Transcription Factories Research Articles

NF-YA promotes invasion and angiogenesis by upregulating EZH2-STAT3 signaling in human melanoma cells.

The process of angiogenesis is essential for tumor development and metastasis. Vascular endothelial growth factor (VEGF), which is overexpressed in most human cancers, has been demonstrated to be a major modulator of angiogenesis. Thus, inhibition of VEGF signaling has the potential for tumor anti-angiogenic therapy. Signal transducer and activator of transcription-3 (STAT3) is a key regulator for angiogenesis by directly binding to the VEGF promoter to upregulate its transcription. Several factors can enhance STAT3 activity to affect angiogenesis. Here, we found that overexpression of nuclear transcription factor-Y alpha (NF-YA) gene could promote cell invasion and angiogenesis accompanying the increase of STAT3 signaling in human melanoma cells. Moreover, the expression and secretion of VEGF was also found to be upregulated by the overexpression of NF-YA gene in melanoma cells. The STAT3 inhibitor was able to attenuate the upregulation of VEGF induced by NF-YA overexpression. Enhancer of zeste homolog2 (EZH2), the catalytic subunit of the Polycomb repressive complex2, enhances STAT3 activity by mediating its lysine methylation. We also showed that NF-YA upregulated the expression of EZH2 and NF-YA‑induced angiogenesis could be inhibited by EZH2 knockdown. Taken together, these findings indicate that overexpression of NF-YA contributes to tumor angiogenesis through EZH2-STAT3 signaling in human melanoma cells, highlighting NF-YA as a potential therapeutic target in human melanoma.

Simulated binding of transcription factors to active and inactive regions folds human chromosomes into loops, rosettes and topological domains.

Biophysicists are modeling conformations of interphase chromosomes, often basing the strengths of interactions between segments distant on the genetic map on contact frequencies determined experimentally. Here, instead, we develop a fitting-free, minimal model: bivalent or multivalent red and green ‘transcription factors’ bind to cognate sites in strings of beads (‘chromatin’) to form molecular bridges stabilizing loops. In the absence of additional explicit forces, molecular dynamic simulations reveal that bound factors spontaneously cluster—red with red, green with green, but rarely red with green—to give structures reminiscent of transcription factories. Binding of just two transcription factors (or proteins) to active and inactive regions of human chromosomes yields rosettes, topological domains and contact maps much like those seen experimentally. This emergent ‘bridging-induced attraction’ proves to be a robust, simple and generic force able to organize interphase chromosomes at all scales.

CTCF-mediated chromatin loops enclose inducible gene regulatory domains.

BackgroundThe CCTC-binding factor (CTCF) protein is involved in genome organization, including mediating three-dimensional chromatin interactions. Human patient lymphocytes with mutations in a single copy of the CTCF gene have reduced expression of enhancer-associated genes involved in response to stimuli. We hypothesize that CTCF interactions stabilize enhancer-promoter chromatin interaction domains, facilitating increased expression of genes in response to stimuli. Here we systematically investigate this model using computational analyses.ResultsWe use CTCF ChIA-PET data from the ENCODE project to show that CTCF-associated chromatin loops have a tendency to enclose regions of enhancer-regulated stimulus responsive genes, insulating them from neighboring regions of constitutively expressed housekeeping genes. To facilitate cell type-specific CTCF loop identification, we develop an algorithm to predict CTCF loops from ChIP-seq data alone by exploiting the CTCF motif directionality in loop anchors. We apply this algorithm to a hundred ENCODE cell line datasets, confirming the universality of our observations as well as identifying a general distinction between primary and immortal cells in loop-enclosed gene content. Finally, we combine the existing evidence to propose a model for the formation of CTCF loops in which partner sites are brought together by chromatin template reeling through stationary RNA polymerases, consistent with the transcription factory hypothesis.ConclusionsWe provide computational evidence that CTCF-mediated chromatin interactions enclose domains of stimulus responsive enhancer-regulated genes, insulating them from nearby housekeeping genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2516-6) contains supplementary material, which is available to authorized users.

Isolation of the protein and RNA content of active sites of transcription from mammalian cells.

