ABSTRACT: THOC5, a member of the mRNA export complex, is essential for hematopoiesis in vivo and is required for CSF-1 induced macrophage differentiation
Project description:THOC5, a member of the mRNA export complex, is essential for hematopoiesis in vivo and is required for CSF-1 induced macrophage differentiation, thereby playing a key role in the mRNA export of immediate early genes induced by CSF-1 stimulation. Hematopoiesis, growth, differentiation, and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their down-stream transcription factor genes. Transcriptional control mechanisms of gene expression during differentiation were mainly studied by focusing on the cis- and trans-element in promoters, however, the role of mRNA export machinery during differentiation have not been adequately examined. A member of the mRNA export complex, THOC5 which is a substrate for the macrophage-colony stimulating factor (CSF-1) receptor, ATM kinase, or protein kinase C, is an essential element in the maintenance of hematopoiesis in adult mice. Using tamoxifen inducible THOC5 knockout mice, we show here that the depletion of THOC5 impaired myeloid differentiation, but does not influence terminally differentiated organs. Furthermore, an in vitro study showed that the depletion of THOC5 in bone marrow cells results in abnormal CSF-1 induced macrophage differentiation. Transcriptome analysis using cytoplasmic RNA derived from macrophages reveals that only 99 genes were down-regulated 3 days after the depletion of THOC5, however, immediate early genes induced by CSF-1 stimulation, such as Ets family genes, and regulators of myeloid differentiation HoxA1, Id1, Id3 were THOC5 direct target mRNAs, suggesting that THOC5 plays a key role in myeloid differentiation.
Project description:THOC5, a member of the mRNA export complex, is essential for hematopoiesis in vivo and is required for CSF-1 induced macrophage differentiation, thereby playing a key role in the mRNA export of immediate early genes induced by CSF-1 stimulation. Hematopoiesis, growth, differentiation, and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their down-stream transcription factor genes. Transcriptional control mechanisms of gene expression during differentiation were mainly studied by focusing on the cis- and trans-element in promoters, however, the role of mRNA export machinery during differentiation have not been adequately examined. A member of the mRNA export complex, THOC5 which is a substrate for the macrophage-colony stimulating factor (CSF-1) receptor, ATM kinase, or protein kinase C, is an essential element in the maintenance of hematopoiesis in adult mice. Using tamoxifen inducible THOC5 knockout mice, we show here that the depletion of THOC5 impaired myeloid differentiation, but does not influence terminally differentiated organs. Furthermore, an in vitro study showed that the depletion of THOC5 in bone marrow cells results in abnormal CSF-1 induced macrophage differentiation. Transcriptome analysis using cytoplasmic RNA derived from macrophages reveals that only 99 genes were down-regulated 3 days after the depletion of THOC5, however, immediate early genes induced by CSF-1 stimulation, such as Ets family genes, and regulators of myeloid differentiation HoxA1, Id1, Id3 were THOC5 direct target mRNAs, suggesting that THOC5 plays a key role in myeloid differentiation. In each of two biological experiments (called rep1 and rep2 below) six different cytoplasmic RNA samples were generated from bone marrow derived macrophages. These different samples (conditions) correspond to: 1) Cells from ERT2 Cre control mice at day 0, 2) Cells from ERT2 Cre control mice at day 3 of differentiation, 3) Cells from ERT2 Cre control mice at day 3 of differentiation stimulated with tamoxifen, 4) Cells from ERT2 Cre Thoc5 (flox/flox) mice at day 0, 5) Cells from ERT2 Cre Thoc5 (flox/flox) mice at day 3 of differentiation, 6) Cells from ERT2 Cre Thoc5 (flox/flox) mice at day 3 of differentiation stimulated with tamoxifen. Day 0 samples of a given genotype were utilized as common reference in two microarrays and were accordingly co-hydridized against both 3-days differentiated samples (treated or none with tamoxifen) of the same genotype and biological experiment. Thus, four dual-color microarrays from each of the two biological replicate series were performed (giving rise to 8 dual-color microarrays in total). The complete set of microarrays from the second biological experiment (rep2) was performed with inverted labeling directions (dye-swap approach).
