Dnmt1 links BCR-ABLp210 to epigenetic tumor stem cell priming in myeloid leukemia

Research in CVD group is partially supported by FEDER, “Miguel Servet” Grant (CP14/00082 - AES 2013-2016) from the Instituto de Salud Carlos III (Ministerio de Economia y Competitividad), “Fondo de Investigaciones Sanitarias/Instituto de Salud Carlos III” (PI17/00167), and by the Lady Tata International Award for Research in Leukaemia 2016–2017. Research in ISG group is partially supported by FEDER and by MINECO (SAF2012-32810, SAF2015-64420-R and Red de Excelencia Consolider OncoBIO SAF2014-57791- REDC), Instituto de Salud Carlos III (PIE14/00066), ISCIII- Plan de Ayudas IBSAL 2015 Proyectos Integrados (IBY15/00003), by Junta de Castilla y Leon (BIO/SA51/15, CSI001U14, UIC-017, and CSI001U16), and by the German Carreras Foundation (DJCLS R13/26). ISG lab is a member of the EuroSyStem and the DECIDE Network funded by the European Union under the FP7 program. IGR was supported by BES-Ministerio de Economia y Competitividad (BES2013-063789). GRH was supported by FSE-Conserjeria de Educacion de la Junta de Castilla y Leon (CSI001-15).

Supplementary Tables   Table S1: RRBS methylation changes in HSPCs of Sca1-BCR-ABLp210 mice compared to wildtype controls. Table S2: RRBS methylation changes in myeloid cells of Sca1-BCR-ABLp210 mice compared to wild-type controls.

Generation of Sca1-BCR-ABLp210 and Sca1-Dnmt1 transgenic mouse strains
All animal work has been conducted according to relevant national and international guidelines and it has been approved by the Bioethics Committee of University of Salamanca and by the Bioethics Subcommittee of Consejo Superior de Investigaciones Cientificas (CSIC).
The Sca1-Dnmt1 vector was generated by inserting the Dnmt1 cDNA into the ClaI site of the pLy6 vector. The transgene fragment was excised from its vector by restriction digestion with NotI, purified and injected (2 ng/mL) into CBAxC57BL/6J fertilized eggs. Sca1-BCR-ABLp210 transgenic mice have been previously described 1 . Transgenic mice were identified by Southern blot analysis of tail snip DNA after EcoRI digestion, using Dnmt1 and BCR-ABLp210 cDNA respectively to detect the transgene. Upon signs of disease, mice were euthanized and subjected to standard necropsy procedures. All major organs were examined under the dissecting microscope. Tissue samples were taken from homogenous portions of the resected organ and fixed immediately after excision. Samples of each organ were processed into paraffin, sectioned and examined histologically including routinely standard haematoxylin and eosin. Differences in Kaplan-Meier survival plots of transgenic and WT mice were analyzed using the log-rank (Mantel-Cox) test.

Flow cytometry
Nucleated cells were obtained from total mouse bone marrow (flushing from the long bones), peripheral blood, thymus, or spleen. In order to prepare cells for flow cytometry, contaminating For each analysis, a total of at least 50,000 viable (PI-) cells were assessed.
The following antibodies were used for flow cytometry: anti-B220 (RA3-6B2) (1:100), CD4 (RM4- were not considered). The number of mismatches in the induced alignment was then counted between the unconverted read and reference, ignoring cases in which a T in the unconverted read is matched to a C in the unconverted reference. For a given read, the best alignment was kept if the second-best alignment had 2 more mismatches; otherwise the read was discarded as non-unique. The mean CpG coverages ranged between 5-11X and total number of unique mapped reads ranged between 1,573,821-21,361,676 for each condition (see table). The methylation level of each sampled cytosine was estimated as the number of reads reporting a C, divided by the total number of reads reporting a C or T. A bioinformatics pipeline was used to score epigenetic alterations according to strength and significance, and links them to potentially affected genes. To that end, we collected a comprehensive set of regions of interest, which includes promoters, CpG islands and repetitive elements. For each of these regions, the number of methylated and unmethylated CpG observations is determined, and a p-value is assigned using Fisher's exact test. Once all p-values are calculated, multiple-testing correction is performed separately for each region type using the q-value method, which controls the false discovery rate to be below a user-specified threshold (typically 10%). The software pipeline is implemented in Python (alignment processing module) and R (statistical analysis module). Unsupervised clustering was performed on methylation ratio data from each condition using Kendall tau rank correlation.   Illustrative examples of hematoxylin and eosin staining of sections from the spleen and liver of diseased Sca1-Dnmt1 mice shows a loss of normal architecture resulting from a myeloid tumor. Specifically, atrophic white pulp and hyperplasic red pulp is infiltrated by myeloid cells in the spleen and accompanied by the deposition of an eosinophilic hyaline substance. The liver is also infiltrated by the myeloid tumor formed by mature myeloid cells with a round to oval nucleus that may be flattened on one side and myeloblast with a larger rounder nucleus. Images are representative of 3 replicates. Scale bar represents 200 µm (=200X) and 100 µm (=400X) for inset.