Genome-Wide Analysis of NOTCH1, ETS Family Factors, and RUNX1 Binding in Human T Lymphoblastic Leukemia Cells Reveals Distinct Regulatory Elements

Abstract Abstract 1277 NOTCH1 regulates gene expression by forming transcription activation complexes with the DNA-binding factor RBPJ and gain-of-function NOTCH1 mutations are common in human and murine T lymphoblastic leukemia/lymphoma (T-LL). Via ChIP-seq studies of T-LL cells with constitutive Notch activation, we previously showed that NOTCH1/RBPJ binding sites in T-LL genomes are highly enriched for motifs corresponding to Ets factors and Runx factors. In this study, we determined the relationship of NOTCH1, RBPJ, ETS1, GABPA and RUNX1 binding sites in human T-LL cells by performing ChIP-Seq for each of these factors, as well as the chromatin marks H3K4me1, H3K4me3, and H3K27me3, and aligning the resulting sequences to human genome reference hg19 using programs available through Cistrome. Peak calling was performed with MACS2, and motif analysis was performed using SeqPos, which relies on JASPAR, TRANSFAC, Protein Binding Microarray (PBM), Yeast-1-hybrid (y1h), and human protein-DNA interaction (hPDI) databases to find known motifs and can also perform de novo motif discovery. Our analysis showed even more pervasive overlap of NOTCH1/RBPJ binding with ETS1/GABPA and RUNX1 factor binding than was predicted by motif analysis, in part due to binding of Ets factors and RUNX1 to non-canonical sequences. Heat-map analysis with K-means clustering on NOTCH1 binding regions identified three major classes of RBPJ/NOTCH1: class 1, characterized by high NOTCH/RBPJ signals, binding of the cofactors ZNF143, ETS1 and GABPA, high H3K4me3 signals, localization to promoters, and binding motifs for ZNF143; class 2, characterized by low NOTCH/RBPJ signals, binding of the cofactors ETS1, GABPA and RUNX1, high H3K4me3 signals, and Ets factor and CREB binding motifs; and class 3, characterized by high NOTCH/RBPJ signals, binding of RUNX1 and ETS1 cofactors, high H3K4me1 signals, intergenic localization (consistent with enhancers), and motifs for RUNX factors, ETS factors, and RBPJ. Of note, the nearest binding sites to the most responsive NOTCH1 target genes (defined as >2 fold stimulation when NOTCH1 was activated following release of gamma-secretase inhibitor (GSI) blockade by drug washout) were preferentially associated with Class 3 sites. Furthermore, shRNA knockdown of Ets factors and RUNX1 in T-LL cell lines induced apoptosis and reduced cell proliferation, implicating these factors in maintenance of T-LL growth and survival. Combination of knockdown of either Ets factors or RUNX1 with GSI treatment resulted in more severe phenotype in terms of apoptosis and cell growth compared to the knockdown or GSI treatment alone. In summary, our studies represent a step forward towards genome-wide understanding of how Notch works in concerts with other transcription factors to regulate the transcriptome of T-LL cells. Disclosures: No relevant conflicts of interest to declare.

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C/ebpα and Pu.1 Are Members of a Critical Regulatory Network Required for Hoxa9-Mediated Immortalization

Blood ◽

10.1182/blood.v120.21.650.650 ◽

2012 ◽

Vol 120 (21) ◽

pp. 650-650

Author(s):

Cailin Collins ◽

Jingya Wang ◽

Joel Bronstein ◽

Jay L. Hess

Keyword(s):

