TALE Proteins Research Articles

CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA

The all-protein cytosine base editor DdCBE uses TALE proteins and a double-stranded DNA-specific cytidine deaminase (DddA) to mediate targeted C•G-to-T•A editing. To improve editing efficiency and overcome the strict TC sequence-context constraint of DddA, we used phage-assisted non-continuous and continuous evolution to evolve DddA variants with improved activity and expanded targeting scope. Compared to canonical DdCBEs, base editors with evolved DddA6 improved mitochondrial DNA (mtDNA) editing efficiencies at TC by 3.3-fold on average. DdCBEs containing evolved DddA11 offered a broadened HC (H = A, C or T) sequence compatibility for both mitochondrial and nuclear base editing, increasing average editing efficiencies at AC and CC targets from less than 10% for canonical DdCBE to 15–30% and up to 50% in cell populations sorted to express both halves of DdCBE. We used these evolved DdCBEs to efficiently install disease-associated mtDNA mutations in human cells at non-TC target sites. DddA6 and DddA11 substantially increase the effectiveness and applicability of all-protein base editing.

Analysis of lamprey meis genes reveals that conserved inputs from Hox, Meis and Pbx proteins control their expression in the hindbrain and neural tube

Meis genes are known to play important roles in the hindbrain and neural crest cells of jawed vertebrates. To explore the roles of Meis genes in head development during evolution of vertebrates, we have identified four meis genes in the sea lamprey genome and characterized their patterns of expression and regulation, with a focus on the hindbrain and pharynx. Each of the lamprey meis genes displays temporally and spatially dynamic patterns of expression, some of which are coupled to rhombomeric domains in the developing hindbrain and select pharyngeal arches. Studies of Meis loci in mouse and zebrafish have identified enhancers that are bound by Hox and TALE (Meis and Pbx) proteins, implicating these factors in the direct regulation of Meis expression. We examined the lamprey meis loci and identified a series of cis-elements conserved between lamprey and jawed vertebrate meis genes. In transgenic reporter assays we demonstrated that these elements act as neural enhancers in lamprey embryos, directing reporter expression in appropriate domains when compared to expression of their associated endogenous meis gene. Sequence alignments reveal that these conserved elements are in similar relative positions of the meis loci and contain a series of consensus binding motifs for Hox and TALE proteins. This suggests that ancient Hox and TALE-responsive enhancers regulated expression of ancestral vertebrate meis genes in segmental domains in the hindbrain and have been retained in the meis loci during vertebrate evolution. The presence of conserved Meis, Pbx and Hox binding sites in these lamprey enhancers links Hox and TALE factors to regulation of lamprey meis genes in the developing hindbrain, indicating a deep ancestry for these regulatory interactions prior to the divergence of jawed and jawless vertebrates.

Divalent cations promote TALE DNA-binding specificity.

Recent advances in gene editing have been enabled by programmable nucleases such as transcription activator-like effector nucleases (TALENs) and CRISPR–Cas9. However, several open questions remain regarding the molecular machinery in these systems, including fundamental search and binding behavior as well as role of off-target binding and specificity. In order to achieve efficient and specific cleavage at target sites, a high degree of target site discrimination must be demonstrated for gene editing applications. In this work, we studied the binding affinity and specificity for a series of TALE proteins under a variety of solution conditions using in vitro fluorescence methods and molecular dynamics (MD) simulations. Remarkably, we identified that TALEs demonstrate high sequence specificity only upon addition of small amounts of certain divalent cations (Mg2+, Ca2+). However, under purely monovalent salt conditions (K+, Na+), TALEs bind to specific and non-specific DNA with nearly equal affinity. Divalent cations preferentially bind to DNA over monovalent cations, which attenuates non-specific interactions between TALEs and DNA and further stabilizes specific interactions. Overall, these results uncover new mechanistic insights into the binding action of TALEs and further provide potential avenues for engineering and application of TALE- or TALEN-based systems for genome editing and regulation.

