ETS Family Transcriptional Repressor Research Articles

Abstract 1461: ETV6 deficiency and microsatellite enhancers drive transcriptional dysregulation in B-lymphoblastic leukemia

Abstract Subtypes of B-lymphoblastic leukemia (B-ALL) are defined by unique genetic aberrations and gene expression programs. ETV6-RUNX1+ (E-R+) B-ALL, a common pediatric subtype, shares a gene activation signature with “ETV6-RUNX1-like” B-ALL that lacks the E-R fusion gene. Both E-R+ and E-R-like B-ALL show frequent inactivating mutations or deletions of ETV6, which encodes an ETS family transcriptional repressor (core binding motif GGAA/T). Signature genes overexpressed in E-R+ B-ALL, such as EPOR and PIK3C3, were shown to sustain the malignant phenotype. We performed H3K27ac ChIP-Seq and ATAC-Seq on 26 B-cell cancer cell lines to identify subtype-specific active enhancer-like elements. Unbiased K-means clustering identified distal elements that were selectively acetylated in four E-R+ B-ALL cell lines (all with inactivation of the second ETV6 allele) and in a fifth B-ALL cell line with biallelic ETV6 deletion. Motif analysis of this cluster showed strong enrichment for tandem repeats of the sequence “GGAA” (p<10-100). Repeats with ≥6 tandem copies of “GGAA” were selectively acetylated and accessible in H3K27ac ChIP-Seq and ATAC-Seq datasets from primary patient samples of E-R+ B-ALL, but not from other genetic subtypes that lack ETV6 aberrations. Doxycycline-inducible expression of ETV6 in ETV6-deficient, E-R+ B-ALL cells led to significant growth inhibition. Restored ETV6 bound extensively to GGAA repeats (80% of ETV6 binding sites), repressed repeat enhancer acetylation, and repressed expression of adjacent E-R+ signature genes (n=40). Repression of a published E-R+ signature gene set was significant by GSEA analysis (p<0.001). Direct CRISPR-interference repression of 6 GGAA repeat enhancers led to significant downregulation of their associated E-R+ signature genes, including EPOR and PIK3C3. GGAA repeat enhancers are a known driver of Ewing sarcoma, where they are activated by oncogenic fusions of the ETS activators FLI1 or ERG. Active GGAA repeat enhancers and repeat-regulated genes in E-R+ B-ALL show only partial overlap with repeat-regulated genes in Ewings, possibly due to tissue-specific factors. Wild-type ERG and FLI1 are highly expressed in B-ALL. We found that knockdown of ERG, but not FLI1, led to significant downregulation of repeat enhancer-regulated E-R+ signature genes in B-ALL. ChIP-Seq showed that the ERG binds nearly all active GGAA repeat enhancers in three ETV6-deficient B-ALL cell lines, but not in ETV6-intact B-ALL. We identify GGAA repeat enhancers, sustained by ETV6 deficiency and strong ERG expression, as a unifying mechanism for a subtype-specific oncogenic gene expression program in B-ALL. Our findings may have crucial implications for E-R+ B-ALL modeling, as most GGAA repeats are not conserved between mice and humans. Mechanisms that activate leukemia-specific repeat enhancers may represent appealing targets for future therapeutic development. Citation Format: Joshua W. Goldman, Rohan Kodgule, Alexander C. Monovich, Travis Saari, Cody N. Hall, Niharika Rajesh, Juhi Gupta, Athalee Aguilar, Noah Brown, Mark Y. Chiang, Marcin P. Cieslik, Russell J. Ryan. ETV6 deficiency and microsatellite enhancers drive transcriptional dysregulation in B-lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1461.

Cell geometry, signal dampening, and a bimodal transcriptional response underlie the spatial precision of an ERK-mediated embryonic induction.