Transcription Factor ◽

Binding Sites ◽

Target Genes ◽

De Novo ◽

Conflicts Of Interest ◽

Epigenetic Modifications ◽

Conditional Knockout ◽

Motif Analysis ◽

Myeloid Leukemias

Abstract Abstract 650 HOXA9 is a homeodomain-containing transcription factor that plays important roles in both development and hematopoiesis. Deregulation of HOXA9 occurs in a variety of acute lymphoid and myeloid leukemias and plays a key role in their pathogenesis. More than 50% of acute myeloid leukemia (AML) cases show up-regulation of HOXA9, which correlates strongly with poor prognosis. Nearly all cases of AML with mixed lineage leukemia (MLL) translocations have increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. Despite the crucial role that HOXA9 plays in development, hematopoiesis and leukemia, its transcriptional targets and mechanisms of action are poorly understood. Previously we identified Hoxa9 and Meis1 binding sites in myeloblastic cells, profiled their epigenetic modifications, and identified the target genes regulated by Hoxa9. Hoxa9 and Meis1 co-bind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/p300 binding characteristic of enhancers. Hoxa9 association at these sites correlates strongly with increases in histone H3K27 acetylation and activation of downstream target genes, including many proleukemic gene loci. De novo motif analysis of Hoxa9 binding sites shows a marked enrichment of motifs for the transcription factors in the C/EBP and ETS families, and C/ebpα and the ETS transcription factor Pu.1 were found to cobind at Hoxa9-regulated enhancers. Both C/ebpα and Pu.1 are known to play critical roles in the establishment of functional enhancers during normal myeloid development and are mutated or otherwise deregulated in various myeloid leukemias. To determine the importance of co-association of Hoxa9, C/ebpα and Pu.1 at myeloid enhancers, we generated cell lines from C/ebpα and Pu.1 conditional knockout mice (kindly provided by Dr. Daniel Tenen, Harvard University) by immortalization with Hoxa9 and Meis1. In addition we transformed bone marrow with a tamoxifen-regulated form of Hoxa9. Strikingly, loss of C/ebpα or Pu.1, or inactivation of Hoxa9, blocks proliferation and leads to myeloid differentiation. ChIP experiments show that both C/ebpα and Pu.1 remain bound to Hoxa9 binding sites in the absence of Hoxa9. After the loss of Pu.1, both Hoxa9 and C/ebpα dissociate from Hoxa9 binding sites with a corresponding decrease in target gene expression. In contrast, loss of C/ebpα does not lead to an immediate decrease in either Hoxa9 or Pu.1 binding, suggesting that C/ebpα may be playing a regulatory as opposed to a scaffolding role at enhancers. Current work focuses on performing ChIP-seq analysis to assess how C/ebpα and Pu.1 affect Hoxa9 and Meis1 binding and epigenetic modifications genome-wide, and in vivo leukemogenesis assays to confirm the requirement of both Pu.1 and C/ebpα in the establishment and maintenance of leukemias with high levels of Hoxa9. Collectively, our findings implicate C/ebpα and Pu.1 as members of a critical transcription factor network required for Hoxa9-mediated transcriptional regulation in leukemia. Disclosures: No relevant conflicts of interest to declare.

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Identification and Characterization By ChIP-Seq Of Genomic Sites Bound By E2A-PBX1 In Acute Lymphoblastic Leukemia Demonstrates Associations With p300 Recruitment, Transcriptionally Active Chromatin and Abundant Transcription

Blood ◽

10.1182/blood.v122.21.2501.2501 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2501-2501

Author(s):

Kyster Nanan ◽

David P LeBrun

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Binding Sites ◽

Target Genes ◽

Conflicts Of Interest ◽

Specific Binding ◽

Lymphoblastic Leukemia ◽

Regulatory Elements ◽

Negative Control ◽

Active Chromatin ◽

P300 Recruitment

Abstract E2A-PBX1 (EP1) is a chimeric oncogenic transcription factor expressed consequent to the 1;19 chromosomal translocation in cases of acute lymphoblastic leukemia (ALL). EP1 can induce transcription of reporter genes and EP1-driven oncogenesis requires direct binding of EP1 with the transcriptional co-activator and histone acetyltransferase p300. Therefore, we hypothesized that EP1 recruits p300 and other co-activators to cis-acting regulatory elements throughout the genome thereby inducing or maintaining transcription of target genes some of which contribute to the neoplastic phenotype. Here we have used chromatin immunoprecipitation followed by next generation DNA sequencing (ChIP-seq) to identify and characterize EP1-bound sites across the genome of the t(1;19)-associated, ALL-derived cell line RCH-ACV. ChIP was performed with an anti-FLAG antibody using sheared chromatin prepared from RCH-ACV cells that stably expressed FLAG-tagged EP1; ChIP from parent RCH-ACV cells not expressing FLAG-EP1 served as a negative control for peak calling. Parallel immunoprecipitations were performed with antibodies for p300 and the chromatin marks H3K4me3, H3K4me1 and H3K27me3. Sequencing of DNA purified from the immunoprecipitated material and of total RNA (RNA-seq) was carried out commercially by BGI whereas bioinformatic analyses were performed in-house. Bioinformatic analysis of data from replicate samples identified 3166 EP1 binding peaks across the RCH-ACV genome (irreproducible discovery rate threshold <0.01). Most EP1 binding sites were located in intronic (1408 sites) or intergenic (1346 sites) regions. Binding site consensus analysis showed overrepresentation of binding motifs for REST, CTCF, MYC, PAX5 and other transcription factors suggesting indirect recruitment of EP1 to DNA mediated by protein-protein interactions. EP1-bound regions were enriched for p300 binding (Figure 1), consistent with the documented importance of p300 recruitment in EP1 oncogenesis. A particular association with H3K4me3 relative to H3K4me1 or H3K27me3 (Figure 2) suggested association with active promoters. Three hundred and forty-two genes had EP1 binding sites within 1000 bp of their transcriptional start sites and these genes were associated with differentially abundant transcription (Figure 3, P<0.001). Querying the online Mammalian Phenotype Ontology tool with genes associated with EP1 binding generated terms that were obviously rich in phenotypes pertaining to B-lymphopoiesis. In summary, our results suggest that EP1 recruits p300 and other co-activators to transcriptionally active chromatin in ALL cells. Results from studies currently underway to confirm the dependency of target gene expression and p300 recruitment upon binding of EP1 at specific binding sites will be presented. Disclosures: No relevant conflicts of interest to declare.

