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Genes & Development Aug 2019Precise spatio-temporal control of gene activity is essential for organismal development, growth, and survival in a changing environment. Decisive steps in gene... (Review)
Review
Precise spatio-temporal control of gene activity is essential for organismal development, growth, and survival in a changing environment. Decisive steps in gene regulation involve the pausing of RNA polymerase II (Pol II) in early elongation, and the controlled release of paused polymerase into productive RNA synthesis. Here we describe the factors that enable pausing and the events that trigger Pol II release into the gene. We also discuss open questions in the field concerning the stability of paused Pol II, nucleosomes as obstacles to elongation, and potential roles of pausing in defining the precision and dynamics of gene expression.
Topics: Animals; Enzyme Stability; Gene Expression Regulation, Developmental; Humans; Nucleosomes; Promoter Regions, Genetic; RNA Polymerase II; Transcription Elongation, Genetic
PubMed: 31123063
DOI: 10.1101/gad.325142.119 -
Bone Research Jun 2023As the major cell precursors in osteogenesis, mesenchymal stem cells (MSCs) are indispensable for bone homeostasis and development. However, the primary mechanisms...
As the major cell precursors in osteogenesis, mesenchymal stem cells (MSCs) are indispensable for bone homeostasis and development. However, the primary mechanisms regulating osteogenic differentiation are controversial. Composed of multiple constituent enhancers, super enhancers (SEs) are powerful cis-regulatory elements that identify genes that ensure sequential differentiation. The present study demonstrated that SEs were indispensable for MSC osteogenesis and involved in osteoporosis development. Through integrated analysis, we identified the most common SE-targeted and osteoporosis-related osteogenic gene, ZBTB16. ZBTB16, positively regulated by SEs, promoted MSC osteogenesis but was expressed at lower levels in osteoporosis. Mechanistically, SEs recruited bromodomain containing 4 (BRD4) at the site of ZBTB16, which then bound to RNA polymerase II-associated protein 2 (RPAP2) that transported RNA polymerase II (POL II) into the nucleus. The subsequent synergistic regulation of POL II carboxyterminal domain (CTD) phosphorylation by BRD4 and RPAP2 initiated ZBTB16 transcriptional elongation, which facilitated MSC osteogenesis via the key osteogenic transcription factor SP7. Bone-targeting ZBTB16 overexpression had a therapeutic effect on the decreased bone density and remodeling capacity of Brd4 Prx1-cre mice and osteoporosis (OP) models. Therefore, our study shows that SEs orchestrate the osteogenesis of MSCs by targeting ZBTB16 expression, which provides an attractive focus and therapeutic target for osteoporosis. Without SEs located on osteogenic genes, BRD4 is not able to bind to osteogenic identity genes due to its closed structure before osteogenesis. During osteogenesis, histones on osteogenic identity genes are acetylated, and OB-gain SEs appear, enabling the binding of BRD4 to the osteogenic identity gene ZBTB16. RPAP2 transports RNA Pol II from the cytoplasm to the nucleus and guides Pol II to target ZBTB16 via recognition of the navigator BRD4 on SEs. After the binding of the RPAP2-Pol II complex to BRD4 on SEs, RPAP2 dephosphorylates Ser5 at the Pol II CTD to terminate the transcriptional pause, and BRD4 phosphorylates Ser2 at the Pol II CTD to initiate transcriptional elongation, which synergistically drives efficient transcription of ZBTB16, ensuring proper osteogenesis. Dysregulation of SE-mediated ZBTB16 expression leads to osteoporosis, and bone-targeting ZBTB16 overexpression is efficient in accelerating bone repair and treating osteoporosis.
PubMed: 37280207
DOI: 10.1038/s41413-023-00267-8 -
Molecular Cell Nov 2021Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps...
Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps from enhancer activation to termination. Here we utilize a rapid degradation system and reveal crucial functions of SPT5 in maintaining cellular and chromatin RNA polymerase II (Pol II) levels. Rapid SPT5 depletion causes a pronounced reduction of paused Pol II at promoters and enhancers, distinct from negative elongation factor (NELF) degradation resulting in short-distance paused Pol II redistribution. Most genes exhibit downregulation, but not upregulation, accompanied by greatly impaired transcription activation, altered chromatin landscape at enhancers, and severe Pol II processivity defects at gene bodies. Phosphorylation of an SPT5 linker at serine 666 potentiates pause release and is antagonized by Integrator-PP2A (INTAC) targeting SPT5 and Pol II, while phosphorylation of the SPT5 C-terminal region links to 3' end termination. Our findings position SPT5 as an essential positive regulator of global transcription.
