RESUMEN
Retinitis pigmentosa (RP) is the most common inherited retinal disease (IRD) and is characterized by photoreceptor degeneration and progressive vision loss. We report 4 patients presenting with RP from 3 unrelated families with variants in TBC1D32, which to date has never been associated with an IRD. To validate TBC1D32 as a putative RP causative gene, we combined Xenopus in vivo approaches and human induced pluripotent stem cell-derived (iPSC-derived) retinal models. Our data showed that TBC1D32 was expressed during retinal development and that it played an important role in retinal pigment epithelium (RPE) differentiation. Furthermore, we identified a role for TBC1D32 in ciliogenesis of the RPE. We demonstrated elongated ciliary defects that resulted in disrupted apical tight junctions, loss of functionality (delayed retinoid cycling and altered secretion balance), and the onset of an epithelial-mesenchymal transition-like phenotype. Last, our results suggested photoreceptor differentiation defects, including connecting cilium anomalies, that resulted in impaired trafficking to the outer segment in cones and rods in TBC1D32 iPSC-derived retinal organoids. Overall, our data highlight a critical role for TBC1D32 in the retina and demonstrate that TBC1D32 mutations lead to RP. We thus identify TBC1D32 as an IRD-causative gene.
Asunto(s)
Células Madre Pluripotentes Inducidas , Degeneración Retiniana , Retinitis Pigmentosa , Humanos , Retina , Retinitis Pigmentosa/genética , Degeneración Retiniana/genética , Epitelio Pigmentado de la Retina , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
BACKGROUND: Human-induced pluripotent stem cell-derived retinal organoids are a valuable tool for disease modelling and therapeutic development. Many efforts have been made over the last decade to optimise protocols for the generation of organoids that correctly mimic the human retina. Most protocols use common media supplements; however, protocol-dependent variability impacts data interpretation. To date, the lack of a systematic comparison of a given protocol with or without supplements makes it difficult to determine how they influence the differentiation process and morphology of the retinal organoids. METHODS: A 2D-3D differentiation method was used to generate retinal organoids, which were cultured with or without the most commonly used media supplements, notably retinoic acid. Gene expression was assayed using qPCR analysis, protein expression using immunofluorescence studies, ultrastructure using electron microscopy and 3D morphology using confocal and biphoton microscopy of whole organoids. RESULTS: Retinoic acid delayed the initial stages of differentiation by modulating photoreceptor gene expression. At later stages, the presence of retinoic acid led to the generation of mature retinal organoids with a well-structured stratified photoreceptor layer containing a predominant rod population. By contrast, the absence of retinoic acid led to cone-rich organoids with a less organised and non-stratified photoreceptor layer. CONCLUSIONS: This study proves the importance of supplemented media for culturing retinal organoids. More importantly, we demonstrate for the first time that the role of retinoic acid goes beyond inducing a rod cell fate to enhancing the organisation of the photoreceptor layer of the mature organoid.
Asunto(s)
Células Madre Pluripotentes Inducidas , Organoides , Diferenciación Celular , Humanos , Organoides/metabolismo , Retina/metabolismo , Tretinoina/farmacologíaRESUMEN
The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) was developed in 2006 and represented a major breakthrough in stem cell research. A more recent milestone in biomedical research was reached in 2013 when the CRISPR/Cas9 system was used to edit the genome of mammalian cells. The coupling of both human (h)iPSCs and CRISPR/Cas9 technology offers great promise for cell therapy and regenerative medicine. However, several limitations including time and labor consumption, efficiency and efficacy of the system, and the potential off-targets effects induced by the Cas9 nuclease still need to be addressed. Here, we describe a detailed method for easily engineering genetic changes in hiPSCs, using a nucleofection-mediated protocol to deliver the CRISPR/Cas9 components into the cells, and discuss key points to be considered when designing your experiment. The clonal, genome-edited hiPSC line generated via our method can be directly used for downstream applications.
