Abstract
Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, we employed CRISPR to delete a short interspersed nuclear element (SINE) in Malat1, a ...cancer-associated lncRNA, to investigate its significance in cellular physiology. We show that Malat1 with a SINE deletion forms diffuse nuclear speckles and is frequently translocated to the cytoplasm. SINE-deleted cells exhibit an activated unfolded protein response and PKR and markedly increased DNA damage and apoptosis caused by dysregulation of TDP-43 localization and formation of cytotoxic inclusions. TDP-43 binds stronger to Malat1 without the SINE and is likely ‘hijacked’ by cytoplasmic Malat1 to the cytoplasm, resulting in the depletion of nuclear TDP-43 and redistribution of TDP-43 binding to repetitive element transcripts and mRNAs encoding mitotic and nuclear-cytoplasmic regulators. The SINE promotes Malat1 nuclear retention by facilitating Malat1 binding to HNRNPK, a protein that drives RNA nuclear retention, potentially through direct interactions of the SINE with KHDRBS1 and TRA2A, which bind to HNRNPK. Losing these RNA–protein interactions due to the SINE deletion likely creates more available TDP-43 binding sites on Malat1 and subsequent TDP-43 aggregation. These results highlight the significance of lncRNA TEs in TDP-43 proteostasis with potential implications in both cancer and neurodegenerative diseases.
We recently discovered that steroid receptor coactivators (SRCs) SRCs-1, 2 and 3, are abundantly expressed in cardiac fibroblasts (CFs) and their activation with the SRC small molecule stimulator ...MCB-613 improves cardiac function and dramatically lowers pro-fibrotic signaling in CFs post-myocardial infarction. These findings suggest that CF-derived SRC activation could be beneficial in the mitigation of chronic heart failure after ischemic insult. However, the cardioprotective mechanisms by which CFs contribute to cardiac pathological remodeling are unclear. Here we present studies designed to identify the molecular and cellular circuitry that governs the anti-fibrotic effects of an MCB-613 derivative, MCB-613-10-1, in CFs. We performed cytokine profiling and whole transcriptome and proteome analyses of CF-derived signals in response to MCB-613-10-1. We identified the NRF2 pathway as a direct MCB-613-10-1 therapeutic target for promoting resistance to oxidative stress in CFs. We show that MCB-613-10-1 promotes cell survival of anti-fibrotic CFs exposed to oxidative stress by suppressing apoptosis. We demonstrate that an increase in HMOX1 expression contributes to CF resistance to oxidative stress-mediated apoptosis via a mechanism involving SRC co-activation of NRF2, hence reducing inflammation and fibrosis. We provide evidence that MCB-613-10-1 acts as a protectant against oxidative stress-induced mitochondrial damage. Our data reveal that SRC stimulation of the NRF2 transcriptional network promotes resistance to oxidative stress and highlights a mechanistic approach toward addressing pathologic cardiac remodeling.
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•Steroid receptor activation by MCB-613-10-1 inhibits fibroblast activation.•Steroid receptor activation enhances antioxidant and cytoprotective signaling.•MCB-613-10-1 protects fibroblast mitochondria from oxidative stress-induced damage.
In this short review we discuss the current view of how the estrogen receptor (ER), a pivotal member of the nuclear receptor superfamily of transcription factors, regulates gene transcription at the ...single cell and allele level, focusing on in vitro cell line models. We discuss central topics and new trends in molecular biology including phenotypic heterogeneity, single cell sequencing, nuclear phase separated condensates, single cell imaging, and image analysis methods, with particular focus on the methodologies and results that have been reported in the last few years using microscopy-based techniques. These observations augment the results from biochemical assays that lead to a much more complex and dynamic view of how ER, and arguably most transcription factors, act to regulate gene transcription.
Estrogen receptor-α (ERα, ESR1) is a pivotal transcriptional regulator of breast cancer physiology and is targeted by endocrine therapies. Loss of ERα activity or expression is an indication of ...endocrine resistance and is associated with increased risk of tumor recurrence and worse prognosis. In this study, we sought to investigate whether elements of the tumor microenvironment, namely macrophages, would impact on ERα and we found that macrophage-derived factors caused loss of ERα expression in breast cancer cells. Conditioned media from macrophages caused activation of several intracellular pathways in breast cancer cells of which c-Src, protein kinase c and mitogen-activated protein kinase (MAPK) were essential for loss of ERα expression. Moreover, a prolonged hyperactivation of MAPK was observed. The activation of this kinase cascade resulted in recruitment of extracellular signal regulated kinase 2 (ERK2) directly to chromatin at the ESR1 gene locus in a process that was dependent upon activation and recruitment of the c-Jun transcription factor. Thus, we identify a novel mechanism for loss of ERα expression in breast cancer cells via macrophage activation of kinase cascades in the cancer cells causing transcriptional repression of the ESR1 gene by a direct chromatin action of a c-Jun/ERK2 complex. The findings in this study support an alternative mechanism, not intrinsic to the tumor cell but derived from the cross-talk with the tumor microenvironment, that could lead to endocrine resistance and might be targeted therapeutically to prevent loss of ERα expression in breast tumors.