Project description:THOC5, a member of the THO complex that is a subcomplex of Transcription/export complex (TREX) 1, is essential for 3´processing of slow kinetics IEGs, and for export of only a subset of genes. Applying nanopore mRNA-sequence technology, we show here that upon depletion of THOC5, 50% of mRNAs showed alteration of 3´end cleavage sites. Moreover, polymerase-II (Pol II)-CHIP-sequencing data reveals that upon depletion of THOC5 Pol II density was increased at gene body and 3´UTR. THOC5 is recruited near to Pol II high density sites except promotor regions, suggesting that THOC5 is involved in transcription elongation. Indeed, 385 THOC5 independent genes in fast Pol II cells became THOC5 dependent in slow Pol II cells. Chromatin associated THOC5 in slow Pol II cells interacts with CDK12, RNA helicases DDX5 and DDX, and THOC6, but not with other members of THO complex. Notably, THOC5 did not form this complex in fast poly II cells. CDK12 was recruited to R-loop (DNA:RNA hybrid) in THOC5/THOC6 dependent manner. Here, THOC6, but not THOC5 directly interacts with R-loop then, recruited DDX5 resolves R-loop and then CDK12 enhances further transcription elongation. These data show for the first time the novel function of THOC5/THOC6 complex during transcription elongation.
Project description:Transcription of immediate early genes (IEGs) in response to extrinsic and intrinsic signals is tightly regulated at multiple stages. THOC5, a member of the TREX (transcription/export) complex plays a role in expression of only a subset of constitutively active genes. In this report, we show that THOC5 plays a role in the 3M-BM-4end-processing of more than 90% of IEGs induced by serum stimulation, however THOC5 depletion does not influence the expression of the most rapidly induced IEGs, such as Fos or Jun. One group of THOC5 target genes, including Id1, Id3 and Wnt11 transcripts were not released from chromatin in THOC5 depleted cells. Another group of genes, such as Myc, and Smad7 transcripts, were released with shortening of 3M-BM-4UTR by alternative cleavage, and were spliced but were exported to a lesser extent in THOC5 depleted cells. Furthermore, upon stimulation with serum THOC5 forms a complex with polyadenylation specific factor 100 (CPSF100). THOC5 is required for recruitment of CPSF100 to 3M-BM-4UTR of THOC5 target genes, but not for THOC5 independent genes. These data suggest the presence of a novel mechanism for the control of immediate early genes response via 3M-BM-4end-processing. In 2 dual-color microarray hybridizations, mRNA expression changes after 1h of FCS treatment were examined. MEF(Rosa26ERT2 Cre: THOC5 (flox/flox)) cells in which a THOC5 knockdown is inducible upon Tamoxifen treatment were analyzed. M4040 represents cells without Taxoxifen pre-treatment (wildtype reference) whereas M4041 represents the same celltype pre-treated for 2 days with Tamoxifen to induce a THOC5 knockdown.
Project description:Transcription of immediate early genes (IEGs) in response to extrinsic and intrinsic signals is tightly regulated at multiple stages. THOC5, a member of the TREX (transcription/export) complex plays a role in expression of only a subset of constitutively active genes. In this report, we show that THOC5 plays a role in the 3´end-processing of more than 90% of IEGs induced by serum stimulation, however THOC5 depletion does not influence the expression of the most rapidly induced IEGs, such as Fos or Jun. One group of THOC5 target genes, including Id1, Id3 and Wnt11 transcripts were not released from chromatin in THOC5 depleted cells. Another group of genes, such as Myc, and Smad7 transcripts, were released with shortening of 3´UTR by alternative cleavage, and were spliced but were exported to a lesser extent in THOC5 depleted cells. Furthermore, upon stimulation with serum THOC5 forms a complex with polyadenylation specific factor 100 (CPSF100). THOC5 is required for recruitment of CPSF100 to 3´UTR of THOC5 target genes, but not for THOC5 independent genes. These data suggest the presence of a novel mechanism for the control of immediate early genes response via 3´end-processing.
Project description:THOC5, a member of the THO complex, is essential for the 3´processing of some inducible genes, the export of a subset of genes and stem cell self-renewal. Utilising nanopore mRNA-sequencing we show that when THOC5 is depleted 50-60% of mRNAs undergo altered 3´end cleavage. Further, THOC5 depletion leads to increased RNA Polymerase II (Pol II) presence at the start site, on the gene body and close to the 3’end. Moreover, THOC5 is recruited close to high density Pol II sites suggesting that THOC5 is involved in transcriptional elongation. Indeed, DRB/TTchem-seq that measures elongation rates in vivo revealed an accumulation of released Pol II near to TSS and a decrease of elongation rates in THOC5 depleted cells. Furthermore, THOC5 is more recruited to its target genes in cells expressing slow Pol II than those expressing fast Pol II. In cells expressing slow Pol II chromatin associated THOC5 interacts with CDK12 (a protein that modulates mRNA elongation rates), RNA helicases DDX5, DDX17, and THOC6. Importantly these interactions were not observed in cells expressing fast Pol II. 3’ cleavage of 50% of THOC5 target genes is also regulated by CDK12 and THOC6. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate that THOC5/THOC6 play a part in transcription elongation. Given the role of THOC5 in primitive cell survival its phosphorylation by agonists, oxidative stress and oncogenes the pathway we identified has relevance in the physiology and pathology of stem cells.