Asunto(s)
Edición Génica , Células Madre Pluripotentes Inducidas , Animales , Sistemas CRISPR-Cas/genética , Células Cultivadas , Edición Génica/métodos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mamíferos/genéticaRESUMEN
Ependymal cells reside in the adult spinal cord and display stem cell properties in vitro. They proliferate after spinal cord injury and produce neurons in lower vertebrates but predominantly astrocytes in mammals. The mechanisms underlying this glial-biased differentiation remain ill-defined. We addressed this issue by generating a molecular resource through RNA profiling of ependymal cells before and after injury. We found that these cells activate STAT3 and ERK/MAPK signaling post injury and downregulate cilia-associated genes and FOXJ1, a central transcription factor in ciliogenesis. Conversely, they upregulate 510 genes, seven of them more than 20-fold, namely Crym, Ecm1, Ifi202b, Nupr1, Rbp1, Thbs2 and Osmr-the receptor for oncostatin, a microglia-specific cytokine which too is strongly upregulated after injury. We studied the regulation and role of Osmr using neurospheres derived from the adult spinal cord. We found that oncostatin induced strong Osmr and p-STAT3 expression in these cells which is associated with reduction of proliferation and promotion of astrocytic versus oligodendrocytic differentiation. Microglial cells are apposed to ependymal cells in vivo and co-culture experiments showed that these cells upregulate Osmr in neurosphere cultures. Collectively, these results support the notion that microglial cells and Osmr/Oncostatin pathway may regulate the astrocytic fate of ependymal cells in spinal cord injury.
Asunto(s)
Linaje de la Célula , Epéndimo/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Oncostatina M/metabolismo , ARN/genética , Traumatismos de la Médula Espinal/genética , Células Madre/patología , Animales , Diferenciación Celular/genética , Proliferación Celular/genética , Cilios/genética , Regulación hacia Abajo/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Subunidad beta del Receptor de Oncostatina M , ARN/metabolismo , Esferoides Celulares/metabolismo , Médula Espinal/patología , Regulación hacia Arriba/genéticaRESUMEN
Human-induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) is a powerful tool for pathophysiological studies and preclinical therapeutic screening, as well as a source for clinical cell transplantation. Thus, it must be validated for maturity and functionality to ensure correct data readouts and clinical safety. Previous studies have validated hiPSC-derived RPE as morphologically characteristic of the tissue in the human eye. However, information concerning the expression and functionality of ion channels is still limited. We screened hiPSC-derived RPE for the polarized expression of a panel of L-type (CaV 1.1, CaV 1.3) and T-type (CaV 3.1, CaV 3.3) Ca2+ channels, K+ channels (Maxi-K, Kir4.1, Kir7.1), and the Cl- channel ClC-2 known to be expressed in native RPE. We also tested the roles of these channels in key RPE functions using specific inhibitors. In addition to confirming the native expression profiles and function of certain channels, such as L-type Ca2+ channels, we show for the first time that T-type Ca2+ channels play a role in both phagocytosis and vascular endothelial growth factor (VEGF) secretion. Moreover, we demonstrate that Maxi-K and Kir7.1 channels are involved in the polarized secretion of VEGF and pigment epithelium-derived factor (PEDF). Furthermore, we show a novel localization for ClC-2 channel on the apical side of hiPSC-derived RPE, with an overexpression at the level of fluid-filled domes, and demonstrate that it plays an important role in phagocytosis, as well as VEGF and PEDF secretion. Taken together, hiPSC-derived RPE is a powerful model for advancing fundamental knowledge of RPE functions.
