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1.
bioRxiv ; 2024 May 22.
Article in English | MEDLINE | ID: mdl-38798402

ABSTRACT

Because most DNA-binding transcription factors (dbTFs), including the architectural regulator CTCF, bind RNA and exhibit di-/multimerization, a central conundrum is whether these distinct properties are regulated post-transcriptionally to modulate transcriptional programs. Here, investigating stress-dependent activation of SIRT1, encoding an evolutionarily-conserved protein deacetylase, we show that induced phosphorylation of CTCF acts as a rheostat to permit CTCF occupancy of low-affinity promoter DNA sites to precisely the levels necessary. This CTCF recruitment to the SIRT1 promoter is eliciting a cardioprotective cardiomyocyte transcriptional activation program and provides resilience against the stress of the beating heart in vivo . Mice harboring a mutation in the conserved low-affinity CTCF promoter binding site exhibit an altered, cardiomyocyte-specific transcriptional program and a systolic heart failure phenotype. This transcriptional role for CTCF reveals that a covalent dbTF modification regulating signal-dependent transcription serves as a previously unsuspected component of the oxidative stress response.

2.
Nat Struct Mol Biol ; 30(2): 148-158, 2023 02.
Article in English | MEDLINE | ID: mdl-36747093

ABSTRACT

Enhancer activation serves as the main mechanism regulating signal-dependent transcriptional programs, ensuring cellular plasticity, yet central questions persist regarding their mechanism of activation. Here, by successfully mapping topoisomerase I-DNA covalent complexes genome-wide, we find that most, if not all, acutely activated enhancers, including those induced by 17ß-estradiol, dihydrotestosterone, tumor necrosis factor alpha and neuronal depolarization, are hotspots for topoisomerase I-DNA covalent complexes, functioning as epigenomic signatures read by the classic DNA damage sensor protein, Ku70. Ku70 in turn nucleates a heterochromatin protein 1 gamma (HP1γ)-mediator subunit Med26 complex to facilitate acute, but not chronic, transcriptional activation programs. Together, our data uncover a broad, unappreciated transcriptional code, required for most, if not all, acute signal-dependent enhancer activation events in both mitotic and postmitotic cells.


Subject(s)
DNA Topoisomerases, Type I , Enhancer Elements, Genetic , DNA , DNA Topoisomerases, Type I/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Ku Autoantigen/metabolism
3.
Proc Natl Acad Sci U S A ; 119(32): e2206216119, 2022 08 09.
Article in English | MEDLINE | ID: mdl-35914133

ABSTRACT

The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of "housekeeping" genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates.


Subject(s)
Cell Compartmentation , Cell Nucleus , Eukaryota , Ribonucleoproteins , Cell Nucleus/chemistry , Cell Nucleus/genetics , Cell Nucleus/metabolism , Chromosomes/genetics , Eukaryota/cytology , Eukaryota/genetics , Genes, Essential , Genome/genetics , Nuclear Speckles/genetics , Ribonucleoproteins/metabolism , Transcription, Genetic
4.
Trends Genet ; 38(10): 1019-1047, 2022 10.
Article in English | MEDLINE | ID: mdl-35811173

ABSTRACT

Gene regulation by transcriptional enhancers is the dominant mechanism driving cell type- and signal-specific transcriptional diversity in metazoans. However, over four decades since the original discovery, how enhancers operate in the nuclear space remains largely enigmatic. Recent multidisciplinary efforts combining real-time imaging, genome sequencing, and biophysical strategies provide insightful but conflicting models of enhancer-mediated gene control. Here, we review the discovery and progress in enhancer biology, emphasizing the recent findings that acutely activated enhancers assemble regulatory machinery as mesoscale architectural structures with distinct physical properties. These findings help formulate novel models that explain several mysterious features of the assembly of transcriptional enhancers and the mechanisms of spatial control of gene expression.


