Initiation Of Terminal Differentiation Research Articles

MicroRNA Profiling in Human Neutrophils during Bone Marrow Granulopoiesis and In Vivo Exudation

The purpose of this study was to describe the microRNA (miRNA) expression profiles of neutrophils and their precursors from the initiation of granulopoiesis in the bone marrow to extravasation and accumulation in skin windows. We analyzed three different cell populations from human bone marrow, polymorphonuclear neutrophil (PMNs) from peripheral blood, and extravasated PMNs from skin windows using the Affymetrix 2.0 platform. Our data reveal 135 miRNAs differentially regulated during bone marrow granulopoiesis. The majority is differentially regulated between the myeloblast/promyelocyte (MB/PM) and myelocyte/metamyelocyte (MC/MM) stages of development. These 135 miRNAs were divided into six clusters according to the pattern of their expression. Several miRNAs demonstrate a pronounced increase or reduction at the transition between MB/PM and MC/MM, which is associated with cell cycle arrest and the initiation of terminal differentiation. Seven miRNAs are differentially up-regulated between peripheral blood PMNs and extravasated PMNs and only one of these (miR-132) is also differentially regulated during granulopoiesis. The study indicates that several different miRNAs participate in the regulation of normal granulopoiesis and that miRNAs might also regulate activities of extravasated neutrophils. The data present the miRNA profiles during the development and activation of the neutrophil granulocyte in healthy humans and thus serves as a reference for further research of normal and malignant granulocytic development.

MiRNA Profiling in Human Granulopoiesis and in Activated Neutrophils From Skin Window.

AbstractAbstract 2136Formation of polymorphonuclear neutrophils (PMN) is a tightly regulated process where the myeloid progenitor cells, myeloblasts (MBs), divide and mature in the bone marrow, along a well defined path. The cells pass through six well defined stages in differentiation ending up with the release of mature PMNs to peripheral blood (granulopoiesis). Expression of essential transcription factors such as RUNX1, C/EBP-a, and C/EBP-e during granulopoiesis has been shown to have great importance for correct neutrophil development. microRNAs (miRNAs) could be important players in the fine-tuning of transcription factor expression due to their ability to regulate protein synthesis.The function of neutrophils is to detect and destroy invading microorganisms. This involves activation of the PMNs in the blood stream causing a release of secretory vesicles and up-regulation of extracellular adhesion molecules followed by migration in the tissue towards the focus of inflammation. Expression of miRNAs might also be regulated during activation and diapedesis of the neutrophils in order to adapt the neutrophil to its new environment and function.A regulatory role for miRNAs has been demonstrated for several biological processes, such as proliferation, differentiation, inflammation and cancer, and dysregulation of miRNA expression has been shown to contribute to disease development.The purpose of this study was to determine the miRNA expression profiles during normal human granulopoiesis starting with the first identifiable granulocytic precursor cell (MB) and ending with activated neutrophils that have migrated into the tissue using an Affymetrix 2,0 miRNA microarray platform. We isolated four populations of cells: Myeloblasts (MB) and promyelocytes (PM), myelocytes (MC) and metamyelocytes (MM), and band cells (BC) and segmented cells (SC) from the bone marrow and PMNs from peripheral blood from three different donors. We found 135 differentially expressed miRNAs in granulopoiesis, which could be divided into six clusters according to their expression pattern. 87% of the 135 miRNAs were differentially regulated between the MB/PM (dividing cells) and the MC/MM stages (cessation of cell proliferation and initiation of terminal differentiation) and could imply a need for miRNA-mediated regulation of the many proteins involved in regulating this process. Interestingly, we also found two distinct clusters of miRNAs that were either up- or down-regulated only in the MC/MM population, indicating the importance of a specific temporary regulation of some proteins during neutrophil development.To determine miRNA expression profiles in activated granulocytes, we examined PMNs and activated neutrophils from skin window (i.e. PMNs migrated to a site of inflammation). We found seven differentially expressed miRNAs, - all of them up regulated in the activated neutrophils.Using microRNA target-prediction software, we found that miRNAs 155, 146a and 130a, all of which are strongly up-regulated in the MB/PM stage, have several targets in the IL1-receptor signalling cascade, indicating the importance of miRNA of dampening an innate immune response in immature neutrophil precursors.miR-146a, 155 and 130a also have predicted targets in either the TGF-βI or the TGF-βII receptor which inhibits proliferation when binding to TGF-β. This finding supports the proliferating profile for the MB/PM cells, and the shift towards cell cycle arrest when the cells differentiate to the next stage, where expression of these three miRNAs is low. miRNA-34c-3p is highly expressed only in the MC/MM stage and has verified targets in many different mRNAs involved in the regulation of cell cycle arrest. All the miRNAs that were up-regulated in the activated neutrophils have several predicted targets in the IL1R pathway, and some of them (miR-212, −132 and −297) have previously been shown to be important in regulating the inflammatory response.The study indicates that several different miRNAs have important roles in the regulation of normal granulopoiesis, and that miRNAs also might be part of a possible negative feed back loop in the inflammatory response in activated neutrophils.Grant acknowledgments: The Danish Cancer Society, Lundbeck foundation, Danish Medical Research Council, Brøchner Mortensen foundation Disclosures:No relevant conflicts of interest to declare.