Mammalian cell nuclei contain three RNA polymerases (RNAP I, RNAP II and RNAP III), which transcribe different gene subsets, and whose active forms are contained in supramolecular complexes known as 'transcription factories.' These complexes are difficult to isolate because they are embedded in the 3D structure of the nucleus. Factories exchange components with the soluble nucleoplasmic pool over time as gene expression programs change during development or disease. Analysis of their content can provide information on the nascent transcriptome and its regulators. Here we describe a protocol for the isolation of large factory fragments under isotonic salt concentrations in <72 h. It relies on DNase I-mediated detachment of chromatin from the nuclear substructure of freshly isolated, unfixed cells, followed by caspase treatment to release multi-megadalton factory complexes. These complexes retain transcriptional activity, and isolation of their contents is compatible with downstream analyses by mass spectrometry (MS) or RNA-sequencing (RNA-seq) to catalog the proteins and RNA associated with sites of active transcription.

Study of RNA Polymerase II Clustering inside Live-Cell Nuclei Using Bayesian Nanoscopy.

Nanoscale spatiotemporal clustering of RNA polymerase II (Pol II) plays an important role in transcription regulation. However, dynamics of individual Pol II clusters in live-cell nuclei has not been measured directly, prohibiting in-depth understanding of their working mechanisms. In this work, we studied the dynamics of Pol II clustering using Bayesian nanoscopy in live mammalian cell nuclei. With 50 nm spatial resolution and 4 s temporal resolution, Bayesian nanoscopy allows direct observation of the assembly and disassembly dynamics of individual Pol II clusters. The results not only provide quantifications of Pol II clusters but also shed light on the understanding of cluster formation and regulation. Our study suggests that transcription factories form on-demand and recruit Pol II molecules in their pre-elongation phase. The assembly and disassembly of individual Pol II clusters take place asynchronously. Overall, the methods developed herein are also applicable to studying a wide realm of real-time nanometer-scale nuclear processes in live cells.

Transcription Factor Clustering in Live Yeast Cells

Genes are regulated by a family of proteins called transcription factors which operate by binding to specific sequences of DNA. These sequences are short and occur frequently in the genome and it is unclear how transcription factors find their correct sites. We have investigated two zinc-finger transcription factors, Mig11 and Msn2, which are both glucose dependant repressors of genes. These proteins have been labelled with the green fluorescent protein (GFP) by chromosomal integration and another protein Nrd1 has been labelled with the mcherry fluorescent protein as a marker for the nucleus. Using single-molecule fluorescence microscopy2 in different extra-cellular glucose conditions, we are able to observe individual molecules of these proteins at different stages: bound to DNA in the nucleus, translocating across the nuclear membrane and diffusing in the cytoplasm. We show that these proteins operate in clusters through all these stages, in the nucleus and cytoplasm. Forming clusters may facilitate transcription factors finding their correct sites3 on the genome and provide possible evidence for transcription factories.1. Wollman, A. & Leake, M. C. FD2015-Single Molecule Microscopy: Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time. Faraday Discuss. (2015). http://dx.doi.org/10.1039/C5FD00077G.2. Wollman, A. J. M., Miller, H., Zhou, Z. & Leake, M. C. Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer. Biochem. Soc. Trans. 43, 139-145 (2015).3. Schmidt, H. G., Sewitz, S., Andrews, S. S. & Lipkow, K. An Integrated Model of Transcription Factor Diffusion Shows the Importance of Intersegmental Transfer and Quaternary Protein Structure for Target Site Finding. 9, (2014).

Phase Behavior of DNA in the Presence of DNA-Binding Proteins

To characterize the thermodynamical equilibrium of DNA chains interacting with a solution of nonspecific binding proteins, we implemented a Flory-Huggins free energy model. We explored the dependence on DNA and protein concentrations of the DNA collapse. For physiologically relevant values of the DNA-protein affinity, this collapse gives rise to a biphasic regime with a dense and a dilute phase; the corresponding phase diagram was computed. Using an approach based on Hamiltonian paths, we show that the dense phase has either a molten globule or a crystalline structure, depending on the DNA bending rigidity, which is influenced by the ionic strength. These results are valid at the thermodynamical equilibrium and therefore should be consistent with many biological processes, whose characteristic timescales range typically from 1 ms to 10 s. Our model may thus be applied to biological phenomena that involve DNA-binding proteins, such as DNA condensation with crystalline order, which occurs in some bacteria to protect their chromosome from detrimental factors; or transcription initiation, which occurs in clusters called transcription factories that are reminiscent of the dense phase characterized in this study.

