Analysis of heat shock-, sodium arsenite- and proteasome inhibitor-induced heat shock protein gene expression in Xenopus laevis

Previous studies have focused on the effect of individual stressors on hsp gene expression in eukaryotic organisms. In the present study, I examined the effect of concurrent low doses of sodium arsenite and mild heat shock temperatures on the expression of hsp30 and hsp70 genes in Xenopus laevis A6 kidney epithelial cells. Northern hybridization and western blot analysis revealed that exposure of A6 cells to 1-10 μM sodium arsenite at a mild heat shock temperature of 30˚C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 μM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26˚C with larger responses at 28 and 30 ˚C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 μM ) and heat shock (30 ˚C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 ˚C. Acquired thermotolerance was not observed with cells treated with the two mild stresses individually. It is likely that the enhanced accumulation of HSPs under these conditions permits the organism to cope with multiple environmental stresses encountered in their natural aquatic habitat. Previous studies have shown that inhibiting the activity of the proteasome also leads to the accumulation of damaged or unfolded proteins within the cell. In the second phase of this study, I report that inhibition of proteasome activity by the inhibitors carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and lactacystin induced the accumulation of HSP30 and HSP70 as well as their respective mRNAs. The accumulation of HSP30 and HSP70 in A6 cells recovering from MG132 exposure was still relatively high 24 h after treatment and it decreased substantially after 48 h. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. HSP30 localization in A6 cells was primarily in the cytoplasm as revealed by immunocytochemistry. In some A6 cells treated with higher concentrations of MG132 and lactacystin, HSP30 was also found to localize in relatively large cytoplasmic foci. In some MG132-treated cells, HSP30 staining was substantially depleted in the cytoplasmic regions surrounding these foci. The activation of HSF1 may be involved in MG132-induced hsp gene expression in A6 cells since KNK437 inhibited the accumulation of HSP30 and HSP70. Lastly, MG132 treatment also conferred a state of thermotolerance in A6 cells such that they were able to survive a subsequent thermal challenge. Analysis of this phenomenon is important given the fact that impaired proteasomal activity has been suggested as an explanation for some of the late-onset neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease

The MMS22L-TONSL Complex Mediates Recovery from Replication Stress and Homologous Recombination

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks

Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism

The genetics of the mood disorder spectrum:genome-wide association analyses of over 185,000 cases and 439,000 controls

Background Mood disorders (including major depressive disorder and bipolar disorder) affect 10-20% of the population. They range from brief, mild episodes to severe, incapacitating conditions that markedly impact lives. Despite their diagnostic distinction, multiple approaches have shown considerable sharing of risk factors across the mood disorders. Methods To clarify their shared molecular genetic basis, and to highlight disorder-specific associations, we meta-analysed data from the latest Psychiatric Genomics Consortium (PGC) genome-wide association studies of major depression (including data from 23andMe) and bipolar disorder, and an additional major depressive disorder cohort from UK Biobank (total: 185,285 cases, 439,741 controls; non-overlapping N = 609,424). Results Seventy-three loci reached genome-wide significance in the meta-analysis, including 15 that are novel for mood disorders. More genome-wide significant loci from the PGC analysis of major depression than bipolar disorder reached genome-wide significance. Genetic correlations revealed that type 2 bipolar disorder correlates strongly with recurrent and single episode major depressive disorder. Systems biology analyses highlight both similarities and differences between the mood disorders, particularly in the mouse brain cell-types implicated by the expression patterns of associated genes. The mood disorders also differ in their genetic correlation with educational attainment – positive in bipolar disorder but negative in major depressive disorder. Conclusions The mood disorders share several genetic associations, and can be combined effectively to increase variant discovery. However, we demonstrate several differences between these disorders. Analysing subtypes of major depressive disorder and bipolar disorder provides evidence for a genetic mood disorders spectrum

Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network

10.1038/s41467-021-23143-7Nature Communications121329