From FISH to Hi-C: The Chromatin Architecture of the Chromosomal Region 7q36.3, Frequently Rearranged in Leukemic Cells, Is Evolutionary Conserved

Abstract

Fluorescence in situ hybridization (FISH) and Hi-C methods are largely used to investigate the three-dimensional organization of the genome in the cell nucleus and are applied here to study the organization of genes (LMBR1, NOM1, MNX1, UBE3C, PTPRN2) localized in the human 7q36.3 band. This region contains the MNX1 gene, which is normally not expressed in human lymphocytes beyond embryonic development. However, this homeobox gene is frequently activated in leukemic cells and its expression is associated with an altered gene positioning in the leukemia cell nuclei. In this study, we used FISH on 3D-preserved nuclei to investigate the nuclear positioning of MNX1 in the leukemia-derived cell line K562. Of the five copies of the MNX1 gene present in K562, four alleles were positioned in the nuclear periphery and only one in the nuclear interior. Using the Juicebox’s Hi-C dataset, we identified five chromatin loops in the 7q36.3 band, with different extensions related to the size and orientation of the genes located here, and independent from their expression levels. We identified similar loops in 11 human and three mouse cell lines, showing that these loops are highly conserved in different human cell lines and during evolution. Moreover, the chromatin loop organization is well conserved also during neuronal cell differentiation, showing consistency in genomic organization of this region in development. In this report, we show that FISH and Hi-C are two different approaches that complement one another and together give complete information on the nuclear organization of specific chromosomal regions in different conditions, including cellular differentiation and genetic diseases.

Engineered model of t(7;12)(q36;p13) AML recapitulates patient-specific features and gene expression profiles

“…By delivery of CRISPR/Cas9 ribonucleoprotein (RNP) complexes targeting clinically accurate breakpoints described by Tosi et al (5) and Simmons et al (4) (Figure 1A; Supplementary Table 1), we were able to achieve the t(7;12) rearrangement in the leukaemia cell line K562, as confirmed by fluorescence in situ hybridisation (FISH) (Figure 1B-C; Supplementary Figure 2A-C) and amplification of genomic fusion junctions by polymerase chain reaction (PCR) (Figure 1D; Supplementary Figure 1). While MNX1 is already expressed in K562 cells (19), the translocation further upregulated its expression (Figure 1E), consistent with ectopic MNX1 overexpression in patients (7,9,11). Altered nuclear positioning has been proposed as a significant mechanism of MNX1 overexpression in t(7;12) leukaemia, with relocalisation of the der (12) containing the translocated MNX1 gene to a more internal transcriptionally active position within the nucleus, where expression of MNX1 can be triggered (7).…”

Engineered model of t(7;12)(q36;p13) AML recapitulates patient-specific features and gene expression profiles

“…While MNX1 is already expressed in K562 cells [ 19 ], the translocation further upregulated its expression (Fig. 1E ), consistent with ectopic MNX1 overexpression in patients [ 7 , 9 , 11 ].…”

Dissecting infant leukemia developmental origins with a hemogenic gastruloid model

“…Experimental models: Cell lines Mus musculus: Flk-1-GFP mouse embryonic stem cell line Alfonso Martinez Arias Lab (Jakobsson et al, 2010) Mus musculus: Sox17-GFP mouse embryonic stem cell line Alfonso Martinez Arias Lab (Niakan et al, 2010) Mus musculus: T/Bra::GFP mouse embryonic stem cell line Alfonso Martinez Arias Lab (Fehling et al, 2003) Mus musculus: E14Tg2a mouse embryonic stem cell line MMRRC, University of California Davis, US (Hooper et al, 1987) Homo sapiens: HEK293T ATCC CRL-3216 Oligonucleotides MNX1 (forward 5-GTTCAAGCTCAACAAGTACC-3; reverse 5-GGTTCTGGAACCAAATCTTC-3) (Gulino et al, 2021) n/a Ppia (forward 5-TTACCCATCAAACCATTCCTTCTG-3; reverse 5-AACCCAAAGAACTTCAGTGAGAGC-3) (Moris, N. et al, 2018) n/a Recombinant DNA pWPT-LSSmOrange-PQR Ghevaert Lab, WT-MRC Cambridge Stem Cell Institute, UK (Dalby et al, 2018) pWPT-LSSmOrange-PQR-MNX1 Cloned by Biomatik Corporation (Kitchener, Canada) n/a Software and algorithms GSEA software v4.2.3 (Subramanian et al, 2005) https://www.gseamsigdb.org/gsea/index.jsp ImageJ (Schneider et al, 2012) https://imagej.nih.gov/ij/ Bowtie2 (Langmead and Salzberg, 2012) http://bowtiebio.sourceforge.net/bowtie 2/index.shtml Samtools (Li et al, 2009) http://samtools.sourceforg e.net/ Tophat2 (Kim, D. et al, 2013) https://ccb.jhu.edu/softwar e/tophat/index.shtml HTseq (Anders et al, 2015) https://htseq.readthedocs.i o/en/master/ DEseq2 (Love et al, 2014) https://bioconductor.org/pa ckages/release/bioc/html/ DESeq2.html ExpressAnalyst n/a https://www.expressanalys t.ca/ PANTHER (Thomas et al, 2022) http://www.pantherdb.org/ EnrichR (Chen et al, 2013;Kuleshov et al, 2016;Xie et al, 2021) https://maayanlab.cloud/E nrichr/ Panglao BP (Franzen et al, 2019) https://panglaodb.se Descartes (Cao et al, 2020)…”