Building Multifunctionality Into a Complex Containing Master Regulators of Hematopoiesis

Building multifunctionality into a complex containing master regulators of hematopoiesis

Tohru Fujiwara1, Hsiang-Ying Lee1, Rajendran Sanalkumar1, and Emery H. Bresnick2

Wisconsin Institutes for Medical Research, University of Wisconsin Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705

Edited by Stuart H. Orkin, Children’s Hospital and The Dana Farber Cancer Institute, Harvard Medical School and The Howard Hughes Medical Institute, Boston, MA, and approved October 14, 2010 (received for review June 3, 2010)

Developmental control mechanisms often use multimeric complexes GATA-1 forms a complex with another master regulator of containing transcription factors, coregulators, and additional non- hematopoiesis, the basic-helix–loop–helix transcription factor Scl/ DNA binding components. It is challenging to ascertain how such TAL1 (22). Scl/TAL1 regulates HSC function, erythropoiesis, and components contribute to complex function at endogenous loci. We is implicated in leukemogenesis (23–25). In vitro DNA binding analyzed the function of components of a complex containing results indicate that the GATA-1-Scl/TAL1 complex includes its master regulators of hematopoiesis (GATA-1 and Scl/TAL1) and the heterodimeric partner E2A and the non-DNA binding compo- non-DNA binding components ETO2, the LIM domain protein LMO2, nents LMO2 and LDB1 (22). The LIM domain protein LMO2 is leukemogenic in humans (26). Retroviral vectors used for gene and the chromatin looping factor LDB1. Surprisingly, we discovered fi that ETO2 and LMO2 regulate distinct target-gene ensembles in therapy of X-linked combined immunode ciency syndrome com- LMO2 erythroid cells. ETO2 commonly repressed GATA-1 function via sup- monly integrate into , activating its expression and inducing T-cell leukemia (26). LDB1 bears similarity to Drosophila Chip, pressing histone H3 acetylation, although it also regulated methyl- fi both implicated in mediating transcriptional control over a long ation of histone H3 at lysine 27 at select loci. Prior studies de ned distance on a chromosome (27, 28). Additional interactors with multiple modes by which GATA-1 regulates target genes with or the GATA-1-Scl/TAL1 complex include the non-DNA binding without the coregulator Friend of GATA-1 (FOG-1). LMO2 selectively component ETO2 (29, 30), which binds histone deacetylases repressed genes that GATA-1 represses in a FOG-1–independent (HDACs) and participates in a chromosomal translocation in GENETICS manner. As LMO2 controls hematopoiesis, its dysregulation is leu- acute myeloid leukemia (31). Single-stranded DNA binding pro- kemogenic, and its influence on GATA factor function is unknown, teins 2 and 3 (32) and the chromatin remodeler BRG1 (33) also this mechanistic link has important biological and pathophysio- bind the GATA-1-Scl/TAL1 complex. logical implications. The demonstration that ETO2 and LMO2 ex- GATA-1 colocalizes with Scl/TAL1 at chromatin sites harbor- ert qualitatively distinct functions at endogenous loci illustrates ing a GATA motif and an E-box, recognized by Scl/TAL1-E2A how components of complexes containing master developmental heterodimers, or solely a GATA motif (34–36). Before GATA-1– regulators can impart the capacity to regulate unique cohorts of mediated displacement of GATA-2 from chromatin, GATA-2 target genes, thereby diversifying complex function. colocalizes with Scl/TAL1 (34). Thus, GATA-2 may also assemble a complex with Scl/TAL1. Many questions remain unanswered blood | hematopoietic | genetic network | leukemia | progenitor cell regarding the functions of the GATA-Scl/TAL1 complex components, and the activities bestowed on the complex by these components. Given the GATA-1-Scl/TAL1 paradigm, it is in- he regulatory machinery orchestrating development includes structive to consider the consequences of perturbing individual multimeric protein complexes containing transcription factors, T constituents of the complex. Interfering with any component might coregulators, and other non-DNA binding proteins. Although elicit similarly devastating consequences. Alternatively, the com- a single complex can contain multiple enzymatic activities, including ponents might function uniquely, and individual perturbations chromatin remodeling and modifying activities, and components would therefore yield qualitatively distinct phenotypes. Herein, we capable of engaging in diverse protein-protein interactions, it is test these models by knocking-down ETO2 and LMO2 in a genetic challenging to elucidate the contribution of the individual compo- complementation assay in GATA-1–null cells. These studies nents to complex function at endogenous loci. identified striking differences in the ETO2- and LMO2-regulated In the context of hematopoiesis, in which stem cells differentiate target-gene ensembles and important mechanistic differences, into progenitors and diverse blood-cell types (1), major progress which highlight how master regulators of development coinha- has been made in defining the transcriptional drivers and their biting the same complex exert