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Motif analysis in co-expression networks reveals regulatory elements in plants: The peach as a model

10.1101/2020.02.28.970137 ◽

2020 ◽

Cited By ~ 1

Author(s):

Najla Ksouri ◽

Jaime A. Castro-Mondragón ◽

Francesc Montardit-Tardà ◽

Jacques van Helden ◽

Bruno Contreras-Moreira ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Motif Discovery ◽

Prunus Persica ◽

De Novo ◽

Transcriptional Start Site ◽

Regulatory Elements ◽

Proximal Promoter ◽

Upstream Region ◽

Motif Analysis ◽

Regulatory Motifs

AbstractIdentification of functional regulatory elements encoded in plant genomes is a fundamental need to understand gene regulation. While much attention has been given to model species as Arabidopsis thaliana, little is known about regulatory motifs in other plant genera. Here, we describe an accurate bottom-up approach using the online workbench RSAT::Plants for a versatile ab-initio motif discovery taking Prunus persica as a model. These predictions rely on the construction of a co-expression network to generate modules with similar expression trends and assess the effect of increasing upstream region length on the sensitivity of motif discovery. Applying two discovery algorithms, 18 out of 45 modules were found to be enriched in motifs typical of well-known transcription factor families (bHLH, bZip, BZR, CAMTA, DOF, E2FE, AP2-ERF, Myb-like, NAC, TCP, WRKY) and a novel motif. Our results indicate that small number of input sequences and short promoter length are preferential to minimize the amount of uninformative signals in peach. The spatial distribution of TF binding sites revealed an unbalanced distribution where motifs tend to lie around the transcriptional start site region. The reliability of this approach was also benchmarked in Arabidopsis thaliana, where it recovered the expected motifs from promoters of genes containing ChIPseq peaks. Overall, this paper presents a glimpse of the peach regulatory components at genome scale and provides a general protocol that can be applied to many other species. Additionally, a RSAT Docker container was released to facilitate similar analyses on other species or to reproduce our results.One sentence summaryMotifs prediction depends on the promoter size. A proximal promoter region defined as an interval of -500 bp to +200 bp seems to be the adequate stretch to predict de novo regulatory motifs in peach

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GimmeMotifs: an analysis framework for transcription factor motif analysis

10.1101/474403 ◽

2018 ◽

Cited By ~ 8

Author(s):

Niklas Bruse ◽

Simon J. van Heeringen

Keyword(s):

