplemented to cultures at early stage of erythroid development is well tolerated. Although the generation of reactive oxygen species (measured by staining with dichlorofluorescein diacetate) was modestly (50 ± 15%, N=4) increased, it was not associated with increased apoptosis, as measured by binding of annexin V, nor necrosis as measured by propidium iodide staining. Having demonstrated the growth and differentiation promoting potential of exogenous hemin on normal erythroid precursors and lack of overt toxicity, we studied the effect of exogenous heme in cultures of erythroid cells derived from six patients with DBA. We show that hemin, added as heme chloride or heme arginate, circumvented the primary defect and significantly stimulated (4 - 20-fold, p<0.001) ) the growth of DBA erythroid cells and their hemoglobinization. In conclusion, our results show that exogenous hemin is taken up by developing erythroid cells and can supplement or substitute endogenously synthesized heme; excess heme stimulates free radical generation moderately but does not cause apoptosis or necrosis; addition of hemin to cultured erythroid precursors derived from normal donors stimulates their growth and hemoglobinization, and in DBA, in contrast to the recently proposed scheme, heme can actually restore the growth and differentiation potential of the DBA-erythroid precursors. The beneficial effect of hemin on DBA erythroid precursors may be related to its effect on translation initiation factors, such as eIF-2 , and suggests a therapeutic potential.

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TAF10 Interacts with GATA1 Transcription Factor and Controls Mouse Erythropoiesis

Blood ◽

10.1182/blood.v124.21.2912.2912 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2912-2912

Author(s):

Petros Papadopoulos ◽

Laura Gutierrez ◽

Jeroen Demmers ◽

Dimitris Papageorgiou ◽

Elena Karkoulia ◽

...

Keyword(s):

Transcription Factor ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Fetal Liver Cells ◽

Gata1 Transcription Factor

Abstract The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs) is a prerequisite for the transcription of protein coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA (Spt-Ada-Gcn5-acetyltransferase), are also necessary to have tightly regulated transcription initiation. However, a new era on the role of the GTFs and specifically on the role of TFIID in tissue specific and promoter specific transcriptional regulation has emerged in the light of novel findings regarding the differentiation programs of different cell types1. TAF10 is a subunit of both the TFIID and the SAGA co-activator HAT complexes2. The role of TAF10 is indispensable for early embryonic transcription and mouse development as knockout (KO) embryos die early in gestation between E3.5 and E5.5, around the stage when the supply of maternal protein becomes insufficient3. However, when analyzing TFIID stability and transcription it was noted that not all cells and tissues were equally affected by the loss of TAF10. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. Ablation of TAF10 specifically in erythroid cells by crossing the TAF10-Lox with the EpoR-Cre mouse led to a differentiation block at around E13.5 with erythroid progenitor cells accumulating at a higher percentage (26% in the KO embryos vs 16% in the WTs at E12.5) at the double positive stage KIT+CD71+ and giving rise to fewer mature TER119+ cells in the fetal liver. At E13.5 embryos were dead with almost no erythroid cells in the fetal liver. Gene expression analysis of the fetal liver cells of the embryos revealed down-regulation of GATA1 expression and its target genes, bh1&bmaj/min globins and KLF1 transcription factor while expression of other genes known to have a role in mouse hematopoiesis remained unaffected (MYB, GATA2, PU.1). In order to get insight to the role of TAF10 during erythropoiesis we analyzed the composition of both TAF10-containing complexes (TFIID and SAGA) by mass spectrometry. We found that their stoichiometry changes slightly but not fundamentally during erythroid differentiation and development (human fetal liver erythroid progenitors, human blood erythroid progenitors and mouse erythroid progenitor cells) and no major rearrangements were generated in the composition of the TFIID as it was reported in other cell differentiation programs (e.g. skeletal differentiation, hepatogenesis). Additionally, we found GATA1 transcription factor only in the fetal liver and not in the adult erythroid cells in the mass spectrometry data of TAF10 immunoprecipitations (IPs), an interaction that we confirmed by reciprocal IP of TAF10 and GATA1 in MEL and mouse fetal liver cells. Most importantly, we checked whether TAF10 binding is enriched on the GATA1 locus in human erythroid cells during the fetal and the adult stage in erythroid proerythroblasts and we found that there is enriched binding of TAF10 in the palindromic GATA1 site in the fetal stage. Our results support a developmental role for TAF10 in GATA1 regulated genes, including GATA1 itself, during erythroid differentiation emphasizing the crosstalk between the transcriptional machinery and activators in erythropoiesis. References 1. Goodrich JA, Tjian R (2010) Unexpected roles for core promoter recognition factors in cell-type-specific transcription and gene regulation. Nature reviews Genetics 11: 549-558 2 .Timmers HT, Tora L (2005) SAGA unveiled. Trends Biochem Sci 30: 7-10 3. Mohan WS, Jr., Scheer E, Wendling O, Metzger D, Tora L (2003) TAF10 (TAF(II)30) is necessary for TFIID stability and early embryogenesis in mice. Mol Cell Biol 23: 4307-4318 Disclosures No relevant conflicts of interest to declare.