Dynamic Behavior of the RNA Polymerase II and the Ubiquitin Proteasome System During the Neuronal DNA Damage Response to Ionizing Radiation.

Neurons are highly vulnerable to genotoxic agents. To restore genome integrity upon DNA lesions, neurons trigger a DNA damage response (DDR) that requires chromatin modifications and transcriptional silencing at DNA damage sites. To study the reorganization of the active RNA polymerase II (Pol II), which transcribes all mRNA-encoding genes, and the participation of the ubiquitin-proteasome system (UPS) in the neuronal DDR, we have used rat sensory ganglion neurons exposed to X-rays (4Gy) ionizing radiation (IR). In control neurons, Pol II appears concentrated in numerous chromatin microfoci identified as transcription factories by the incorporation of 5'-fluorouridine into nascent RNA. Upon IR treatment, numerous IR-induced foci (IRIF), which were immunoreactive for γH2AX and 53BP1, were observed as early as 30min post-IR; their number progressively reduced at 3h, 1day, and 3days post-IR. The formation of IRIF was associated with a decrease in Pol II levels by both immunofluorescence and Western blotting. Treatment with the proteasome inhibitor bortezomib strongly increased Pol II levels in both control and irradiated neurons, suggesting that proteasome plays a proteolytic role by clearing stalled Pol II complexes at DNA damage sites, as a prelude to DNA repair. Neuronal IRIF recruited ubiquitylated proteins, including ubiquitylated histone H2A (Ub-H2A), and the catalytic proteasome 20S. Ub-H2A has been associated with transcriptional silencing at DNA damage sites. On the other hand, the participation of UPS in neuronal DDR may be essential for the ubiquitylation of Pol II and other proteasome substrates of the DNA repair machinery and their subsequent proteasome-mediated degradation.

Wnt signaling induces transcription, spatial proximity, and translocation of fusion gene partners in human hematopoietic cells

AbstractChromosomal translocations are frequently associated with a wide variety of cancers, particularly hematologic malignancies. A recurrent chromosomal abnormality in acute myeloid leukemia is the reciprocal translocation t(8;21) that fuses RUNX1 and ETO genes. We report here that Wnt/β-catenin signaling increases the expression of ETO and RUNX1 genes in human hematopoietic progenitors. We found that β-catenin is rapidly recruited into RNA polymerase II transcription factories (RNAPII-Ser5) and that ETO and RUNX1 genes are brought into close spatial proximity upon Wnt3a induction. Notably, long-term treatment of cells with Wnt3a induces the generation a frequent RUNX1-ETO translocation event. Thus, Wnt/β-catenin signaling induces transcription and translocation of RUNX1 and ETO fusion gene partners, opening a novel window to understand the onset/development of leukemia.

Functional organization of the human 4D Nucleome

The 4D organization of the interphase nucleus, or the 4D Nucleome (4DN), reflects a dynamical interaction between 3D genome structure and function and its relationship to phenotype. We present initial analyses of the human 4DN, capturing genome-wide structure using chromosome conformation capture and 3D imaging, and function using RNA-sequencing. We introduce a quantitative index that measures underlying topological stability of a genomic region. Our results show that structural features of genomic regions correlate with function with surprising persistence over time. Furthermore, constructing genome-wide gene-level contact maps aided in identifying gene pairs with high potential for coregulation and colocalization in a manner consistent with expression via transcription factories. We additionally use 2D phase planes to visualize patterns in 4DN data. Finally, we evaluated gene pairs within a circadian gene module using 3D imaging, and found periodicity in the movement of clock circadian regulator and period circadian clock 2 relative to each other that followed a circadian rhythm and entrained with their expression.