Subject(s)
DNA, Viral , Enhancer Elements, Genetic , Base Sequence , Cell Nucleus/genetics , Gene Expression Regulation/genetics
5.
Nature ; 595(7869): 735-740, 2021 07.
Article in English | MEDLINE | ID: mdl-34040254

ABSTRACT

The functional engagement between an enhancer and its target promoter ensures precise gene transcription1. Understanding the basis of promoter choice by enhancers has important implications for health and disease. Here we report that functional loss of a preferred promoter can release its partner enhancer to loop to and activate an alternative promoter (or alternative promoters) in the neighbourhood. We refer to this target-switching process as 'enhancer release and retargeting'. Genetic deletion, motif perturbation or mutation, and dCas9-mediated CTCF tethering reveal that promoter choice by an enhancer can be determined by the binding of CTCF at promoters, in a cohesin-dependent manner-consistent with a model of 'enhancer scanning' inside the contact domain. Promoter-associated CTCF shows a lower affinity than that at chromatin domain boundaries and often lacks a preferred motif orientation or a partnering CTCF at the cognate enhancer, suggesting properties distinct from boundary CTCF. Analyses of cancer mutations, data from the GTEx project and risk loci from genome-wide association studies, together with a focused CRISPR interference screen, reveal that enhancer release and retargeting represents an overlooked mechanism that underlies the activation of disease-susceptibility genes, as exemplified by a risk locus for Parkinson's disease (NUCKS1-RAB7L1) and three loci associated with cancer (CLPTM1L-TERT, ZCCHC7-PAX5 and PVT1-MYC).


Subject(s)
CCCTC-Binding Factor/genetics , Enhancer Elements, Genetic , Genetic Predisposition to Disease , Promoter Regions, Genetic , CRISPR-Cas Systems , Cell Cycle Proteins/genetics , Cells, Cultured , Chromatin , Chromosomal Proteins, Non-Histone/genetics , Gene Deletion , Gene Expression Regulation, Neoplastic , Genome-Wide Association Study , Humans , MCF-7 Cells , Neoplasms/genetics , Neural Stem Cells , Oncogenes , Parkinson Disease/genetics , Cohesins
6.
Nat Struct Mol Biol ; 26(3): 193-203, 2019 03.
Article in English | MEDLINE | ID: mdl-30833784

ABSTRACT

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17ß-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA-dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.


Subject(s)
Enhancer Elements, Genetic/genetics , Estradiol/metabolism , Ribonucleoproteins/metabolism , Transcriptional Activation/physiology , Cell Line, Tumor , Chromatin , Chromosomes/physiology , Humans , MCF-7 Cells , Protein Conformation , Transcription, Genetic/genetics , Transcriptional Activation/genetics
8.
Nat Commun ; 8: 15908, 2017 06 26.
Article in English | MEDLINE | ID: mdl-28649985

ABSTRACT

Most BRCA1-associated breast tumours are basal-like yet originate from luminal progenitors. BRCA1 is best known for its functions in double-strand break repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions fully explains the cell lineage-specific tumorigenesis. In vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Here we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 5' end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1-knockout mouse mammary epithelium. Our studies show that Pol II pausing is an important contributor to BRCA1-associated R-loop accumulation and breast cancer development.


Subject(s)
BRCA1 Protein/chemistry , BRCA1 Protein/genetics , Breast Neoplasms/enzymology , RNA Polymerase II/metabolism , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics , Animals , BRCA1 Protein/metabolism , Breast/enzymology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Carcinogenesis , Female , Humans , Mice , Mice, Knockout , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleic Acid Conformation , Promoter Regions, Genetic , RNA Polymerase II/genetics , RNA-Binding Proteins , Tumor Suppressor Proteins/metabolism
9.
Nat Commun ; 7: 10913, 2016 Mar 04.
Article in English | MEDLINE | ID: mdl-26941120

ABSTRACT

The breast cancer susceptibility gene BRCA1 is well known for its function in double-strand break (DSB) DNA repair. While BRCA1 is also implicated in transcriptional regulation, the physiological significance remains unclear. COBRA1 (also known as NELF-B) is a BRCA1-binding protein that regulates RNA polymerase II (RNAPII) pausing and transcription elongation. Here we interrogate functional interaction between BRCA1 and COBRA1 during mouse mammary gland development. Tissue-specific deletion of Cobra1 reduces mammary epithelial compartments and blocks ductal morphogenesis, alveologenesis and lactogenesis, demonstrating a pivotal role of COBRA1 in adult tissue development. Remarkably, these developmental deficiencies due to Cobra1 knockout are largely rescued by additional loss of full-length Brca1. Furthermore, Brca1/Cobra1 double knockout restores developmental transcription at puberty, alters luminal epithelial homoeostasis, yet remains deficient in homologous recombination-based DSB repair. Thus our genetic suppression analysis uncovers a previously unappreciated, DNA repair-independent function of BRCA1 in antagonizing COBRA1-dependent transcription programme during mammary gland development.


