Fig 2: Activation of HOTTIP Rescues the HOXA Gene Chromatin Defects in the CBS7/9+/− AML Cells(A) qRT-PCR analysis of HOXA gene expression in WT, CBS7/9+/−, and the dCas9-VP-160-mediated HOTTIP-activated MOLM13 clones.(B) Proliferation curves of the WT, CBS7/9+/−, and the dCas9-VP-160-mediated HOTTIP-activated MOLM13 cells.(C) Cell-cycle analysis of the WT, CBS7/9+/−, and dCas9-VP-160-mediated HOTTIP-activated MOLM13 clones.(D) Heatmap of RNA-seq analysis shows up-and downregulated genes of WT and the dCas9-VP-160-mediated HOTTIP-activated clones compared with the CBS7/9+/− clone.(E) qRT-PCR validation of the key altered genes identified by RNA-seq comparing WT, CBS7/9+/−, and the dCas9-VP-160-mediated HOTTIP-activated clones.(F) Enrichment of upregulated genes involved in HOXA9 (top) and AML (bottom) pathways upon HOTTIP activation in the CBS7/9+/− MOLM13 by GSEA.(G) ATAC-seq analysis of chromatin accessibility in WT, CBS7/9+/−, and the dCas9-VP-160-mediated HOTTIP-activated MOLM13 cells.(H) ChIP-seq analysis of MLL1 recruitment in WT, CBS7/9+/−, and the dCas9-VP-160-mediated HOTTIP-activated MOLM13 cells.(I) Hi-C interacting maps in part of the human chromosome 7p15 region containing the HOXA locus comparing WT, CBS7/9+/−, and the dCas9-VP-160-mediated HOTTIP-activated MOLM13 cells.(J) Kaplan-Meier curve of NSG mice transplanted with WT, CBS7/9+/−, or the dCas9-VP-160-mediated HOTTIP-activated MOLM13 cells.(K) FACS analysis of hCD45+ cell chimerism in BM, spleen (SP), and PB of NSG mice 14 days after transplantation of WT (n = 4), CBS7/9+/− (n = 4), or the dCas9-VP-160-mediated HOTTIP-activated MOLM13 cells (n = 4).Data in (A), (B), (C), (E), and (K) are presented as mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant by Student’s t-test. See also Figure S5.

Fig 3: HOXBLINC is activated in NPM1c+ AML patients and loss of HOXBLINC perturbs NPM1c+-mediated transcription program and leukemogenesis.a RT-qPCR analysis of HOXBLINC RNA expression in BMMNCs (n = 16) and/or CD34+ cells (n = 11) from healthy individuals and in BMMNCs from NPM1wt (n = 40) or NPM1c+ (n = 25) AML patients (Data is presented as Median ± interquartile; P value was calculated by two-sided Mann Whitney nonparametric test). b TCGA database (GSE62944) was used to retrieve RNA expression levels of HOXBLINC in BM cells from AML patients without NPM1 mutations and MLL rearrangements (NPM1c−/MLLr−, n = 128), AML patients with MLL rearrangements (MLLr+, n = 11), or AML patients with NPM1 mutations (NPM1c+, n = 42). Violin plots show mean, interquartile, and 1.5x interquartile. The width shows the probability density. P was calculated by two-tailed t-test. c Volcano plot of RNA-seq analysis of differentially expressed genes in Npm1c+ knock-in (Npm1c/+) vs. WT LSK cells based on two independent experiments. d Heat map of RNA-seq analysis shows the up- and down-regulated genes in HOXBLINC-KRAB vs. WT OCI-AML3 cells based on two independent experiments. Arrows: dysregulated genes implicated in leukemogenesis. e The HOXBLINC-KRAB affected genes in OCI-AML3 cells were analyzed and annotated by the Gene Ontology (GO) analysis. f Enrichment of HOXBLINC-KRAB dysregulated genes in NPM1-mutated (top) and HOXA9 (bottom) oncogenic pathways by Gene Set Enrichment Analysis (GSEA). g Proliferation curves of WT, shScramble-expressing, and two different shHOXBLINC-expressing OCI-AML3 cells upon doxycycline (Dox) treatment for 5 days. (Bars represent the mean ± SD of total viable cells from three independent experiments; two-tailed t-test; **P < 0.01; n = 3 independent experiments). h Kaplan-Meier survival curve of inducible shHOXBLINC OCI-AML3 cell transplanted mice treated with or without Dox (Log-rank test; n = 5 mice/group). i Kaplan-Meier survival curves of mice transplanted with control or HOXBLINC-KRAB (HOXBLINCi) BM cells from 2 AML patients with NPM1 mutation. 1315# patient (NPM1c+;FLT3wt, left), 921# patient (NPM1c+;FLT3mu, right). n = 4 mice/group. The Log-rank test was used to analyze differences between the survival curves. j Representative images of H&E (right) and anti-hCD45 immunostaining (left) of femur sections from 4 mice transplanted with control or HOXBLINCi BM cells from 1315# patients for 18 days (scale = 20 μm).