qualitatively distinct activities. complexes. These drivers include the GATA factors, three of which (GATA-1 to -3) control hematopoiesis (2). GATA-1 regu- Results and Discussion lates the differentiation of red blood cells, platelets, mast cells, and eosinophils (3–7). GATA-2 regulates the genesis and survival of Discriminatory Functions of a Non-DNA Binding Component of a Complex hematopoietic stem cells (HSCs) and multipotent progenitors (8), Containing Master Regulators of Hematopoiesis. The contribution of non-DNA binding components of the GATA-1-Scl/TAL1 complex whereas GATA-3 controls lymphopoiesis (9). fi The canonical mechanism by which GATA-1 activates and to activity of the complex is not well de ned. We analyzed the role of represses transcription involves binding to the coregulator Friend of GATA-1 (FOG-1) (10). FOG-1 has nine zinc fingers, with four implicated in GATA-1 binding (11). Although FOG-1 does not Author contributions: T.F., H.-Y.L., R.S., and E.H.B. designed research; T.F., H.-Y.L., and R.S. appear to contact DNA, it facilitates GATA-1 chromatin occu- performed research; T.F., H.-Y.L., R.S., and E.H.B. analyzed data; and T.F., H.-Y.L., R.S., and pancy at select sites (12, 13), GATA-1–mediated displacement of E.H.B. wrote the paper. GATA-2 from chromatin (GATA switches) (12), and chromatin The authors declare no conflict of interest. looping (14). FOG-1 binds NuRD (15) and CtBP corepressor This article is a PNAS Direct Submission. complexes (16, 17), and NuRD binding mediates certain FOG-1 Data deposition: Gene array datasets have been deposited in the GEO database, www. functions (18, 19). GATA-1 also activates and represses targets in ncbi.nlm.nih.gov/geo (accession no. GSE24359). a FOG-1–independent manner (20, 21). Because this poorly un- 1T.F., H.-Y.L., and R.S. contributed equally to this work. derstood mechanism controls critical genes, including the HSC 2To whom correspondence should be addressed. E-mail: [email protected]. regulator LYL1 and the red cell cytoskeletal protein band 4.9 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (21), it is important to elucidate the underlying mechanisms. 1073/pnas.1007804107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1007804107 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 one of these components, ETO2, in GATA-1 activity using GATA- certain GATA-1 targets were unaffected, ETO2 mediates 1–null G1E cells (37) stably expressing GATA-1 fused to the es- GATA-1 function in a context-dependent manner. trogen receptor ligand-binding domain (G1E-ER-GATA-1 cells) The gene exhibiting the greatest sensitivity to ETO2 knock- (38, 39). We tested whether reducing ETO2 levels influences the down was Slc4a1 (Fig. 1C, Left), a GATA-1 target gene encoding capacity of ER-GATA-1 to regulate target genes. After transfection an anion exchanger with pivotal roles in red cells (41). GATA-1 of G1E-ER-GATA-1 cells with anti-Cbfa2t3 [Cbfa2t3 encodes (Fig. S3B) and ETO2 (Fig. S3C) occupies the Slc4a1 promoter ETO2 (40)] siRNA, ER-GATA-1 was activated with β-estradiol and intron 1 (+1.4 kb) (Fig. S3A) and regulates Slc4a1 tran- (Fig. 1A). Cbfa2t3 mRNA (Fig. S1A) and protein (Fig. S1B, Upper) scription in a FOG-1–dependent manner (36, 42), but other expression strongly decreased, and ER-GATA-1 levels were un- aspects of this transcriptional mechanism are unknown. altered (Fig. S1B, Lower). To establish how knocking-down ETO2 renders Slc4a1 hyper- We compared expression profiles in β-estradiol-induced G1E- sensitive to ER-GATA-1, we analyzed the epigenetic landscape at ER-GATA-1 cells transfected with control and anti-Cbfa2t3 Slc4a1 in untreated and β-estradiol–treated G1E-ER-GATA-1 siRNA. This analysis revealed altered expression of established cells. Histone H3 di-acetylated at K9/K14 was considerably higher GATA-1 targets and those not known to be GATA-1-regulated at sites of ER-GATA-1 occupancy, whereas tetra-acetylated (Fig. 1B). The ETO2-regulated genes included those in which histone H4 was distributed throughout the locus. ER-GATA-1– ER-GATA-1–mediated activation (Fig. 1C) and repression (Fig. mediated activation of Slc4a1 transcription was accompanied 1D) were significantly enhanced by the knockdown (P < 0.05). by increased H3-diacK9/K14 and tetraacetylated H4 (Fig. S3 D An additional cohort of GATA-1 targets was unaffected (Fig. and E). Analogous to H3-diacK9/K14, H3-dimeK4 was highest at 1E). The reduction in ETO2 levels therefore derepressed ER- ER-GATA-1 occupancy sites and increased upon activation, albeit GATA-1 activity, increasing its activation and repression of not at the promoter (Fig. S3F). Two marks associated with re- targets. ETO2 occupied GATA switch sites bound by GATA-2 pression, H3-trimeK9 and H3-trimeK27 (43), were unusually high (Fig. S2E) and GATA-1 (Fig. S2A) in uninduced and induced (comparable or higher than the inactive Necdin promoter) at the G1E-ER-GATA-1 cells, respectively, at representatives of this inactive and active Slc4a1 locus (Fig. S3 G and H). The presence of group (Fig. S2C), indicating a direct function at these loci. As active and repressive marks resembles bivalent chromatin domains,

