PS1168 CHROMATIN TOPOLOGY AND THREE‐DIMENSIONAL ORGANIZATION OF CHROMOSOME TERRITORIES 9 AND 22 IN CD34+ BONE MARROW CELLS FROM CHRONIC MYELOID LEUKEMIA PATIENTS
Authors: Fabian‐Morales E. , Torres Y.L. Rodriguez , Escamilla D.G. Vallejo , Gonzalez‐Barrios R. , De La Torre Lujan A. , Lopez‐Saavedra A. , Hernandez C. Castro , Montalvo L.A. Herrera 
Published: June 2019

Background:

During interphase, chromosomes occupy a limited space within the nucleus, referred to as chromosome territory (CT). The non‐random CT's spatial organization in the nucleus contributes to determining the outcome of chromosomal translocations (t). Comparative analysis of the spatial arrangement of t‐partners and their frequency suggest that this chromosome aberration occurs preferentially among proximally positioned genome regions. The consistence appearance of t(9;22) in hematopoietic stem cells, apart from being etiological factor for chronic myeloid leukemia (CML), is a clear example of what is considered a non‐random chromosome aberration. Besides, the CT's spatial distribution is regulated by epigenetic factors such as post‐translational histone modifications. These modifications are also directly related to the chromatin topology in these CTs, which is relevant since a chromatin misconfiguration has been related to the susceptibility to generate mutations and translocations.

Aims:

‐ Determine the 3D arrangement of interphase CTs 9 and 22 and BCR‐ABL genes in CD34+ bone marrow (BM) cells from CML patients at the time of diagnosis (CML RxDx) and patients with complete response (CML CR) compared to healthy donors’ CD34+ mobilized bone marrow cells (MBM) and peripheral blood mononuclear cells (PBMC).

‐ Map and quantify the distribution of the histone modifications H3K27me3 and H3K9ac across CTs 9 and 22 in our study population.

Methods:

The study population consisted of 5 BM from CML RxDx and CML CR patients, 5 MBM and PBMC from healthy donors. CD34+ hematopoietic cells were isolated and subsequently processed for 3D‐FISH and 3D immuno‐FISH. Super‐resolution imaging was performed by 3D structured illumination microscopy (SIM) and images reconstructed and analysed using Imaris program (Bitplane).

Results:

We found that nuclear volumes were significantly larger in CD34 + cells from MBM and CML patients compared to PBMC controls. The absolute CTs volume analysis shows a major volume of CT9 compared to CT22. To determine the distance between BA genes and their CTs we calculated the nearest distance between the mass center of CT9 and CT22. The shorter distance was between de CML RxDx cells since the translocation presence. When we compare the samples without t(9;22), the shorter distances were observed between BA from MBM and CML CR cells compared to PBMC samples. After normalizing the distance between the CTs and BA genes against the radius of the nucleus. We observed that the location of CT22 tended towards the center, whereas that of CT9 towards the nuclear periphery. Meanwhile BA genes were located closer to the nuclear center than the respective CTs’ geometrical center. We have mapped relevant histone modifications like H3K9ac and H3K27me3 across CT9 and 22 using 3D immuno‐FISH. The enrichment of H3K27me3 seems to be significantly decreased in CT22 from samples of CML CR cells.

Summary/Conclusion:

The consistent appearance of specific chromosomal translocation in leukemias particularly t(9;22) in CML patients has suggested that the positioning and chromatin topology of CTs might play a role in the occurrence of particular chromosomal rearrangements associated with malignant transformation. Using 3D‐FISH and 3D‐SIM we have determined the positions, distances and volumes of CT9 and CT22 and in BCR‐ABL1 genes, in interphase nuclei of CD34+ bone marrow (BM) cells from CML RxDx and in CR patients compared to healthy donors’ CD34+ MBM and PBMC. Likewise, we mapped relevant histone modifications finding a particular difference in TC22 and H3K27me3 histone mark from CML CR CD34+ cells.

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