RESUMO
Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared with non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism used by a transcription factor to boost gene expression.
Assuntos
Núcleo Celular/genética , Regulação da Expressão Gênica/genética , Proteínas Nucleares/genética , RNA/genética , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética , DNA/genética , Células HEK293 , Humanos , Corpos de Inclusão Intranuclear/genética , Ligação Proteica/genética , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
TSA-seq mapping suggests that gene distance to nuclear speckles is more deterministic and predictive of gene expression levels than gene radial positioning. Gene expression correlates inversely with distance to nuclear speckles, with chromosome regions of unusually high expression located at the apex of chromosome loops protruding from the nuclear periphery into the interior. Genomic distances to the nearest lamina-associated domain are larger for loop apexes mapping closest to nuclear speckles, suggesting the possibility of conservation of speckle-associated regions. To facilitate comparison of genome organization by TSA-seq, we reduced required cell numbers 10- to 20-fold for TSA-seq by deliberately saturating protein-labeling while preserving distance mapping by the still unsaturated DNA-labeling. Only â¼10% of the genome shows statistically significant shifts in relative nuclear speckle distances in pair-wise comparisons between human cell lines (H1, HFF, HCT116, K562); however, these moderate shifts in nuclear speckle distances tightly correlate with changes in cell type-specific gene expression. Similarly, half of heat shock-induced gene loci already preposition very close to nuclear speckles, with the remaining positioned near or at intermediate distance (HSPH1) to nuclear speckles but shifting even closer with transcriptional induction. Speckle association together with chromatin decondensation correlates with expression amplification upon HSPH1 activation. Our results demonstrate a largely "hardwired" genome organization with specific genes moving small mean distances relative to speckles during cell differentiation or a physiological transition, suggesting an important role of nuclear speckles in gene expression regulation.
RESUMO
The human genome encodes thousands of long noncoding RNA (lncRNA) genes; the function of majority of them is poorly understood. Aberrant expression of a significant number of lncRNAs is observed in various diseases, including cancer. To gain insights into the role of lncRNAs in breast cancer progression, we performed genome-wide transcriptome analyses in an isogenic, triple negative breast cancer (TNBC/basal-like) progression cell lines using a 3D cell culture model. We identified significantly altered expression of 1853 lncRNAs, including ~500 natural antisense transcript (NATs) lncRNAs. A significant number of breast cancer-deregulated NATs displayed co-regulated expression with oncogenic and tumor suppressor protein-coding genes in cis. Further studies on one such NAT, PDCD4-AS1 lncRNA reveal that it positively regulates the expression and activity of the tumor suppressor PDCD4 in mammary epithelial cells. Both PDCD4-AS1 and PDCD4 show reduced expression in TNBC cell lines and in patients, and depletion of PDCD4-AS1 compromised the cellular levels and activity of PDCD4. Further, tumorigenic properties of PDCD4-AS1-depleted TNBC cells were rescued by exogenous expression of PDCD4, implying that PDCD4-AS1 acts upstream of PDCD4. Mechanistically, PDCD4-AS1 stabilizes PDCD4 RNA by forming RNA duplex and controls the interaction between PDCD4 RNA and RNA decay promoting factors such as HuR. Our studies demonstrate crucial roles played by NAT lncRNAs in regulating post-transcriptional gene expression of key oncogenic or tumor suppressor genes, thereby contributing to TNBC progression.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Estabilidade de RNA , RNA Antissenso/genética , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Proteínas de Ligação a RNA/genética , Neoplasias de Mama Triplo Negativas/genética , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Ligação Proteica , RNA Antissenso/metabolismo , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Adenosine deaminases acting on RNA (ADARs) catalyze the editing of adenosine residues to inosine (A-to-I) within RNA sequences, mostly in the introns and UTRs (un-translated regions). The significance of editing within non-coding regions of RNA is poorly understood. Here, we demonstrate that association of ADAR2 with RNA stabilizes a subset of transcripts. ADAR2 interacts with and edits the 3ÎUTR of nuclear-retained Cat2 transcribed nuclear RNA (Ctn RNA). In absence of ADAR2, the abundance and half-life of Ctn RNA are significantly reduced. Furthermore, ADAR2-mediated stabilization of Ctn RNA occurred in an editing-independent manner. Unedited Ctn RNA shows enhanced interaction with the RNA-binding proteins HuR and PARN [Poly(A) specific ribonuclease deadenylase]. HuR and PARN destabilize Ctn RNA in absence of ADAR2, indicating that ADAR2 stabilizes Ctn RNA by antagonizing its degradation by PARN and HuR. Transcriptomic analysis identified other RNAs that are regulated by a similar mechanism. In summary, we identify a regulatory mechanism whereby ADAR2 enhances target RNA stability by limiting the interaction of RNA-destabilizing proteins with their cognate substrates.