A siRNA siRNA - -estradiol -estradiol (1 M) + -estradiol Time (h) 0424 8 25 B Slc4a1 C 4930449E01Rik - BC056923 600 Epb4.2 n.d. 20 Tmod1 Rragd Slc25a37 Clca1 n.d. 15 Hba-a1 400 Add2 Ak7 n.d. 9130218O11Rik n.d. 10 Nalp6 - 4432416J03Rik 200 Pigq -

Hbb-b1 (Relative Units) 5 ENSMUST00000098674 mRNA Expression mRNA Mybpc2 n.d. Aqp8 Lrrc39 - 0 0 BB736773 n.d. -estradiol Aldoa - 9030617O03Rik n.d. Slc4a1 BC056923 Tmod1 Rragd Slc25a37 2310015A05Rik n.d. 1.2 Rps23 - D Ptprz1 n.d. Gpx1 - Fold change Fau - Odc1 n.d. 5.0 Cdkl1 n.d. 4.0 Vangl1 - 0.8 3.0 Cdc25b - 2.0 Tmem16a n.d. 1.0 Rpl32 - Ccdc102a n.d. 0 Tdrd7 n.d. -1.0 Ypel4 n.d. 0.4 -2.0 Msi2 n.d. -3.0 Cotl1 -4.0 Mical1 (Relative Units) Ifngr2 - Expression mRNA -5.0 Rgs18 Gsn - BC013561 n.d. 0 Fes - -estradiol Mapre2 - 6430527G18Rik n.d. Plekhg3 Lilrb3 Hdc Hmga1 Rgs18 c-kit Gata2 CK347075 n.d. 25 1.6 Fhod1 - E 5033405K12Rik - Zfp1 ENSMUST00000070860 n.d. 20 Fcgr3 - 1.2 Fig. 1. ETO2 controls a small subset of GATA-1 target Psen2 - Cep55 - genes. (A) Experimental strategy for siRNA-mediated 9130213B05Rik 15 LOC668139 n.d. knockdown of Cbfa2t3 in G1E-ER-GATA-1 cells. (B) Bhlhb8 0.8 ﬁ β Tec Pro les were compared in -estradiol-treated G1E-ER- Serpine1 - 10 GATA-1 cells transfected with control or Cbfa2t3 siRNA. AK141415 n.d. Adora3 - The heat map depicts the mean fold-change resulting - Fcgr2b (Relative Units) 0.4 n.d. NAP057003-1 Expression mRNA 5 from ETO2 knockdown (n = 2). The GATA-1 respon- Lincr - Alox5ap - siveness in G1E-ER-GATA-1 cells, based on prior array LOC675624 n.d. ▲ Gng2 0 0 analyses (36, 56), is indicated on the right: , GATA-1 9830134C10Rik n.d. -estradiol ▼ − Itgal activated; , GATA-1-repressed; , not GATA-1 regu- Cd200r3 n.d. Abcb10 Hebp1 Lyl1 c-myb – Socs1 - lated; n.d., expression not detected. (C E) Quantitative Hmga1 Control siRNA Cbfa2t3 siRNA Rasgrp4 n.d. RT-PCR validation of array results. (C) Cbfa2t3 siRNA- Hdc activated genes; (D) Cbfa2t3 siRNA-repressed genes; (E) Lilrb3 Plekhg3 Cbfa2t3 siRNA-insensitive genes (mean ± SE, n =4).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1007804107 Fujiwara et al. Downloaded by guest on September 29, 2021 which may reflect a state in which the gene is poised to be activated regulated GATA-1 targets. Although ETO2-regulated targets (44), but such domains have not been described in erythroid cells. (BC056923, Tmod1, and Rragd), but not the nonresponsive tar- We asked whether the ETO2 knockdown altered all or specific get (Abcb10), have high-level H3-trimeK27 (Fig. S4), ETO2 did components of this nucleoprotein architecture in untreated and not regulate H3-trimeK27 at all targets (Fig. 2F, Right). ETO2 β-estradiol–treated G1E-ER-GATA-1 cells. In untreated cells, suppressed H3-diacK9/K14 at all responsive targets, but not the knocking-down ETO2 significantly, but modestly, increased ER- nonresponsive targets (Fig. 2C, Right). ETO2 also suppressed GATA-1 (Fig. 2A) and Scl/TAL1 (Fig. 2B) occupancy at the ER-GATA-1–mediated repression at select loci, and this action promoter and the +1.4-kb site. ER-GATA-1 (Fig. 2A) and Scl/ at Plekhg3 involved suppressing H3-trimeK27 and maximizing TAL1 (Fig. 2B) occupancy was unaffected in β-estradiol–treated H3-dimeK4 (Fig. S5). Thus, ETO2-dependent transcriptional cells. The knockdown significantly increased H3-diacK9/K14 at repression involves a widely used epigenetic mechanism to sup- the promoter and intron 1, without influencing this mark at the press H3-diacK9/K14 and a locus-selective action to regulate active and inactive RPII215 and Necdin promoters, respectively H3-trimeK27 (Fig. 2G). (Fig. 2C). Whereas the knockdown did not affect H3-dimeK4 (Fig. 2D) and H3-trimeK9 (Fig. 2E)atSlc4a1 and other targets, GATA-1-Scl/TAL1 Complex Structure/Function: Qualitatively Distinct in the presence of active ER-GATA-1, the knockdown strongly Mechanistic and Biological Functions of Individual Complex Compo- reduced H3-trimeK27 at the promoter and intron 1, without nents. Although ETO2, LMO2, and LDB1 are components of affecting this mark at control promoters (Fig. 2F). Although GATA-1-Scl/TAL1 complexes (22, 29, 30, 46), whether these ETO2 regulation of histone modifications had not been de- components function as a unit or independently to regulate scribed, given its binding to HDACs (45), increased H3-diacK9/ GATA-1 target genes is unknown. We knocked-down LMO2 K14 is consistent with this mechanism. In contrast, its regula- mRNA (Fig. S1C) and protein (Fig. S1D, Upper), which did not tion of H3-trimeK27 was surprising. It is attractive to consider alter ER-GATA-1 levels (Fig. S1D, Lower), and conducted ex- that high-level H3-trimeK27 might uniquely characterize ETO2- pression profiling to establish the LMO2-regulated gene ensemble (Fig. 3A). Surprisingly, the knockdown significantly up-regulated all but five of the LMO2-regulated genes, indicating an unexpected predominant repressive function of LMO2. As LMO2 A overexpression inhibited erythropoiesis, our loss-of-function analy- 0.20 sis provides a conceptual framework for understanding this result 0.15

