Search

This study aimed to identify a urinary metabolite panel for the detection/diagnosis of pancreatic ductal adenocarcinoma (PDAC). PDAC continues to have poor survival outcomes. One of the major reasons for poor prognosis is the advanced stage of the disease at diagnosis. Hence, identification of a novel and cost-effective biomarker signature for early detection/diagnosis of PDAC could lead to better survival outcomes. Untargeted metabolomics was employed to identify a novel metabolite-based biomarker signature for PDAC diagnosis. Urinary metabolites from 92 PDAC patients (56 discovery cohort and 36 validation cohort) were compared with 56 healthy volunteers using 1 H-nuclear magnetic resonance (1 H-NMR) spectroscopy. Multivariate (Partial-least squares discriminate analysis) and univariate (Mann-Whiney's U-test) analysis were performed to identify a metabolite panel which can be used to detect PDAC. The selected metabolites were further validated for their diagnostic potential using the area under the receiver operating characteristic (AUROC) curve. Statistical analysis identified a six-metabolite panel (trigonelline, glycolate, hippurate, creatine, myoinositol and hydroxyacetone) which demonstrated high potential to diagnose PDAC, with AUROC of 0.933 and 0.864 in the discovery and validation cohort, respectively. Notably, the identified panel also demonstrated very high potential to diagnose early stage (I and II) PDAC patients with AUROC of 0.897. These results demonstrate that the selected metabolite signature could be used to detect PDAC and will pave the way for the development of a urinary test for detection/diagnosis of PDAC.

Significance Large DNA-encoded libraries of cyclic peptides are emerging as powerful sources of molecules to tackle challenging drug targets. The structural and functional diversity contained within these libraries is, however, little explored. Here we demonstrate that one such library contains members that use unexpectedly diverse mechanisms to recognize the same surface on the same target proteins with high affinity and specificity. This range of binding modes is much larger than observed in natural ligands of the same proteins, demonstrating the power and versatility of the technology. Our data also reveal opportunities for the development of more sophisticated approaches to achieving specificity when trying to selectively target one member of a family of closely related proteins., Cyclic peptide library screening technologies show immense promise for identifying drug leads and chemical probes for challenging targets. However, the structural and functional diversity encoded within such libraries is largely undefined. We have systematically profiled the affinity, selectivity, and structural features of library-derived cyclic peptides selected to recognize three closely related targets: the acetyllysine-binding bromodomain proteins BRD2, -3, and -4. We report affinities as low as 100 pM and specificities of up to 106-fold. Crystal structures of 13 peptide–bromodomain complexes reveal remarkable diversity in both structure and binding mode, including both α-helical and β-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural preorganization. Our data demonstrate the enormous potential within these libraries to provide diverse binding modes against a single target, which underpins their capacity to yield highly potent and selective ligands.

Protein aggregation constitutes a recurring complication in the manufacture and clinical use of therapeutic monoclonal antibodies (mAb) and mAb derivatives. Antibody aggregates can reduce production yield, cause immunogenic reactions, decrease the shelf-life of the pharmaceutical product and impair the capacity of the antibody monomer to bind to its cognate antigen. A common strategy to tackle protein aggregation involves the identification of surface-exposed aggregation-prone regions (APR) for replacement through protein engineering. It was shown that the insertion of N-glycosylation sequons on amino acids proximal to an aggregation-prone region can increase the physical stability of the protein by shielding the APR, thus preventing self-association of antibody monomers. We recently implemented this approach in the Fab region of full-size adalimumab and demonstrated that the thermodynamic stability of the Fab domain increases upon N-glycosite addition. Previous experimental data reported for this technique have lacked appropriate confirmation of glycan occupancy and structural characterization of the ensuing glycan profile. Herein, we mutated previously identified candidate positions on the Fab domain of Trastuzumab and employed tandem mass spectrometry to confirm attachment and obtain a detailed N-glycosylation profile of the mutants. The Trastuzumab glycomutants displayed a glycan profile with significantly higher structural heterogeneity compared to the HEK Trastuzumab antibody, which contains a single N-glycosylation site per heavy chain located in the CH2 domain of the Fc region. These findings suggest that Fab N-glycosites have higher accessibility to enzymes responsible for glycan maturation. Further, we have studied effects on additional glycosylation on protein stability via accelerated studies by following protein folding and aggregation propensities and observed that additional glycosylation indeed enhances physical stability and prevent protein aggregation. Our findings shed light into mAb glycobiology and potential implications in the application of this technique for the development of “biobetter” antibodies.