Precise control of lineage segregation is critical for the development of multicellular organisms, but our quantitative understanding of how variable signaling inputs are integrated to activate lineage-specific gene programs remains limited. Here, we show how precisely two out of eight ectoderm cells adopt neural fates in response to ephrin and FGF signals during ascidian neural induction. In each ectoderm cell, FGF signals activate ERK to a level that mirrors its cell contact surface with FGF-expressing mesendoderm cells. This gradual interpretation of FGF inputs is followed by a bimodal transcriptional response of the immediate early gene, Otx, resulting in its activation specifically in the neural precursors. At low levels of ERK, Otx is repressed by an ETS family transcriptional repressor, ERF2. Ephrin signals are critical for dampening ERK activation levels across ectoderm cells so that only neural precursors exhibit above-threshold levels, evade ERF repression, and "switch on" Otx transcription.

A Spic/BCLAF1 Complex Suppresses Pre-B Cell Receptor Signaling in Response to RAG-Mediated DNA Breaks

B cell development is carefully orchestrated to generate a diverse antibody repertoire while preventing malignant transformation. At early developmental stages, the pre-B cell receptor (pre-BCR) is an essential regulator of B cell maturation, coordinating both clonal expansion and immunoglobulin light chain gene (Igl) assembly. Igl gene rearrangement proceeds through V(D)J recombination, which requires the generation of DNA double-stranded breaks (DSBs) and the subsequent joining of two distant DNA segments. The DSBs necessary for Igl gene rearrangements are generated by the RAG1 and RAG2 proteins, which form the RAG endonuclease. Once RAG DSBs are generated, cells must be prevented from re-entering cell cycle where DSBs can be aberrantly repaired as deleterious translocations. Thus, temporal segregation of pre-BCR driven proliferation and Igl gene assembly is essential for normal maturation and prevention of errors that could lead to leukemic transformation. The pre-BCR signals through the SYK tyrosine kinase to both drive proliferation and initiate Igl gene rearrangement. Unopposed activation of the pre-BCR, particularly increased SYK activity, blocks B cell development and supports leukemic transformation. Furthermore, pre-B leukemic blasts depend on SYK for proliferation and survival. Tight regulation of pre-BCR signaling is essential for enforcing normal maturation and suppressing leukemic transformation.We previously showed that pre-BCR signaling is inhibited by signals from RAG DSBs. Specifically, RAG DSBs trigger ATM-dependent induction of SPIC, an ETS family transcriptional repressor with significant homology to PU.1 and SPIB. SPIC inhibits expression of the SYK tyrosine kinase resulting in suppression of pre-BCR signaling, inhibition of pre-B cell proliferation, and limitation of additional RAG DSB generation. In order to determine the mechanism of SPIC-mediated gene regulation and to determine the role of SPIC in early B cell development, we compared SPIC and PU.1 binding throughout the genome in early B cells. Chromatin-immunoprecipitation followed by sequencing (ChIP-seq) identified shared binding sites for SPIC and PU.1 throughout the genome in pre-B cells suggesting that both transcription factors regulate a similar cohort of genes. Interestingly, binding of SPIC to the Syk promoter and the Igl(kappa) enhancer results in reduction of PU.1 binding at these sites and suppression of transcription of the respective genes. These results demonstrate that SPIC antagonizes PU.1 function to repress pre-BCR signaling in early B cells.SPIC shares a DNA binding domain motif with the other ETS-family transcription factors, PU.1 and SPIB, but has a unique transactivation domain, which could recruit transcriptional repressors to gene regulatory regions. To determine the unique binding partners of SPIC and PU.1, we generated pre-B cells expressing tetracycline-inducible FLAG-SPIC and FLAG-PU.1. The tagged proteins were purified from nuclei using tandem affinity purification. Mass spectrometry of the precipitated samples identified BCLAF1 (Bcl2 associated transcription factor 1) as a unique SPIC interactor. In contrast, IRF4 binds to PU.1 but not to SPIC. In pre-B cells, BCLAF1 binds to regulatory regions of a similar set of genes as SPIC. Consistent with this, BCLAF1 binds to the Syk promoter only in cells with RAG DSBs, which correlates with binding of SPIC and loss of PU.1 at this site. RNAi-mediated knockdown of BCLAF1 results in increased Syk mRNA expression. However, loss of BCLAF1 does not affect SPIC occupancy at the Syk promoter. Thus, SPIC binds DNA but only represses transcription if BCLAF1 is also recruited. Collectively, our findings reveal that, in response to RAG DSBs, SPIC and BCLAF1 cooperate to antagonize PU.1 function and regulate expression of a broad genetic program that modulates pre-BCR signaling to enforce normal B cell maturation. DisclosuresNo relevant conflicts of interest to declare.