0.10 (47). Genes encoding prototypical erythroid proteins (e.g., heme GENETICS * (Relative Units) 0.05 biosynthetic enzymes and red cell cytoskeletal proteins) were

ER-GATA-1 Occupancy ER-GATA-1 0 Control siRNA Cbfa2t3 siRNA conspicuously absent from this gene ensemble. LMO2 targets B 0.5 * included genes shown previously to be FOG-1–independent, 0.4 – 0.3 GATA-1 repressed genes (21), including Lyl1, Rgs18, Clec4d, 0.2 * Vim, Sept9, Lgals9, and Klf10 (Fig. 3A). Because the mechanism 0.1 (Relative Units) – –

Scl/TAL1 Occupancy Scl/TAL1 0 underlying FOG-1 independent GATA-1 mediated repression C 10 * 3.0 * * has been elusive, and these targets include the important regu- 8 * 2.0 * lator of HSCs LYL1 (48), we focused on this set of genes. 6 * * ﬁ 4 Quantitative RT-PCR analysis con rmed that the LMO2 knock- H3-acK9/14 * * 1.0 (Relative Units) 2 down up-regulated FOG-1–independent, GATA-1–repressed genes 0 0 (Rgs18, Vim, Clec4d, Lyl1)(Fig.3B). Even in uninduced G1E-ER- D 2.0 1.5 GATA-1 cells before GATA-1 occupancy, the knockdown up-regu- 1.5 1.0 1.0 lated Rgs18, Vim, Clec4d,andLyl1 (Fig. 3B). In contrast, the ETO2

