The severe phenotype of Diamond-Blackfan anemia is modulated by heat shock protein 70

Key Points Proteasomal HSP70 degradation results in cleavage of GATA1, decrease in erythroid progenitors, and apoptosis in severe DBA phenotype. HSP70 plays a role not only during terminal erythroid differentiation, but also in the earlier proliferation of erythroid progenitor cells.

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Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2

Blood ◽

10.1182/blood.v88.5.1576.1576 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1576-1582 ◽

Cited By ~ 243

Author(s):

M Silva ◽

D Grillot ◽

A Benito ◽

C Richard ◽

G Nunez ◽

...

Keyword(s):

Progenitor Cells ◽

Apoptotic Cell ◽

Ectopic Expression ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Survival Factor ◽

Erythroid Progenitor Cells ◽

Survival Signal ◽

Proliferation And Differentiation

Abstract Erythropoietin (Epo), the hormone that is the principal regulator of red blood cell production, interacts with high-affinity receptors on the surface of erythroid progenitor cells and maintains their survival. Epo has been shown to promote cell viability by repressing apoptosis; however, the molecular mechanism involved is unclear. In the present studies we have examined whether Epo acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. We addressed this issue in HCD-57, a murine erythroid progenitor cell line that requires Epo for proliferation and survival. When HCD-57 cells were cultured in the absence of Epo, Bcl-2 and Bcl-XL but not Bax were downregulated, and the cells underwent apoptotic cell death. HCD-57 cells infected with a retroviral vector encoding human Bcl-XL or Bcl-2 rapidly stopped proliferating but remained viable in the absence of Epo. Furthermore, endogenous levels of bcl-2 and bcl-XL were downregulated after Epo withdrawal in HCD-57 cells that remained viable through ectopic expression of human Bcl-XL, further indicating that Epo specifically maintains the expression of bcl-2 and bcl-XL. We also show that HCD-57 rescued from apoptosis by ectopic expression of Bcl-XL can undergo erythroid differentiation in the absence of Epo, demonstrating that a survival signal but not Epo itself is necessary for erythroid differentiation of HCD-57 progenitor cells. Thus, we propose a model whereby Epo functions as a survival factor by repressing apoptosis through Bcl-XL and Bcl-2 during proliferation and differentiation of erythroid progenitors.

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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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Runx1 promotes murine erythroid progenitor proliferation and inhibits differentiation by preventing Pu.1 downregulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1901122116 ◽

2019 ◽

Vol 116 (36) ◽

pp. 17841-17847 ◽

Cited By ~ 2

Author(s):

Michael A. Willcockson ◽

Samuel J. Taylor ◽

Srikanta Ghosh ◽

Sean E. Healton ◽

Justin C. Wheat ◽

...

Keyword(s):

Ectopic Expression ◽

Terminal Differentiation ◽

Erythroid Differentiation ◽

Chromatin Accessibility ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Down Regulation ◽

Erythroid Progenitor ◽

Terminal Erythroid Differentiation ◽

In Erythroid Cells

Pu.1 is an ETS family transcription factor (TF) that plays critical roles in erythroid progenitors by promoting proliferation and blocking terminal differentiation. However, the mechanisms controlling expression and down-regulation of Pu.1 during early erythropoiesis have not been defined. In this study, we identify the actions of Runx1 and Pu.1 itself at the Pu.1 gene Upstream Regulatory Element (URE) as major regulators of Pu.1 expression in Burst-Forming Unit erythrocytes (BFUe). During early erythropoiesis, Runx1 and Pu.1 levels decline, and chromatin accessibility at the URE is lost. Ectopic expression of Runx1 or Pu.1, both of which bind the URE, prevents Pu.1 down-regulation and blocks terminal erythroid differentiation, resulting in extensive ex vivo proliferation and immortalization of erythroid progenitors. Ectopic expression of Runx1 in BFUe lacking a URE fails to block terminal erythroid differentiation. Thus, Runx1, acting at the URE, and Pu.1 itself directly regulate Pu.1 levels in erythroid cells, and loss of both factors is critical for Pu.1 down-regulation during terminal differentiation. The molecular mechanism of URE inactivation in erythroid cells through loss of TF binding represents a distinct pattern of Pu.1 regulation from those described in other hematopoietic cell types such as T cells which down-regulate Pu.1 through active repression. The importance of down-regulation of Runx1 and Pu.1 in erythropoiesis is further supported by genome-wide analyses showing that their DNA-binding motifs are highly overrepresented in regions that lose chromatin accessibility during early erythroid development.