A Multipronged Screening Approach Targeting Inhibition of ETV6 PNT Domain Polymerization

ETV6 is an ETS family transcriptional repressor for which head-to-tail polymerization of its PNT domain facilitates cooperative binding to DNA by its ETS domain. Chromosomal translocations frequently fuse the ETV6 PNT domain to one of several protein tyrosine kinases. The resulting chimeric oncoproteins undergo ligand-independent self-association, autophosphorylation, and aberrant stimulation of downstream signaling pathways, leading to a variety of cancers. Currently, no small-molecule inhibitors of ETV6 PNT domain polymerization are known and no assays targeting PNT domain polymerization have been described. In this study, we developed complementary experimental and computational approaches for identifying such inhibitory compounds. One mammalian cellular approach utilized a mutant PNT domain heterodimer system covalently attached to split Gaussia luciferase fragments. In this protein–fragment complementation assay, inhibition of PNT domain heterodimerization reduces luminescence. A yeast assay took advantage of activation of the reporter HIS3 gene upon heterodimerization of mutant PNT domains fused to DNA-binding and transactivation domains. In this two-hybrid screen, inhibition of PNT domain heterodimerization prevents cell growth in medium lacking histidine. The Bristol University Docking Engine (BUDE) was used to identify virtual ligands from the ZINC8 library predicted to bind the PNT domain polymerization interfaces. More than 75 hits from these three assays were tested by nuclear magnetic resonance spectroscopy for binding to the purified ETV6 PNT domain. Although none were found to bind, the lessons learned from this study may facilitate future approaches for developing therapeutics that act against ETV6 oncoproteins by disrupting PNT domain polymerization.

Novel Congenital Platelet Disorders

The processes of megakaryocyte differentiation, proplatelet formation, and the daily release of 1011 platelets into the bloodstream are tightly regulated. Genetic disturbances can lead to a cascade of downstream molecular alterations that markedly affect the function of megakaryocytes and platelets. Therefore, identifying new genes and their function in megakaryocytes and platelets is critical for understanding how these unique cells contribute to health and disease. Over the last decade advances in genomics, specifically next generation sequencing, have allowed for the discovery of several mutations and genetic variants that cause disease or influence associated hematological traits. By performing platelet RNA-Seq we were among the first to identify NBEAL2 as the causative gene for gray platelet syndrome (GPS) and showed that NBEAL2 regulates megakaryocyte development and platelet function.1-3 Mice carrying targeted Nbeal2 null alleles not only replicated the thrombocytopenia and lack of alpha granules observed in humans, but also provided new information about the role of platelets in thromboinflammation, wound healing, myelofibrosis and metastasis dissemination.4-7 More recently, we and others found that germline mutations in ETV6 lead to thrombocytopenia, red cell macrocytosis, and predisposition to lymphoblastic leukemia.8,9ETV6 encodes an ETS family transcriptional repressor, which exerts its activity by binding a consensus sequence in the promoter regions of DNA. Mice with conditional Etv6 knockout in megakaryocytic-erythroid cells are thrombocytopenic indicating the involvement of Etv6 in thrombopoiesis.10 Several of the families recently described have a missense mutation in the central domain of ETV6 (p.P214L). This mutation results in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired megakaryocyte maturation. The bone marrow of individuals affected by this mutation show hyperplasia of immature megakaryocytes suggesting a differentiation arrest. Deep sequencing of the platelet transcriptome also revealed significant differences in mRNA expression levels between patients with the ETV6 p.P214L mutation and non-affected family members, indicating that ETV6 is critically involved in defining the molecular phenotype and function of platelets. Consistent with this notion, individuals with the ETV6 p.P214L mutation experience bleeding that is disproportionate to their mild thrombocytopenia. We have also used CRISPR/Cas9 technology to generate a mouse colony where the human p.P214L ETV6 mutation was inserted into the conserved site of Etv6. Mice with this mutation (Etv6H.P214L) have reduced platelet counts. In summary, advances in human genetics that led to the discovery of novel congenital platelet disorders coupled with relevant animal models will likely contribute to our understanding of megakaryopoiesis and platelet function.