H3-dimeK4 0.5

(Relative Units) 0.5 knockdown decreased Rgs18 expression (Fig. 1D). Although knock- 0 0 ing down ETO2 yielded ER-GATA-1–mediated hyperinduction of E Slc4a1 (Fig. 1C), knocking-down LMO2 signiﬁcantly inhibited 1.5 3.0 – 1.0 2.0 GATA-1 mediated induction of Slc4a1 (Fig. 3C, Left). Although

H3-trimeK9 0.5 1.0 ETO2 and LMO2 exert qualitatively distinct responses, both occupy (Relative Units)

0 0 Lyl1, Rgs18,andSlc4a1 (Fig. S2 C and D). Because LMO2 facilitates ER-GATA-1–mediated repression of FOG-1–independent genes, F * * 1.5 1.5 we tested whether it also affects FOG-1–independent activation of 1.0 * 1.0 * genes. The LMO2 knockdown did not affect ER-GATA-1–mediated H3-trimeK27 0.5 0.5 (Relative Units) induction of the prototypical FOG-1–independent gene Fog1 nor 0 0 – -estradiol repression of the prototypical FOG-1 dependent gene Gata2 (Fig. Slc4a1 Slc4a1 RPII215 Necdin BC056923 Tmod1 Rragd Abcb10 promoter +1.4kb promoter promoter promoter promoter promoter promoter 3C, Right).

Widely Utilized Locus-Selective Because ETO2 and LMO2 knockdowns yielded qualitatively G distinct outputs, and both factors bind Scl/TAL1, we asked whether the factor-specific activities are Scl/TAL1-dependent or -independent. If Scl/TAL1 is critical for ETO2 or LMO2 to regulate their gene ensembles, knocking-down Scl/TAL1 should re- capitulate the gene dysregulation resulting from ETO2 or LMO2 knockdowns. Knocking-down Scl/TAL1 (Fig. S1 E and F), which – Fig. 2. ETO2-dependent epigenetic mechanisms. (A F) Quantitative ChIP did not alter ER-GATA-1 levels (Fig. S1F), significantly reduced analysis at ETO2-regulated genes (Slc4a1, BC056923, Tmod1,andRragd) and ER-GATA-1–mediated activation of Slc4a1, resembling the LMO2 ETO2-insensitive genes (Abcb10, RPII215, and Necdin) in G1E-ER-GATA-1 cells knockdown (Fig. 3C, Left), although the reduction was greater transfected with control and Cbfa2t3 siRNA: GATA-1 (A), Scl/TAL1 (B), acetyl with the Scl/TAL1 knockdown (Fig. 3D, Left). Also resembling the H3 (C), H3-dimeK4 (D), H3-trimeK9 (E), and H3-trimeK27 (F). For histone fi modifications (C–F), the result of uninduced G1E-ER-GATA-1 cells transfected LMO2 knockdown, knocking-down Scl/TAL1 signi cantly reduced ± < ER-GATA-1–mediated repression of the FOG-1–independent with control siRNA was set to 1.0 (mean SE, n = 3). *P 0.05. (G) Models – depicting the broadly used mechanism in which ETO2 suppresses H3-diacK9/ gene Lyl1, but not the FOG-1 dependent gene Gata2 (Fig. 3D, K14 near the ETO2-associated GATA-1-Scl/TAL1 complex and the locus- Right). As LMO2 and Scl/TAL1 exert qualitatively similar influences selective mechanism at Slc4a1 in which ETO2 confers high-level H3-trimeK27. on GATA-1 targets, this finding supports the notion that LMO2 HDAC, histone deacetylase; HMT, histone methyltransferase. Preimmune and Scl/TAL1 function collectively in the FOG-1–independent values did not exceed 0.0035. GATA-1 repression mechanism. In contrast, knocking-down Scl/

