Analysis of disease-causing GATA1 mutations in murine gene complementation systems

Abstract

Missense mutations in transcription factor GATA1 underlie a spectrum of congenital red blood cell and platelet disorders. We investigated how these alterations cause distinct clinical phenotypes by combining structural, biochemical, and genomic approaches with gene complementation systems that examine GATA1 function in biologically relevant cellular contexts. Substitutions that disrupt FOG1 cofactor binding impair both gene activation and repression and are associated with pronounced clinical phenotypes. Moreover, clinical severity correlates with the degree of FOG1 disruption. Surprisingly, 2 mutations shown to impair DNA binding of GATA1 in vitro did not measurably affect in vivo target gene occupancy. Rather, one of these disrupted binding to the TAL1 complex, implicating it in diseases caused by GATA1 mutations. Diminished TAL1 complex recruitment mainly impairs transcriptional activation and is linked to relatively mild disease. Notably, different substitutions at the same amino acid can selectively inhibit TAL1 complex or FOG1 binding, producing distinct cellular and clinical phenotypes. The structure-function relationships elucidated here were not predicted by prior in vitro or computational studies. Thus, our findings uncover novel disease mechanisms underlying GATA1 mutations and highlight the power of gene complementation assays for elucidating the molecular basis of genetic diseases. (Blood. 2013;121(26):5218-5227) IntroductionErythrocyte and megakaryocyte development are under the control of transcription factor GATA1.1,2 GATA1 promotes differentiation by activating all known erythroid-and megakaryocytespecific genes and silencing genes associated with the immature, proliferative state and alternative lineages (for review, see Ferreira et al 3 ). GATA1 contains 2 highly conserved zinc finger (ZF) domains. The C-terminal ZF primarily binds to the sequence (A/T)GATA(A/G) while the N-terminal ZF (NF) stabilizes DNA interactions by contacting noncanonical GATC and palindromic ATC(A/T)GATA(A/G) motifs. [4][5][6] The NF also binds coregulators, including the multi-ZF protein FOG1.7 Like GATA1, FOG1 is required for erythroid and megakaryocyte development, and disrupting the GATA1-FOG1 interaction impairs maturation of these lineages. [8][9][10] Activation and repression of most GATA1-regulated genes requires FOG1, 11,12 as does silencing of mast cell-specific genes.13-15 FOG1 also modulates GATA1 chromatin occupancy at a subset of genomic sites. [15][16][17] Additionally, the TAL1 complex, composed of TAL1, E2A, LMO2, and Ldb1, interacts via LMO2 with the GATA1 NF.18,19 TAL1, LMO2, and Ldb1 are essential for erythrocyte and megakaryocyte differentiation.20-22 TAL1 complex recruitment occurs predominantly at GATA1-activated genes and tends to be depleted at sites where GATA1 functions as a repressor. 23,24 The distinct interaction surfaces of GATA1 that contact DNA, FOG1, and LMO2 have been defined previously. 19,25,26 Missense mutations in the GATA1 NF cause distinct forms of congenital anemia and thrombocy...

Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus

“…However, several disease-associated amino acid substitutions within the N-finger inhibit GATA1 binding to target genes in vivo, yet have no effect on in vitro binding to naked DNA templates. These regions of the N-finger do not interact with DNA directly, but rather are believed to promote GATA1 chromatin occupancy by recruiting essential cofactors, including FOG1 and the TAL1/LMO2/LDB1 complex (35,38). Similarly, loss of the Kruppel-like factor 3 transcription N-terminus, which also does not bind DNA directly, alters chromatin occupancy selectively at a subset of in vivo target genes in mouse embryonic fibroblasts (54).…”

Insight into GATA1 transcriptional activity through interrogation ofciselements disrupted in human erythroid disorders

“…In several Mendelian erythroid disorders (MEDs), NC genetic variants have been identified in the DNA-binding motif (WGATAR) of the hematopoietic master regulator, GATA1 (20)(21)(22)(23)(24)(25). GATA1 is both necessary for proper erythropoiesis (3,26) and sufficient to reprogram alternative lineages toward an erythroid fate (27,28). Moreover, numerous studies have shown that GATA1 acts in a…”

Role of LDB1 in the transition from chromatin looping to transcription activation

“…7A, middle; Supplemental Fig. S10B), including a-globin, b-globin, Alas2, and Gypa, whose transcription is known to be FOG1-dependent (Crispino et al 1999;Anguita et al 2004;Letting et al 2004;Pal et al 2004; Campbell et al 2013). FOG1 occupied these genes or their regulatory regions in cells expressing LDB1 FL but was significantly decreased in cells expressing LDB1D4/5 (Fig.…”