Ets transcription factor Pointed promotes the generation of intermediate neural progenitors in Drosophila larval brains

Intermediate neural progenitor (INP) cells are transient amplifying neurogenic precursor cells generated from neural stem cells. Amplification of INPs significantly increases the number of neurons and glia produced from neural stem cells. In Drosophila larval brains, INPs are produced from type II neuroblasts (NBs, Drosophila neural stem cells), which lack the proneural protein Asense (Ase) but not from Ase-expressing type I NBs. To date, little is known about how Ase is suppressed in type II NBs and how the generation of INPs is controlled. Here we show that one isoform of the Ets transcription factor Pointed (Pnt), PntP1, is specifically expressed in type II NBs, immature INPs, and newly mature INPs in type II NB lineages. Partial loss of PntP1 in genetic mosaic clones or ectopic expression of the Pnt antagonist Yan, an Ets family transcriptional repressor, results in a reduction or elimination of INPs and ectopic expression of Ase in type II NBs. Conversely, ectopic expression of PntP1 in type I NBs suppresses Ase expression the NB and induces ectopic INP-like cells in a process that depends on the activity of the tumor suppressor Brain tumor. Our findings suggest that PntP1 is both necessary and sufficient for the suppression of Ase in type II NBs and the generation of INPs in Drosophila larval brains.

Sterile Alpha Motif Domain-Mediated Self-Association Plays an Essential Role in Modulating the Activity of the Drosophila ETS Family Transcriptional Repressor Yan

The ETS family transcriptional repressor Yan is an important downstream target and effector of the receptor tyrosine kinase (RTK) signaling pathway in Drosophila melanogaster. Structural and biochemical studies have shown that the N-terminal sterile alpha motif (SAM) of Yan is able to self associate to form a helical polymeric structure in vitro, although the extent and functional significance of self-association of full-length Yan remain unclear. In this study, we demonstrated that full-length Yan self associates via its SAM domain to form higher-order complexes in living cells. Introduction of SAM domain missense mutations that restrict Yan to a monomeric state reduces Yan's transcriptional repression activity and impairs its function during embryonic and retinal development. Coexpression of combinations of SAM domain mutations that permit the formation of Yan dimers, but not higher-order oligomers, increases activity relative to that of monomeric Yan, but not to the level obtained with wild-type Yan. Mechanistically, self-association directly promotes transcriptional repression of target genes independent of its role in limiting mitogen-activated protein kinase (MAPK)-mediated phosphorylation and nuclear export of Yan. Thus, we propose that the formation of higher-order Yan oligomers contributes to proper repression of target gene expression and RTK signaling output in developing tissues.