Fujiwara et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 TAL1 did not yield hyperactivation of Slc4a1 in response to ER- moters similarly in uninduced and induced cells (Fig. S2B), GATA-1 activation. The distinct inﬂuences of ETO2 and Scl/TAL1 consistent with its colocalization with GATA-2, before GATA-1 on the FOG-1–dependent, GATA-1–activated gene Slc4a1 are in- occupancy (34, 36). LMO2 occupancy resembled that of Scl/ consistent with ETO2 contributing to a solitary function of the TAL1 (Fig. S2D), and coupled with dysregulated transcription GATA-1-Scl/TAL1 complex. upon LMO2 knockdown, these results indicate that LMO2 The different ETO2 and LMO2 mechanisms (Fig. 4A) raises functions directly to control these FOG-1–independent, GATA- the question as to whether the respective gene ensembles mediate 1–repressed genes. similar or distinct biological pathways. Gene Ontology analysis revealed that the majority of ETO2- and LMO2-regulated genes Multimeric Complexes Containing Master Developmental Regulators: control distinct pathways (Fig. 4B). Most ETO2- and LMO2- Mechanistic Principles and Biological Implications. Critical develop- regulated genes (61 and 138, respectively) differed, but both mental regulators that establish and modulate genetic networks factors coregulated only 16 genes (Fig. 4C). The coregulated often reside in multimeric complexes. Many questions remain genes included four ER-GATA-1–activated (Hba-a1, Hbb-b1, unanswered regarding how individual components contribute to Cotl1, Gng2), four ER-GATA-1–repressed (Mical1, Hdc, Lilrb3, complex function. Perhaps the simplest mechanism involves the Plekhg3), and eight not regulated by ER-GATA-1 (Aldoa, Fau, coordination of the actions of all components of a complex to yield Gsn, Fes, Fcgr3, Fcgr2b, Alox5ap, and Ptprz1). ETO2 and LMO2 a sole molecular output, such as activation or repression. Individual coregulate a minority of GATA-1–regulated and –insensitive components may have dedicated functions to mediate recruitment genes (Fig. 4C). Thus, a comparable reduction in the level of two of the complex to factors occupying target genes, to stabilize components of the GATA-1-Scl/TAL1 complex dysregulated intracomplex interactions, to catalyze chromatin modiﬁcation/ distinct gene ensembles, and the genes comprising these ensem- remodeling, to interact with the transcriptional machinery, or to bles differed considerably vis-à-vis their cellular functions. integrate cell signals. Given these intertwined activities, perturba- LMO2 had not been implicated in FOG-1–independent GATA tions of individual components would yield qualitatively similar factor pathways or any other mode of GATA-1 function. We defects. Alternatively, distinct components may function in unique tested whether LMO2 occupies FOG-1–independent, GATA-1– pathways. Rather than collectively mediating complex recruitment repressed genes, which would imply a direct function at these loci. and activity, individual components might selectively mediate re- ER-GATA-1 occupied the Lyl1 and Rgs18 promoters, but not the cruitment to distinct cohorts of targets. Similarly, components may control Necdin promoter (Fig. S2A). Scl/TAL1 occupied the pro- have intrinsic enzymatic activities essential for the control of select