Topics: Animals; Antigens, Differentiation, B-Lymphocyte; Chromatin; Chromosomal Proteins, Non-Histone; Enhancer Elements, Genetic; Fibroblasts; Genome; HEK293 Cells; Histocompatibility Antigens Class II; Humans; Mice; Mutation; Nuclear Proteins; Phosphorylation; Promoter Regions, Genetic; RNA Polymerase II; RNA-Seq; Regulatory Sequences, Nucleic Acid; Transcription, Genetic; Transcriptional Activation; Transcriptional Elongation Factors
PubMed: 34534457
DOI: 10.1016/j.molcel.2021.08.029 -
The Plant Cell Mar 2023Fruit ripening relies on the precise spatiotemporal control of RNA polymerase II (Pol II)-dependent gene transcription, and the evolutionarily conserved Mediator (MED)...
Fruit ripening relies on the precise spatiotemporal control of RNA polymerase II (Pol II)-dependent gene transcription, and the evolutionarily conserved Mediator (MED) coactivator complex plays an essential role in this process. In tomato (Solanum lycopersicum), a model climacteric fruit, ripening is tightly coordinated by ethylene and several key transcription factors. However, the mechanism underlying the transmission of context-specific regulatory signals from these ripening-related transcription factors to the Pol II transcription machinery remains unknown. Here, we report the mechanistic function of MED25, a subunit of the plant Mediator transcriptional coactivator complex, in controlling the ethylene-mediated transcriptional program during fruit ripening. Multiple lines of evidence indicate that MED25 physically interacts with the master transcription factors of the ETHYLENE-INSENSITIVE 3 (EIN3)/EIN3-LIKE (EIL) family, thereby playing an essential role in pre-initiation complex formation during ethylene-induced gene transcription. We also show that MED25 forms a transcriptional module with EIL1 to regulate the expression of ripening-related regulatory as well as structural genes through promoter binding. Furthermore, the EIL1-MED25 module orchestrates both positive and negative feedback transcriptional circuits, along with its downstream regulators, to fine-tune ethylene homeostasis during fruit ripening.
Topics: Transcription Factors; Solanum lycopersicum; Fruit; Plant Proteins; Ethylenes; Gene Expression Regulation, Plant
PubMed: 36471914
DOI: 10.1093/plcell/koac349 -
Nature Communications Jan 2022Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a regulator of oxygen (O) homeostasis in metazoan species by binding to hypoxia response...
Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a regulator of oxygen (O) homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions but pauses after approximately 30-60 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1α and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain as well as the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies reveal a molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.
Topics: Amino Acid Motifs; Cell Line, Tumor; Cyclin-Dependent Kinase 9; DNA-Activated Protein Kinase; Gene Expression Regulation; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Phosphorylation; Promoter Regions, Genetic; Protein Binding; RNA Polymerase II; Response Elements; Transcription, Genetic; Tripartite Motif-Containing Protein 28
PubMed: 35031618
DOI: 10.1038/s41467-021-27944-8 -
Frontiers in Plant Science 2020LTR-retrotransposons share a common genomic organization in which the 5' long terminal repeat (LTR) is followed by the and genes and terminates with the 3' LTR.... (Review)
Review
LTR-retrotransposons share a common genomic organization in which the 5' long terminal repeat (LTR) is followed by the and genes and terminates with the 3' LTR. Although GAG-POL-encoded proteins are considered sufficient to accomplish the LTR-retrotransposon transposition, a number of elements carrying additional open reading frames (aORF) have been described. In some cases, the presence of an aORF can be explained by a phenomenon similar to retrovirus gene transduction, but in these cases the aORFs are present in only one or a few copies. On the contrary, many elements contain aORFs, or derivatives, in all or most of their copies. These aORFs are more frequently located between and 3' LTR, and they could be in sense or antisense orientation with respect to -. Sense aORFs include those encoding for ENV-like proteins, so called because they have some structural and functional similarities with retroviral ENV proteins. Antisense aORFs between and 3' LTR are also relatively frequent and, for example, are present in some characterized LTR-retrotransposon families like maize Grande, rice RIRE2, or Retand, although their possible roles have been not yet determined. Here, we discuss the current knowledge about these sense and antisense aORFs in plant LTR-retrotransposons, suggesting their possible origins, evolutionary relevance, and function.
PubMed: 32528484
DOI: 10.3389/fpls.2020.00555 -
Molecular Cell Sep 2022It is unclear how various factors functioning in the transcriptional elongation by RNA polymerase II (RNA Pol II) cooperatively regulate pause/release and productive...