Almost all eukaryotic proteins receive diverse post-translational modifications (PTMs) that modulate protein activity. Many histone PTMs are well characterized, heavily influence gene regulation, and are often predictors of distinct transcriptional programs. Although our understanding of the histone PTM network has matured, much is yet to be understood about the roles of transcription factor (TF) PTMs, which might well represent a similarly complex and dynamic network of functional regulation. Members of the bromodomain and extra-terminal domain (BET) family of proteins recognize acetyllysine residues and relay the signals encoded by these modifications. Here, we have investigated the acetylation dependence of several functionally relevant BET-TF interactions in vitro using surface plasmon resonance, nuclear magnetic resonance, and X-ray crystallography. We show that motifs known to be acetylated in TFs E2F1 and MyoD1 can interact with all bromodomains of BRD2, BRD3, and BRD4. The interactions are dependent on diacetylation of the motifs and show a preference for the first BET bromodomain. Structural mapping of the interactions confirms a conserved mode of binding for the two TFs to the acetyllysine binding pocket of the BET bromodomains, mimicking that of other already established functionally important histone- and TF-BET interactions. We also examined a motif from the TF RelA that is known to be acetylated but were unable to observe any interaction, regardless of the acetylation state of the sequence. Our findings overall advance our understanding of BET-TF interactions and suggest a physical link between the important diacetylated motifs found in E2F1 and MyoD1 and the BET-family proteins.

Graphical abstract, Highlights • The mechanisms by which ISL1 and LHX3 specify neuronal cell identity are unknown. • EMSA/SPR data show ISL1 and LHX3 have markedly different DNA-binding behaviours. • SAXS shows ISL1/LHX3:DNA complexes are flexible in nature. • ISL1 binds DNA poorly but appears to modulate the DNA-binding specificity of LHX3., The roles of ISL1 and LHX3 in the development of spinal motor neurons have been well established. Whereas LHX3 triggers differentiation into interneurons, the additional expression of ISL1 in developing neuronal cells is sufficient to redirect their developmental trajectory towards spinal motor neurons. However, the underlying mechanism of this action by these transcription factors is less well understood. Here, we used electrophoretic mobility shift assays (EMSAs) and surface plasmon resonance (SPR) to probe the different DNA-binding behaviours of these two proteins, both alone and in complexes mimicking those found in developing neurons, and found that ISL1 shows markedly different binding properties to LHX3. We used small angle X-ray scattering (SAXS) to structurally characterise DNA-bound species containing ISL1 and LHX3. Taken together, these results have allowed us to develop a model of how these two DNA-binding modules coordinate to regulate gene expression and direct development of spinal motor neurons.

A thermostable drug suppresses a pERK-FosB/ΔFosB-VCAM-1 axis, endothelial activation, angiogenesis, and retinal permeability., Vascular permeability and angiogenesis underpin neovascular age-related macular degeneration and diabetic retinopathy. While anti-VEGF therapies are widely used clinically, many patients do not respond optimally, or at all, and small-molecule therapies are lacking. Here, we identified a dibenzoxazepinone BT2 that inhibits endothelial cell proliferation, migration, wound repair in vitro, network formation, and angiogenesis in mice bearing Matrigel plugs. BT2 interacts with MEK1 and inhibits ERK phosphorylation and the expression of FosB/ΔFosB, VCAM-1, and many genes involved in proliferation, migration, angiogenesis, and inflammation. BT2 reduced retinal vascular leakage following rat choroidal laser trauma and rabbit intravitreal VEGF-A165 administration. BT2 suppressed retinal CD31, pERK, VCAM-1, and VEGF-A165 expression. BT2 reduced retinal leakage in rats at least as effectively as aflibercept, a first-line therapy for nAMD/DR. BT2 withstands boiling or autoclaving and several months’ storage at 22°C. BT2 is a new small-molecule inhibitor of vascular permeability and angiogenesis.