Dna Sequences ◽

Motif Discovery ◽

High Throughput Sequencing ◽

Performance Metrics ◽

De Novo ◽

Expression Patterns ◽

Regulatory Elements ◽

Ensemble Method ◽

Regulatory Sequences ◽

Motif Analysis

AbstractBackgroundTranscription factors (TFs) bind to specific DNA sequences, TF motifs, in cis-regulatory sequences and control the expression of the diverse transcriptional programs encoded in the genome. The concerted action of TFs within the chromatin context enables precise temporal and spatial expression patterns. To understand how TFs control gene expression it is essential to model TF binding. TF motif information can help to interpret the exact role of individual regulatory elements, for instance to predict the functional impact of non-coding variants.FindingsHere we present GimmeMotifs, a comprehensive computational framework for TF motif analysis. Compared to the previously published version, this release adds a whole range of new functionality and analysis methods. It now includes tools for de novo motif discovery, motif scanning and sequence analysis, motif clustering, calculation of performance metrics and visualization. Included with GimmeMotifs is a non-redundant database of clustered motifs. Compared to other motif databases, this collection of motifs shows competitive performance in discriminating bound from unbound sequences. Using our de novo motif discovery pipeline we find large differences in performance between de novo motif finders on ChIP-seq data. Using an ensemble method such as implemented in GimmeMotifs will generally result in improved motif identification compared to a single motif finder. Finally, we demonstrate maelstrom, a new ensemble method that enables comparative analysis of TF motifs between multiple high-throughput sequencing experiments, such as ChIP-seq or ATAC-seq. Using a collection of ~200 H3K27ac ChIP-seq data sets we identify TFs that play a role in hematopoietic differentiation and lineage commitment.ConclusionGimmeMotifs is a fully-featured and flexible framework for TF motif analysis. It contains both command-line tools as well as a Python API and is freely available at: https://github.com/vanheeringen-lab/gimmemotifs.

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Genome-wide identification and expression analysis of Raffinose synthetase family in cotton

BMC Bioinformatics ◽

10.1186/s12859-021-04276-4 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Ruifeng Cui ◽

Xiaoge Wang ◽

Waqar Afzal Malik ◽

Xuke Lu ◽

Xiugui Chen ◽

...

Keyword(s):

Abiotic Stress ◽

Expression Patterns ◽

Regulatory Elements ◽

Plant Leaves ◽

Motif Analysis ◽

Expression Levels ◽

Genome Wide ◽

Salt And Drought Stress ◽

Key Genes ◽

Stress Gene

Abstract Background The Raffinose synthetase (RAFS) genes superfamily is critical for the synthesis of raffinose, which accumulates in plant leaves under abiotic stress. However, it remains unclear whether RAFS contributes to resistance to abiotic stress in plants, specifically in the Gossypium species. Results In this study, we identified 74 RAFS genes from G. hirsutum, G. barbadense, G. arboreum and G. raimondii by using a series of bioinformatic methods. Phylogenetic analysis showed that the RAFS gene family in the four Gossypium species could be divided into four major clades; the relatively uniform distribution of the gene number in each species ranged from 12 to 25 based on species ploidy, most likely resulting from an ancient whole-genome polyploidization. Gene motif analysis showed that the RAFS gene structure was relatively conservative. Promoter analysis for cis-regulatory elements showed that some RAFS genes might be regulated by gibberellins and abscisic acid, which might influence their expression levels. Moreover, we further examined the functions of RAFS under cold, heat, salt and drought stress conditions, based on the expression profile and co-expression network of RAFS genes in Gossypium species. Transcriptome analysis suggested that RAFS genes in clade III are highly expressed in organs such as seed, root, cotyledon, ovule and fiber, and under abiotic stress in particular, indicating the involvement of genes belonging to clade III in resistance to abiotic stress. Gene co-expressed network analysis showed that GhRFS2A-GhRFS6A, GhRFS6D, GhRFS7D and GhRFS8A-GhRFS11A were key genes, with high expression levels under salt, drought, cold and heat stress. Conclusion The findings may provide insights into the evolutionary relationships and expression patterns of RAFS genes in Gossypium species and a theoretical basis for the identification of stress resistance materials in cotton.

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Evolution of mouse circadian enhancers from transposable elements

Genome Biology ◽

10.1186/s13059-021-02409-9 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Julius Judd ◽

Hayley Sanderson ◽

Cédric Feschotte

Keyword(s):

Gene Expression ◽

Transposable Elements ◽

Binding Sites ◽

Mouse Liver ◽

Integration Site ◽

Point Mutations ◽

Regulatory Elements ◽

Circadian Gene ◽

Binding Motifs ◽

Reporter Assays

Abstract Background Transposable elements are increasingly recognized as a source of cis-regulatory variation. Previous studies have revealed that transposons are often bound by transcription factors and some have been co-opted into functional enhancers regulating host gene expression. However, the process by which transposons mature into complex regulatory elements, like enhancers, remains poorly understood. To investigate this process, we examined the contribution of transposons to the cis-regulatory network controlling circadian gene expression in the mouse liver, a well-characterized network serving an important physiological function. Results ChIP-seq analyses reveal that transposons and other repeats contribute ~ 14% of the binding sites for core circadian regulators (CRs) including BMAL1, CLOCK, PER1/2, and CRY1/2, in the mouse liver. RSINE1, an abundant murine-specific SINE, is the only transposon family enriched for CR binding sites across all datasets. Sequence analyses and reporter assays reveal that the circadian regulatory activity of RSINE1 stems from the presence of imperfect CR binding motifs in the ancestral RSINE1 sequence. These motifs matured into canonical motifs through point mutations after transposition. Furthermore, maturation occurred preferentially within elements inserted in the proximity of ancestral CR binding sites. RSINE1 also acquired motifs that recruit nuclear receptors known to cooperate with CRs to regulate circadian gene expression specifically in the liver. Conclusions Our results suggest that the birth of enhancers from transposons is predicated both by the sequence of the transposon and by the cis-regulatory landscape surrounding their genomic integration site.