A B + -estradiol 4 * Fzd7 Hbb-b1 4 * * Lilrb3 n.d. Gm6607 n.d. * Gsn n.d. Stk10 Lgals3 n.d. A_51_P356871 n.d. 2 Fcgr3 - Slc39a4 - Rgs18* Taldo1 3 P2rx1 Rps23 - 0 Rnase6 Gchfr Ahnak - Tinagl1 n.d. Rgs18 Vim Clec4d Lyl1 Pdxk n.d. Oxct1 Dok2 Aldoa - Vim* Gp5 - 2 Hey1 - A_51_P516125 n.d. Polr1c - Rps27 - Plekhg3 - Aldh2 - Fcgr2b - H2afj n.d. (Relative Units) mRNA Expression mRNA Arhgap15 Fam46a - 1 Clec4d * Soat1 n.d. Alox5ap - Btk - Myo1g Lpar6 n.d. Camkk1 Gnb2l1 - Acot11 - Rps29 - Lcp2 - Pros1 0 Fold change St8sia4 Siglec5 n.d. -estradiol Sept9 * n.d. Rps20 - 8.0 Lgals1 - Aaas - Rgs18 Vim Clec4d Lyl1 - - 6.0 Gm1966 Daglb 250 Lyl1 * Zbtb4 - 4.0 Gmfg Rps8 - C 2.0 Aim1 - Maoa n.d. 4 0 Anxa2 Iqce 200 -2.0 Tubb2b n.d. Prdx2 n.d. n.d. -4.0 Rell1 Rps2 Spnb2 - Dennd1c - 3 -6.0 Frmd5 - Bcl2 150 -8.0 Ddc n.d. Hcls1 - Paqr7 Tnfrsf13c n.d. Me1 Ikzf2 n.d. 2 Grap2 - Rnf128 - 100 Ptgir n.d. Mmp14 A_52_P576499 n.d. Rps24 - Prg3 - Uba52 - n.d. - 1 A_52_P764477 2310014H01Rik (Relative Units) 50 Rab44 n.d. Erdr1 mRNA Expression mRNA A_51_P311648 n.d. Abi2 n.d. Nme4 Pgm2 Rcsd1 - Rgs19 0 0 Fig. 3. A distinct component of the GATA-Scl/TAL1 Itgb7 Rpl27 - -estradiol Hba-a1 Rps4x - complex, LMO2, regulates a unique target gene ensem- Mras n.d. Rpl12 - Slc4a1 Gata2 Fog1 ﬁ β Cugbp2 - Rps6ka1 - ble. (A) Expression pro les were compared in -estradiol- Gvin1 n.d. Sox6 Control siRNA Lmo2 siRNA Amhr2 Rbm20 treated G1E-ER-GATA-1 cells transfected with control or Abca1 n.d. Rpl8 - 100 2.0 Lmo2 siRNA. The heat map depicts the mean fold-change Plac8 Sdsl n.d. D Arap3 Furin resulting from LMO2 knockdown (n = 2). The GATA-1 A_52_P98452 n.d. Spry1 - Rpl7a - Ptprz1 n.d. 80 responsiveness in G1E-ER-GATA-1 cells, based on prior ar- Gm8995 n.d. Selenbp2 1.5 Fau - Rn18s n.d. ray analyses (36, 56), is indicated on the right: ▲,GATA-1 Adrb2 - Pde6h Hist1h2bc Frmpd1 - 60 activated; ▼,GATA-1repressed;−, not GATA-1 regulated; Cotl1 Gp1ba Clec10a n.d. Rpl38 1.0 n.d., expression not detected. *Genes demonstrated pre- A_52_P186362 n.d. Pabpc1 - – – Ptgr1 - Rps3 - 40 viously (21) to be FOG-1 independent, GATA-1 repressed Mical1 Klf10 * genes. (B and C) Quantitative RT-PCR validation of array Hist1h1c - Rac1 Mfge8 - Man2b1 0.5 < (Relative Units) 20 results. (B) Lmo2 siRNA-activated genes; *P 0.05; (C) Fes - Meis1 Prmt2 Eya1 Expression mRNA Lmo2 siRNA-repressed (Slc4a1) and -insensitive (Gata2, A_51_P250597 n.d. A_52_P361462 n.d. Lgals9 * Tug1 n.d. 0 Fog1) genes. (D) Quantitative RT-PCR analysis of Slc4a1, Inpp4b - Zdhhc25 - 0 Hdc Lmo2 -estradiol Lyl1,andGata2 mRNA in G1E-ER-GATA-1 cells transfected Rplp0 n.d. Crlf1 n.d. Slc4a1 Lyl1 Gata2 Gng2 Lin9 - with siRNA against Scl/TAL1 or control siRNA. Gapdh mRNA Control siRNA Scl/TAL1 siRNA was analyzed as a control.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1007804107 Fujiwara et al. Downloaded by guest on September 29, 2021 Cell protein metabolic process independent functions, ETO2 and LMO2 coregulate a small A B Immune system process + regulation of cell processes cohort of genes (Fig. 4C). Thus, the independent actions of these Regulation of signal transduction Cytoskeletal protein binding factors do not preclude their collective function, at least not at 1 ETO2 Suppression of GATA-1 Regulation of response to stimulus at FOG-1-Dependent Genes Actin binding select loci. In principle, an alternative possibility is that ETO2 Hematopoiesis Myeloid leukocyte activation and LMO2 function both within and independent of the GATA- Regulation of inflammatory response FOG-1 Myeloid cell differentiation 1-Scl/TAL1 complex. ETO2 interacts with the intracellular do- ETO2 + regulation of signal transduction LMO2 + regulation of cell communication main of Notch1 and the Notch-regulated transcription factor GATA-1 - Scl/TAL1 Mast cell activation Complex Chloride transport Carbon-carbon lyase activity CBF1, thereby repressing Notch target genes (52). - regulation of response to stimulus Considering parameters dictating the composition of multi- 0 510 Cell protein metabolic process protein complexes at endogenous chromatin sites, at the most ru- - regulation of biological process 2 LMO2 Facilitation of GATA-1- Translation dimentary level, the requisite DNA and protein interaction modules Mediated FOG-1-Independent Structural constituent of ribosome Immune system process are critically important. The precise conﬁguration of cis-elements Repression Cell adhesion Regulation of cell component organization ﬂ Alcohol metabolic process may in uence the conformation of DNA-bound components, fa- Homeostasis of number of cells ETO2 LMO2 ? Hematopoiesis voring or disfavoring nucleation of scaffold constituents. Regarding Membrane organization GATA-1 - Scl/TAL1 Skeletal system development the GATA-1-Scl/TAL1 complex, a deeper understanding of the Complex Regulation of phosphorylation - regulation of signal transduction - regulation of cell communication molecular structure of the complex in solution and when bound to Phagocytosis, engulfment Blood coagulation chromatin is required to assess the relative importance of cis-ele- Myeloid cell differentiation B cell homeostasis ment–instigated conformational transitions. Differences in the sig- Mast cell activation Regulation of GPCR signaling pathway naling milieu characteristic of distinct subnuclear compartments Catecholamine metabolic process 0 10 20 30 may also control scaffold composition in a spatially restricted man- Number of Genes ner. Although GATA-1 target genes regulated via distinct mecha- ETO2/LMO2-shared ETO2-selective LMO2-selective nisms can reside in different subnuclear compartments (53), whether ETO2 LMO2 ETO2 LMO2 ETO2 LMO2 C regulated regulated regulated regulated regulated regulated (77) (154) (24) (55) (53) (99) subnuclear organization and spatially restricted signaling affect GATA-1-Scl/TAL1 complex structure and function is unknown.

