GATA-related hematologic disorders

Accepted Manuscript GATA-related Hematological Disorders Ritsuko Shimizu, Masayuki Yamamoto PII:

S0301-472X(16)30135-7

DOI:

10.1016/j.exphem.2016.05.010

Reference:

EXPHEM 3409

To appear in:

Experimental Hematology

Received Date: 5 April 2016 Revised Date:

15 May 2016

Accepted Date: 17 May 2016

Please cite this article as: Shimizu R, Yamamoto M, GATA-related Hematological Disorders, Experimental Hematology (2016), doi: 10.1016/j.exphem.2016.05.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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GATA-related Hematological Disorders

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Ritsuko Shimizua* and Masayuki Yamamotob

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Department of aMolecular Hematology and bMedical Biochemistry,

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Tohoku University Graduate School of Medicine, Sendai, Japan

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* To whom correspondence should be addressed: Ritsuko Shimizu, MD & PhD Professor Department of Molecular Hematology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan, Phone 81-22-717-7952; Fax 81-22-717-8083 E-mail: [email protected]

Key words: GATA1, leukemia Text word count: 4574 words The number of figures and tables: 4 The number of references: 75

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ACCEPTED MANUSCRIPT ABSTRACT The transcription factors GATA1 and GATA2 are fundamental regulators of hematopoiesis and show overlapping expression profiles. GATA2 is expressed in hematopoietic stem cells and

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early erythroid-megakaryocytic progenitors and activates a certain set of early-phase genes, including the GATA2 gene itself. GATA2 also initiates GATA1 gene expression. In contrast, GATA1 is expressed in relatively mature erythroid progenitors and facilitates the expression of

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genes associated with differentiation, including the GATA1 gene itself; however, GATA1 represses the expression of GATA2. Switching of the GATA factors from GATA2 to GATA1

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appears to be one of the key regulatory mechanisms underlying erythroid differentiation. Loss-of-function analyses using mice in vivo have shown that GATA2 and GATA1 are functionally non-redundant and that neither can compensate for the absence of the other. However, transgenic expression of GATA2 under the transcriptional regulation of the Gata1

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gene rescues lethal dyserythropoiesis in GATA1-deficient mice, demonstrating that the dynamic expression profiles of these GATA factors are critically important for the maintenance of hematopoietic homeostasis. Analysis of naturally occurring leukemias in GATA1-knockdown

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mice revealed that leukemic stem cells undergo functional alterations in response to exposure to chemotherapeutic agents. This mechanism may also underlie the aggravating features of

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relapsing leukemias. Recent hematological analyses have suggested that disturbances in the balance of the GATA factors are associated with specific types of hematopoietic disorders. Here, we describe GATA1- and GATA2-related hematological diseases, focusing on the regulation of GATA factor gene expression.

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ACCEPTED MANUSCRIPT INTRODUCTION The GATA family transcription factors are principal regulators of the differentiation and functions of specific cell lineages that act by binding to a unique consensus motif,

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(A/T)GATA(A/G), and by regulating of a set of target genes [1]. In vertebrates, six GATA factors have been identified that share highly conserved dual zinc fingers located in the middle regions of the molecules [2]. N-terminal and C-terminal zinc fingers are referred to as N-fingers

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and C-fingers, respectively. C-fingers play an indispensable role in the transcriptional activity of GATA factors by directly binding to the GATA motif [2]. The C-finger of GATA factors is

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evolutionarily conserved from invertebrates to vertebrates [3]. In contrast, the N-finger has diverged during molecular evolution, indicating that it possesses certain evolutionarily acquired features.

Of the 6 GATA factors identified in vertebrates, GATA1, GATA2 and GATA3 are expressed

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mainly in hematopoietic tissues and are referred to as hematopoietic GATA factors [2]. Hematopoietic GATA factors possess two transactivation domains in the N-terminal and C-terminal regions [4]. The expression profile of GATA2 during erythropoiesis partly overlaps

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with that of GATA1. GATA2 is mainly expressed in hematopoietic stem cells and early progenitors and activates genes essential for early progenitor function and its own expression, as

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well as those required for initiating GATA1 gene expression. Once GATA1 gene expression is activated, GATA1 activates genes involved in erythroid differentiation and also facilitates its own gene expression. Importantly, GATA1 suppresses GATA2 gene expression. Consequently, the expression of GATA2 is downregulated during erythroid maturation, whereas that of GATA1 is unregulated. This process has been referred to as “GATA factor switching” during erythroid differentiation [4, 5]. Thus, the dynamic expression profiles of GATA2 and GATA1 are linked to a key aspect of the transcription factor network that coordinates hematopoietic homeostasis. In

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ACCEPTED MANUSCRIPT this chapter, we will describe the pathogenesis of GATA-related disorders, with special emphasis on the regulation of GATA factor expression.