The development of low-affinity fragment hits into higher affinity leads is a major hurdle in fragment-based drug design. Here we demonstrate an approach for the Rapid Elaboration of Fragments into Leads (REFiL) applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure-activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. Upon completion of a fragment screen against Bromodomain-3 extra terminal (BRD3-ET) domain we applied the REFiL workflow, which allowed us to develop a series of tetrahydrocarbazole ligands that bind to the peptide binding site of BRD3-ET. With REFiL we were able to rapidly improve binding affinity >30-fold. The REFiL workflow can be applied readily to a broad range of protein targets without the need of a structure, allowing the efficient evolution of low-affinity fragments into higher affinity leads and chemical probes.

Members of the bromodomain and extra-terminal domain (BET) family of proteins (bromodomain-containing (BRD) 2, 3, 4, and T) are widely expressed and highly conserved regulators of gene expression in eukaryotes. These proteins have been intimately linked to human disease, and more than a dozen clinical trials are currently underway to test BET-protein inhibitors as modulators of cancer. However, although it is clear that these proteins use their bromodomains to bind both histones and transcription factors bearing acetylated lysine residues, the molecular mechanisms by which BET family proteins regulate gene expression are not well defined. In particular, the functions of the other domains such as the ET domain have been less extensively studied. Here, we examine the properties of the ET domain of BRD3 as a protein/protein interaction module. Using a combination of pulldown and biophysical assays, we demonstrate that BRD3 binds to a range of chromatin-remodeling complexes, including the NuRD, BAF, and INO80 complexes, via a short linear “KIKL” motif in one of the complex subunits. NMR-based structural analysis revealed that, surprisingly, this mode of interaction is shared by the AF9 and ENL transcriptional coregulators that contain an acetyl-lysine-binding YEATS domain and regulate transcriptional elongation. This observation establishes a functional commonality between these two families of cancer-related transcriptional regulators. In summary, our data provide insight into the mechanisms by which BET family proteins might link chromatin acetylation to transcriptional outcomes and uncover an unexpected functional similarity between BET and YEATS family proteins.

The aim of the present study was to determine the relationships between the blood metabolome and (1) carcass traits with a focus on intramuscular fat (marbling), and (2) the length of time cattle consumed a high-starch diet in feedlot cattle. Blood samples were obtained from 181 Wagyu-crossbred steers between 300–400 days before slaughter when carcass data was collected. 1H NMR spectroscopy identified 35 metabolites with 7 positively associated with marbling (3-hydroxybutyrate, propionate, acetate, creatine, histidine, valine, and isoleucine; P ≤ 0.05). Subcutaneous rump fat thickness was positively associated with glucose, leucine and lipids (P ≤ 0.05) and negatively associated with anserine and arabinose (P ≤ 0.05). Carcass weight and growth rate were negatively associated with 3-hydroxybutyrate (P 0.05), and growth rate was negatively associated with creatine (P P 0.05). Glucose and arginine showed a significant interaction between marbling and number of days animals consumed a high-starch diet (P 0.05). Sire was the single variable with the largest effect on the relative concentration of metabolites and carcass and production traits. Blood metabolomics helps understand fat and muscle metabolism, and is associated with genotype, and carcass and production traits in cattle offering potential biomarkers suitable to select animals for management and genetic improvement.