Subject(s)
DNA Repair/physiology , Mammary Glands, Animal/growth & development , Nuclear Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Aging , Animals , BRCA1 Protein , DNA Breaks, Double-Stranded , Epithelial Cells , Estrogens/metabolism , Female , Gene Expression Regulation, Developmental/physiology , Homeostasis , Mice , Mice, Knockout , Nuclear Proteins/genetics , Progestins/metabolism , RNA-Binding Proteins , Sexual Maturation , Transcriptome , Tumor Suppressor Proteins/genetics
10.
Exp Hematol Oncol ; 1(1): 31, 2012 Oct 08.
Article in English | MEDLINE | ID: mdl-23210696

ABSTRACT

Radiation therapy (RT) after breast conservation therapy has recently been linked with significant reduction in risk of ipsilateral breast cancer among BRCA1 mutation carriers. However, the exact mechanism by which RT reduces incidence of BRCA1-associated cancer remains unclear. Here we studied fresh breast tissue from a BRCA1 mutation carrier who was initially treated with a lumpectomy and RT for a unilateral cancer and two years later chose a prophylactic bilateral mastectomy while remaining cancer-free. Flow cytometry analysis demonstrated a strikingly lower luminal cell population in the irradiated breast as compared to the non-irradiated breast, which was confirmed by immunohistochemistry. Furthermore, the irradiated breast tissue exhibited very low progenitor cell activity in vitro. Given the emerging evidence that BRCA1 tumors originate from luminal progenitor cells, our observations suggest that preferential and long-lasting elimination of luminal ductal epithelium may partly underlie the mechanism of RT-associated reduction in recurrence of BRCA1-associated cancer.

11.
J Biol Chem ; 286(42): 36248-57, 2011 Oct 21.
Article in English | MEDLINE | ID: mdl-21865163

ABSTRACT

Many mammalian genes are occupied by paused RNA polymerase II (pol II) in the promoter-proximal region on both sides of the transcription start site. However, the impact of pol II pausing on gene expression and cell biology is not fully understood. In this study, we used a Cre-Lox system to conditionally knock out the b subunit of mouse negative elongation factor (Nelf-b), a key pol II-pausing factor, in mouse embryonic fibroblasts. We found that Nelf-b was associated with the promoter-proximal region of the majority of expressed genes, yet genetic ablation of Nelf-b only affected the steady-state mRNA levels of a small percentage of the Nelf-b-associated genes. Interestingly, Nelf-b deletion also increased levels of transcription start site upstream transcripts at multiple negative elongation factor-associated genes. The direct target genes of Nelf-b were highly enriched with those involved in the control of cell growth and cell death. Correspondingly, Nelf-b knock-out mouse embryonic fibroblasts exhibited slower progression from quiescence to proliferation, as well as in a cycling cell population. Furthermore, Nelf-b deletion also resulted in increased apoptosis. Thus, the genetic and genomic studies provide new physiological and molecular insight into Nelf-mediated pol II pausing.


Subject(s)
Cell Proliferation , Embryo, Mammalian/metabolism , Fibroblasts/metabolism , Nuclear Proteins/metabolism , RNA Polymerase II/metabolism , Animals , Apoptosis/genetics , Cell Line , Embryo, Mammalian/cytology , Fibroblasts/cytology , Gene Deletion , Genome/physiology , Mice , Nuclear Proteins/genetics , RNA Polymerase II/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA-Binding Proteins
12.
PLoS One ; 4(4): e5034, 2009.
Article in English | MEDLINE | ID: mdl-19340312