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Diseases caused by haploinsufficiency of GATA2 The human GATA2 gene consists of two alternative first exons encoding untranslated regions, both of which are conserved in mouse and chicken [6, 7], and five translated exons (Fig. 1). The

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distal first exon is specifically transcribed in hematopoietic and neural tissues, whereas the proximal first exon generally plays a housekeeping role. Therefore, the distal and proximal first

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exons are referred to as “IS” (specific) and “IG” (general) exons, respectively. Given that mice lacking the IS exon do not show an apparent phenotype [8], the function of the distal first exon of the Gata2 gene can be compensated for by that of the IG exon and promoter. In the mouse Gata2 gene, the GATA sites that function during erythroid maturation are

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located at -77, -3.9, -2.8, -1.8 and +9.5 kb from the transcription start site of the IS exon (Fig. 1) [9]. Studies utilizing transgenic reporter mice carrying the -3.1-kb Gata2 gene regulatory domain upstream of the IS exon (G2EHRD; Gata2 early hematopoietic regulatory domain) have

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shown that G2EHRD recapitulates Gata2 gene expression in hematopoietic and endothelial bi-potential precursors in the aorta-gonad-mesonephros (AGM) region. Based on the results of

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these G2EHRD transgenic mouse assays, GATA-binding motifs clustered in the 2.8-kb upstream region appear to be essential for G2EHRD activity [10]. However, mice lacking either the 3.9-, 2.8- or 1.8-kb upstream region are born alive without significant hematopoietic abnormalities (Fig. 1) [11-13], suggesting that the enhancer activities of the promoter-neighboring regions of the IS exon may compensate for each other. In contrast, disruption of either the -77-kb or the +9.5-kb region causes mice to die in utero with specific hematological abnormalities [14, 15]. Dysfunction of the -77-kb enhancer region decreases Gata2 gene expression in myeloerythroid

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ACCEPTED MANUSCRIPT progenitors but not in hematopoietic stem cells (HSCs), whereas the +9.5-kb element is required for GATA2 gene expression in HSCs (Fig. 1) [14, 15]. Given that Gata2 gene expression is supported by multiple cis-acting elements that exhibit distinct properties, we envisage that

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various types of hematopoietic diseases may arise as a result of aberrant GATA2 gene regulation. Familial acute myeloid leukemias and myelodysplastic syndromes (familial AML/MDS), DCML deficiency (loss of dendritic cells, monocytes and natural killer and B lymphoid cells),

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MonoMAC syndrome (monocytopenia with atypical mycobacterial infection) and Emberger syndrome (lymphedema with MDS) have been described as major GATA2-associated

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hematological diseases provoked by inherited or sporadic GATA2 gene mutations [16]. Various types of monoallelic mutations, including substitutions and indel mutations that are scattered among five translated exons of the GATA2 gene, have been identified in patients with GATA2-associated hematological diseases, although the mutation types do not correspond well

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with the clinical diagnoses [16, 17]. As a result of missense, nonsense or frameshift mutations, either a mutant GATA2 protein or no GATA2 protein is produced [16]. Interestingly, whereas heterozygous Gata2-knockout mice have normal life spans and do

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not develop hematological disease although they display a slight defect in stem cell function [18], functional haploinsufficiency for GATA2 appears to result in dominant traits of

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immunodeficiency and hematological disease in humans. Indeed, monoallelic mutations in the composite cis-regulatory elements, which consist of E-box and GATA motifs followed by an ETS-binding motif, within the +9.5-kb region of Gata2, have recently been identified in patients with GATA2-associated hematological diseases (Fig. 1) [19, 20]. Whereas homozygous disruption of the +9.5-kb region causes embryonic lethality in mice due to a lack of GATA2 in HSCs [14], reduced expression of the GATA2 gene in HSCs due to a monoallelic mutation appears to play a role in the onset of GATA2-associated hematological disease.

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Leukemogenesis associated with GATA2 gene regulation Chromosomal rearrangements are frequently observed in the context of hematologic malignancy.

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In particular, translocations leading to the production of chimeric proteins composed of parts of two independent proteins have been extensively studied in relation to leukemogenesis. However, it has been very difficult to identify the cause of leukemogenesis in patients with chromosomal

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rearrangements that do not produce chimeric proteins. One salient example of the latter case is lymphocytic malignancies driven by the translocation of oncogenes in proximity with

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immunoglobulin or T-cell receptor genes.

