Biomedical subjects

L I Rothblum

Publications and source records attributed to L I Rothblum.

At least 19 recordsLinked to original sources

PubMed2001

The role of acetylation in rDNA transcription.

Treatment of NIH 3T3 cells with trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), resulted in a dose-dependent increase in transcription from a rDNA reporter and from endogenous rRNA genes. Chromatin immunoprecipitation using anti-acetyl-histone H4 antibodies demonstrated a direct effect of TSA on the acetylation state of the ribosomal chromatin. TSA did not reverse inhibition of transcription from the rDNA reporter by retinoblastoma (Rb) protein, suggesting that the main mechanism by which Rb blocks rDNA transcription may not involve recruitment of deacetylases to rDNA chromatin. Overexpression of histone transacetylases p300, CBP and PCAF stimulated transcription in transfected NIH 3T3 cells. Recombinant p300, but not PCAF, stimulated rDNA transcription in vitro in the absence of nucleosomes, suggesting that the stimulation of rDNA transcription by TSA might have a chromatin-independent component. We found that the rDNA transcription factor UBF was acetylated in vivo. Finally, we also demonstrated the nucleolar localization of CBP. Our results suggest that the organization of ribosomal chromatin of higher eukaryotes is not static and that acetylation may be involved in affecting these dynamic changes directly through histone acetylation and/or through acetylation of UBF or one of the other components of rDNA transcription.

3T3 Cells↗

PubMed2001

An immediate response of ribosomal transcription to growth factor stimulation in mammals is mediated by ERK phosphorylation of UBF.

Ribosomal transcription in mammals is regulated in response to growth, differentiation, disease, and aging, but the mechanisms of this regulation have remained unresolved. We show that epidermal growth factor induces immediate, ERK1/2-dependent activation of endogenous ribosomal transcription, while inactivation of ERK1/2 causes an equally immediate reversion to the basal transcription level. ERK1/2 was found to phosphorylate the architectural transcription factor UBF at amino acids 117 and 201 within HMG boxes 1 and 2, preventing their interaction with DNA. Mutation of these sites inhibited transcription activation and abrogated the transcriptional response to ERK1/2. Thus, growth factor regulation of ribosomal transcription likely acts by a cyclic modulation of DNA architecture. The data suggest a central role for ribosome biogenesis in growth regulation.

Animals↗

PubMed2001

Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes.

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant (P < or = 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.

Adenoviridae↗

PubMed2001

Revealing the unseen: the organizer region of the nucleolus.

We carried out a high-resolution ultrastructural analysis of the nucleolus in mouse P815 cells by combining specific DNA and RNA staining, anti-fibrillarin immunolabeling, contrast enhancement by energy filtering TEM and phosphorus mapping by ESI to visualize nucleic acids. We demonstrated that specifically contrasted DNA, fibrillarin and phosphorus overlap within the nucleolar dense fibrillar component. Moreover, we describe a 'DNA cloud' consisting of an inner core of DNA fibers (fibrillar center) and a periphery made of extremely thin fibrils overlapping the anti-fibrillarin immunolabeling (dense fibrillar component). This highly sensitive approach has allowed us to demonstrate, for the first time, the exact distribution of DNA within the decondensed interphase counterpart of the NOR, which includes both the fibrillar center and the dense fibrillar component.

Animals↗

PubMed2000

Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the interaction between UBF and SL-1.

We have previously demonstrated that the protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a regulator of transcription by RNA polymerase I (rDNA transcription) by inhibiting UBF-mediated transcription. In the present study, we have examined the mechanism by which Rb represses UBF-dependent rDNA transcription and determined if other Rb-like proteins have similar effects. We demonstrate that authentic or recombinant UBF and Rb interact directly and this requires a functional A/B pocket. DNase footprinting and band-shift assays demonstrated that the interaction between Rb and UBF does not inhibit the binding of UBF to DNA. However, the formation of an UBF/Rb complex does block the interaction of UBF with SL-1, as indicated by using the 48 kDa subunit as a marker for SL-1. Additional evidence is presented that another pocket protein, p130 but not p107, can be found in a complex with UBF. Interestingly, the cellular content of p130 inversely correlated with the rate of rDNA transcription in two physiological systems, and overexpression of p130 inhibited rDNA transcription. These results suggest that p130 may regulate rDNA transcription in a similar manner to Rb.

Animals↗

PubMed2000

RNA polymerase I transcription in confluent cells: Rb downregulates rDNA transcription during confluence-induced cell cycle arrest.