Increased Frataxin Expression Induced in Friedreich Ataxia Cells by Platinum TALE-VP64s or Platinum TALE-SunTag

Frataxin gene (FXN) expression is reduced in Friedreich’s ataxia patients due to an increase in the number of GAA trinucleotides in intron 1. The frataxin protein, encoded by that gene, plays an important role in mitochondria’s iron metabolism. Platinum TALE (plTALE) proteins targeting the regulatory region of the FXN gene, fused with a transcriptional activator (TA) such as VP64 or P300, were used to increase the expression of that gene. Many effectors, plTALEVP64, plTALEp300, and plTALESunTag, targeting 14 sequences of the FXN gene promoter or intron 1 were produced. This permitted selection of 3 plTALEVP64s and 2 plTALESunTag that increased FXN gene expression by up to 19-fold in different Friedreich ataxia (FRDA) primary fibroblasts. Adeno-associated viruses were used to deliver the best effectors to the YG8R mouse model to validate their efficiencies in vivo. Our results showed that these selected plTALEVP64s or plTALESunTag induced transcriptional activity of the endogenous FXN gene as well as expression of the frataxin protein in YG8R mouse heart by 10-fold and in skeletal muscles by up to 35-fold. The aconitase activity was positively modulated by the frataxin level in mitochondria, and it was, thus, increased in vitro and in vivo by the increased frataxin expression.

HOXA1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on HOXA1 targets.

Hoxa1 has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins.

Single-molecule biophysics: TALEs spin along, but not around.

TALE proteins search for target sequences along a DNA substrate in a uniquely one-dimensional and non-rotating fashion.

Improvement of Ethanol Production in Saccharomyces cerevisiae by High-Efficient Disruption of the ADH2 Gene Using a Novel Recombinant TALEN Vector.

Bioethanol is becoming increasingly important in energy supply and economic development. However, the low yield of bioethanol and the insufficiency of high-efficient genetic manipulation approaches limit its application. In this study, a novel transcription activator-like effector nuclease (TALEN) vector containing the left and right arms of TALEN was electroporated into Saccharomyces cerevisiae strain As2.4 to sequence the alcohol dehydrogenase gene ADH2 and the hygromycin-resistant gene hyg. Western blot analysis using anti-FLAG monoclonal antibody proved the successful expression of TALE proteins in As2.4 strains. qPCR and sequencing demonstrated the accurate knockout of the 17 bp target gene with 80% efficiency. The TALEN vector and ADH2 PCR product were electroporated into ΔADH2 to complement the ADH2 gene (ADH2+ As2.4). LC–MS and GC were employed to detect ethanol yields in the native As2.4, ΔADH2 As2.4, and ADH2+ As2.4 strains. Results showed that ethanol production was improved by 52.4 ± 5.3% through the disruption of ADH2 in As2.4. The bioethanol yield of ADH2+ As2.4 was nearly the same as that of native As2.4. This study is the first to report on the disruption of a target gene in S. cerevisiae by employing Fast TALEN technology to improve bioethanol yield. This work provides a novel approach for the disruption of a target gene in S. cerevisiae with high efficiency and specificity, thereby promoting the improvement of bioethanol production in S. cerevisiae by metabolic engineering.

137. Analysis of Cas9-FokI and TALE-MutH Endonuclease Activity and Toxicity as Key Elements in the Development of a Gene Therapeutic Approach to Treat XLRP