In this regard, the mechanism underlying leukemogenesis resulting from the chromosomal rearrangements inv(3)(q21q26) and t(3;3)(q21;q26) remains to be elucidated because chimeric fusion proteins are not produced in patients with these translocations or inversion. This type of

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leukemia is defined as 3q21q26 syndrome and is recognized as a distinct clinicopathologic entity with abnormal megakaryocytopoiesis, an elevated platelet count, and an extremely poor prognosis [21]. It has been proposed that aberrant overexpression of the EVI1 gene at the 3q26

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locus is involved in the onset of 3q21q26 syndrome. Recently, a breakthrough was achieved with the discovery that in this syndrome, the EVI1 gene translocates to an area close to the

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-77-kb enhancer of the GATA2 gene and takes over its enhancer activity, resulting in aberrant overexpression of EVI1 in hematopoietic progenitor populations (Fig. 1) [22, 23]. The abovementioned translocation and inversion result in characteristic alterations in the GATA2 enhancer, transforming it into a “super-enhancer” [22]. Indeed, in a study in which a rearranged transgene was prepared by linking two bacterial artificial chromosomes (BACs) harboring the 3q21 and 3q26 loci and transgenic mice carrying the rearranged transgene were generated, the mice developed a leukemia that faithfully mimicked the human disease. In

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ACCEPTED MANUSCRIPT contrast, a rearranged BAC transgene lacking the -77-kb enhancer region failed to drive leukemia development in mice [23]. TGFβ signaling activates Gata2 gene expression via binding of Smad4 to the -77-kb enhancer of the GATA2 gene [24], suggesting that the TGFβ

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signaling pathway may influence the pathogenesis of 3q21q26 syndrome.

Regulation of Gata1 gene expression

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Similar to the GATA2 gene, the GATA1 gene consists of one untranslated first exon and five translated exons. An alternative first exon has been reported in the mouse and rat Gata1 genes,

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i.e., rodent Gata1 genes harbor distal testis-specific (IT) and proximal hematopoietic (IE) promoters and first exons [25]. Germline deletion of the IE exon abrogates GATA1 expression in the hematopoietic lineage, indicating that the contribution of the distal first exon is insufficient for hematopoiesis [26, 27].

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Gata1 gene regulation has been extensively studied using conventional reporter transfection assays and transgenic mouse reporter assays. These analyses have revealed that functional GATA sites are located at both the 3.9-kb upstream and promoter upstream regions of

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the IE exon and that these sites are well conserved between humans and mice [28]. A regulatory region consisting of the 3.9-kb upstream region of the IE exon plus the IE exon and the first

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intron was found to drive reporter gene expression in transgenic mice, a situation that faithfully recapitulates endogenous Gata1 gene expression. This region is referred to as the Gata1 hematopoietic regulatory domain (GIHRD). Indeed, transgenic expression of Gata1 cDNA linked to the G1HRD rescues GATA1-deficient mice from embryonic lethality [29]. Transgenic mouse reporter assays based on GIHRD regulation have been utilized to assess cis-acting regulatory elements of the Gata1 gene. A series of analyses has revealed that the GATA sites in the 3.9-kb upstream region and the upstream promoter region are both essential

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ACCEPTED MANUSCRIPT for G1HRD activity [30]. The analyses have shown that there are at least three independent cis-regulatory elements in the vicinity of the IE promoter, i.e., double GATA-binding motifs in a palindromic orientation (dbGATA), CP2-binding motifs, and CACCC motifs [28, 31]. These

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elements are highly conserved in the human and mouse GATA1 genes. Available evidence from transgenic reporter gene expression analyses indicates that these cis-regulatory elements contribute differentially and cooperatively to the regulation of G1HRD activity during primitive

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and definitive erythropoiesis [31].

Elaborate analyses of the Gata1 gene were conducted using a BAC-DNA clone of

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approximately 200 kb in length including the entire Gata1 gene locus. In this BAC clone, the mouse Gata1 gene was homologously recombined with the green fluorescent protein (GFP) gene (G1-BAC-GFP clone). Transgenic mouse reporter assays utilizing the G1-BAC-GFP clone have demonstrated that the GATA sites in the 3.9-kb upstream region and the proximal enhancer

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region are specifically required for the initiation of Gata1 gene expression in early-stage progenitor cells but are less important for Gata1 gene expression in erythroid-committed progenitors [32, 33]. Indeed, mice lacking one of the two GATA binding motifs are born alive

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with a slight defect in erythropoiesis [34, 35].