It is unclear how various factors functioning in the transcriptional elongation by RNA polymerase II (RNA Pol II) cooperatively regulate pause/release and productive elongation in living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in the release of paused RNA Pol II into gene bodies through an impaired recruitment of PAF1C. Short genes demonstrate a release with increased mature transcripts, whereas long genes are released but fail to yield mature transcripts, due to a reduced processivity resulting from both SPT6 and PAF1C loss. Unexpectedly, SPT6 depletion causes an association of NELF with the elongating RNA Pol II on gene bodies, without any observed functional significance on transcriptional elongation pattern, arguing against a role for NELF in keeping RNA Pol II in the paused state. Furthermore, SPT6 depletion impairs heat-shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through PAF1C recruitment.
Topics: Heat-Shock Response; Promoter Regions, Genetic; RNA Polymerase II; Transcription Factors; Transcription, Genetic
PubMed: 35973425
DOI: 10.1016/j.molcel.2022.06.037 -
Science Advances Jul 2023Transcription factor (TF) IIIC recruits RNA polymerase (Pol) III to most of its target genes. Recognition of intragenic A- and B-box motifs in transfer RNA (tRNA) genes...
Transcription factor (TF) IIIC recruits RNA polymerase (Pol) III to most of its target genes. Recognition of intragenic A- and B-box motifs in transfer RNA (tRNA) genes by TFIIIC modules τA and τB is the first critical step for tRNA synthesis but is mechanistically poorly understood. Here, we report cryo-electron microscopy structures of the six-subunit human TFIIIC complex unbound and bound to a tRNA gene. The τB module recognizes the B-box via DNA shape and sequence readout through the assembly of multiple winged-helix domains. TFIIIC220 forms an integral part of both τA and τB connecting the two subcomplexes via a ~550-amino acid residue flexible linker. Our data provide a structural mechanism by which high-affinity B-box recognition anchors TFIIIC to promoter DNA and permits scanning for low-affinity A-boxes and TFIIIB for Pol III activation.
Topics: Humans; Cryoelectron Microscopy; Transcription Factors, TFIII; Transcription, Genetic; DNA; RNA, Transfer
PubMed: 37418517
DOI: 10.1126/sciadv.adh2019 -
Journal of Molecular Biology Jul 2021Although we have made significant progress, we still possess a limited understanding of how genomic and epigenomic information directs gene expression programs through... (Review)
Review
Although we have made significant progress, we still possess a limited understanding of how genomic and epigenomic information directs gene expression programs through sequence-specific transcription factors (TFs). Extensive research has settled on three general classes of TF targets in metazoans: promoter accessibility via chromatin regulation (e.g., SAGA), assembly of the general transcription factors on promoter DNA (e.g., TFIID), and recruitment of RNA polymerase (Pol) II (e.g., Mediator) to establish a transcription pre-initiation complex (PIC). Here we discuss TFs and their targets. We also place this in the context of our current work with Saccharomyces (yeast), where we find that promoters typically lack an architecture that supports TF function. Moreover, yeast promoters that support TF binding also display interactions with cofactors like SAGA and Mediator, but not TFIID. It is unknown to what extent all genes in metazoans require TFs and their cofactors.
Topics: Animals; Chromatin; Enhancer Elements, Genetic; Gene Expression Regulation; Humans; Multiprotein Complexes; Promoter Regions, Genetic; RNA Polymerase II; Transcription Factor TFIID; Transcription Factors; Transcription, Genetic
PubMed: 33621520
DOI: 10.1016/j.jmb.2021.166883 -
Nature Communications Sep 2023Telomerase RNA (TERC) has a noncanonical function in myelopoiesis binding to a consensus DNA binding sequence and attracting RNA polymerase II (RNA Pol II), thus...
Telomerase RNA (TERC) has a noncanonical function in myelopoiesis binding to a consensus DNA binding sequence and attracting RNA polymerase II (RNA Pol II), thus facilitating myeloid gene expression. The CR4/CR5 domain of TERC is known to play this role, since a mutation of this domain found in dyskeratosis congenita (DC) patients decreases its affinity for RNA Pol II, impairing its myelopoietic activity as a result. In this study, we report that two aptamers, short single-stranded oligonucleotides, based on the CR4/CR5 domain were able to increase myelopoiesis without affecting erythropoiesis in zebrafish. Mechanistically, the aptamers functioned as full terc; that is, they increased the expression of master myeloid genes, independently of endogenous terc, by interacting with RNA Pol II and with the terc-binding sequences of the regulatory regions of such genes, enforcing their transcription. Importantly, aptamers harboring the CR4/CR5 mutation that was found in DC patients failed to perform all these functions. The therapeutic potential of the aptamers for treating neutropenia was demonstrated in several preclinical models. The findings of this study have identified two potential therapeutic agents for DC and other neutropenic patients.
Topics: Humans; Animals; Aptamers, Nucleotide; Myelopoiesis; RNA Polymerase II; Syndrome; Zebrafish; Dyskeratosis Congenita
PubMed: 37737237
DOI: 10.1038/s41467-023-41472-7