The IgH 3′ regulatory region influences lymphomagenesis in Igλ-Myc mice

The IgH 3'regulatory region (3'RR), encompassing the four transcriptional enhancers hs3a-hs1,2-hs3b-hs4, has a key role on class switch recombination, somatic hypermutation, IgH transcription and B-cell fate. In plasma cells, transcribed IgH and IgL loci often colocalized in transcription factories and an IgL transcription defect might translate into lowered IgH transcription. We explored whether the 3'RR would affect lymphomagenesis in Igλ-Myc transgenic mice prone to lymphoproliferations. Breeding Igλ-Myc transgenics in a background deficient for the 3'RR influences lymphomagenesis toward less mature lymphomas (16% vs 54%, p = 0.01, Z test for two population proportions). In a 3'RR-deficient background mature tumors less often expressed the CD43 antigen (54% vs 0%, p = 0.02), a membrane glycoprotein expressed on activated mature B-cells. In contrast, in a 3'RR-deficient background tumors more often expressed the CD5 antigen (32% vs 12%, p = 0.05) that may serve to control autoimmunity and that is suspected to play a role in leukemic transformation. Lymphoma myc transcript levels, the Ki67 index of proliferation, the clonality, the usage of V(D)J segments, and their somatic hypermutation status were not affected in the 3'RR-deficient background. In conclusion, most probably through its action during the maturation process, the 3'RR can influence lymphomagenesis even when not linked with an oncogene.

Dissecting the nascent human transcriptome by analysing the RNA content of transcription factories.

While mapping total and poly-adenylated human transcriptomes has now become routine, characterizing nascent transcripts remains challenging, largely because nascent RNAs have such short half-lives. Here, we describe a simple, fast and cost-effective method to isolate RNA associated with transcription factories, the sites responsible for the majority of nuclear transcription. Following stimulation of human endothelial cells with the pro-inflammatory cytokine TNFα, we isolate and analyse the RNA content of factories by sequencing. Comparison with total, poly(A)+ and chromatin RNA fractions reveals that sequencing of purified factory RNA maps the complete nascent transcriptome; it is rich in intronic unprocessed transcript, as well as long intergenic non-coding (lincRNAs) and enhancer-associated RNAs (eRNAs), micro-RNA precursors and repeat-derived RNAs. Hence, we verify that transcription factories produce most nascent RNA and confer a regulatory role via their association with a set of specifically-retained non-coding transcripts.

Enhancer-promoter interaction facilitated by transiently forming G-quadruplexes

G-quadruplexes are guanine-rich nucleic acid sequences capable of forming a four-stranded structure through Hoogsteen hydrogen bonding. G-quadruplexes are highly concentrated near promoters and transcription start sites suggesting a role in gene regulation. They are less often found on the template than non-template strand where they either inhibit or enhance transcription, respectively. However, their potential role in enhancers and other distal regulatory elements has not been assessed yet. Here we show that DNAse hypersensitive (DHS) cis-regulatory elements are also enriched in Gs and their G-content correlate with that of their respective promoters. Besides local G4s, the distal cis regions may form G-quadruplexes together with the promoters, each contributing half a G4. This model is supported more for the non-template strand and we hypothesised that the G4 forming capability of the promoter and the enhancer non-template strand could facilitate their binding together and making the DHS regions accessible for the transcription factory.

Abundance and distribution of RNA polymerase II in Arabidopsis interphase nuclei.

RNA polymerase II (RNAPII) is responsible for the transcription of most eukaryotic protein-coding genes. Analysing the topological distribution and quantification of RNAPII can contribute to understanding its function in interphase nuclei. Previously it was shown that RNAPII molecules in plant nuclei form reticulate structures within euchromatin of differentiated Arabidopsis thaliana nuclei rather than being organized in distinct 'transcription factories' as observed in mammalian nuclei. Immunosignal intensity measurements based on specific antibody labelling in maximum intensity projections of image stacks acquired by structured illumination microscopy (SIM) suggested a relative proportional increase of RNAPII in endopolyploid plant nuclei. Here, photoactivated localization microscopy (PALM) was applied to determine the absolute number and distribution of active and inactive RNAPII molecules in differentiated A. thaliana nuclei. The proportional increase of RNAPII during endopolyploidization is confirmed, but it is also shown that PALM measurements are more reliable than those based on SIM in terms of quantification. The single molecule localization results show that, although RNAPII molecules are globally dispersed within plant euchromatin, they also aggregate within smaller distances as described for mammalian transcription factories.