Given the increasing incidence of antibiotic resistance, antibiotics that employ new strategies are urgently needed. Bacterial survival is dependent on proper function of the signal recognition particle (SRP) and its receptor (FtsY). A unique set of interactions in FtsY:SRP-RNA represents a promising candidate for new antibiotic development as no antibiotic targets this complex and these interactions are functionally replaced by protein:protein interactions in eukaryotes. We used a Fragment Based Drug Design (FBDD) approach to search for new compounds that can bind FtsY, and have identified three lead fragments. In vitro and in vivo analyses have shown that despite a high micromolar binding affinity, one fragment has some antimicrobial properties. X-ray structures of E. coli FtsY:fragments reveal the fragments bind in the targeted RNA interaction site. Our results show that FBDD is a suitable approach for targeting FtsY:SRP-RNA for antibiotic development and opens the possibility of targeting protein:RNA interactions in general.

The transcription factor GATA1 helps regulate the expression of thousands of genes involved in blood development, by binding to single or double GATA sites on DNA. An important part of gene activation is chromatin looping, the bringing together of DNA elements that lie up to many thousands of basepairs apart in the genome. It was recently suggested, based on studies of the closely related protein GATA3, that GATA-mediated looping may involve interactions of each of two zinc fingers (ZF) with distantly spaced DNA elements. Here we present a structure of the GATA1 ZF region bound to pseudopalindromic double GATA site DNA, which is structurally equivalent to a recently-solved GATA3-DNA complex. However, extensive analysis of GATA1-DNA binding indicates that although the N-terminal ZF (NF) can modulate GATA1-DNA binding, under physiological conditions the NF binds DNA so poorly that it cannot play a direct role in DNA-looping. Rather, the ability of the NF to stabilize transcriptional complexes through protein-protein interactions, and thereby recruit looping factors such as Ldb1, provides a more compelling model for GATA-mediated looping.

Peptide aptamers of LIM-only protein 2 (Lmo2) were previously used to successfully treat Lmo2-induced tumours in a mouse model of leukaemia. Here we show that the Lmo2 aptamer PA207, either as a free peptide or fused to thioredoxin Trx-PA207, causes purified Lmo2 to precipitate rather than binding to a defined surface on the protein. Stabilisation of Lmo2 through interaction with LIM domain binding protein 1 (Ldb1), a normal binding partner of Lmo2, abrogates this effect. The addition of free zinc causes Trx-PA207 to self associate, suggesting that PA207 destabilises Lmo2 by modulating normal zinc-coordination in the LIM domains. GST-pulldown experiments with other Lmo and Gata proteins indicates that PA207 can bind to a range of zinc finger proteins. Thus, PA207 and other cysteine-containing peptide aptamers for Lmo2 may form a class of general zinc finger inhibitors.

Lmo2 is a LIM-only protein involved in hematopoiesis and the development of T-cell acute lymphoblastic leukaemia. Here we report backbone and side chain NMR assignments for an engineered intramolecular complex of the C-terminal LIM domain from Lmo2 tethered to the LIM interaction domain (LID) from LIM domain binding protein 1 (Ldb1).

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to alpha-synuclein of approximately 1 x 10(9)M(-1) was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).

Although metal ions can promote amyloid formation from many proteins, their effects on the formation of amyloid from transthyretin have not been previously studied. We therefore screened the effects of Cu(II), Zn(II), Al(III), and Fe(III) on amyloid formation from wild-type (WT) transthyretin as well as its V30M, L55P, and T119M mutants. Cu(II) and Zn(II) promoted amyloid formation from the L55P mutant of transthyretin at pH 6.5 but had little effect on amyloid formation from the other forms of the protein. Zn(II) promoted L55P amyloid formation at pH 7.4 but Cu(II) inhibited it. Cu(II) gave dose-dependent quenching of the tryptophan fluorescence of transthyretin and the fluorescence of 1-anilino-8-naphthalene sulfonate bound to it. Zn(II) gave dose-dependent quenching of the tryptophan but not the 1-anilino-8-naphthalene sulfonate fluorescence. Apparent dissociation constants for Cu(II) and Zn(II) binding at pH 7.4 of approximately 10 nM and approximately 1 microM (approximately 0.4 microM and approximately 5 microM at pH 6.5), respectively, were obtained from the quenching data. Zn(II) enhanced urea-mediated the dissociation of the L55P but not the WT transthyretin tetramer. Cu(II), depending on its concentration, either had no effect or stabilized the WT tetramer but could enhance urea-mediated dissociation of L55P.