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause severe X-linked retinitis pigmentosa (XLRP). More than 80% of the mutations are located in the terminal exon ORF15 of the RPGR gene. Genome editing, which represents a novel approach to treat monogenic disorders, is based on highly specific nucleases that cleave or nick at a chosen position within the complex genome followed by the repair of the double or single strand break (DSB or SSB) by endogenous repair mechanisms. The major pathways include error prone non-homologous end joining (NHEJ), microhomology-mediated end-joining (MMEJ), and homologous recombination (HR), the latter two with the help of a donor template. Currently, endonucleases for inducing the DSB are based on the CRISPR-Cas9 system or TALE proteins fused to the non-specific FokI nuclease (TALEN). However, specificity and toxicity of both endonuclease types raise concerns about their use for therapeutic in vivo applications. In order to study in vivo genome editing for the treatment of XLRP, our lab has generated a mouse model containing a point mutation in the ORF15 exon. In the present study, we characterize advanced variants of both endonuclease types (Cas9-FokI and TALE-MutH) for their activity and toxicity at the murine Rpgr-ORF15 locus for later usage in the mouse model. In total, ten sequences within or near the ORF15 exon have been targeted for the induction of DSB or SSB. Nine target sites for CRISPR/Cas9-FokI were chosen: three before, within, and behind the exon, respectively, and one target site for TALE-MutH within the exon. These sequences have been cloned into the traffic light reporter (TLR) gene expression system at the homing endonuclease I-SceI site. The TLR system has been modified to express either GFP in case of successful HR or BFP in case of NHEJ. Plasmids containing substrate, nucleases and template DNA were transfected into HEK293T cells. Efficiency of DNA modification was measured by FACS analysis and T7 surveyor assay, and toxicity was assessed by cell survival assay. In addition to the episomal TLR system within a human cell line (HEK293T), the genome of murine C2C12 cells was targeted by all endonuclease variants and toxicity was analysed via the T7 surveyor assay. Toxicity of Cas9-FokI and TALE-MutH are comparable to the golden standard ISceI while standard Cas9 nucleases showed slightly increased toxicity in HEK293T cells. Two different concentrations of the nucleases were used in a toxicity assay and were equally tolerated. Cas9-FokI showed preferences in its activity at the nine target sites with activities well above ISce-I level, while the one target site of TALE-MutH was as efficient as ISceI. Activity results were confirmed in the murine cell line C2C12. Off target toxicity in C2C12 cells was non-detectable. The characterization of the activity and toxicity of the tested endonucleases helped us to identify the most promising tailored nuclease and its target sequence in our gene targeting approach to treat XLRP. With the help of mouse retinal explants and subsequently in vivo experiments, we will confirm the efficacy of endonuclease mediated genome editing in photoreceptors.

The Development of TALE Nucleases for Biotechnology.

The development of a facile genome engineering technology based on transcription activator-like effector nucleases (TALENs) has led to significant advances in diverse areas of science and medicine. In this review, we provide a broad overview of the development of TALENs and the use of this technology in basic science, biotechnology, and biomedical applications. This includes the discovery of DNA recognition by TALEs, engineering new TALE proteins to diverse targets, general advances in nuclease-based editing strategies, and challenges that are specific to various applications of the TALEN technology. We review examples of applying TALENs for studying gene function and regulation, generating disease models, and developing gene therapies. The current status of genome editing and future directions for other uses of these technologies are also discussed.

RiboTALE: A modular, inducible system for accurate gene expression control.

A limiting factor in synthetic gene circuit design is the number of independent control elements that can be combined together in a single system. Here, we present RiboTALEs, a new class of inducible repressors that combine the specificity of TALEs with the ability of riboswitches to recognize exogenous signals and differentially control protein abundance. We demonstrate the capacity of RiboTALEs, constructed through different combinations of TALE proteins and riboswitches, to rapidly and reproducibly control the expression of downstream targets with a dynamic range of 243.7 ± 17.6-fold, which is adequate for many biotechnological applications.

604. TALE-VP64 Targeting the Frataxin Promoter Increase the Expression of That Gene in Friedreich Fibroblasts

Friedreich's ataxia (FRDA) is due to a reduced frataxin expression due to a trinucleotide repeat. It is 30% in patients with 200 GAA repeats and only 5% in patients with 900 GAA repeats 33. However, carriers of this disease produce about only 50% of the normal level of frataxin but do not develop symptoms. We have engineered 12 genes coding for TALE proteins targeting different nucleotide sequences present in the frataxin promoter. Each expression plasmid contains a TALEFrat gene fused with a transcription activator, VP64 under the EF1α promoter, a 2A peptide and an EGFP. When one of these plasmids was transfected alone in human cells, only green fluorescence was detected by FACS indirectly confirming the expression of the TALEFrat/VP64 protein. To identify which of our 12 different TALEFrat/VP64 proteins were able to better induce the expression of the frataxin gene, we have constructed a reporter plasmid containing the proximal region of the frataxin promoter, followed by a minimal CMV promoter and a mCherry reporter gene (pCR3.1 proximal-promoter-frataxin-miniCMV-mCherry). This reporter plasmid was initially transfected in human cells alone. Very few cells expressed the red fluorescence because the promoter was not effective without transactivation by binding factors. When the reporter plasmid was co-transfected in human cells with one of the pCR3.1-TALEFrat/VP64-2A-EGFP plasmids, a much higher number of cells expressed the red fluorescence because the TALEFrat/VP64 attached to the proximal frataxin promoter and induced the transcription of mCherry. The 3 TALEFrat/VP64, which induced the strongest expression of mCherry, were targeting promoter sequences close to each other.A plasmid coding for TALEFrat#8/VP64 was nucleofected in normal fibroblasts. Using quantitative RT-PCR, we have confirmed in 3 independent experiments that the expression of the frataxin mRNA (relative to GAPDH mRNA) in human cells was doubled or triple by TALEFrat#8/VP64 when results were normalized with cells transfected with EGFP or non-transfected cells. We have also shown that this TALE also increased by 2 folds the frataxin protein in fibroblasts from a FRDA patient. In a recent preliminary result, we have shown that the transfection of TALEFrat#8/VP64 plasmid in YG8R fibroblasts also increases frataxin mRNA (by about 1.4 to 1.9 fold) and protein (by about 1.4 fold). Such increases would be in the therapeutic range (i. e., 50% of normal frataxin level) for many patients. However, for patients that have less than 25% of the normal level of frataxin expression, a further increase of frataxin would be required and this project may permit to obtain higher frataxin increases and thus this would lead to an increased number of FRDA patients who would benefit from our therapy.