Asunto(s)
Canales de Calcio Tipo T/metabolismo , Canales de Cloruro/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Canales de Potasio/metabolismo , Epitelio Pigmentado de la Retina/fisiología , Canales de Calcio Tipo T/genética , Diferenciación Celular , Canales de Cloruro/genética , Regulación de la Expresión Génica , Humanos , Canales de Potasio/genéticaRESUMEN
Inherited retinal dystrophies (IRDs) are characterized by progressive photoreceptor degeneration and vision loss. Usher syndrome (USH) is a syndromic IRD characterized by retinitis pigmentosa (RP) and hearing loss. USH is clinically and genetically heterogeneous, and the most prevalent causative gene is USH2A. USH2A mutations also account for a large number of isolated autosomal recessive RP (arRP) cases. This high prevalence is due to two recurrent USH2A mutations, c.2276G>T and c.2299delG. Due to the large size of the USH2A cDNA, gene augmentation therapy is inaccessible. However, CRISPR/Cas9-mediated genome editing is a viable alternative. We used enhanced specificity Cas9 of Streptococcus pyogenes (eSpCas9) to successfully achieve seamless correction of the two most prevalent USH2A mutations in induced pluripotent stem cells (iPSCs) of patients with USH or arRP. Our results highlight features that promote high target efficacy and specificity of eSpCas9. Consistently, we did not identify any off-target mutagenesis in the corrected iPSCs, which also retained pluripotency and genetic stability. Furthermore, analysis of USH2A expression unexpectedly identified aberrant mRNA levels associated with the c.2276G>T and c.2299delG mutations that were reverted following correction. Taken together, our efficient CRISPR/Cas9-mediated strategy for USH2A mutation correction brings hope for a potential treatment for USH and arRP patients.
RESUMEN
Anamniotes, rodents, and young humans maintain neural stem cells in the ependymal zone (EZ) around the central canal of the spinal cord, representing a possible endogenous source for repair in mammalian lesions. Cell diversity and genes specific for this region are ill defined. A cellular and molecular resource is provided here for the mouse and human EZ based on RNA profiling, immunostaining, and fluorescent transgenic mice. This uncovered the conserved expression of 1,200 genes including 120 transcription factors. Unexpectedly the EZ maintains an embryonic-like dorsal-ventral pattern of expression of spinal cord developmental transcription factors (ARX, FOXA2, MSX1, and PAX6). In mice, dorsal and ventral EZ cells express Vegfr3 and are derived from the embryonic roof and floor plates. The dorsal EZ expresses a high level of Bmp6 and Gdf10 genes and harbors a subpopulation of radial quiescent cells expressing MSX1 and ID4 transcription factors.
Asunto(s)
Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , ARN/genética , Médula Espinal/metabolismo , Células Madre/metabolismo , Animales , Células Madre Embrionarias/citología , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Femenino , Humanos , Factor de Transcripción MSX1/genética , Factor de Transcripción MSX1/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Persona de Mediana Edad , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , ARN/metabolismo , Médula Espinal/citología , Nicho de Células Madre , Células Madre/citología , Adulto JovenRESUMEN
The proliferation and differentiation of neural stem cells are tightly controlled by intrinsic and extrinsic cues. Cell adhesion molecules are increasingly recognized as regulators of these processes. Here we report the expression of the olfactory cell adhesion molecule (OCAM/NCAM2/RNCAM) during mouse spinal cord development and in neural stem cells cultured as neurospheres. OCAM is also weakly expressed in the dormant adult stem cell niche around the central canal and is overexpressed after spinal cord injury. Both transmembrane (TM) and glycosylphosphatidylinositol (GPI)-linked isoforms are present in neurospheres. Electron microscopy and internalisation experiments revealed a dynamic trafficking of OCAM between the membrane and intracellular compartments. After differentiation, OCAM remains in neurons and oligodendrocytes whereas no expression is detected in astrocytes. Using OCAM knockout (KO) mice, we found that mutant spinal cord stem cells showed an increased proliferation and self-renewal rates although no effect on differentiation was observed. This effect was reversed by lentivirus-mediated re-introduction of OCAM. Mechanistically, we identified the ErbB2/Neu/HER2 protein as being implicated in the enhanced proliferation of mutant cells. ErbB2 protein expression and phosphorylation level were significantly increased in KO cells whereas no difference was observed at the mRNA level. Overexpression of ErbB2 in wild-type and mutant cells also increased their growth while reintroduction of OCAM in mutant cells reduced the level of phosphorylated ErbB2. These results indicate that OCAM exerts a posttranscriptional control on the ErbB2 signalling in spinal cord stem cells. This study adds further support for considering cell adhesion molecules as regulators of the ErbB signalling.