Surprisingly, expression of the Gata1 gene in HSCs was shown to be negatively regulated

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by DNA methylation of the region referred to as the Gata1 methylation-determining region (G1MDR) (Fig. 2) [36]. Methylation of the G1MDR appears to interfere with GATA2 access to the GATA-binding sites in the 3.9-kb upstream region and the proximal enhancer region; the G1MDR is located between these two GATA sites. Thus, expression of the Gata1 gene is meticulously regulated by multiple mechanisms that use the G1MDR for silencing and two GATA motifs for upregulation (Fig. 2), and it seems plausible that a high level of GATA1 expression during the early stage of hematopoiesis leads to an unfavorable outcome. We surmise

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ACCEPTED MANUSCRIPT that the Gata1 gene locus may contain additional regulatory regions that have not yet been described that contribute to Gata1 gene regulation. Because GATA1 contributes critically to the regulation of erythropoiesis, these regulatory elements act in an overlapping and redundant

for Gata1 gene expression (Fig. 2).

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Pathology provoked by Gata1 hypomorphic expression in mice

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manner in erythroid-committed progenitors, leading to construction of a reliable regulatory loop

Because the GATA1/Gata1 (human/mouse) genes are located on X chromosomes, hemizygous

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male mice carrying a Gata1-knockout allele (Gata1KO/Y) die in utero due to failure of primitive erythropoiesis [37]. A Gata1-knockdown allele referred to as GATA1.05 has been established by insertion of a neomycin-resistance gene cassette into the promoter proximal regulatory region [38]. The strong enhancer activity within the neomycin cassette interferes with endogenous IE

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promoter activity, resulting in reduction of Gata1 gene expression to approximately 5% of the endogenous level. Gata1-knockdown male mice (Gata11.05/Y) die during a similar stage of embryonic development, as do complete Gata1-knockout embryos, indicating that 5% of normal

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GATA1 expression is insufficient to sustain erythropoiesis [38]. Further investigations were conducted by conditional genome engineering in mice using the

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Cre-loxP system. Conditional deletion of all coding exons of the Gata1 gene in adult mice (Gata1CKO/Y; Gata1 gene expression is completely removed) leads to aplastic erythropoiesis and a reduced number of erythroid progenitors [39]. In contrast, mice harboring a conditional IE exon deletion (Gata1IECKO/Y) exhibit anemia with accumulation of CD71+c-KIT+ erythroid progenitors [40]. In erythroid progenitors of the latter mice, aberrant Gata1 transcripts are produced in which the IE exon is replaced with IT and a few other cryptic/alternative first exons. The aberrant transcripts lead to the production of a short form of GATA1 lacking the N-terminal

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ACCEPTED MANUSCRIPT transactivation domain (∆NT-GATA1). Importantly, ∆NT-GATA1 is a mouse homolog of GATA1-S, which is produced in patients with Diamond-Blackfan anemia (DBA) who carry GATA1 gene mutations [41-43]. GATA1-S/∆NT-GATA1 fails to support the erythroid

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differentiation of hematopoietic progenitors; consequently, the progenitors accumulate in mice. In contrast with erythroid progenitors, aberrant Gata1 transcripts are not produced in megakaryocytes of Gata1IECKO/Y mice. Therefore, due to GATA1-deficiency, hyper-lobulated

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megakaryocytes accumulate in Gata1IECKO/Y mice, as well as in Gata1CKO/Y mice.

Another Gata1-knockdown (Gatallow) allele was generated by replacing the 8-kb DNA

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fragment upstream of the IE exon that contains the GATA-binding motif at 3.9-kb upstream with a neomycin resistance cassette. This replacement reduces Gata1 gene expression to 20% of endogenous GATA1 expression [35]. Male mice carrying the Gatallow allele are born alive at a lower frequency than predicted by Mendelian genetics. Newborn Gatallow male mice have

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anemia and thrombocytopenia. Gatallow pups that do not succumb to early postnatal lethality recover from anemia by adulthood, indicating that the expression of GATA1 is important for supporting embryonic hematopoiesis. Although the 20% GATA1 expression conferred by the

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mutant allele is insufficient to support a normal level of embryonic erythropoiesis, it seems to be sufficient for adult erythropoiesis. In contrast, the expression of GATA1 appears to be almost

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completely abrogated in megakaryocytes of Gatallow mice. These mice suffer from severe thrombocytopenia with accumulation of poorly developed megakaryocytes throughout their lives [44]. Gatallow mice have features resembling those of patients with idiopathic myelofibrosis [45, 46].