Enhancing stem cell survival in an ischemic heart by CRISPR-dCas9-based gene regulation

Ischemic heart disease has remained the number one killer around the world for over the past 20years. While stem cell therapy has become a promising new frontier to repair the damaged heart, limited stem cell survivability post-transplantation has precluded widespread use of this therapy. Strategies to genetically modify stem cells to activate pro-survival and anti-apoptotic and anti-inflammatory pathways, such as Akt and heme oxygenase-1, have been shown to improve the lifespan of transplanted stem cells within the ischemic myocardium, but constitutive overexpression of these pathways at high levels has been shown to have side effects. Therefore, more specific and controlled gene activation would be necessary. Current techniques used for gene regulation include zinc finger and TALE proteins, but there are still disadvantages to each of these methods, such as ease and cost of use. Also, those methods use synthesized promoters to express synthesized cDNA, which lack regulatory elements, including introns and 3′ untranslated regions for microRNA mediated post-transcriptional regulation. A new novel technique, the CRISPR/dCas9 system, was recently developed as a simple and efficient method for endogenous gene regulation. With its use of single guide chimeric RNA’s (sgRNA’s), this system has been shown to provide a high level of specificity and efficiency. When targeting different loci, past studies have found that the CRISPR/dCas9 system can activate gene expression at varying levels. In addition, this system makes use of the genome’s endogenous regulatory elements, such as the aforementioned introns and 3′ UTR’s, which can help provide a safer method of gene activation. If targeted to a gene promoting cellular survival or decreasing cell death, it could potentially improve stem cell longevity in a more efficient and controllable manner. As a result, our hypothesis is to use the CRISPR/dCas9 system to activate expression of an anti-inflammatory and anti-apoptotic gene, such as heme oxygenase-1 (HO-1), to an optimal level to increase transplanted stem cell survival while also mitigating its cytotoxic effects due to lack of internal regulation, thus prolonging its effects within the ischemic myocardium leading to greater therapeutic benefit.

A simple TALEN-based protocol for efficient genome-editing in Drosophila

Drosophila is a well-established genetic model organism: thousands of point mutations, deficiencies or transposon insertions are available from stock centres. However, to date, it is still difficult to modify a specific gene locus in a defined manner. A potential solution is the application of transcription activator-like effector nucleases (TALENs), which have been used successfully to mutate genes in various model organisms. TALENs are constructed by fusion of TALE proteins to the endonuclease FokI, resulting in artificial, sequence-specific endonucleases. They induce double strand breaks, which are either repaired by error-prone non-homologous end joining (NHEJ) or homology directed repair (HDR). We developed a simple TALEN-based protocol to mutate any gene of interest in Drosophila within approximately 2months. We inject mRNA coding for two TALEN pairs targeting the same gene into embryos, employ T7 endonuclease I screening of pooled F1 flies to identify mutations and generate a stable mutant stock in the F3 generation. We illustrate the efficacy of our strategy by mutating CG11617, a previously uncharacterized putative transcription factor with an unknown function in Drosophila. This demonstrates that TALENs are a reliable and efficient strategy to mutate any gene of interest in Drosophila.