Asunto(s)
Células Madre Embrionarias/metabolismo , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Receptor ErbB-2/biosíntesis , Médula Espinal/metabolismo , Animales , Adhesión Celular/genética , Proliferación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Moléculas de Adhesión de Célula Nerviosa/genética , ARN Mensajero/biosíntesis , Receptor ErbB-2/genética , Transducción de Señal/genética , Médula Espinal/crecimiento & desarrolloRESUMEN
BACKGROUND: The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin+ Sox2+ neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium). RESULTS: Here we report the isolation and long term propagation of another population of Nestin+ cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges. CONCLUSION: Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.
Asunto(s)
Calcificación Fisiológica/fisiología , Proteínas de Homeodominio/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Cultivo Primario de Células/métodos , Médula Espinal/irrigación sanguínea , Médula Espinal/metabolismo , Adulto , Adhesión Celular/fisiología , Separación Celular/métodos , Proteínas de Homeodominio/sangre , Humanos , Proteínas de Filamentos Intermediarios/sangre , Miocitos del Músculo Liso/citología , Proteínas del Tejido Nervioso/sangre , Nestina , Médula Espinal/citologíaRESUMEN
BACKGROUND: The dual specificity phosphatase cdc25C was the first human cdc25 family member found to be essential in the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline-dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/activation of cdc25C would be part of an amplification loop with cdk1/cyclin B1. METHODOLOGY/PRINCIPAL FINDINGS: Using highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130, we show here that these two phospho-forms of cdc25C represent distinct pools with differential localization during human mitosis. Phosphorylation on T67 occurs from prophase and the cdc25C-pT67 phospho-isoform closely localizes with condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in living cells, that the sequence around pT130 acts as a true Polo Box Domain (PBD) binding site as previously identified from in vitro peptide screening studies. Overexpression of non-phosphorylatable alanine mutant forms for each isoform, but not wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylation of cdc25C at mitosis. CONCLUSIONS/SIGNIFICANCE: These results show for the first time that in human mitosis, distinct phospho-isoforms of cdc25C exist with different localizations and interacting partners, thus implying that the long-standing model of a cdc25C/cdk1 multi-site auto amplification loop is implausible.
Asunto(s)
Mitosis/fisiología , Fosfatasas cdc25/metabolismo , Células Cultivadas , Centrosoma/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Mitosis/genética , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Treonina/metabolismo , Fosfatasas cdc25/genéticaRESUMEN
IGF-I and its receptor IGF-IR are seen as critical effectors of muscle hypertrophy, a notion recently questioned. Using MKR transgenic mice that express a dominant negative IGF-IR only in skeletal muscle, we have examined the role of the IGF-IR signaling in differentiation and repair of muscle fibers after damage-induced muscle regeneration. This process is impaired in MKR muscle, with incomplete regeneration, persistence of infiltrating cells and sustained expression of differentiation markers. Analysis of MKR and WT muscle-derived progenitor stem cells and myoblasts showed twice as many such cells in MKR muscle and an incomplete in vitro differentiation, that is, despite similar levels of myogenin expression, the level of fusion of MKR myoblasts was significantly reduced in comparison to WT myoblasts. These data show IGF-IR signaling is not only required at early hyperplasia stages of muscle differentiation, but also for late stages of myofiber maturation and hypertrophy.