Possible links of GATA1 gene mutations to human diseases Over the past two decades, a variety of mutations in the GATA1 and GATA2 genes have been

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ACCEPTED MANUSCRIPT identified as causes of inherited and sporadic hematopoietic disorders. Contributions of GATA1 and GATA2 to hematopoiesis in vivo have been investigated by means of mouse gene manipulations and genetic engineering. The genotype-phenotype correlations for Gata1 and

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Gata2 mutations in mice are summarized in Table 1. Although the phenotypes of these mouse models do not always correlate with the human phenotypes even if similar gene mutations are identified in human diseases, analyses of mouse models have provided important insights for

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guiding human case analyses and for increasing the understanding of the pathophysiological mechanisms of human diseases. For instance, GATA1-related leukemias share many common

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features between mice and humans, but they are not identical. In this regard, if the mutations result in embryonic lethal phenotypes in mice, a transgenic complementation rescue approach involving expression of mutant proteins at several-fold higher levels than the endogenous levels is a useful way to circumvent this problem. Salient examples of this approach will be described

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in later sections.

To our knowledge, human diseases caused by mutations in the cis-regulatory regions of the GATA1 gene have not been reported to date. Because the GATA1 and Gata1 genes are located on

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the X chromosomes of humans and mice, respectively, human disorders caused by GATA1 gene mutations show X-chromosome inheritance and severe phenotypes in males. Further, regulatory

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and non-synonymous mutations in the GATA1 gene that are linked to severe dyserythropoiesis are predicted to cause embryonic lethality. In contrast, females with loss-of-function mutations in the GATA1 gene appear as asymptomatic carriers unless the mutant GATA1 acquires a dominant negative function (Fig. 3). Female carriers of GATA1 mutations harbor two types of cells that differ depending on which X chromosome is developmentally inactivated. One type of cell retains a chromosome with an activate wild-type GATA1 gene, whereas the other retains a chromosome with an active GATA1 mutant allele. Erythropoiesis and platelet production in the

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ACCEPTED MANUSCRIPT latter type of cell is hindered due to the aberrant expression of GATA1, whereas the former type exhibits terminal maturation of erythroid and megakaryocytic progenitors in females (Fig. 3).

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Perturbations of GATA1/Gata1 gene expression and leukemogenesis As mentioned above, GATA1.05 heterozygous female mice (Gata11.05/X) are prone to developing leukemia with the c-Kit+CD71+ immunophenotype [47]. Intriguingly, heterozygous female mice

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with complete knockout of Gata1 (Gata1KO/X) never develop this type of leukemia. Erythroid progenitors recovered from Gata11.05/X mice harbor an X chromosome with an active mutant

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allele, and their differentiation is arrested. This feature has also been observed in progenitor cells recovered from Gata1KO/X mice. However, a clear-cut difference exists between these two types of GATA1-mutant progenitors; in Gata11.05/X mice, which display residual GATA1 expression equivalent to 5% of the normal level, apoptotic elimination of immature erythroid

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progenitors is blocked, whereas apoptotic elimination of these progenitors is not blocked in Gata1KO/X mice. Therefore, progenitors with an active GATA1.05 allele continue to survive in hematopoietic organs under arrested conditions, and these progenitors are prone to acquiring

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extra genetic aberrations implicated in stepwise leukemogenesis (Fig. 3) [47]. Female mice with heterozygous deletions of the IE exon are also prone to developing c-Kit+CD71+-leukemia [27].

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We surmise that this predisposition may be the result of ∆NT-GATA1 formation. Naturally occurring leukemias in Gata11.05/X mice provide insights into a deeper understanding of leukemic stem cells (LSCs) [48]. LSCs that possess the capacity to reinitiate leukemia represent a small minority of the quiescent population of leukemic cells and are usually resistant to chemotherapeutic agents, similar to normal HSCs [49]. Therefore, once chemotherapeutic agents eradicate leukemic cells and normal chemo-sensitive cells, LSCs and HSCs exit quiescence and enter the cell cycle, leading to the production of numerous offspring

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ACCEPTED MANUSCRIPT to return to the previous steady-state. LSCs in Gata11.05/X mice are morphologically indistinguishable from other leukemic cells, with a proerythroblast-like appearance [48]. Unlike normal HSCs, LSCs that enter the cell cycle fail to return to the previous quiescent state and

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continue to replicate [48]. Thus, this difference between LSCs and HSCs is possibly implicated in malignant evolution of relapsing leukemias and might be an effective new therapeutic target of refractory leukemias.

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It has been shown that GATA1 coordinates the expression of diversified target genes that play important roles in promoting differentiation, controlling proliferation, and preventing

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apoptosis. Therefore, perturbation and imbalance of GATA1 function brought about by various mutations in the GATA1 gene result in alterations of the physiological features of the affected progenitors, leading to pathogenesis in hematological lineages.