The Hox-TALE has been wagging for a long time

Hox and TALE proteins interact in a sea anemone, just as they do in flies and mice, indicating that the Hox-TALE system originated very early in animal evolution.

TALE Factors Poise Promoters for Activation by Hox Proteins

Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we findmaternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. Bygastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription.

Glimpse into Hox and tale regulation of cell differentiation and reprogramming

During embryonic development, cells become gradually restricted in their developmental potential and start elaborating lineage-specific transcriptional networks to ultimately acquire a unique differentiated state. Hox genes play a central role in specifying regional identities, thereby providing the cell with critical information on positional value along its differentiation path. The exquisite DNA-binding specificity of the Hox proteins is frequently dependent upon their interaction with members of the TALE family of homeodomain proteins. In addition to their function as Hox-cofactors, TALE homeoproteins control multiple crucial developmental processes through Hox-independent mechanisms. Here, we will review recent findings on the function of both Hox and TALE proteins in cell differentiation, referring mostly to vertebrate species. In addition, we will discuss the direct implications of this knowledge on cell plasticity and cell reprogramming.

TALE transcription factors during early development of the vertebrate brain and eye

Our brain's cognitive performance arises from the coordinated activities of billions of nerve cells. Despite a high degree of morphological and functional differences, all neurons of the vertebrate central nervous system (CNS) arise from a common field of multipotent progenitors. Cell fate specification and differentiation are directed by multistep processes that include inductive/external cues, such as the extracellular matrix or growth factors, and cell-intrinsic determinants, such as transcription factors and epigenetic modulators of proteins and DNA. Here we review recent findings implicating TALE-homeodomain proteins in these processes. Although originally identified as HOX-cofactors, TALE proteins also contribute to many physiological processes that do not require HOX-activity. Particular focus is, therefore, given to HOX-dependent and -independent functions of TALE proteins during early vertebrate brain development. Additionally, we provide an overview about known upstream and downstream factors of TALE proteins in the developing vertebrate brain and discuss general concepts of how TALE proteins function to modulate neuronal cell fate specification.

Structure-Based Models to Understand the Cipher that Governs Tale Protein Affinity with DNA.

Recent use of TALEs, transcription activator like effectors as a novel DNA binding domain, has raised the interest in deciphering the mechanisms of DNA recognition by these multimeric TALE proteins. TALE proteins can be fused with the catalytic domain of FOKI nucleases to act as artificial restriction enzymes to create a targeted DNA double strand break in-vivo for genome editing. The TAL effectors are characterized by a central domain of 30 tandem repeats of 33 to 35 amino acid sequence motifs. The amino acid sequence of each repeat is largely invariant, with the exception of two adjacent amino acid at position 12th and 13th (the repeat variable diresidue or RVD). Repeats with different RVD recognize different DNA base pairs, and it is hypothesized that there is one-to-one correspondence between the RVDs in the repeat domain and the nucleotide in the target DNA sequence. But how this central domain contacts DNA and what determines the specificity has remained enigmatic so far. Here we use structure-based models to investigate the structure of TALENS and their interaction with DNA. Simulations with biophysical models for multimeric protein in complex with DNA are presently too computationally expensive to help, but structure-based simulations been used to study protein folding and protein-protein interaction mechanisms for over a decade. We performed homology modeling to construct a structure of single repeat and 1.5 repeat using the PthA (TPR domain) as a template protein, which predicts only alpha helices and turns for both the repeat domains. Strucuture based models are generated by inspecting the native contacts of DNA and individual repeat domain from previous experimental knowledge and can be easily adapted to different DNA sequences and multidomain of TALE, as long as the binding motives are conserved.