Asunto(s)
Diferenciación Celular/fisiología , Músculo Esquelético/fisiología , Mioblastos/fisiología , Receptor IGF Tipo 1/metabolismo , Regeneración/fisiología , Animales , Células Cultivadas , Técnicas de Inactivación de Genes , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Músculo Esquelético/citología , Músculo Esquelético/patología , Mioblastos/citología , Receptor IGF Tipo 1/genética , Transducción de Señal/fisiología , Células Madre/fisiologíaRESUMEN
Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal beta III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.
Asunto(s)
Linaje de la Célula , Músculo Esquelético/citología , Miocardio/citología , Neuronas/citología , Células Madre/citología , Animales , Western Blotting , Adhesión Celular , Diferenciación Celular , Separación Celular , Células Cultivadas , Técnicas de Cocultivo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas , Cadenas Pesadas de Miosina/metabolismo , Fenotipo , Regeneración , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante de Células MadreRESUMEN
Akt1 and Akt2 are the major isoforms of Akt expressed in muscle cells and muscle tissue. We have performed siRNA silencing of Akt1 and Akt2 in C2 myoblasts to characterize their specific implication in muscle differentiation. Whereas silencing Akt2, and not Akt1, inhibited cell cycle exit and myoblast differentiation, Akt2 overexpression led to an increased proportion of differentiated myoblasts. In addition, we demonstrate that Akt2 is required for myogenic conversion induced by MyoD overexpression in fibroblasts. We show Akt2, but not Akt1, binds Prohibitin2/Repressor of Estrogen Activator, PHB2/REA, a protein recently implicated in transcriptionnal repression of myogenesis. Co-immunoprecipitation experiments on endogenous proteins showed the Akt2-REA complex does not contain Prohibitin1. We have analyzed expression and localization of PHB2/REA during proliferation and differentiation of mouse and human myoblasts. PHB2/REA shows punctated nuclear staining which partially co-localizes with Akt2 in differentiated myotubes and PHB2 levels decrease at the onset of myogenic differentiation concomitant with an increase in Akt2. There appears to be an inverse correlation between Akt2 and PHB2 protein levels where cells silenced for Akt2 expression show increased level of PHB2/REA and overexpression of Akt2 resulted in decreased Prohibitin2/REA. Taken together, these results, along with our previous observations, clearly show that Akt2 and not Akt1 plays a major and early role in cell cycle exit and myogenic differentiation and this function involves its specific interaction with PHB2/REA.
Asunto(s)
Diferenciación Celular/fisiología , Músculo Esquelético/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Represoras/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular , Núcleo Celular/metabolismo , Medios de Cultivo , Citoplasma/metabolismo , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Directa , Perfilación de la Expresión Génica , Ratones , Microinyecciones , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/enzimología , Miogenina/metabolismo , Pruebas de Precipitina , Prohibitinas , Unión Proteica , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , TransfecciónRESUMEN
Citrobacter freundii cells produce L-methionine gamma-lyase when grown on a medium containing L-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded L-methionine gamma-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3'-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found.
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Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Citrobacter freundii/enzimología , Citrobacter freundii/genética , Genoma Bacteriano , Secuencia de Bases , Liasas de Carbono-Azufre/aislamiento & purificación , Clonación Molecular , Evolución Molecular , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
The sigma(70) subunit of Escherichia coli RNA polymerase (RNAP) is a transcription initiation factor that can also be associated with RNAP during elongation. We provide biochemical evidence that sigma(70) induces a transcription pause at the lacUV5 promoter after RNAP has synthesized a 17-nucleotide transcript. The sigma(70)-dependent pausing requires an interaction between sigma(70) and a part of the lac repressor operator sequence resembling a promoter -10 consensus. The polysaccharide heparin triggers the release of sigma(70) from the paused complexes, supporting the view that during the transition from initiation to elongation the interactions between sigma(70) and core RNAP are weakened. We propose that the binding and retention of sigma(70) in elongation complexes are stabilized by its ability to form contacts with DNA of the transcription bubble. In addition, we suggest that the sigma(70) subunit in the elongation complex may provide a target for regulation of gene expression.