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Diseases caused by imbalance of N-finger function of GATA1

Because DNA binding via the C-finger is a requisite for GATA1 function, transgenic expression of GATA1 lacking the C-finger cannot rescue the phenotype of GATA1-deficient mice. However,

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the situation with the N-finger is different. Transgenic overexpression of ∆NF-GATA1 (GATA1 lacking the complete N-finger) can compensate, albeit partly, for the functional defect caused by

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loss of NF function in mice [50, 51]. Of note, point mutations in the NF domain of GATA1 have been identified in patients with dyserythropoiesis and thrombocytopenia [52]. This is in clear contrast with the situation with the C-finger; to our knowledge, no such mutation in the C-finger region has been reported as a cause of human disease. One of the important functions of the N-finger is interaction with friend of GATA1 (FOG1), a specific co-factor for GATA factors [53]. Several families have been reported with substitution mutations in amino acids that interact with FOG1 [52]. A mouse model carrying the

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ACCEPTED MANUSCRIPT GATA1 mutation has been developed that lacks this interaction with FOG1, and it shows a decreased potential to interact with FOG1. Analyses of this line of mice have revealed that the GATA1-FOG1 interaction specifically contributes to erythropoiesis and megakaryopoiesis

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through the expression of genes involved in platelet production and erythroid membrane structure [54-56].

Another important function of the N-finger is participation in the transactivation of GATA1

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by eliciting conformational changes in GATA1 that increase its binding to DNA [57]. Lack of DNA interaction through the N-finger of GATA1 causes porphyria and thalassemia in humans

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[58, 59]. The amino acid residues responsible for the interaction of DNA with two GATA motifs aligned in a palindromic orientation reside on the DNA-binding face of the N-finger domain [60, 61], which is located on the GATA1 face opposite the face that interacts with FOG1 [62].

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Pathogenesis due to imbalance of the transactivation function of GATA1 It has been well documented that two types of GATA1 isoforms are generated depending on whether the second exon is skipped. Full-length GATA1 and a short form of GATA1 (GATA-S)

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are produced that utilize alternative initiation codons located in the second and third exons, respectively, even in healthy humans [63]. GATA1-S differs from full-length GATA1 in absence

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of the N-terminal 83 amino acids. Full-length and ∆NT-GATA1 (mouse homolog of GATA1-S) proteins are also produced in mice that utilize two alternative in-frame translation initiation codons in a single transcript [64]. Importantly, although GATA-S/∆NT-GATA1 lacks the N-terminal transactivation domain of GATA1 (corresponding to the 83-amino-acid fragment), it contains another transactivation domain located in the C-terminal region [65]. These two transactivation domains of GATA1 independently and/or cooperatively participate in the regulation of diverse target genes [65].

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ACCEPTED MANUSCRIPT Several families with germline mutations at the boundary of the second exon have been identified in which some members suffer from inherited dyserythropoiesis and DBA [41-43]. In these cases, GATA1-S, instead of full-length GATA1, is exclusively produced by means of

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second-exon skipping or due to the presence of a missense mutation in the first initiation codon in the second exon. Intriguingly, although the dyserythropoiesis phenotype that occurs due to defective GATA1 function is remarkable, symptoms related to megakaryopoiesis and platelet

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production are rarely observed [41-43]. GATA1-S exerts its effects on immature progenitors to bias differentiation toward the myeloid lineage rather than toward erythroid cells, and immature

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progenitors carrying a GATA1-S mutation exhibit specifically impaired erythroid differentiation [66, 67]. These findings suggest that GATA1 supports megakaryopoiesis and platelet production during adulthood without requiring extensive contribution of the N-terminal transactivation domain.

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In contrast, many lines of evidence support the notion that the function of the N-terminal transactivation domain of GATA1 is involved in the onset of acute megakaryocytic leukemia (AMKL) in Down syndrome (DS) children. DS children are at 20- and 500-fold greater risks of

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developing acute lymphoblastic leukemias and AMKLs, respectively, than the general population [68]. AMKLs in DS children follow a characteristic clinical course. Circulating

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blasts with the erythroid-megakaryocytic immunophenotype are found in approximately 5-30% of DS babies at birth, with accompanying organopathy caused by blast cell infiltration. The symptoms of affected DS babies are quite similar to those of acute leukemia, except that the blasts spontaneously disappear within 6 months after birth. The transient hyperleukocytosis observed in DS babies is referred to as transient myeloproliferative disease (TMD). Of note, 10-20% of DS children with a TMD history develop genuine AMKL after several years of asymptomatic latency.

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ACCEPTED MANUSCRIPT An intriguing finding related to the above information is that GATA1 gene mutations leading to the production of GATA1-S can be found in almost all TMD cases [69, 70]. In analyses of TMD/AMKL in non-DS children, it has been shown that blasts often carry an extra

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copy of chromosome 21 or exhibit partial trisomy 21 due to mosaic DS or acquired trisomy 21 [71]. Occasionally, multiple clones carrying different GATA1 gene mutations are simultaneously found in an individual with TMD, suggesting that the GATA1 locus is prone to acquiring genetic

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events in the presence of trisomy 21.