Project description:THOC5, a member of the THO complex, is essential for the 3´processing of some inducible genes, the export of a subset of genes and stem cell self-renewal. Utilising nanopore mRNA-sequencing we show that when THOC5 is depleted 50% of mRNAs undergo altered 3´end cleavage. Further, THOC5 depletion leads to increased RNA Polymerase II (Pol II) presence on the gene body and close to the 3’end. Moreover, THOC5 is recruited close to high density Pol II sites except those at the promotor regions suggesting that THOC5 is involved in transcriptional elongation. Indeed, THOC5 independent genes in cells expressing fast Pol II became THOC5 dependent in cells expressing slow Pol II. In cells expressing slow Pol II chromatin associated THOC5 interacts with CDK12 (a protein that modulates mRNA elongation rates), RNA helicases DDX5, DDX17, and THOC6. Importantly these interactions were not observed in cells expressing fast Pol II. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate that THOC5/THOC6 play a part in transcription elongation. Given the role of THOC5 in primitive cell survival its phosphorylation by agonists, oxidative stress and oncogenes the pathway we identified has relevance in the physiology and pathology of stem cells
Project description:Hepatocellular carcinoma (HCC) is a frequent cancer with poor prognosis and with limited possibilities for anti-cancer treatment. Evidence has recently accumulated suggesting that multiple signaling pathways are activated in human cancer. It has been recently shown by exosome sequencing of HCC that 161 putative driver genes are associated with 11 recurrently altered pathways, suggesting that we need to inhibit these multiple pathways for HCC therapy. THOC5, a member of transcription/export (TREX) complex, that plays a role in less than 1% of mRNA processing in normal cells such as fibroblasts or macrophages, is not required for maintenance of mature hepatocytes. In this study, we have examined the role of THOC5 in human HCC. Enhanced THOC5 expression was not observed in differentiation grading 1 (G1) human HCC, but 78% of G2 and G3 exhibited increased levels of THOC5. Furthermore, the 50% depletion of THOC5 in hepatocellular carcinoma cell lines, Huh7 and HepG2 causes lipid accumulation and apoptosis. Transcriptome analysis using THOC5 depleted Huh7 and HepG2 cells revealed that 1049 and 837 genes were downregulated upon depletion of THOC5 in Huh7 and HepG2 cells, respectively. Among these genes, 396 were commonly downregulated in both cell lines. Some of these, such as tissue inhibitor of metalloproteinase 3 (TIMP3), tripartite motif containing 24 (TRIM24), ribosomal protein S6 kinase polypeptide 3 (RPS6KA3) and transmembrane emp24-like trafficking protein 10 (TMED10) are known to be fine tuners for several signal transduction pathways. The expression of these proteins was correlated with the THOC5 expression level in HCC. To inhibit multiple signaling pathways we are currently examining the effects of a combination of siRNAs against THOC5 target genes in HCC cell lines. Our data suggest that THOC5, which controls a set of genes that are involved in HCC malignancy can be a potential biomarker for HCC. In addition, THOC5 target gene, TMED10 may also be a novel biomarker of HCC. Furthermore, the suppression of the THOC5 gene per se or multiple THOC5 target genes may represent a novel strategy for cancer therapy. Huh7 and HepG2 cells were infected with lentiviral THOC5 and control shRNAs. Five days after infection, total RNAs were isolated and subjected to microarray analysis.
Project description:Embryonic stem cell (ESC) self-renewal and differentiation are governed by a broad-ranging regulatory network. Although the transcriptional regulatory mechanisms involved have been investigated extensively, post-transcriptional regulation is still poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5, and is required for self-renewal at least in part by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, leading to decreased expression of pluripotency proteins that facilitates exit from self-renewal. THO is also important for the establishment of pluripotency, as its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicate that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and therefore uncover a role for this aspect of post-transcriptional regulation in stem cell fate specification. mouse J1 cells were transfected with non-targeting (NT), Thoc2, and Thoc5 siRNAs. Total RNA was isolated 96 hours after transfection.