Assuntos
Adenosina Desaminase/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Exorribonucleases/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Transportador 2 de Aminoácidos Catiônicos/genética , Transportador 2 de Aminoácidos Catiônicos/metabolismo , Camundongos , Edição de RNA , RNA Longo não Codificante/metabolismoRESUMO
Breast cancer (BC) is a highly heterogeneous disease, both at the pathological and molecular level, and several chromatin-associated proteins play crucial roles in BC initiation and progression. Here, we demonstrate the role of PSIP1 (PC4 and SF2 interacting protein)/p75 (LEDGF) in BC progression. PSIP1/p75, previously identified as a chromatin-adaptor protein, is found to be upregulated in basal-like/triple negative breast cancer (TNBC) patient samples and cell lines. Immunohistochemistry in tissue arrays showed elevated levels of PSIP1 in metastatic invasive ductal carcinoma. Survival data analyses revealed that the levels of PSIP1 showed a negative association with TNBC patient survival. Depletion of PSIP1/p75 significantly reduced the tumorigenicity and metastatic properties of TNBC cell lines while its over-expression promoted tumorigenicity. Further, gene expression studies revealed that PSIP1 regulates the expression of genes controlling cell-cycle progression, cell migration and invasion. Finally, by interacting with RNA polymerase II, PSIP1/p75 facilitates the association of RNA pol II to the promoter of cell cycle genes and thereby regulates their transcription. Our findings demonstrate an important role of PSIP1/p75 in TNBC tumorigenicity by promoting the expression of genes that control the cell cycle and tumor metastasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ciclo Celular/genética , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromatina/genética , Cromatina/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Oncogenes , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Análise Serial de Tecidos , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Models of nuclear genome organization often propose a binary division into active versus inactive compartments, yet they overlook nuclear bodies. Here we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Whereas gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.
RESUMO
A significant number of the genetic alterations observed in cancer patients lie within nonprotein-coding segments of the genome, including regions coding for long noncoding RNAs (lncRNAs). LncRNAs display aberrant expression in breast cancer (BrCa), but the functional implications of this altered expression remain to be elucidated. By performing transcriptome screen in a triple negative BrCa (TNBC) isogenic 2D and 3D spheroid model, we observed aberrant expression of >1000 lncRNAs during BrCa progression. The chromatin-associated lncRNA MANCR shows elevated expression in metastatic TNBC. MANCR is upregulated in response to cellular stress and modulates DNA repair and cell proliferation. MANCR promotes metastasis as MANCR-depleted cells show reduced cell migration, invasion, and wound healing in vitro, and reduced metastatic lung colonization in xenograft experiments in vivo. Transcriptome analyses reveal that MANCR modulates expression and pre-mRNA splicing of genes, controlling DNA repair and checkpoint response. MANCR promotes the transcription of NET1A, a Rho-GEF that regulates DNA damage checkpoint and metastatic processes in cis, by differential promoter usage. Experiments suggest that MANCR regulates the expression of cancer-associated genes by modulating the association of various transcription factors and RNA-binding proteins. Our results identified the metastasis-promoting activities of MANCR in TNBC by cis-regulation of gene expression.
RESUMO
Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated TSA-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in hESCs. Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology.
RESUMO
Adenosine deaminases acting on RNA (ADARs) are proteins that catalyse widespread A-to-I editing within RNA sequences. We recently reported that ADAR2 edits and stabilizes nuclear-retained Cat2 transcribed nuclear RNA (Ctn RNA). Here, we report that ADAR1 coordinates with ADAR2 to regulate editing and stability of Ctn RNA. We observe an RNA-dependent interaction between ADAR1 and ADAR2. Furthermore, ADAR1 negatively regulates interaction of Ctn RNA with RNA-destabilizing proteins. We also show that breast cancer (BC) cells display elevated ADAR1 but not ADAR2 levels, compared to nontumourigenic cells. Additionally, BC patients with elevated levels of ADAR1 show low survival. Our findings provide insights into overlapping substrate preferences of ADARs and potential involvement of ADAR1 in BC.
Assuntos
Adenosina Desaminase/metabolismo , Edição de RNA/genética , Estabilidade de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Regiões 3' não Traduzidas/genética , Adenosina Desaminase/genética , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Imunoprecipitação , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genéticaRESUMO
BACKGROUND: Expression of miR-122 is highly specific to hepatocytes of the liver. This miRNA is involved in lipid hemostasis of the tissue; however, there is no comprehensive understanding of its function in lipid hemostasis. MATERIALS AND METHODS: Since hepatocytes are responsible for part of Triacylglycerol (TAG) synthesis in the body, we hypothesized that miR-122, as the most abundant miRNA in the tissue, might regulate TAG metabolism by targeting key enzymes that are involved in its production pathway. A systematic computational analysis of putative targets of miR-122 identified CTDNEP1 and LPIN1 genes in the TAG pathway. We used dual-luciferase reporter assay, quantitative RT-PCR as well as western blot to confirm the repressive effect of miR-122 on CTDNEP1 and LPIN1 in TAG pathway. RESULTS: Real time PCR on liver needle biopsies with hepatosteatosis showed that miR-122 is up-regulated in hepatosteatosis. Surprisingly, the protein and RNA level of identified targets of miR-122 are also up-regulated in clinical samples, probably as a disproportionate feedback response to the high level of miR-122. CONCLUSION: Our findings suggest that up-regulation of miR-122 can trigger the compensatory response of LPIN1 and CTDNEP1 in hepatosteatosis.