Quantification of Interphase Chromatin Dynamics in Fission Yeast

In mammals and other higher organisms, interphase chromosomes remain separated from each other and compartmentalized into chromosome territories. In yeast, chromosomes adopt a Rabl configuration, with arms extending from centromeres tethered at the spindle pole body(SPB), functionally equivalent to the centrosome, to telomeres at the opposite nuclear envelope. These organizations generally constrain chromatin motion, and contribute to gene positioning inside the nucleus. On the other hand, active genes escape from such physical constraints and are located at active regions such as transcription factories and nuclear pore complexes(NPCs). In spite of extensive studies for these phenomena, how gene loci sustain and change their positioning during the cell cycle still remains unclear. In order to elucidate chromatin dynamics during interphase, we visualized several gene loci and the centromeres (and the SPB) on the fission yeast S. pombe chromosomes that show a Rabl configuration, and then tracked the positions of the loci on a longer time scale than conventional one. The statistical analysis of the physical distance from the SPB to each locus suggested that the gene loci show not only restricted diffusion due to the physical constraint of the Rabl configuration, but also a novel dynamic property, that is quite different from simple diffusive behaviors reported so far. In this presentation, we will discuss functional roles that such chromatin dynamics possibly play within the interphase nucleus.

Spatial Organization of Transcription in E. coli Cells

RNA polymerase (RNAP) has been shown to exhibit clustered distribution in bacterial cells. These clusters were hypothesized to be active transcription factories in which concentrated RNAP molecules are engaged in ribosomal RNA synthesis. To examine this hypothesis, in this study we used photoactivated localization microscopy (PALM) to map the spatial distribution of RNAP in correlation with active transcription in superresolution. We monitored the colocalization of RNAP with single mRNA or rRNA gene sites, with nascent transcripts, and with elongation factors NusA and NusB. We further examined the cellular positioning of rRNA and mRNA genes sharing similar regulatory controls under different transcription conditions. Our results provide evidence rigorously examining the transcription factory model.

Compartmentalization of the cell nucleus and spatial organization of the genome

The eukaryotic cell nucleus is one of the most complex cell organelles. Despite the absence of membranes, the nuclear space is divided into numerous compartments where different processes in- volved in the genome activity take place. The most important nuclear compartments include nucleoli, nuclear speckles, PML bodies, Cajal bodies, histone locus bodies, Polycomb bodies, insulator bodies, transcription and replication factories. The structural basis for the nuclear compartmentalization is provided by genomic DNA that occupies most of the nuclear volume. Nuclear compartments, in turn, guide the chromosome folding by providing a platform for the spatial interaction of individual genomic loci. In this review, we discuss fundamental principles of higher order genome organization with a focus on chromosome territories and chromosome domains, as well as consider the structure and function of the key nuclear compartments. We show that the func- tional compartmentalization of the cell nucleus and genome spatial organization are tightly interconnected, and that this form of organization is highly dynamic and is based on stochastic processes.

Spatial organization of housekeeping genes in interphase nuclei

Spatial organization of the eukaryotic genome is tightly connected to its functioning. In particular, the interaction of gene promoters with remote enhancer elements in active chromatin hubs, as well as the recruitment of genes to the common transcription factories plays an important role in regulation of gene transcription. Most of works related to the analysis of spatial interaction of genome regulatory elements relies on models of tissue-specific genes. Meanwhile, it remains unclear to which extent the spatial organization of chromosomes is guided by house-keeping genes that are transcribed in most of cell types and outnumber the transcribed tissue-specific genes. To address this question, we used the 4C technique to characterize genome-wide the spatial contacts of the chicken house-keeping genes CARHSP1 and TRAP1 situated on chromosome 14. The promoters of these genes had an increased frequency of interaction with chromosome regions enriched in CpG islands and binding motifs for the ubiquitous transcription factor Sp1, both of which mark promoters of house-keeping genes, and overall with transcriptionally active regions. By contrast, the analysis of interaction of a gene poor region of chromosome 14 revealed no such preferences. The evidence for the interaction of house-keeping gene promoters were also obtained in independent cytological experiments aimed at visualization of non-methylated CpG islands in individual nuclei of human cells, which showed clustering of CpG islands in the nuclear space. Altogether, the results of our work suggest that the interaction of house-keeping genes constitutes an important factor that determines the spatial organization of interphase chromosomes.

RanBP9 modulates AICD localization and transcriptional activity via direct interaction with Tip60.