79% 90% 67% 85% 85% 92% Given our strong evidence that components of a multimeric (16) (8) (8) (61) (138) (16) (47) (45) (91) complex containing master regulators of development exert qualitatively distinct functions, it is instructive to consider the patho- Total GATA-1 regulated GATA-1 insensitive

physiological implications of this work. As disruptions of different GENETICS Fig. 4. Mechanistic and biological diversity of ETO2 versus LMO2 target genes. GATA-1-Scl/TAL1 complex constituents yielded qualitatively dis- (A) Models of ETO2 and LMO2 function. The models depict the qualitatively tinct phenotypes, this may explain why different complex compo- distinct activities of ETO2 and LMO2 within the GATA-1-Scl/TAL1 complex at nents are linked to the genesis of distinct leukemias (26, 31, 54). To different target genes. Because knocking down LMO2 reduces but does not understand how perturbations of individual components of a com- prevent FOG-1–independent repression, an unidentified factor may also me- plex containing multiple developmental regulators yield diverse diate repression. (B) Gene Ontology analysis. Genes showing ≥ 2.2-fold change pathophysiologies, it will be important to define the structure and based on microarray analysis (Figs. 1B and 3A) were analyzed using DAVID function of the complex in distinct cellular contexts, and importantly Bioinformatics Program (http://david.abcc.ncifcrf.gov/). A P value of 0.05 was in distinct neighborhoods within the 3D confines of the nucleus. used as the standard cutoff level. (C) Relationship between ETO2- and LMO2- regulated genes, based on expression changes ≥ 2.2-fold. The 16 coregulated Materials and Methods genes are fewer than that suggested by B, as the ETO2/LMO2-shared GO cat- Cell Culture. Cultures were grown as described in SI Materials and Methods. egory contains genes coregulated by ETO2- and LMO2 and those uniquely regulated by ETO2 or LMO2. Antibodies. Antibodies are described in SI Materials and Methods.

targets, and therefore building multiple components with special- Quantitative ChIP Assay. Real-time PCR-based quantitative ChIP analysis was ized activities into the complex would diversify complex function. conducted as described (55) and in SI Materials and Methods. Because the GATA-1-Scl/TAL1 complex has been defined bio- chemically (15, 22, 29, 46, 47, 49), its components colocalize at Small Interfering RNA-Mediated Knockdown. ETO2 knockdown in G1E-ER- chromatin sites (36, 50, 51), and it has critical biological func- GATA-1 cells was as described previously (36). The SMARTpool siRNA tions (1), this is a particularly attractive system for addressing sequences are described in SI Materials and Methods. The siGENOME Non- Targeting siRNA pool (D-001206–13, Dharmacon/Thermo Fisher Scientific) mechanisms underlying the function of multimeric complexes. × 6 High-efficiency knockdowns of two components of this complex was used as a control. Small interfering RNA was transfected into 3 10 G1E- ER-GATA-1 cells by Amaxa Nucleofector kit R (Amaxa Inc.). The siRNA was revealed major differences in their activities to control target genes. transfected twice at 0 and 24 h, cells were induced with β-estradiol at 24 h, and Gene Ontology analysis revealed extreme differences in the spec- harvested after 48 h (Fig. 1A). trum of biological activities attributed to ETO2 vs. LMO2 targets. ETO2 and LMO2 therefore exert qualitatively distinct functions, Quantitative RT-PCR Analysis and Transcription Profiling. Total RNA was ana- inconsistent with a model in which the tightly linked actions of these lyzed as described in SI Materials and Methods. components confer a solitary activity. The qualitatively distinct activities indicate that these components can convey independent Western Blotting. Western blotting was conducted as described in SI Materials molecular and cellular functions, entirely unexpected based on the and Methods. GATA-1-Scl/TAL1 complex paradigm. Our results illustrate how trans-acting factors that bind DNA ACKNOWLEDGMENTS. This work was supported by National Institutes of sequence specifically can serve as a scaffold to build a complex Health Grants DK68634 and DK50107 (to E.H.B.). H.-Y.L. was supported by with multifunctionality. Although ETO2 and LMO2 clearly exert a predoctoral fellowship from the American Heart Association.

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