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Two-phase culture in Diamond Blackfan anemia: localization of erythroid defect

Blood ◽

10.1182/blood-2004-03-1016 ◽

2005 ◽

Vol 105 (2) ◽

pp. 838-846 ◽

Cited By ~ 59

Author(s):

Yaw Ohene-Abuakwa ◽

Karen A. Orfali ◽

Carine Marius ◽

Sarah E. Ball

Keyword(s):

Phase 1 ◽

Clinical Severity ◽

Phase 2 ◽

Erythroid Progenitors ◽

Two Phase ◽

Erythroid Progenitor ◽

Diamond Blackfan Anemia ◽

Erythroid Progenitor Cells ◽

Adenosine Deaminase Activity ◽

Molecular Pathophysiology

AbstractThe erythroid defect in Diamond Blackfan anemia (DBA) is known to be intrinsic to the stem cell, but its molecular pathophysiology remains obscure. Using a 2-phase liquid erythroid culture system, we have demonstrated a consistent defect in DBA, regardless of clinical severity, including 3 first-degree relatives with normal hemoglobin levels but increased erythrocyte adenosine deaminase activity. DBA cultures were indistinguishable from controls until the end of erythropoietin (Epo)–free phase 1, but failed to demonstrate the normal synchronized wave of erythroid expansion and terminal differentiation on exposure to Epo. Dexamethasone increased Epo sensitivity of erythroid progenitor cells, and enhanced erythroid expansion in phase 2 in both normal and DBA cultures. In DBA cultures treated with dexamethasone, Epo sensitivity was comparable to normal, but erythroid expansion remained subnormal. In clonogenic phase 2 cultures, the number of colonies did not significantly differ between normal cultures and DBA, in the presence or absence of dexamethasone, and at both low and high Epo concentrations. However, colonies were markedly smaller in DBA under all conditions. This suggests that the Epo-triggered onset of terminal maturation is intact in DBA, and the defect lies down-stream of the Epo receptor, influencing survival and/or proliferation of erythroid progenitors.

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An RNA Interference Model of RPS19 Deficiency in Diamond Blackfan Anemia Recapitulates Defective Erythropoiesis and Rescue by Dexamethasone: Identification of Dexamethasone Responsive Genes by Microarray.

Blood ◽

10.1182/blood.v104.11.721.721 ◽

2004 ◽

Vol 104 (11) ◽

pp. 721-721 ◽

Cited By ~ 1

Author(s):

Benjamin L. Ebert ◽

Michele Lee ◽

Jennifer Pretz ◽

Todd R. Golub ◽

Colin Sieff

Keyword(s):

Progenitor Cells ◽

Molecular Basis ◽

Cellular Proliferation ◽

Erythroid Differentiation ◽

Regulatory Genes ◽

Cell Surface Expression ◽

Erythroid Progenitor ◽

Diamond Blackfan Anemia ◽

Erythroid Progenitor Cells ◽

Cd34 Cells

Abstract Diamond Blackfan Anemia (DBA), a congenital erythroblastopenia, is a model disease for the study of erythroid differentiation, but is poorly understood. RPS19 is the only gene yet to have been associated with DBA, but the relevance of this ubiquitously expressed ribosomal gene to erythroid differentiation is unclear. Glucocorticoids are the primary pharmacological therapy for patients with DBA, but the molecular basis for the activity of glucocorticoids in erythroid differentiation has not been identified. Through retroviral expression of short hairpin RNAs (shRNAs), we demonstrate that targeted degradation of the RPS19 gene in cultured human CD34+ cells blocks the proliferation and differentiation of erythroid progenitor cells. Decreased RPS19 expression limited production of erythroid colony formation on methylcellulose, decreased cell surface expression of glycophorin-A, and decreased cellular proliferation. Treatment of RPS19 deficient cells with dexamethasone restored erythroid differentiation to normal levels. We investigated the molecular basis of pharmacologic therapies for DBA using oligonucleotide microarrays to survey gene expression in CD34+ cells treated with combinations of erythropoietin, dexamethasone, IL-3, and SCF. None of these agents had a direct effect on the expression of RPS19 or on the coordinate expression of other ribosomal genes. Instead, dexamethasone activated a genetic program that includes a set of key hematopoietic regulatory genes, including Flt-3 and PLZF, that increase proliferation of hematopoietic progenitor cells. Genes specific to erythroid progenitor cells were up-regulated by dexamethasone, while genes specific to non-erythroid lineages were powerfully down-regulated. Deficiency of RPS19 therefore blocks proliferation of immature erythroid progenitor cells, and dexamethasone activates proliferation of the same cell population through mechanisms independent of RPS19. Identification of the key regulatory genes affected by dexamethasone may aid in the development of novel therapeutics that de-couple the beneficial hematopoietic effects of dexamethasone from detrimental non-hematopoietic side effects.