Atypical E2F activity restrains APC/C CCS52A2 function obligatory for endocycle onset

The endocycle represents an alternative cell cycle that is activated in various developmental processes, including placental formation, Drosophila oogenesis, and leaf development. In endocycling cells, mitotic cell cycle exit is followed by successive doublings of the DNA content, resulting in polyploidy. The timing of endocycle onset is crucial for correct development, because polyploidization is linked with cessation of cell division and initiation of terminal differentiation. The anaphase-promoting complex/cyclosome (APC/C) activator genes CDH1, FZR, and CCS52 are known to promote endocycle onset in human, Drosophila, and Medicago species cells, respectively; however, the genetic pathways governing development-dependent APC/C(CDH1/FZR/CCS52) activity remain unknown. We report that the atypical E2F transcription factor E2Fe/DEL1 controls the expression of the CDH1/FZR orthologous CCS52A2 gene from Arabidopsis thaliana. E2Fe/DEL1 misregulation resulted in untimely CCS52A2 transcription, affecting the timing of endocycle onset. Correspondingly, ectopic CCS52A2 expression drove cells into the endocycle prematurely. Dynamic simulation illustrated that E2Fe/DEL1 accounted for the onset of the endocycle by regulating the temporal expression of CCS52A2 during the cell cycle in a development-dependent manner. Analogously, the atypical mammalian E2F7 protein was associated with the promoter of the APC/C-activating CDH1 gene, indicating that the transcriptional control of APC/C activator genes by atypical E2Fs might be evolutionarily conserved.

Developmental signals in skin morphogenesis.

Developmental signals in skin morphogenesis.

Transient activation of FOXN1 in keratinocytes induces a transcriptional programme that promotes terminal differentiation: contrasting roles of FOXN1 and Akt.

The forkhead transcription factor FOXN1 is required for normal cutaneous and thymic epithelial development. Mutations in FOXN1 give rise to the nude phenotype in mice, rats and man. However, the genes that are regulated by FOXN1 are unknown. To investigate FOXN1 function we expressed an inducible form of the protein, FOXN1ER, that is activated by 4-hydroxytamoxifen in primary human epidermal keratinocytes. Transient activation of FOXN1 decreased the proportion of keratinocytes that formed actively growing clones attributable to stem cell founders and increased the number of abortive clones, without inducing apoptosis. Within 24 hours the majority of cells had initiated terminal differentiation, as assessed by involucrin expression. We performed a cDNA microarray experiment to analyse changes in the transcription of approximately 6,000 genes. Following FOXN1 activation we detected increases of two fold or greater in the RNA levels of over 30 genes. Genes promoting growth arrest, survival and differentiation featured prominently and markers of early events in keratinocyte differentiation were also detected. Since one of the induced genes was Akt we investigated whether Akt played a role in terminal differentiation. Activation of PI 3-kinase but not Akt was necessary for FOXN1-induced differentiation. In reconstituted epidermis FOXN1 promoted early stages of terminal differentiation whereas Akt activation was sufficient to induce late stages, including formation of the cornified layers. These results establish a role for FOXN1 in initiation of terminal differentiation and implicate Akt in subsequent events.

Immunohistochemical analyses of DNA topoisomerase II isoforms in developing rat cerebellum.