In extensive analysis of individual cases in which AMKL developed after a history of TMD,

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the GATA1 mutation in AMKL blasts was frequently found to be identical to the mutation identified in TMD blasts. Of note, whereas TMD blasts rarely carry mutations other than the GATA1 mutation, mutations in multiple genes accumulate in AMKL cells [72]. Therefore, aberrant GATA1 function due to the GATA1 gene mutation seems to be related to the onset of

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TMD and seems to occur prior to the additional genetic event(s) that triggers AMKL, indicating that AMKL development in DS children follows a typical multi-step leukemogenesis program. It is unclear how the extra copy of chromosome 21 contributes to the formation of

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GATA1-S and to TMD/AMKL development. Several DS model mice with increased dosages of identical genes on human chromosome 21 have been established. These animals have

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phenotypes that are partially similar to those observed in DS patients, including cranio-vertebral malformations and mental retardation, as well as hematological abnormalities. However, the model mice never acquire somatic Gata1 gene mutations [73]. In addition, introduction of Gata1 gene mutations into DS model mice never drives leukemia [74]. Thus, the mouse model of DS does not accurately mimic the molecular pathology of human TMD and AMKL. We have previously established mice carrying the ∆NT-GATA1 mutation using the transgenic complementation rescue method. Consistent with our expectations, transgenic

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ACCEPTED MANUSCRIPT expression of ∆NT-GATA1 under Gata1 gene regulation rescues GATA1-deficient mice from embryonic lethality. Intriguingly, the rescued mice suffer from transient massive megakaryocytosis during the embryonic stage, which resembles that observed in association

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with the TMD phenotype [74]. Showing very good agreement, ∆NT-GATA1 knock-in animals have been demonstrated to exhibit transient hyper-megakaryopoiesis during the early embryonic stage but to remain healthy until adulthood [75]. Thus, the ∆NT-GATA1 mutation alone drives

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hyperproliferation of embryonic megakaryocytes in disomic mice. We envisage that an imbalance of N- and C-terminal transactivation domains and resulting alteration in GATA1

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function is related to the hyperproliferative phenotype of embryonic megakaryocytes.

Conclusions

Despite the advances in molecular dissection of GATA1 and GATA2, the pathogenesis of

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GATA-related disorders remains enigmatic due to our limited understanding of the diverse regulatory mechanisms of GATA1 and GATA2 gene expression. Cross-regulation of the GATA1 and GATA2 genes and their redundant and competitive roles in hematopoiesis hinder

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straightforward analysis and interpretation. Thus, we surmise that currently unknown GATA-related disorders must exist. Although animal models of diseases cannot always be

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extrapolated to humans, efforts in line with analyses of model mice will clarify particular aspects of GATA-related hematopoietic disorders.

Acknowledgments We thank Ms. Aya Goto for the technical help. This work was supported in part by JSPS KAKENHI (RS; grant numbers 15H04759, MS; 15H02507), the Platform for Drug Discovery, Informatics, and Structural Life Science of the Japan Agency for Medical Research and

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ACCEPTED MANUSCRIPT Development (AMED) (RS and MY), AMED-Core Research for Evolutional Science and Technology (CREST) (RS and MY), the Naito Foundation (MY) and the Takeda Medical

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Research Foundation (MY).

Conflicts of interest disclosure

No financial interests/relationships with financial interests related to the topic of this review are

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declared.

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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. Gene structure and cis-regulatory elements of the GATA2 gene. Notably, these elements contain GATA sites. The -77-kb region, promoter-neighboring region, and +9.5-kb

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region, which are involved in GATA2 gene expression in myeloerythroid progenitors, bi-potential precursors in the AGM region, and hematopoietic stem cells, respectively, are depicted in yellow. Human hematological diseases caused by mutations in the cis-regulatory

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elements of the GATA2 gene have been reported [16, 17, 19, 20].

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Figure 2. Gene structure and cis-regulatory elements of the GATA1 gene. Notably, these elements contain GATA sites. The GATA sites located in the -3.9-kb region and the promoter proximal enhancer region are depicted in red. GATA2 access to the GATA sites is blocked through the G1MDR in hematopoietic stem cells. When GATA2 binds and initiates Gata1 gene

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expression in early progenitor cells, a positive regulatory loop of GATA1 transcription involving GATA sites that are widely scattered throughout the Gata1 gene is generated. Some of these sites may not yet have been identified; these potentially unidentified sites are depicted in pink.

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lineage.