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Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2

Blood ◽

10.1182/blood.v88.5.1576.bloodjournal8851576 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1576-1582 ◽

Cited By ~ 10

Author(s):

M Silva ◽

D Grillot ◽

A Benito ◽

C Richard ◽

G Nunez ◽

...

Keyword(s):

Progenitor Cells ◽

Apoptotic Cell ◽

Ectopic Expression ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Survival Factor ◽

Erythroid Progenitor Cells ◽

Survival Signal ◽

Proliferation And Differentiation

Erythropoietin (Epo), the hormone that is the principal regulator of red blood cell production, interacts with high-affinity receptors on the surface of erythroid progenitor cells and maintains their survival. Epo has been shown to promote cell viability by repressing apoptosis; however, the molecular mechanism involved is unclear. In the present studies we have examined whether Epo acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. We addressed this issue in HCD-57, a murine erythroid progenitor cell line that requires Epo for proliferation and survival. When HCD-57 cells were cultured in the absence of Epo, Bcl-2 and Bcl-XL but not Bax were downregulated, and the cells underwent apoptotic cell death. HCD-57 cells infected with a retroviral vector encoding human Bcl-XL or Bcl-2 rapidly stopped proliferating but remained viable in the absence of Epo. Furthermore, endogenous levels of bcl-2 and bcl-XL were downregulated after Epo withdrawal in HCD-57 cells that remained viable through ectopic expression of human Bcl-XL, further indicating that Epo specifically maintains the expression of bcl-2 and bcl-XL. We also show that HCD-57 rescued from apoptosis by ectopic expression of Bcl-XL can undergo erythroid differentiation in the absence of Epo, demonstrating that a survival signal but not Epo itself is necessary for erythroid differentiation of HCD-57 progenitor cells. Thus, we propose a model whereby Epo functions as a survival factor by repressing apoptosis through Bcl-XL and Bcl-2 during proliferation and differentiation of erythroid progenitors.

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Mcph1, Mutated in Primary Microcephaly, Is Required to Complete Terminal Erythroid Differentiation

Blood ◽

10.1182/blood-2019-126475 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1192-1192

Author(s):

Yoann Vial ◽

Jeannette Nardelli ◽

Justine Rousselot ◽

Emilie Lepeu ◽

Michele Souyri ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Cell Division ◽