In mammalian cells, two isoforms of DNA topoisomerase II (topo IIalpha and topo IIbeta) have been identified. Topo IIalpha is essential in mitotic cells, whereas the function of topo IIbeta remains unclear. In the present study, we investigated the developmental control of topo II isoforms in two different neuronal lineages, cerebellar Purkinje cells and granule cells, by immunohistochemical analysis with isoform-specific monoclonal antibodies. As expected, proliferating cells in the neuroepithelium and in the external germinal layer (EGL) were topo IIalpha immunopositive. The migrating as well as differentiating Purkinje cells and granule cells showed an enhanced topo IIbeta immunoreactivity. The postmitotic granule cells in the postnatal EGL showed an abrupt transition of expressed topo II isoforms from IIalpha to IIbeta. The transition was clearly coincident with the completion of final cell division and the initiation of terminal differentiation because no increase of the topo IIbeta immunoreactivity was observed in the spreading EGL cells that are still in the cell division cycle. The topo IIbeta signal was detected in both nucleoplasm and nucleolus of differentiating cells. However, the nucleoplasmic signal decreased significantly as the cells reached terminal differentiation. The residual topo IIbeta in nucleoli was shown to occupy an unique location with respect to other nucleolar proteins, nucleolin and DNA topoisomerase I. Our findings indicate that both Purkinje cells and granule cells express the topo II isoforms in a similar timing during the cerebellar development and also suggest that topo IIbeta localized in nucleoplasm is the functional entity involved in neuronal differentiation.

Transgene expression in human epidermal keratinocytes: cell cycle arrest of productively transfected cells.

We have analysed the consequences of liposome mediated gene transfer into human primary epidermal keratinocytes and compared non-Epstein-Barr Virus (EBV) and EBV based expression vectors that carry the genes encoding human Growth Hormone (hGH) or Enhanced Green Fluorescent Protein (EGFP). Different kinetics between the non-EBV and EBV based vectors were revealed upon subcultivation of hGH transfected keratinocytes. The keratinocytes transfected with non-EBV based vector showed a rapid reduction in hGH production. Although the EBV based vector resulted in more stable expression, this was also reduced over time. Chromatin inactivation by deacetylation was investigated by treatment with sodium butyrate and found not to be the reason for the decreasing expression. Keratinocytes divided into subpopulations enriched for either stem cells or transit amplifying cells, based on beta1-integrin expression and function, do not differ significantly with respect to susceptibility to productive transfection. However, when the keratinocytes were transfected with the EGFP gene and sorted live by FACS into EGFP negative and positive populations, only the negative cells were capable of forming significant numbers of colonies. This is consistent with the observation that the ability to incorporate BrdU was dramatically reduced in the EGFP expressing population within 24-48 h post transfection indicating an almost complete cell cycle arrest. p53 levels were unaffected by the procedures, and the keratinocyte cell line HaCat, mutated in both p53 alleles, also shows a marked reduction in clonogenic potency upon transfection. There was a slight increase of TUNEL positive apoptotic nuclei in the positive population at early time points. However, the apoptotic index was still very low. When we measured the frequency of involucrin expressing cells, we found an increase in the productively transfected population over time indicating an initiation of terminal differentiation. In contrast to the transfected cultures, keratinocytes that were transduced using a retroviral vector showed no decrease in colony forming efficiency. In conclusion we find that transgene expressing cells from transfected cultures of epidermal keratinocytes undergo cell cycle arrest and initiate terminal differentiation by mechanisms which are independent of p53 levels.

Elevated cyclin E levels, inactive retinoblastoma protein, and suppression of the p27(KIP1) inhibitor characterize early development of promyeloid cells into macrophages.