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Gene-specific mechanisms direct glucocorticoid-receptor-driven repression of inflammatory response genes in macrophages

eLife ◽

10.7554/elife.34864 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 26

Author(s):

Maria A Sacta ◽

Bowranigan Tharmalingam ◽

Maddalena Coppo ◽

David A Rollins ◽

Dinesh K Deochand ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Binding Sites ◽

De Novo ◽

Elongation Factor ◽

Myeloid Cell ◽

Genome Wide Analysis ◽

Genome Wide ◽

Mouse Macrophages ◽

Underlying Mechanisms ◽

Temporal Events

The glucocorticoid receptor (GR) potently represses macrophage-elicited inflammation, however, the underlying mechanisms remain obscure. Our genome-wide analysis in mouse macrophages reveals that pro-inflammatory paused genes, activated via global negative elongation factor (NELF) dissociation and RNA Polymerase (Pol)2 release from early elongation arrest, and non-paused genes, induced by de novo Pol2 recruitment, are equally susceptible to acute glucocorticoid repression. Moreover, in both cases the dominant mechanism involves rapid GR tethering to p65 at NF-kB-binding sites. Yet, specifically at paused genes, GR activation triggers widespread promoter accumulation of NELF, with myeloid cell-specific NELF deletion conferring glucocorticoid resistance. Conversely, at non-paused genes, GR attenuates the recruitment of p300 and histone acetylation, leading to a failure to assemble BRD4 and Mediator at promoters and enhancers, ultimately blocking Pol2 initiation. Thus, GR displays no preference for a specific pro-inflammatory gene class; however, it effects repression by targeting distinct temporal events and components of transcriptional machinery.

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The Integrity of piRNA Clusters is Abolished by Insulators in the Drosophila Germline

Genes ◽

10.3390/genes10030209 ◽

2019 ◽

Vol 10 (3) ◽

pp. 209 ◽

Cited By ~ 1

Author(s):

Elizaveta Radion ◽

Olesya Sokolova ◽

Sergei Ryazansky ◽

Pavel Komarov ◽

Yuri Abramov ◽

...

Keyword(s):

Binding Sites ◽

Regulatory Elements ◽

Evolutionary Constraints ◽

Heterochromatin Protein 1 ◽

Heterochromatin Protein ◽

Genome Wide Analysis ◽

Genome Wide ◽

Pirna Cluster ◽

A Genome ◽

Cluster Activity

Piwi-interacting RNAs (piRNAs) control transposable element (TE) activity in the germline. piRNAs are produced from single-stranded precursors transcribed from distinct genomic loci, enriched by TE fragments and termed piRNA clusters. The specific chromatin organization and transcriptional regulation of Drosophila germline-specific piRNA clusters ensure transcription and processing of piRNA precursors. TEs harbour various regulatory elements that could affect piRNA cluster integrity. One of such elements is the suppressor-of-hairy-wing (Su(Hw))-mediated insulator, which is harboured in the retrotransposon gypsy. To understand how insulators contribute to piRNA cluster activity, we studied the effects of transgenes containing gypsy insulators on local organization of endogenous piRNA clusters. We show that transgene insertions interfere with piRNA precursor transcription, small RNA production and the formation of piRNA cluster-specific chromatin, a hallmark of which is Rhino, the germline homolog of the heterochromatin protein 1 (HP1). The mutations of Su(Hw) restored the integrity of piRNA clusters in transgenic strains. Surprisingly, Su(Hw) depletion enhanced the production of piRNAs by the domesticated telomeric retrotransposon TART, indicating that Su(Hw)-dependent elements protect TART transcripts from piRNA processing machinery in telomeres. A genome-wide analysis revealed that Su(Hw)-binding sites are depleted in endogenous germline piRNA clusters, suggesting that their functional integrity is under strict evolutionary constraints.