Erythroid Differentiation ◽

Rna Seq ◽

Wild Type ◽

Erythroid Progenitors ◽

Primary Microcephaly ◽

Erythroid Progenitor ◽

Terminal Erythroid Differentiation

Introduction: Microcephaly is a recurrent feature in patients with inherited bone marrow failure (iBMF) and DNA damage response (DDR) disorders suggesting that common cellular pathways regulate the proliferation and differentiation of neural and hematopoietic progenitors. However, while several studies addressed the role of iBFM or DDR genes in brain development, a possible role for microcephaly genes in hematopoietic development has not been investigated. To address this issue, we studied a mouse model of primary microcephaly with biallelic loss-of-function in Mcph1.MCPH1 mutations are found in 10% of patients with isolated forms of genetic microcephaly (MCPH). Interestingly, MCPH1 helps to maintain genomic integrity during cell division by interacting with proteins involved in cell cycle progression, apoptosis or DNA repair, all cellular processes being also involved in iBMF and DDR syndromes. Methods: Mcph1 null mice were generated by germline deletion of Mcph1 exon 2 (Mcph1tm1.2Kali) (Liang et al., PLoS Genet., 2010). The subpopulations of erythroid progenitors S0 to S5 were phenotypically defined and sorted by flow cytometry according to CD71 and Ter119 expression in the Lin- compartment from mouse liver obtained at birth and during fetal development (E12.5). RNA sequencing (RNA-Seq) was performed on sorted erythroid progenitor fractions obtained from E12.5 fetal livers (SMARTer® Stranded Total RNA-Seq Kit V2-Pico Input library preparation kit). Cell division was studied by multiplexing erythroid specific antibodies with EdU flow cytometry cell proliferation assay. Results: Null mice recapitulated the microcephaly phenotype seen in MCPH patients, but also showed a striking anemic pallor. Numeration and cytomorphologic examination of peripheral blood at birth confirmed macrocytic anemia with low red blood cell count and anisopoikilocytosis. These observations were consistent with congenital dyserythropoiesis in Mcph1-/- mice and prompted us to further characterize the erythroid lineage. Quantification of erythroid progenitor populations in liver at birth showed a significant decreased from the S3 subset (Lin-, CD71High, Ter119High) suggesting impaired terminal differentiation. Similar results were obtained in fetal livers at E12.5 indicating that the defect arose early in hematopoietic ontogeny. Transcriptome analysis of wild-type progenitor populations (S0 to S3) confirmed that Mcph1 is expressed during normal erythropoiesis following a Gata1-like expression profile. This is consistent with the presence in the Mcph1 gene promoter of a binding site for Gata1 and Ldb1 (ENCODE project), supporting an activation by the main erythroid differentiation complex. Strikingly, RNA-Seq analysis revealed deregulation of p53 pathway associated genes in all subsets of Mcph1-/- erythroid progenitors as compared to their wild-type counterparts. Two transcriptional p53 targets involved in cell cycle control, Cdkn1a coding the cyclin-dependent kinase inhibitor (p21) and Ccng1 coding Cyclin G1, were among the most upregulated genes. Cell cycle analysis performed on sorted erythroid progenitors revealed an endoreduplication phenomenon restricted to the S3 subset with subsequent accumulation of tetraploid cells. Interestingly, physiological endoreduplication is initiated by p21 and E2Fs transcription factors, and Mcph1 functionally interacts with E2f1. Our findings suggest that, in the absence of Mcph1, Cdkn1a overexpression possibly combined to a decreased E2f1 activity may lead to endoreduplication in S3 progenitors, impairing further differentiation into mature red blood cells. Few data are available for patients with MCPH1 mutations, hematological defects being possibly outlooked due to the severity of the neurological phenotype. However, CBC performed in one of our patients revealed a macrocytosis consistent with dyserythropoiesis evidenced in mice. Conclusion: We demonstrate for the first time that Mcph1 expression is critical during terminal erythroid differentiation in mice. Altogether our findings provide additional evidence of a unique link between hematopoiesis and neuronal development. Disclosures No relevant conflicts of interest to declare.

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Failure of Terminal Erythroid Differentiation in EKLF-Deficient Mice Is Associated with Cell Cycle Perturbation and Reduced Expression of E2F2

Molecular and Cellular Biology ◽

10.1128/mcb.01087-08 ◽

2008 ◽

Vol 28 (24) ◽

pp. 7394-7401 ◽

Cited By ~ 62

Author(s):

Andre M. Pilon ◽

Murat O. Arcasoy ◽

Holly K. Dressman ◽

Serena E. Vayda ◽

Yelena D. Maksimova ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Target Genes ◽

Transcriptional Profiling ◽

Erythroid Differentiation ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Chromatin Modifier ◽

Terminal Erythroid Differentiation

ABSTRACT Erythroid Krüppel-like factor (EKLF) is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf −/− ) mice die at day 14.5 of gestation from severe anemia. In this study, we demonstrate that early progenitor cells fail to undergo terminal erythroid differentiation in Eklf −/− embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis, transcriptional profiling was performed with RNA from wild-type and Eklf −/− early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation, with the critical regulator of the cell cycle, E2f2, at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf −/− early erythroid progenitor cells, which showed a delay in the G1-to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier, EKLF binding sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation.