Cyclin-dependent kinase inhibitors such as p27(KIP1) have recently been shown to lead to cellular differentiation by causing cell cycle arrest, but it is unknown whether similar events occur in differentiating promyeloid cells. Hematopoietic progenitor cells undergo lineage-restricted differentiation, which is accompanied by expression of distinct maturation markers. Here we show that the classical growth factor insulin-like growth factor I (IGF-I) potently promotes vitamin D(3)-induced macrophage differentiation of promyeloid cells, as assessed by measurement of a coordinate increase in expression of the integrin alpha subunit CD11b, the CD14 lipopolysaccharide receptor, and the macrophage-specific esterase, alpha-naphthyl acetate esterase, as early as 24 h following initiation of terminal differentiation. Addition of IGF-I to cells undergoing vitamin D(3)-induced differentiation also leads to an early increase in expression of cyclin E, phosphorylation of the retinoblastoma tumor suppressor protein, and a doubling of the cell number. Early expression of CD11b (24 h) is simultaneously accompanied by inhibition in the expression of p27(KIP1). Cell cycle analysis with propidium iodide revealed that CD11b expression at 24 h following initiation of differentiation occurs at all phases of the cell cycle instead of only those cells arrested in G(0)/G(1). Similarly, development of a novel double-labeling intra- and extracellular flow-cytometric technique demonstrated that single cells expressing the mature leukocyte differentiation antigen CD11b can also incorporate the thymidine analog bromodeoxyuridine. Likewise, expression of the intracellular DNA polymerase delta cofactor/proliferating-cell nuclear antigen at 24 h is also simultaneously expressed with the surface marker CD11b, indicating that these cells continue to proliferate early in their differentiation program. Finally, at 24 h following induction of differentiation, IGF-I promoted a fourfold increase in the uptake of [(3)H]thymidine by purified populations of CD11b-expressing cells. Taken together, these data demonstrate that the initial steps associated with terminal macrophage differentiation occur concomitantly with progression through the cell cycle and that these very early differentiation events do not require the accumulation of p27(KIP1).

Diverse developing mouse lineages exhibit high-level c-Myb expression in immature cells and loss of expression upon differentiation.

The c-myb gene encodes a sequence specific transactivator that is required for fetal hematopoiesis, but its potential role in other tissues is less clear because of the early fetal demise of mice with targeted deletions of the c-myb gene and incomplete of knowledge about c-myb's expression pattern. In the hematopoietic system, c-Myb protein acts on target genes whose expression is restricted to individual lineages, despite Myb's presence and role in multiple immature lineages. This suggests that c-Myb actions within different cell type-specific contexts are strongly affected by combinatorial interactions. To consider the possibility of similar c-Myb actions could extend into non-hematopoietic systems in other cell and tissue compartments, we characterized c-myb expression in developing and adult mice using in situ hybridization and correlated this with stage-specific differentiation and mitotic activity. Diverse tissues exhibited strong c-myb expression during development, notably tooth buds, the thyroid primordium, developing trachea and proximal branching airway epithelium, hair follicles, hematopoietic cells, and gastrointestinal crypt epithelial cells. The latter three of these all maintained high expression into adulthood, but with characteristic restriction to immature cell lineages prior to their terminal differentiation. In all sites, during fetal and adult stages, loss of c-Myb expression correlated strikingly with the initiation of terminal differentiation, but not the loss of mitotic activity. Based on these data, we hypothesize that c-Myb's function during cellular differentiation is both an activator of immature gene expression and a suppressor of terminal differentiation in diverse lineages.

Potassium chloride inhibits proliferation of cerebellar granule neuron progenitors

CNS neurogenesis involves a critical transition where neuronal progenitors exit the cell cycle and initiate terminal differentiation. Recent experiments have suggested that depolarization inhibits DNA synthesis in cortical progenitors. Depolarization of proliferating neuronal progenitors may thus activate mechanisms that prevent proliferation and allow the initiation of terminal differentiation. We present evidence that depolarizing concentrations of KCl (25–50 mM) reduce proliferation of developing postnatal cerebellar granule cells in culture. These studies show that KCl antagonizes the mitogenic response of granule cells to insulin-like growth factor-I (IGF-I) and that this reduction in proliferating cells is not the result of a selective cell death. We also examined the differentiation of granule cell cultures using Brn-5 expression as an early differentiation marker. In vivo Brn-5 expression occurs soon after developing granule cells exit the cell cycle and begin their final differentiation. In control cultures and cultures treated with high concentrations of KCl Brn-5 expression increased over 24–48 h of culture. Our results suggest depolarizing concentrations of KCl antagonize proliferation of cerebellar granule neuron progenitors however allow their continued differentiation.

Targeted disruption of the MYC antagonist MAD1 inhibits cell cycle exit during granulocyte differentiation.