The transcription factor GATA1 helps regulate the expression of thousands of genes involved in blood development, by binding to single or double GATA sites on DNA. An important part of gene activation is chromatin looping, the bringing together of DNA elements that lie up to many thousands of basepairs apart in the genome. It was recently suggested, based on studies of the closely related protein GATA3, that GATA-mediated looping may involve interactions of each of two zinc fingers (ZF) with distantly spaced DNA elements. Here we present a structure of the GATA1 ZF region bound to pseudopalindromic double GATA site DNA, which is structurally equivalent to a recently-solved GATA3-DNA complex. However, extensive analysis of GATA1-DNA binding indicates that although the N-terminal ZF (NF) can modulate GATA1-DNA binding, under physiological conditions the NF binds DNA so poorly that it cannot play a direct role in DNA-looping. Rather, the ability of the NF to stabilize transcriptional complexes through protein–protein interactions, and thereby recruit looping factors such as Ldb1, provides a more compelling model for GATA-mediated looping.

Genetic disorders resulting from defects in the adult globin genes are among the most common inherited diseases. Symptoms worsen from birth as fetal γ-globin expression is silenced. Genome editing could permit the introduction of beneficial single-nucleotide variants to ameliorate symptoms. Here, as proof of concept, we introduce the naturally occurring Hereditary Persistance of Fetal Haemoglobin (HPFH) -175TC point mutation associated with elevated fetal γ-globin into erythroid cell lines. We show that this mutation increases fetal globin expression through de novo recruitment of the activator TAL1 to promote chromatin looping of distal enhancers to the modified γ-globin promoter.

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.

LIM-only protein 2, Lmo2, is a regulatory protein that is essential for hematopoietic development and inappropriate overexpression of Lmo2 in T-cells contributes to T-cell leukemia. It exerts its functions by mediating protein-protein interactions and nucleating multicomponent transcriptional complexes. Lmo2 interacts with LIM domain binding protein 1 (Ldb1) through the tandem LIM domains of Lmo2 and the LIM interaction domain (LID) of Ldb1. Here, we present the solution structure of the LIM2 domain of Lmo2 bound to Ldb1(LID) . The ordered regions of Ldb1 in this complex correspond well with binding hotspots previously defined by mutagenic studies. Comparisons of this Lmo2(LIM2) -Ldb1(LID) structure with previously determined structures of the Lmo2/Ldb1(LID) complexes lead to the conclusion that modular binding of tandem LIM domains in Lmo2 to tandem linear motifs in Ldb1 is accompanied by several disorder-to-order transitions and/or conformational changes in both proteins.

The fact that GATA-1 binds to gene regulatory elements that contain GATA sites, and thereby ultimately regulates the expression of hundreds of genes involved in hematopoietic development is well established. However, at a molecular level, the steps between DNA binding and changes in the expression levels of target genes are only partly understood, and the goal of our work is understand at a mechanistic level the changes in chromatin structure and occupancy and the protein interactions that are made to drive these changes in gene expression. We have recently shown that, surprisingly, the localization of GATA1 to its target sites in chromatin is dependent on acetylation of specific lysines in the protein, and have elucidated the structural basis for this requirement [1,2]. Our data point towards a model for gene regulation in which not only are the post-translational modifications of histones essential for establishing gene expression patterns, but equivalent modifications in DNA-binding transcription factors are also an integral part of whatever ‘code’ underlies these patterns. Concurrently, we have also determined the molecular details underlying the recruitment of the Nucleosome Remodeling and Deacetylase (NuRD) complex to GATA1-dependent gene promoters [3], an event that is essential for normal GATA1 activity. Combined with data that explore the molecular makeup of the NuRD complex, these results provide a glimpse into the mechanisms through which complex coregulator complexes are recruited to target genes and begin to map out the molecular events that drive gene regulation both in erythropoiesis and, by extension, in a range of other tissues.