Proteolytic processing of the amyloid-β protein precursor (AβPP) occurs via alternative pathways, culminating with the production of the AβPP intracellular domain (AICD). AICD can translocate to the nucleus and regulate transcription, but its activity is modulated by interactions with other proteins. In the nucleus, AICD, FE65, and Tip60 associate into AFT complexes, which are targeted to nuclear spots which correspond to transcription factories. Here we report that RanBP9 interacts with the cytoplasmic domain of AβPP, through the NPXY internalization motif. Moreover, RanBP9 interaction with Tip60 is also described. The RanBP9-Tip60 interaction dramatically relocated RanBP9 from a widespread cellular distribution to nuclear speckles. AβPP processing is a central aspect in determining the protein's function and that of its resulting proteolytic fragments, among them AICD. The latter results from the amyloidogenic pathway and is the peptidic species predominantly involved in nuclear signaling. Of note RanBP9 transfection was previously demonstrated to increase amyloid-β generation. Here we show that RanBP9 relocates AICD to the Tip60-enriched nuclear speckles, and prevented the formation of nuclear spots formation, having therefore a negative effect on AICD mediated nuclear signaling and consequently AFT complex formation. Furthermore, by transfecting cells with increasing amounts of RanBP9, the expression of AICD-regulated genes, including AβPP itself, was reduced. Given the data presented, one can deduce that RanBP9 has an inhibitory regulatory effect on AICD-mediated transcription and the effect is mediated by relocating AICD away from transcription factories.

Abstract B35: Estradiol drives MLL gene fusions in infant acute leukemia

Abstract Translocation breakpoints involving the MLL gene linked to Infant Acute Leukemia (IAL) and therapy related acute leukemia (tAL) are tightly clustered between MLL exons 8 and 12. Exon 12 also marks the location of a well-described cleavage hotspot that is synchronous with a sharp decline in total MLL fusions observed in clinical samples. Though multiple MLL fusion partners have been identified, fusions to MLLT3 (AF9) and AFF1 (AF4) comprise 56% of all clinical rearrangements so far assayed. Epidemiological data has linked maternal exposure to birth control formulations with an increased risk of IAL involving MLL gene rearrangements. Subsequent in-vitro studies suggested a role of estradiol (E2) in the generation of such rearrangements. In order to probe the action of E2 in generating these lesions, the ability of E2 to impact MLL rearrangement formation was studied, focusing on the exon 12 hotspot and using immortalized but not transformed TK6 lymphoblastoid cells. Real-time PCR studies showed that in this cell line, transient exposure to 10 nM E2 enhanced transcription of MLL eight fold over controls. E2 treatment also increased transcription of MLL partner genes, MLLT3 and AFF1, through to a lesser degree. To determine if the process of transcription led to gene co-localization, chromatin conformation capture (3C) experiments were performed. Here, brief exposure to 10 nM E2 led to the co-localization of MLL with MLLT3, using primer sets targeting both MLL introns 9 and 13 and MLLT3 introns 4 and 8. These data indicated contact between these two genes, over a substantial region, consistent with their occupation of an operationally defined “transcription factory”. Surprisingly, low levels of E2 also stimulated the generation of de-novo MLL-MLLT3 fusion transcripts, without the application of any genotoxic stressors. To identify the process whereby each gene is fragmented, an essential precursor to any rearrangement, RNAi knockdown of activation induced cytidine deaminase (AICDA) was studied. AICDA activity is normally associated with class switch recombination and somatic hypermutation, but has recently been identified as the fragmenting agent associated with c-myc/IgH fusions in Burkitts lymphoma. RNAi knockdown of AICDA suppressed the induction of MLL-MLLT3 fusion transcript formation observed with E2 treatment, suggesting AICDA involvement in MLL and partner gene fragmentation. To probe this association in more detail, a ChIP analysis was performed targeting AICDA recruitment to MLL intron 11 (hot spot for MLL rearrangements) or intron 12 (few MLL rearrangements). Here, E2 dependent localization of AICDA was noted upstream of the DNA cleavage hotspot and within the region of elevated MLL fusions in intron 11, but not in a region showing few rearrangements. The differential accumulation of AICDA either side of the exon 12 fragmentation hot spot may be explained by the presence of a DNA palindromic sequence that restricts transcriptional progression and triggers AICDA recruitment. Combined, these studies show that concentrations of E2 that occur during pregnancy, or during use of oral contraceptives, have the potential to initiate MLL fusions through an endogenous AICDA-mediated mechanism, that is enhanced by gene proximity associated with synchronous transcription of both MLL and partner genes. If correct, then this mechanism may provide an index of potential leukemogenic risk, and a target for intervention. Citation Format: Andrew T. Vaughan, Rebecca Wright, Katrina Slemmons. Estradiol drives MLL gene fusions in infant acute leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B35.