Proteasome inhibition alters mitotic progression through the upregulation of centromeric α-Satellite RNAs.

Cell cycle progression requires control of the abundance of several proteins and RNAs over space and time to properly transit from one phase to the next and to ensure faithful genomic inheritance in daughter cells. The proteasome, the main protein degradation system of the cell, facilitates the establishment of a proteome specific to each phase of the cell cycle. Its activity also strongly influences transcription. Here, we detected the upregulation of repetitive RNAs upon proteasome inhibition in human cancer cells using RNA-seq. The effect of proteasome inhibition on centromeres was remarkable, especially on α-Satellite RNAs. We showed that α-Satellite RNAs fluctuate along the cell cycle and interact with members of the cohesin ring, suggesting that these transcripts may take part in the regulation of mitotic progression. Next, we forced exogenous overexpression and used gapmer oligonucleotide targeting to demonstrate that α-Sat RNAs have regulatory roles in mitosis. Finally, we explored the transcriptional regulation of α-Satellite DNA. Through in silico analyses, we detected the presence of CCAAT transcription factor-binding motifs within α-Satellite centromeric arrays. Using high-resolution three-dimensional immuno-FISH and ChIP-qPCR, we showed an association between the α-Satellite upregulation and the recruitment of the transcription factor NFY-A to the centromere upon MG132-induced proteasome inhibition. Together, our results show that the proteasome controls α-Satellite RNAs associated with the regulation of mitosis.

Large-scale topological disruption of chromosome territories 9 and 22 is associated with nonresponse to treatment in CML.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm defined by the presence of t(9;22) translocation whose origin has been associated with the tridimensional genome organization. This rearrangement leads to the fusion of BCR and ABL1 genes giving rise to a chimeric protein with constitutive kinase activity. Imatinib, a tyrosine kinase inhibitor (TKI), is used as a first-line treatment for CML, though ~40% of CML patients do not respond. Here, using structured illumination microscopy (SIM) and 3D reconstruction, we studied the 3D organization patterns of the ABL1 and BCR genes, and their chromosome territories (CTs) CT9 and CT22, in CD34+ cells from CML patients that responded or not to TKI. We found that TKI resistance in CML is associated with high levels of structural disruption of CT9 and CT22 in CD34+ cells, increased CT volumes (especially for CT22), intermingling between CT9 and CT22, and an open-chromatin epigenetic mark in CT22. Altogether our results suggest that large-scale disruption of CT9 and CT22 correlates with the clinical response of CML patients, which could be translated into a potential prognostic marker of response to treatment in this disease and provide novel insights into the mechanisms underlying resistance to TKI in CML.

Genotoxicity of Marijuana in Mono-Users.

Marijuana (Cannabis sp.) is among the most recurred controlled substances in the world, and there is a growing tendency to legalize its possession and use; however, the genotoxic effects of marijuana remain under debate. A clear definition of marijuana's genotoxic effects remains obscure by the simultaneous consumption of tobacco and other recreational substances. In order to assess the genotoxic effects of marijuana and to prevent the bias caused by the use of substances other than cannabis, we recruited marijuana users that were sub-divided into three categories: (1) users of marijuana-only (M), (2) users of marijuana and tobacco (M+T), and (3) users of marijuana plus other recreative substances or illicit drugs (M+O), all the groups were compared against a non-user control group. We quantified DNA damage by detection of γH2AX levels and quantification of micronuclei (MN), one of the best-established methods for measuring chromosomal DNA damage. We found increased levels of γH2AX in peripheral blood lymphocytes from the M and M+T groups, and increased levels of MNs in cultures from M+T group. Our results suggest a DNA damage increment for M and M+T groups but the extent of chromosomal damage (revealed here by the presence of MNs and NBuds) might be related to the compounds found in tobacco. We also observed an elevated nuclear division index in all marijuana users in comparison to the control group suggesting a cytostatic dysregulation caused by cannabis use. Our study is the first in Mexico to assess the genotoxicity of marijuana in mono-users and in combination with other illicit drugs.