The switch from transcriptionally activating MYC-MAX to transcriptionally repressing MAD1-MAX protein heterodimers has been correlated with the initiation of terminal differentiation in many cell types. To investigate the function of MAD1-MAX dimers during differentiation, we disrupted the Mad1 gene by homologous recombination in mice. Analysis of hematopoietic differentiation in homozygous mutant animals revealed that cell cycle exit of granulocytic precursors was inhibited following the colony-forming cell stage, resulting in increased proliferation and delayed terminal differentiation of low proliferative potential cluster-forming cells. Surprisingly, the numbers of terminally differentiated bone marrow and peripheral blood granulocytes were essentially unchanged in Mad1 null mice. This imbalance between the frequencies of precursor and mature granulocytes was correlated with a compensatory decrease in granulocytic cluster-forming cell survival under apoptosis-inducing conditions. In addition, recovery of the peripheral granulocyte compartment following bone marrow ablation was significantly enhanced in Mad1 knockout mice. Two Mad1-related genes, Mxi1 and Mad3, were found to be expressed ectopically in adult spleen, indicating that functional redundancy and cross-regulation between MAD family members may allow for apparently normal differentiation in the absence of MAD1. These findings demonstrate that MAD1 regulates cell cycle withdrawal during a late stage of granulocyte differentiation, and suggest that the relative levels of MYC versus MAD1 mediate a balance between cell proliferation and terminal differentiation.

Integrin expression in normal, hyperplastic, dysplastic, and malignant oral epithelium.

We have examined the distribution of a range of integrin subunits in normal and lesional oral mucosa. The alpha 2, alpha 3, alpha 6, beta 1, and beta 4 subunits were highly expressed in normal epithelium, and there was weaker, more variable expression of alpha 5 and alpha v. Expression of all subunits was highest in the basal layer of normal epithelium, but extensive staining above the basal layer was also observed, particularly in the floor of the mouth and the lateral margin of the tongue. In dysplastic lesions and hyperplastic epithelium adjacent to ulcers, suprabasal staining was even more pronounced. Staining patterns in squamous cell carcinomas showed considerable variation, both within and between individual tumours: in some areas there was staining reminiscent of normal epithelium, but uniform staining throughout tumour islands, and patchy and variable cytoplasmic and pericellular staining were also seen. Thirteen out of 17 carcinomas showed some loss of integrin expression: six out of ten moderately well differentiated tumours and all the poorly differentiated tumours. Focal loss of alpha 6 and beta 4 was most commonly observed, but loss of alpha 2 and alpha 3 also occurred. Since integrins regulate not only keratinocyte adhesion, but also the initiation of terminal differentiation, the changes in integrin expression that we have observed may have significance for the behaviour of individual tumours.

α5 Integrin Is a Critical Component of Adhesion Plaques in Myogenesis

We investigated the distribution and expression of α5β1 and α3β1 integrin in differentiating myogenic cells in culture. The myogenic cells expressed both α5 and α3 integrin subunits with the same molecular sizes as those expressed by chicken embryo fibroblasts (CEF). However, the ratio of total α5 to α3 was threefold higher in the muscle cultures than that in CEF cultures. A new method is described whereby adhesion plaque-associated integrin was cross-linked to its extracellular matrix ligand on the substrate using a nonpenetrating cross-linker, BS e, and integrin not involved in substrate adhesion as well as cytoskeletal proteins were removed with a zwitterionic detergent. α5 and β1 integrin, but not α3 could be cross-linked to fibronectin at adhesion plaques throughout myogenesis in culture. α5β1 integrin was found only at the edge of myoblasts 4 hr after plating but became distributed under their entire surface by 1 day in culture. When the muscle cells became elongated, a morphology they express after the initiation of terminal differentiation, and as they began to fuse, α5 was found redistributed in small adhesion plaques along the lateral edges of the postmitotic myocytes and early myotubes. In mature myotubes, which are large multinucleated branched structures, α5β1 integrin was localized to parallel streaks underneath their entire substrate surface. Throughout the different stages of myogenesis, vinculin colocalized with α5β1 integrin in adhesion plaques, but α-actinin only colocalized to the adhesion plaques in myoblasts, not in myotubes. These studies suggest that α5β1 integrin through its dynamic interaction with both fibronectin and the cytoskeleton is important for both the signals which initiate the differentiation process and for subsequent morphological and structural changes during the differentiation process.

Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis.