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Consequently, GATA1 accumulates in progenitor cells that are committed to the erythroid

Figure 3. X-linked or aberrant GATA1 gene expression is associated with hematological diseases. Because Gata1 is located on the X chromosome and hematopoietic cells with an X chromosome harboring an active wild-type GATA1 allele can support normal hematopoiesis, females carrying inherited loss-of-function mutations are usually asymptomatic carriers. Affected males in a family tree show anemia and thrombocytopenia, the severity and specificity of which depend on the imbalance in GATA1 function, determined by the mutation type and

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ACCEPTED MANUSCRIPT expression level of GATA1. In addition, embryonic lethality is predicted in severe cases. In contrast, hematopoietic cells with aberrantly activated GATA1 function are prone to transform

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into leukemic cells.

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ACCEPTED MANUSCRIPT Table 1. Genotype-phenotype correlations for Gata1 and Gata2 mutations in mice Types of gene mutations in mice

Ref.

Phenotypes in mice

Homozygotes: die at E10 Heterozygotes: do not show MonoMAC-like phenotype, although the HSC function is slightly disturbed. Targeted deletion of Gata2 IS Homozygotes: normal at birth without significant exon hematopoietic abnormalities. Targeted deletion of either -3.9, Homozygotes: born alive without significant -2.8 or -1.8 cis-element of hematopoietic abnormalities. Gata2 gene Homozygotes: die by E14.5 without long-term repopulating HSC activity Targeted deletion of +9.5 Heterozygotes: do not show MonoMAC-like phenotype, cis-element of Gata2 gene although long-term repopulating HSC activity is reduced. Homozygotes: survive beyond E15.5 but die before Targeted deletion of -77 weaning, probably due to defective myeloid progenitor cis-element of Gata2 gene cell function. Linked BAC transgene Transgenic mice: predisposed to leukemia resembling recapitulating 3q21q26 3q21q26 syndrome in humans. translocation Males: die by E11.5 due to failure of primitive erythropoiesis. Null mutation in Gata1 gene Females: born with slight anemia but grow up normally. Males: die by E11.5 due to failure of primitive erythropoiesis. Disruption of Gata1 IE exon Females: born with slight anemia and are predisposed to leukemia. Targeted deletion of GATA-site, either in the 3.9-kb upstream Males: born alive with a slight defect in erythropoiesis region or the proximal enhancer region Males: die by E11.5 due to failure of primitive Gata1-knockdown (GATA1.05; erythropoiesis. reduced to 5% of endogenous Female: born with slight anemia and are predisposed to GATA1 expression) leukemia Males: show thrombocytopenia with increased Gata1-knockdown (Gata1low; megakaryocytes and develop myelofibrosis during reduced to 20% of endogenous adulthood, and pups show anemia that resolves by GATA1 expression) adulthood. Gata1 gene knockout during Males: show anemia with reduced erythroid progenitors adulthood and thrombocytopenia with increased megakaryocytes. Males: show anemia and thrombocytopenia with IE exon deletion during accumulation of erythroid progenitors and adulthood megakaryocytes.

8

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Null mutation in Gata2 gene

V205G-GATA1 knock-in

Males: die at E11.5-perinatal stage due to anemia.

V205G-GATA1 transgene

Gata1-knockdown males carrying the transgene: show anemia and thrombocytopenia resembling the familial diseases and suffer from spherocytic hemolytic anemia.

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8 11-13

14, 19

15

23

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34, 35

38, 47

35, 45, 46 39 40 54 55, 56

ACCEPTED MANUSCRIPT Gata1-knockdown males carrying the transgene: die in utero due to failure of definitive erythropoiesis. (It is unknown whether mice show porphyria and thalassemia-like phenotype). Male embryos: show TMD-like transient megakaryocyte hyperplasia even without trisomy. Gata1-knockdown males carrying the transgene: show transient megakaryocyte hyperplasia in newborns even without trisomy, which mimics TMD in DS children, and show anemia throughout of life resembling DBA.

R216Q-GATA1 transgene

∆NT-GATA1 knock-in

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50, 69

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∆NT-GATA1 transgene

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Transgene of GATA1 lacking Gata1-knockdown males carrying the transgene: die in C-terminal transactivation utero due to failure of definitive erythropoiesis. domain

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ACCEPTED MANUSCRIPT [74] Shimizu R, Kobayashi E, Engel JD, Yamamoto M. Induction of hyperproliferative fetal megakaryopoiesis by an N-terminally truncated GATA1 mutant. Genes Cells. 2009;14:1119-1131. [75] Li Z, Godinho FJ, Klusmann JH, Garriga-Canut M, Yu C, Orkin SH. Developmental

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