ABSTRACT

BACKGROUND: Negative elongation factor (NELF) is a four-subunit protein complex conserved from Drosophila to humans. In vitro biochemical and tissue culture-based studies have demonstrated an important role of NELF in controlling RNA polymerase II (Pol II) pausing in transcription. However, the physiological significance of NELF function is not clear due to the lack of any genetic systems for studying NELF. PRINCIPAL FINDINGS: Here we show that disruption of the mouse B subunit of NELF (NELF-B), also known as cofactor of BRCA1 (Cobra1), causes inner cell mass (ICM) deficiency and embryonic lethality at the time of implantation. Consistent with the phenotype of the Cobra1 knockout (KO) embryos, knockdown of Cobra1 in mouse embryonic stem cells (ESCs) reduces the efficiency of colony formation and increases spontaneous differentiation. Cobra1-depleted ESCs maintain normal levels of Oct4, Nanog, and Sox2, master regulators of pluripotency in ESCs. However, knockdown of Cobra1 leads to precocious expression of developmental regulators including lymphoid enhancer-binding factor 1 (Lef1). Chromatin immunoprecipitation (ChIP) indicates that Cobra1 binds to the Lef1 promoter and modulates the abundance of promoter-bound RNA polymerase. CONCLUSIONS: Cobra1 is essential for early embryogenesis. Our findings also indicate that Cobra1 helps maintain the undifferentiated state of mESCs by preventing unscheduled expression of developmental genes.


Subject(s)
Embryonic Development/physiology , Nuclear Proteins/physiology , Animals , Chromatin Immunoprecipitation , Embryonic Development/genetics , Embryonic Stem Cells/cytology , Female , Fetal Death , Gene Deletion , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Mice , Mice, Knockout , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , RNA, Small Interfering , RNA-Binding Proteins
13.
Anim Reprod Sci ; 96(1-2): 21-9, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16337101

ABSTRACT

A comparative study was conducted to monitor the activities of some antioxidant enzymes, lipid peroxidation and viability of cattle and buffalo bull spermatozoa during storage of semen at refrigeration temperature over a period of 72 h. Semen samples, collected from six cross bred cattle bulls (group I) and six Murrah buffalo bulls (group II), were diluted in egg-yolk-citrate and the spermatozoa were separated from seminal plasma by centrifugation at 4 degrees C in a refrigerated centrifuge. The malondialdehyde (MDA) production in group I increased from 1.17+/-0.29 at 0 h to 7.50+/-0.52 nmol/10(8)spermatozoa after 72 h of storage while in group II it increased from 1.99+/-0.26 to 8.70+/-0.10 nmol/10(8)spermatozoa in the same period. However, buffalo bull spermatozoa had a significantly higher (p<0.05) lipid peroxidation at 0 h as well as at 12, 24 and 48 h (p<0.01) periods. The activities of antioxidant enzymes viz. SOD, GPx and G6PD in both the groups showed a similar pattern of change i.e. the activities declined successively in spermatozoa and increased in the seminal plasma. However, the activities of these three enzymes remained significantly higher in the cattle bull spermatozoa than that in buffalo bull spermatozoa. Amount of MDA produced in spermatozoa of both the groups was negatively correlated while SOD, GPx and G6PD activities in spermatozoa were positively correlated to the motility and viability of spermatozoa. Sperm motility as well as viability was significantly less (p<0.05) in group II than that in group I. SOD, GPx and G6PD activities in spermatozoa of both the groups were negatively correlated to lipid peroxidation of spermatozoa cell membrane. The results showed that the less activities of antioxidant enzymes in buffalo bull spermatozoa was due to higher lipid peroxidation that indicated that they were more prone to oxidative stress as compared to cattle bull spermatozoa when stored at refrigeration temperature.


Subject(s)
Antioxidants/metabolism , Buffaloes , Cattle , Lipid Peroxidation , Semen Preservation/veterinary , Spermatozoa/physiology , Animals , Cell Separation , Cell Survival , Centrifugation , Cold Temperature , Glucosephosphate Dehydrogenase/metabolism , Glutathione Peroxidase/metabolism , Male , Malondialdehyde/analysis , Semen Preservation/methods , Sperm Motility , Spermatozoa/enzymology , Superoxide Dismutase/metabolism , Time Factors
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