We have examined integrin expression during the remodeling of the epidermis that takes place during wound healing, using a suction blister model in which the epidermis is detached from the dermis, leaving the basement membrane intact. By immunofluorescence microscopy, we found that the same integrin subunits were expressed during wound healing as in normal epidermis with very little change in the relative intensity or distribution of staining at the leading edge of the migrating epidermis. However, at the time of wound closure, when the epidermis is still hyperproliferative, alpha 2, alpha 3, alpha 6, and beta 1 were no longer confined to the basal layer, as in normal epidermis, but were also found in all the living suprabasal cell layers, coexpressed with the terminal differentiation markers involucrin, keratin 10, and keratin 16. Strong suprabasal staining for alpha v was also found in one specimen. beta 4, which normally forms a heterodimer with alpha 6, and alpha 5 remained predominantly basal. Three of the integrin ligands, fibronectin, type IV collagen, and laminin, remained largely confined to the basement membrane zone and dermis. By 14 d after wounding, the integrins were once more restricted to the basal layer. Suprabasal integrin expression was also observed in involved psoriatic lesions. Thus, in two situations in which the epidermis is hyperproliferative, there is a failure to downregulate integrin expression on initiation of terminal differentiation. The functional consequences of this aberrant integrin expression remain to be explored.

Changes in the abundance and distribution of actin and associated proteins during terminal differentiation of human epidermal kératinocytes

We have examined the abundance and distribution of actin and several actin-associated proteins in human epidermal keratinocytes before and after initiation of terminal differentiation. Keratinocytes were placed in suspension in methylcellulose for 1 h or 24 h and then extracted for immunoblotting. At 24 h, when the proportion of cells expressing the terminal differentiation marker, involucrin, had increased approximately 3-fold, there were marked decreases in the levels of vinculin, talin, filamin and gelsolin. The level of actin was unchanged and the level of alpha-actinin decreased only slightly. To complement the immunoblot analysis, we also examined the distribution of each protein in basal (involucrin-negative) and suprabasal (involucrin-positive) cells in stratified colonies, using confocal microscopy. Gelsolin, filamin, vinculin, talin, alpha-actinin and filamentous actin were all less abundant in suprabasal cells than in basal cells. There were also differences in the distribution of all the proteins in the basal compared to the suprabasal layers. In addition to the changes associated with terminal differentiation, there was variation in the distribution of focal contacts and stress fibres and in gelsolin levels between basal cells at the periphery of colonies and those in the centre. These results are discussed in the context of the known association of the actin cytoskeleton with receptors of the integrin family, the loss of integrins that occurs during keratinocyte terminal differentiation, and the possible role of the cytoskeleton in signalling between integrins and the nucleus.

Glyceraldehyde‐3‐Phosphate Dehydrogenase Is a Nonhistone Protein and a Possible Activator of Transcription in Neurons

A single-stranded DNA-binding protein of Mr 35,000 (35K protein) was isolated from calf cerebral cortex by affinity chromatography on immobilized double-stranded and single-stranded DNA. Its localization in the nuclear compartment was demonstrated by immunohistochemistry. Previous studies had uncovered a homologous nonhistone chromosomal protein in the nuclei of rat cerebral cortex neurons, cerebellar neurons, oligodendrocytes, and liver cells. The rat protein accumulated in the nuclear compartment of neurons in exact temporal coincidence with the arrest of cell division and the initiation of terminal differentiation. Therefore, in the present work, the 35K protein was tested for an activating role in RNA transcription. During the course of this study we became aware that the 35K protein was identical to a glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). When authentic GAPDH from rabbit skeletal muscle was injected into Xenopus laevis oocytes, it greatly stimulated RNA polymerase II transcription, whereas the 35K protein from calf brain did not. This apparent discrepancy was partially resolved by the finding that rabbit muscle GAPDH could be fractionated into two components by affinity chromatography on single-stranded DNA cellulose. Only 5% of the applied protein was retained on the column and could be eluted with a shallow salt gradient identical to the one used for the isolation of the 35K protein. This single-stranded DNA-binding component of rabbit muscle GAPDH did not stimulate transcription. Apparently, the 35K protein from calf brain corresponded to this single-stranded DNA-binding subfraction, which explained its failure to activate transcription.(ABSTRACT TRUNCATED AT 250 WORDS)