Xia–Gibbs syndrome (XGS) is a rare Mendelian disease typically caused by de novo stop‐gain or frameshift mutations in the AT‐hook DNA binding motif containing 1 (AHDC1) gene. Patients usually present ...in early infancy with hypotonia and developmental delay and later exhibit intellectual disability (ID). The overall presentation is variable, however, and the emerging clinical picture is still evolving. A detailed phenotypic analysis of 34 XGS individuals revealed five core phenotypes (delayed motor milestones, speech delay, low muscle tone, ID, and hypotonia) in more than 80% of individuals and an additional 12 features that occurred more variably. Seizures and scoliosis were more frequently associated with truncations that arise before the midpoint of the protein although the occurrence of most features could not be predicted by the mutation position. Transient expression of wild type and different patient truncated AHDC1 protein forms in human cell lines revealed abnormal patterns of nuclear localization including a diffuse distribution of a short truncated form and nucleolar aggregation in mid‐protein truncated forms. Overall, both the occurrence of variable phenotypes and the different distribution of the expressed protein reflect the heterogeneity of this syndrome.
Xia‐Gibbs syndrome individuals studied thus far share 5 core phenotypes occurring in more than 80% of individuals. Seizures and scoliosis were associated with truncations occurring before the midpoint of the protein with other features occurring variably. Both phenotypic analysis and differential mutant protein localization are consistent with the heterogeneity of the disorder.
Diverse toxicants and mixtures that affect hormone responsive cells endocrine disrupting chemicals (EDCs) are highly pervasive in the environment and are directly linked to human disease. They often ...target the nuclear receptor family of transcription factors modulating their levels and activity. Many high-throughput assays have been developed to query such toxicants; however, single-cell analysis of EDC effects on endogenous receptors has been missing, in part due to the lack of quality control metrics to reproducibly measure cell-to-cell variability in responses.
We began by developing single-cell imaging and informatic workflows to query whether the single cell distribution of the estrogen
(ER), used as a model system, can be used to measure effects of EDCs in a sensitive and reproducible manner.
We used high-throughput microscopy, coupled with image analytics to measure changes in single cell ER nuclear levels on treatment with
toxicants, over a large number of biological and technical replicates.
We developed a two-tiered quality control pipeline for single cell analysis and tested it against a large set of biological replicates, and toxicants from the EPA and Agency for Toxic Substances and Disease Registry lists. We also identified a subset of potentially novel EDCs that were active only on the endogenous ER level and activity as measured by single molecule RNA fluorescence
hybridization (RNA FISH).
We demonstrated that the distribution of ER levels per cell, and the changes upon chemical challenges were remarkably stable features; and importantly, these features could be used for quality control and identification of endocrine disruptor toxicants with high sensitivity. When coupled with orthogonal assays, ER single cell distribution is a valuable resource for high-throughput screening of environmental toxicants. https://doi.org/10.1289/EHP9297.
By integrating growth pathways on which cancer cells rely, steroid receptor coactivators (SRC-1, SRC-2, and SRC-3) represent emerging targets in cancer therapeutics. High-throughput screening for SRC ...small molecule inhibitors (SMIs) uncovered MCB-613 as a potent SRC small molecule “stimulator” (SMS). We demonstrate that MCB-613 can super-stimulate SRCs’ transcriptional activity. Further investigation revealed that MCB-613 increases SRCs’ interactions with other coactivators and markedly induces ER stress coupled to the generation of reactive oxygen species (ROS). Because cancer cells overexpress SRCs and rely on them for growth, we show that we can exploit MCB-613 to selectively induce excessive stress in cancer cells. This suggests that over-stimulating the SRC oncogenic program can be an effective strategy to kill cancer cells.
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•The small molecule compound MCB-613 super-stimulates SRC transcriptional activity•Acute hyper-activation of SRCs by MCB-613 leads to aberrant cellular stress•MCB-613 selectively kills cancer cells and inhibits tumor growth in vivo•Over-activation of SRCs is a potential alternative strategy for cancer therapy
Wang et al. identify MCB-613 as a potent steroid receptor coactivator (SRC) small molecule stimulator. They show that, paradoxically, acute super-activation of SRCs specifically and effectively kills cancer cells by inducing aberrant cellular stress, suggesting an alternative strategy against cancer.
Upon stimulation by extrinsic stimuli, stem cells initiate a programme that enables differentiation or self-renewal. Disruption of the stem state exit has catastrophic consequences for embryogenesis ...and can lead to cancer. While some elements of this stem state switch are known, major regulatory mechanisms remain unclear. Here we show that this switch involves a global increase in splicing efficiency coordinated by DNA methyltransferase 3α (DNMT3A), an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and haematopoietic stem cells depends on messenger RNA processing, influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukaemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem state towards differentiation.
Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of ...hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.
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