Abstract 3439 Missense mutations in the gene encoding hematopoietic transcription factor GATA1 cause congenital anemias and/or thrombocytopenias. All seven reported mutations give rise to amino acid substitutions within the amino-terminal zinc finger (NF), but produce a range of phenotypes. The clinical severity depends on the site and type of substitution, and different substitutions of the same residue can produce disparate phenotypes. We combined structural, biochemical, in vivo conditional rescue approaches, and genomic analyses to systematically characterize all known GATA1 mutations with the goal of determining how they alter GATA1 function to result in disease. Introducing mutant forms of GATA1 into GATA1-null erythroid or bipotential erythromegakaryocytic cell lines essentially recapitulated patient phenotypes. The V205M, G208S, G208R, and D218Y mutations severely impaired both erythroid and megakaryocyte maturation, while the R216Q, R216W, and D218G mutations had only a mild effect on the maturation of these lineages. Global differentiation defects were reproduced at the level of individual GATA1 target genes. Moreover, the former mutants greatly impaired both the transcriptional activation and repression functions of GATA1, while the latter moderately impaired gene activation but had no effect on repression. It had been suggested previously that GATA1 mutations could be categorized into two classes, those that impair binding of the NF to the essential GATA1 cofactor FOG1 (V205M, G208S, G208R) and those that diminish binding of the NF to DNA (R216Q and R216W). The impact of the final two mutations (D218G and D218Y) remained uncertain, as this residue is not part of any known interaction face. Our work led to the following novel conclusions: Binding studies using isothermal titration calorimetry (ITC) and chromatin immunoprecipitation (ChIP) produced concurrent results showing that the V205M, G208S, G208R, and D218Y mutations diminish the GATA1-FOG1 interaction in vitro and FOG1 recruitment to GATA1 target genes in vivo. Interestingly, in contrast to D218Y, D218G did not affect FOG1 binding in vitro or in vivo. Furthermore, G208S had a less pronounced impact on FOG1 binding than the other three mutations, thus correlating the severity of the clinical presentation with the degree of FOG1 disruption. This confirms and extends previous work linking impaired FOG1 binding to the disease phenotypes associated with this class of mutations.ITC showed that R216Q and R216W disrupt DNA binding in vitro, consistent with previous in vitro studies. However, remarkably, ChIP assays revealed that neither mutation impaired in vivo GATA1 target site occupancy at any examined simple or palindromic GATA elements, suggesting that failure to bind DNA does not account for the associated clinical phenotypes.Notably, the R216Q and D218G mutations selectively diminished recruitment of Tal1/SCL without affecting the interaction with FOG1 or DNA. This implicates for the first time the Tal1/SCL complex in the pathogenesis of disorders caused by GATA1 mutations. Since the Tal1/SCL complex functions mostly during GATA1 gene activation, this also explains the observation that these GATA1 mutants largely retain their ability to repress transcription. Moreover, changes in the gene expression profiles of R216Q and D218G expressing cells are highly correlated with each other but clearly distinct from the gene expression changes associated with different substitutions at the same residues (R216W or D218Y), revealing a specific subset of genes that are most sensitive to disruption of the GATA1-Tal1/SCL interaction.An unexpected finding from our studies is that different substitutions of the same residue can disrupt binding to distinct cofactors (e.g. D218G impairs Tal1/SCL binding while D218Y impairs FOG1 binding), thus accounting for variable disease presentation. In concert, our work on GATA1 mutations in their native environment reveals critical new insights not obtainable from in vitro studies. This highlights the usefulness of gene complementation studies in the relevant lineages for the dissection of transcription pathways to better understand and ultimately diagnose and treat hematologic disease. Disclosures: No relevant conflicts of interest to declare.