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Distinct roles for TET family proteins in regulating human erythropoiesis

Blood ◽

10.1182/blood-2016-08-736587 ◽

2017 ◽

Vol 129 (14) ◽

pp. 2002-2012 ◽

Cited By ~ 24

Author(s):

Hongxia Yan ◽

Yaomei Wang ◽

Xiaoli Qu ◽

Jie Li ◽

John Hale ◽

...

Keyword(s):

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Key Points ◽

Terminal Erythroid Differentiation

Key Points TET3 knockdown impairs terminal erythroid differentiation, whereas TET2 knockdown leads to accumulation of erythroid progenitors. Global levels of 5mC are not altered by knockdown of either TET2 or TET3.

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SATB1 Regulates GATA1 Protein Expression in Early Hematopoiesis and Is Deregulated in Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2020-140437 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 3-3

Author(s):

Mark C Wilkes ◽

Hee-Don Chae ◽

Ethan Patrick Wentworth ◽

Toshinobu Nishimura ◽

Anupama Narla ◽

...

Keyword(s):

Protein Expression ◽

Fetal Liver ◽

Gene Mutations ◽

Erythroid Differentiation ◽

Ribosomal Gene ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Enhancer Element ◽

Diamond Blackfan Anemia

The induction of the master erythroid transcription factor, GATA1 during early erythropoiesis is critical for efficient red blood cell production. However, GATA1 is expressed at low levels in hematopoietic stem cells (HSCs) and is moderately induced at both the common myeloid progenitor (CMP) and megakaryocyte/erythroid progenitor (MEP) stages prior to lineage commitment. Diamond Blackfan Anemia is a rare disease, usually associated with ribosomal gene mutations, leading to significant decrease in GATA1 expression and block in early committed erythroid differentiation. Mild defects in other myeloid lineages are also observed, with limited clinical relevance. The importance of GATA1 downregulation in disease pathogenesis is manifested by rare patients with DBA carrying GATA1 mutations. To understand signaling pathways that contribute to the pathogenesis DBA, we perform RNA-seq with mRNA from human CD34+ fetal liver cells and found that the chromatin organizer, Special AT-rich sequence binding protein 1 (SATB1) was prematurely downregulated. Our results further demonstrated that sustained SATB1 expression is critical to maintain required levels of GATA1 protein at both the CMP and MEP stages of differentiation, but not in committed erythroid progenitors. In mice, SATB1 is modestly expressed in HSCs and upregulated during lymphopoiesis. SATB1 is downregulated during myeloid and erythroid differentiation and antagonizes myeloid and erythroid expansion. However, in human hematopoietic stem and progenitor cells (HSPCs), SATB1 is required for efficient expansion of these lineages. SATB1 maintains 78% expression in human MEPs, but is undetectable in early committed erythroid progenitors. In RPS19-insufficient human HSPCs, SATB1 was downregulated to 22% in MEPs (p=0.02). Re-expression of SATB1 corrected a significant subset of deregulated mRNAs, including GATA1 regulators. In the absence of SATB1, one such GATA1 regulator, heat shock protein 70 (HSP70), failed to be induced in ribosome-competent human MEPs, reducing GATA1 protein expression by 35.7% (p= 0.026). Concurrently, MEP expansion was inhibited by 64.5% (p=0.023), reducing erythroid and megakaryocyte expansion by 18.2% (p=0.024) and 20.4% (p=0.183) respectively. SATB1 facilitated the formation of chromatin loops linking together an enhancer element with HSP70 promoters required for HSP70 induction in early differentiation. Although GATA1 is significantly upregulated in committed erythroid progenitors, RPS19-insufficient human CD235+ erythrocytes express GATA1 28.4% of controls (p= 0.011). SATB1 re-expression increased GATA1 expression to 31.4% (p=0.089). Similarly, SATB1 re-expression increased CD235+ expansion from 13.9% to 39.5% (p=0.02) compared to controls. Our data indicate that premature SATB1 downregulation contributes to erythroid failure in DBA by reducing MEP expansion, but aberrant GATA1 expression observed in more mature erythrocytes is predominantly SATB1-independent. However, SATB1-re-expression improved CD11b+ myeloid expansion from 81.2% to 90.4% (p=0.045) and CD41a+ megakaryocyte expansion from 76.7% to 214.7% (p=0.038) respectively. Our results demonstrate that SATB1 plays an important role in human hematopoiesis and is an important regulator of GATA1. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy.

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