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Cell-intrinsic requirement for pRb in erythropoiesis

Blood ◽

10.1182/blood-2004-02-0618 ◽

2004 ◽

Vol 104 (5) ◽

pp. 1324-1326 ◽

Cited By ~ 42

Author(s):

Allison J. Clark ◽

Kathryn M. Doyle ◽

Patrick O. Humbert

Keyword(s):

Erythroid Differentiation ◽

Published Data ◽

Erythroid Cells ◽

Cell Cycle Exit ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation ◽

A Cell ◽

Rb Gene

Abstract Retinoblastoma (Rb) and family members have been implicated as key regulators of cell proliferation and differentiation. In particular, accumulated data have suggested that the Rb gene product pRb is an important controller of erythroid differentiation. However, current published data are conflicting as to whether the role of pRb in erythroid cells is cell intrinsic or non–cell intrinsic. Here, we have made use of an in vitro erythroid differentiation culture system to determine the cell-intrinsic requirement for pRb in erythroid differentiation. We demonstrate that the loss of pRb function in primary differentiating erythroid cells results in impaired cell cycle exit and terminal differentiation. Furthermore, we have used coculture experiments to establish that this requirement is cell intrinsic. Together, these data unequivocally demonstrate that pRb is required in a cell-intrinsic manner for erythroid differentiation and provide clarification as to its role in erythropoiesis.

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Elucidation of the Role of LMO2 (LIM-only protein 2) in Erythroid Cells

Blood ◽

10.1182/blood.v120.21.3443.3443 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3443-3443

Author(s):

AI Inoue ◽

Tohru Fujiwara ◽

Yoko Okitsu ◽

Noriko Fukuhara ◽

Yasushi Onishi ◽

...

Keyword(s):

Dna Binding ◽

Cord Blood ◽

Western Blotting ◽

Target Genes ◽

K562 Cells ◽

Erythroid Differentiation ◽

Dependent Manner ◽

Erythroid Cells ◽

In Erythroid Cells

Abstract Abstract 3443 Background: Developmental control mechanisms often utilize multimeric complexes containing transcription factors, coregulators, and additional non-DNA binding components. It is challenging to ascertain how such components contribute to complex function at endogenous loci. LMO2 (LIM-only protein 2) is a non-DNA binding transcriptional coregulator, and is an important regulator of hematopoietic stem cell development and erythropoiesis, as mice lacking this gene show defects in blood formation as well as fetal erythropoiesis (Warren et al. Cell. 1994). In the context of erythropoiesis, LMO2 has been demonstrated to be a part of multimetric complex, including master regulators of hematopoiesis (GATA-1 and SCL/TAL1), chromatin looping factor LDB1 and hematopoietic corepressor ETO2 (referred as GATA-SCL/TAL1 complex). As LMO2 controls hematopoiesis, its dysregulation is leukemogenic, and its influence on GATA factor function is still not evident, we investigated here the transcriptional regulatory mechanism via LMO2 in erythroid cells. Methods: For LMO2 knockdown, anti-LMO2 siRNA (Thermo Scientific Dharmacon) and pGIPZ lentiviral shRNAmir system (Open Biosystems) were used. Western blotting and Quantitative ChIP analysis were performed using antibodies for GATA-1, LMO2 (abcam), GATA-2, TAL1 and LDB1 (Santa Cruz). To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in liquid suspension culture. For transcription profiling, human whole expression array was used (Agilent), and the data was analyzed with GeneSpring GX software. To induce erythroid differentiation of K562 cells, hemin was treated at a concentration of 30 uM for 24h. Results: siRNA-mediated LMO2 knockdown in hemin-treated K562 cells results in significantly decreased ratio of benzidine-staining positive cells, suggesting that LMO2 has an important role in the erythroid differentiation of K562 cells. Next, we conducted microarray analysis to characterize LMO2 target gene ensemble in K562 cells. In contrast to the predominantly repressive role of LMO2 in murine G1E-ER-GATA-1 cells (Fujiwara et al. PNAS. 2010), the analyses (n = 2) demonstrated that 177 and 78 genes were upregulated and downregulated (>1.5-fold), respectively, in the LMO2-knockdowned K562 cells. Downregulated gene ensemble contained prototypical erythroid genes such as HBB and SLC4A1 (encodes erythrocyte membrane protein band 3). To test what percentages of LMO2-regulated genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n= 5,749, Fujiwara et al. Mol Cell. 2009), and demonstrated that 26.4% and 23.1% of upregulated and downregulated genes, respectively, contained significant GATA-1 peaks in their loci. Furthermore, whereas LMO2 knockdown in K562 cells did not affect the expression of GATA-1, GATA-2 and SCL/TAL1 based on quantitative RT-PCR as well as Western blotting, the knockdown resulted in the significantly decreased chromatin occupancy of GATA-1, GATA-2, SCL/TAL1 and LDB1 at beta-globin locus control region and SLC4A1 locus. We subsequently analyzed the consequences of LMO2 knockdown in primary erythroblasts. Endogeneous LMO2 expression was upregulated along with the differentiation of cord blood cell-derived primary erythroblasts. shRNA-mediated knockdown of LMO2 in primary erythroblasts resulted in significant downregulation of HBB, HBA and SLC4A1. Conclusion: Our results suggest that LMO2 contributes to the expression of GATA-1 target genes in a context-dependent manner, through modulating the assembly of the components of GATA-SCL/TAL1 complex at endogeneous loci. Disclosures: No relevant conflicts of interest to declare.