Therapeutic Potential of Targeted HOXA-TALE Knockdown in AML,

Abstract 3460HOX genes are master regulators of hematopoietic stem and progenitor cell (HSPC) development. HOX interaction with TALE (PBX or MEIS) proteins is pivotal to their function. The prevailing hypothesis is that HOX/TALE expression underlies HSPC self-renewal while dysregulated HOX/TALE expression underpins maintenance of the leukemia initiating cell. Expression profiling studies support this hypothesis and highlight the importance of the HOXA cluster and TALE genes in hematopoiesis and leukemogenesis. The aim of this study was to identify and target clinically relevant HOX and TALE genes in AML cells representative of the particular disease subtype.HOXA cluster and leukemia-associated TALE gene expression was evaluated in 166 favourable and intermediate AML patient samples by Affymetrix microarray and expression of the significantly different genes was confirmed by RQ-PCR assays in an independent patient cohort. HOXA1-A2, A4-A10, PBX3 and MEIS1 displayed significant differential expression (p’0.01) between favourable and intermediate AML subgroups and confirmed an association of HOXA/TALE with nucleophosmin gene mutations (NPM1-mut) previously identified as a favourable prognostic marker in AML. HOXA9 was shown to be differentially expressed and HOXA6 was identified as the most consistently and highly expressed member of the previously reported ‘HOX code’ by microarray analysis. These two HOX genes together with MEIS1 and PBX3 were selected for further study. Immunoprecipitation assays identified HOXA6/9-PBX3-MEIS1 protein interactions in AML cell lines demonstrating potential functional benefit of targeting members of these complexes in the disease setting.The HOXA/TALE axis was targeted in OCI AML3 (NPM1-mut) and U937 (NPM1-WT) AML cells using specific shRNAs (at least two per target) with Nucleofection or viral delivery followed by puromycin selection. Knockdown was validated by RQ-PCR as greater than 70% and western blotting where compatible antibodies were available. Cell viability was assessed using CellTiter-Glo® and direct cell counting whilst cell death was determined using the Caspase-Glo™assay. Cell responses were examined following targeted single gene knockdown of HOXA6, HOXA9, MEIS1 and PBX3, dual knockdown of HOXA6+HOXA9 either alone or in combination with standard-of-care therapeutic agents (Cytarabine or Mylotarg). Altered expression of genes associated with cell cycle and adhesion was identified following targeted gene knockdown using the Roche AmpliChip containing 1500 leukemia-associated probesets.Targeted reduction of HOXA6, HOXA9, MEIS1 PBX3 and combined HOXA6+HOXA9 levels greatly reduced cell growth compared to controls and was additive when combined with IC50 values of the chemotherapies used in the treatment of AML. This effect on cell growth may be due in part to increased apoptosis since increased caspase3/7 activity (up to 3.3 fold for PBX3 in U937 cells) was observed following HOX or TALE knockdown in either cell line. Furthermore, HOX or TALE knockdown resulted in impaired colony formation in both cell lines either due to reduction in number, size or metabolic activity compared to non-silenced controls. In addition, altered cell morphology with reduced blast-like features, was observed by HOXA6, HOXA9, MEIS1 or PBX3 knockdown in both cell lines. Together the data support a key role for HOX-TALE in AML and demonstrate dependency on this axis in leukemia maintenance.In summary, HOXA/TALE knockdown impairs growth and sensitises leukemic cells to chemotherapy independent of NPM1 mutation status. These findings suggest targeting of clinically significant HOXA cluster genes and their critical co-factors or modulation of downstream pathways could offer a novel strategy in the treatment of AML. Disclosures:No relevant conflicts of interest to declare.

Regulation of CCL2 Expression by an Upstream TALE Homeodomain Protein-Binding Site That Synergizes with the Site Created by the A-2578G SNP

CC Chemokine Ligand 2 (CCL2) is a potent chemoattractant produced by macrophages and activated astrocytes during periods of inflammation within the central nervous system. Increased CCL2 expression is correlated with disease progression and severity, as observed in pulmonary tuberculosis, HCV-related liver disease, and HIV-associated dementia. The CCL2 distal promoter contains an A/G polymorphism at position -2578 and the homozygous -2578 G/G genotype is associated with increased CCL2 production and inflammation. However, the mechanisms that contribute to the phenotypic differences in CCL2 expression are poorly understood. We previously demonstrated that the -2578 G polymorphism creates a TALE homeodomain protein binding site (TALE binding site) for PREP1/PBX2 transcription factors. In this study, we identified the presence of an additional TALE binding site 22 bp upstream of the site created by the -2578 G polymorphism and demonstrated the synergistic effects of the two sites on the activation of the CCL2 promoter. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated increased binding of the TALE proteins PREP1 and PBX2 to the -2578 G allele, and binding of IRF1 to both the A and G alleles. The presence of TALE binding sites that form inverted repeats within the -2578 G allele results in increased transcriptional activation of the CCL2 distal promoter while the presence of only the upstream TALE binding site within the -2578 A allele exerts repression of promoter activity.