Fig 4: HOTTIP Reprograms AML Chromatin and Regulates Leukemic Specific Transcription Networks(A) ChIRP-seq analysis of HOTTIP binding in WT and HOTTIP KO MOLM13 cells.(B) Pie chart shows global HOTTIP binding distribution in the human AML genome.(C) GO analysis of HOTTIP regulated transcription and signal pathways.(D) Changes in HOTTIP binding (ChIRP-seq), chromatin accessibility (ATAC-seq), and MLL1 recruitment and H3K4me3 enrichment (ChIP-seq) upon HOTTIP KO.(E) Shared top TF binding motifs enriched by the HOTTIP ChIRP-seq (top) and the ATAC-seq altered peaks (bottom) upon HOTTIP−/−by the de novo motif analysis.(F) HOTTIP and protein interactions detected by RIP.(G) ChIP-seq analysis of changes in H3K4me3, H3K27me3, and H3K79me2 modification levels upon HOTTIP−/− in MOLM13 cells.(H) Altered ATAC-seq accessibility of genomic regions upon HOTTIP−/−. Box plots show horizontal line (zero Z score, mean), box indicating the median with upper and lower quartiles, and whiskers indicating the highest and lowest values. p value is calculated by Kolmogorov-Smirnov test.(I) ATAC-seq analysis of altered chromatin accessibility upon HOTTIP−/− in MOLM13 cells.See also Figure S3; Tables S1 and S2.

Fig 5: Taspase1 is a co-activator of estrogen-driven transcription. (A) Downregulation of Taspase1 affects the transcriptional estrogen response. Western Blot analysis of whole cell lysates of untransfected 293T cells or 293T cells 24 h and 48 h after transfection with Taspase1 siRNA or control siRNA unravels a partial Taspase1 knock-out coupled with an attenuated expression of estrogene-responsive genes. (B) Model of Taspase1-influenced estrogen-driven transcription: In an uninduced state, the co-repressor complex (Nucleolin, NPM1, PARP1 and Topo IIß) is bound to promoter regions of estrogen target genes, thereby preventing transcriptional initiation. In addition, Taspase1 is associated with this complex via Nucleolin and NPM1. Estrogen-stimulation leads to a spatial rearrangement of the co-repressor complex and enables Taspase1 to bind and successively promote Topo IIß to induce transient DNA double strand breaks (DSBs), labeled by ?H2AX which is presumably associated with the complex via DNA damage response (DDR) proteins. Topological barriers such as DNA knots or supercoiling are solved, allowing unhindered access for the transcription machinery. Moreover, Taspase1 cleaves different substrates, e.g., TFIIA and USF2 to adapt their function to changing physiological conditions. Furthermore, Taspase1 enables H3K4me3 epigenetic labelling via cleavage-triggered MLL activation in an estrogen-dependent manner. This epigenetic signature may thus allow to maintain distinct hormone-activated transcriptional programs. This illustration was created with BioRender.com.