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Deciphering the Cis- and Trans-regulatory Roles of KLF6 in Primitive Hematopoiesis

Blood ◽

10.1182/blood.v120.21.4730.4730 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4730-4730

Author(s):

Qian Xiong ◽

Zhaojun Zhang ◽

Hongzhu Qu ◽

Xiuyan Ruan ◽

Hai Wang ◽

...

Keyword(s):

Expression Pattern ◽

K562 Cells ◽

Erythroid Differentiation ◽

Hek293 Cells ◽

Luciferase Reporter ◽

Erythroid Cells ◽

Dynamic Expression ◽

Primitive Hematopoiesis ◽

Cis And Trans

Abstract Abstract 4730 Krüppel-like factors (KLFs) are a conserved family of Cys2His2 zinc finger proteins which are important components of eukaryotic cellular transcriptional machinery that controls many biological processes including erythroid differentiation and development. As a transcriptional activator and a tumor suppressor, KLF6 was also involved in hematopoiesis. Klf6−/− mice is embryonic lethal by embryonic day 12.5 and associated with markedly reduced hematopoiesis as well as poorly organized yolk sac vascularization. Moreover, the expression of erythroid differentiation markers including Klf1, Gata1 and Scl are delayed and hematopoietic differentiation is impaired in klf6−/− ES cells. However, the detailed mechanism that KLF6 regulates hematopoiesis is not fully understood. To characterize the role of KLF6 in hematopoiesis, we firstly detected the dynamic expression pattern of KLF6 during erythroid differentiation by mRNA-seq in undifferentiated human embryonic stem cells (hESC), three primary erythroid cells at different developmental stages including ES-derived erythroid cells (ESER), fetal- and adult-type erythroid cells (FLER, PBER). The transcriptome analysis showed that KLF6 expressed at significantly higher level in ESER cells compared with that in other cells. Meanwhile, chromatin immunoprecipitation (ChIP) studies in human K562 cells demonstrated the enrichment of KLF6 on the promoter region of embryonic epsilon-globin gene. These results probably indicate that KLF6 play an important role in primitive hematopoiesis. To clarify whether the erythroid-specific enhancers in the genomic region of KLF6 participate in the regulation of primitive hematopoiesis, we extensively screened the erythroid-specific DNaseI hypersensitive sites (DHSs) in the KLF6 locus, from 70 kb upstream of the transcription start site to 20 kb downstream of the poly(A) site, from DNase-seq data in four erythroid cells including ESER, FLER, PBER, K562 and seven non-erythroid cells. The enhancer activity of these erythroid-specific DHSs was comprehensively characterized by dual-luciferase reporter assay in K562 cells as well as non-erythroid HeLa and HEK293 cells. Three erythroid-specific enhancers located 18–24 kb upstream of human KLF6 were finally characterized, which not only helps to understand the higher expression of KLF6 in ESER, but also hints that KLF6 could participate in primitive hematopoiesis through erythroid-specific enhancers. In conclusion, we depicted the dynamic expression pattern of KLF6 during erythroid differentiation, characterized three erythroid-specific enhancers in KLF6 gene locus, and disclosed the potential role of KLF6 in primitive hematopoiesis. Next, the overexpression and depletion of KLF6 in K562 cells will be executed to further explore whether the abnormal KLF6 will affect the expression and functions of globin genes as well as erythroid-specific transcription factors. Chromosome conformation capture (3C) analysis will be performed to evaluate the interactions between the erythroid-specific enhancers and the cis-regulatory elements of hematopoiesis related genes. Moreover, we will establish morpholino-based klf6 knockdown zebrafish model and study the target genes, interacting networks and pathways in which KLF6 involved. Collectively, these results will address the detailed cis- and trans- regulatory functions and molecular mechanism of KLF6 in regulating hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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