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Analysis of the human CD10/neutral endopeptidase 24.11 promoter region: two separate regulatory elements

Blood ◽

10.1182/blood.v85.11.3199.bloodjournal85113199 ◽

1995 ◽

Vol 85 (11) ◽

pp. 3199-3207 ◽

Cited By ~ 34

Author(s):

F Ishimaru ◽

MA Shipp

Keyword(s):

Binding Sites ◽

Lymphoblastic Leukemia ◽

Regulatory Region ◽

Neutral Endopeptidase ◽

Regulatory Elements ◽

Control Element ◽

Regulatory Regions ◽

Endopeptidase 24.11

The cell surface zinc metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) is expressed on normal and malignant lymphoid progenitors, granulocytes, and a variety of epithelial cells. To further define the tissue-specific and developmentally related expression of CD10/NEP, we have characterized two separate regulatory regions that control the transcription of 5′ alternatively spliced CD10/NEP transcripts. These type 1 and 2 CD10/NEP regulatory regions are both characterized by the presence of multiple transcription initiation sites and the absence of classic TATA boxes and consensus initiator elements. The purine-rich type 1 regulatory region, which includes 5′ UTR exon 1 sequence, is characterized by multiple putative PU.1 binding sites and consensus ets-binding motifs. In marked contrast, the GC-rich type 2 regulatory region contains multiple putative Sp1 binding sites, a potential consensus retinoblastoma control element (RCE), and an inverted CCAAT box. In the majority of tissues examined to date, type 2 CD10/NEP transcripts were more abundant; the abundance of type 1 transcripts was more variable, with the highest type 1 levels in fetal thymus and certain lymphoblastic leukemia cell lines.

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Pediatric Precursor-B ALL with BTG1 Deletions Show a Distinct Genomic Profile.

Blood ◽

10.1182/blood.v114.22.3244.3244 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3244-3244

Author(s):

Esmee Waanders ◽

Frank N. van Leeuwen ◽

Eugene Verwiel ◽

Simon V van Reijmersdal ◽

Marloes R Levers ◽

...

Keyword(s):

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

B Cell Differentiation ◽

Chi Square ◽

Genome Wide ◽

Intron 1 ◽

Number Variation ◽

Unselected Cohort

Abstract Abstract 3244 Poster Board III-181 Recent genome-wide profiling studies have revealed that childhood acute lymphoblastic leukemia (ALL) is characterized by recurrent microdeletions, including the cell cycle regulator CDKN2A, the B-cell differentiation genes PAX5, EBF1 and IKZF1 (Ikaros) and the anti-proliferative gene B-cell translocation gene 1 (BTG1). In a previous study, we have shown that BTG1 is an important determinant of glucocorticoid sensitivity (Van Galen et al. Blood/ ASH Annual Meeting Abstracts, 2008). In the present study we have characterized these cases in more detail and elucidated the frequency of recurrent lesions in BTG1 deletion cases. Using locus-specific MLPA screening of an unselected cohort of 305 precursor B-ALL cases, we identified 26 microdeletions (8.5%). All deletions encompassed BTG1 only. We were able to genomically profile 22 diagnosis samples using Affimetrix SNP6.0 arrays. Of these, 12 did not develop a relapse during a minimal of 4,5 years of follow up. The mean number of CNVs was 29.6 of which 10.3 gains and 22.5 losses (median size 512 kb and 115 kb respectively). BTG1 deletions were generally focal, varying in size from 104 kb to 1,4 Mb. In all but one patient the breakpoints at the 5' end of the deletion tightly clustered and subsequent fine-mapping using qPCR revealed that this breakpoint cluster was located within intron 1 of the BTG1 gene. At the 3'end of the deletion, four breakpoint clusters could be identified. Analysis of the copy number variation (CNV) profiles showed that patients with a BTG1 deletion more often harbored a deletion in IKZF1 compared to an unselected cohort of pre-B ALL cases (27% vs 7%, chi-square p=0.042). In contrast, recurrent CNVs like PAX5, EBF1 and CDKN2A/B occur in similar frequencies (23%, 9% and 32% vs 17%, 0% and 23% respectively). In addition, the BTG1 deletion cases that developed into a relapse showed significantly more often a deletion in CDKN2A/B compared to the BTG1 deletion cases that did not develop a relapse (60% vs 8%, p=0.02). Together, these data indicate that pediatric precursor-B ALL carrying BTG1 deletions have distinct genomic profiles, showing increased frequencies of deletions in IKZF1 and CDKN2A. Disclosures No relevant conflicts of interest to declare.

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