Cutting Edge: A Transcriptional Repressor and Corepressor Induced by the STAT3-Regulated Anti-Inflammatory Signaling Pathway

IL-10 regulates anti-inflammatory signaling via the activation of STAT3, which in turn controls the induction of a gene expression program whose products execute inhibitory effects on proinflammatory mediator production. In this study we show that IL-10 induces the expression of an ETS family transcriptional repressor, ETV3, and a helicase family corepressor, Strawberry notch homologue 2 (SBNO2), in mouse and human macrophages. IL-10-mediated induction of ETV3 and SBNO2 expression was dependent upon both STAT3 and a stimulus through the TLR pathway. We also observed that ETV3 expression was strongly induced by the STAT3 pathway regulated by IL-10 but not by STAT3 signaling activated by IL-6, which cannot activate the anti-inflammatory signaling pathway. ETV3 and SBNO2 repressed NF-kappaB- but not IFN regulatory factor 7 (IRF7)-activated transcriptional reporters. Collectively our data suggest that ETV3 and SBNO2 are components of the pathways that contribute to the downstream anti-inflammatory effects of IL-10.

Identification of the nuclear localization motif in the ETV6 (TEL) protein

ETV6, or Translocation-Ets-Leukemia (TEL), is an ETS family transcriptional repressor that is essential for establishing hematopoiesis in neonatal bone marrow, and is frequently a target of chromosomal translocations in human cancer. ETV6 is predominantly a nuclear phosphoprotein that represses transcription by binding directly to the promoters of target genes. The nuclear localization mechanism of ETV6, however, is not well understood. In this report, we provide evidence that a nuclear localization signal (NLS) exists in the C-terminal region of ETV6. ETV6 proteins with mutations outside of amino acids 332–452 localize to the nucleus, whereas proteins with mutations within amino acids 332–452 remain in the cytoplasm. Furthermore, when a fragment of ETV6 comprised of amino acids 332–452 was fused to cytoplasmic β-galactosidase protein, the fusion protein was able to enter the nucleus. These results strongly indicate that residues 332–452 mediate nuclear localization of ETV6.

Derepression by Depolymerization: Structural Insights into the Regulation of Yan by Mae

Yan, an ETS family transcriptional repressor, is regulated by receptor tyrosine kinase signaling via the Ras/MAPK pathway. Phosphorylation and downregulation of Yan is facilitated by a protein called Mae. Yan and Mae interact through their SAM domains. We find that repression by Yan requires the formation of a higher order structure mediated by Yan-SAM polymerization. Moreover, a crystal structure of the Yan-SAM/Mae-SAM complex shows that Mae-SAM specifically recognizes a surface on Yan-SAM that is also required for Yan-SAM polymerization. Mae-SAM binds to Yan-SAM with ∼1000-fold higher affinity than Yan-SAM binds to itself and can effectively depolymerize Yan-SAM. Mutations on Mae that specifically disrupt its SAM domain-dependent interactions with Yan disable the derepression function of Mae in vivo. Depolymerization of Yan by Mae represents a novel mechanism of transcriptional control that sensitizes Yan for regulation by receptor tyrosine kinases.

TEL, a Putative Tumor Suppressor, Induces Apoptosis and Represses Transcription of Bcl-XL

The ETS family transcriptional repressor TEL is frequently disrupted by chromosomal translocations, including the t(12;21) in which the second allele of TEL is deleted in up to 90% of the cases. Consistent with its role as a putative tumor suppressor, TEL expression inhibits colony formation by Ras-transformed NIH 3T3 cells and hinders proliferation of a variety of cell types. Although we observed no alteration in the cell cycle of TEL-expressing cells, we did find a marked increase in apoptosis of serum-starved TEL-expressing NIH 3T3 cells. This decrease in cell survival required the DNA binding domain of TEL, suggesting that TEL repressed an anti-apoptotic gene. These observations prompted us to search for genes regulated by ETS family proteins that regulate apoptosis. The anti-apoptotic molecule Bcl-XL contains multiple ets-factor binding sites within its promoters, and TEL repressed a Bcl-XL promoter-linked reporter gene. Moreover, the enforced expression of TEL decreased the endogenous expression of both Bcl-XL mRNA and protein. TEL-mediated repression of Bcl-XL likely affects cell survival via regulation of the apoptotic pathway.