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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 -
Molecular Cell Nov 2023The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental... (Review)
Review
The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental cues in metazoan. Dysregulated transcriptional elongation has been associated with developmental defects as well as disease and aging processes. Decades of genetic and biochemical studies have painstakingly identified and characterized an ensemble of factors that regulate RNA Pol II elongation. This review summarizes recent findings taking advantage of genetic engineering techniques that probe functions of elongation factors in vivo. We propose a revised model of elongation control in this accelerating field by reconciling contradictory results from the earlier biochemical evidence and the recent in vivo studies. We discuss how elongation factors regulate promoter-proximal RNA Pol II pause release, transcriptional elongation rate and processivity, RNA Pol II stability and RNA processing, and how perturbation of these processes is associated with developmental disorders, neurodegenerative disease, cancer, and aging.
Topics: Animals; Humans; RNA Polymerase II; Transcriptional Elongation Factors; Neurodegenerative Diseases; Transcription, Genetic; Gene Expression Regulation; Aging; Genes, Developmental
PubMed: 37922911
DOI: 10.1016/j.molcel.2023.10.004 -
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 -
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 -
BioRxiv : the Preprint Server For... Oct 2023During heart development, a well-characterized network of transcription factors initiates cardiac gene expression and defines the precise timing and location of cardiac...
During heart development, a well-characterized network of transcription factors initiates cardiac gene expression and defines the precise timing and location of cardiac progenitor specification. However, our understanding of the post-initiation transcriptional events that regulate cardiac gene expression is still incomplete. The PAF1C component Rtf1 is a transcription regulatory protein that modulates pausing and elongation of RNA Pol II, as well as cotranscriptional histone modifications. Here we report that Rtf1 is essential for cardiogenesis in fish and mammals, and that in the absence of Rtf1 activity, cardiac progenitors arrest in an immature state. We found that Rtf1's Plus3 domain, which confers interaction with the transcriptional pausing and elongation regulator Spt5, was necessary for cardiac progenitor formation. ChIP-seq analysis further revealed changes in the occupancy of RNA Pol II around the transcription start site (TSS) of cardiac genes in morphants reflecting a reduction in transcriptional pausing. Intriguingly, inhibition of pause release in morphants and mutants restored the formation of cardiac cells and improved Pol II occupancy at the TSS of key cardiac genes. Our findings highlight the crucial role that transcriptional pausing plays in promoting normal gene expression levels in a cardiac developmental context.
PubMed: 37873297
DOI: 10.1101/2023.10.13.562296