When 3T6 cells are confluent, they withdraw from the cell cycle. Concomitant with cell cycle arrest a significant reduction in RNA polymerase I transcription (80% decrease at 100% confluence) is observed. In the present study, we examined mechanism(s) through which transcription of the ribosomal genes is coupled to cell cycle arrest induced by cell density. Interestingly with an increase in cell density (from 3 - 43% confluence), a significant accumulation in the cellular content of hyperphosphorylated Rb was observed. As cell density increased further, the hypophosphorylated form of Rb became predominant and accumulated in the nucleoli. Co-immunoprecipitation experiments demonstrated there was also a significant rise in the amount of hypophosphorylated Rb associated with the rDNA transcription factor UBF. This increased interaction between Rb and UBF correlated with the reduced rate of rDNA transcription. Furthermore, overexpression of recombinant Rb inhibited UBF-dependent activation of transcription from a cotransfected rDNA reporter in either confluent or exponential cells. The amounts or activities of the rDNA transcription components we examined did not significantly change with cell cycle arrest. Although the content of PAF53, a polymerase associated factor, was altered marginally (decreased 38%), the time course and magnitude of the decrease did not correlate with the reduced rate of rDNA transcription. The results presented support a model wherein regulation of the binding of UBF to Rb and, perhaps the cellular content of PAF53, are components of the mechanism through which cell cycle and rDNA transcription are linked. Oncogene (2000) 19, 3487 - 3497

Animals↗

PubMed2000

Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP.

In situ sites of nucleolar transcription in cells microinjected with 5-bromo-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4-90 min at 37 degrees C, fixed, and embedded in LR White resin. Postembedding immunoelectron microscopic visualization with colloidal gold has been used for localizing both Br-labeled precursor incorporated into pre-rRNA and different nucleolar transcription or processing factors. This high resolution approach allowed us to identify significant signal as early as after 4-min incubation periods following BrUTP microinjection. It revealed the dense fibrillar component (DFC) as being the first nucleolar compartment labeled with anti-bromodeoxyuridine antibody. Moreover, RNA polymerase I, nucleolar transcription factor UBF, and fibrillarin were also detected almost exclusively in this same nucleolar compartment. From 30 min onward, following microinjection, Br-labeled rRNA occurred also in the granular component. The results indicate that the DFC is the site of pre-rRNA transcription and of initial steps of pre-rRNA processing. Moreover, it demonstrates that BrUTP microinjection followed by postembedding detection of Br-labeled RNA is a useful technique for high resolution studies of structure-function associations in the nucleolus.

Cell Nucleolus↗

PubMed2000

Competitive recruitment of CBP and Rb-HDAC regulates UBF acetylation and ribosomal transcription.

RNA polymerase I (PolI) transcription is activated by the HMG box architectural factor UBF, which loops approximately 140 bp of DNA into the enhancesome, necessitating major chromatin remodeling. Here we show that the acetyltransferase CBP is recruited to and acetylates UBF both in vitro and in vivo. CBP activates PolI transcription in vivo through its acetyltransferase domain and acetylation of UBF facilitates transcription derepression and activation in vitro. CBP activation and Rb suppression of ribosomal transcription by recruitment to UBF are mutually exclusive, regulating in vivo PolI transcription through an acetylation-deacetylation "flip-flop." Thus, PolI transcription is regulated by protein acetylation, and the competitive recruitment of CBP and Rb.

3T3 Cells↗

PubMed2000

Sprint training normalizes Ca(2+) transients and SR function in postinfarction rat myocytes.

Previous studies have shown that myocytes isolated from sedentary (Sed) rat hearts 3 wk after myocardial infarction (MI) undergo hypertrophy, exhibit altered intracellular Ca(2+) concentration ([Ca(2+)](i)) dynamics and abnormal contraction, and impaired sarcoplasmic reticulum (SR) function manifested as prolonged half-time of [Ca(2+)](i) decline. Because exercise training elicits positive adaptations in cardiac contractile function and myocardial Ca(2+) regulation, the present study examined whether 6-8 wk of high-intensity sprint training (HIST) would restore [Ca(2+)](i) dynamics and SR function in MI myocytes toward normal. In MI rats, HIST ameliorated myocyte hypertrophy as indicated by significant (P </= 0.05) decreases in whole cell capacitances [Sham-Sed 179 +/-12 (n = 20); MI-Sed 226 +/- 7 (n = 20); MI-HIST 183 +/- 11 pF (n = 19)]. HIST significantly (P < 0.0001) restored both systolic [Ca(2+)](i) [Sham-Sed 421 +/- 9 (n = 79); MI-Sed 350 +/- 6 (n = 70); MI-HIST 399 +/- 9 nM (n = 70)] and half-time of [Ca(2+)](i) decline (Sham-Sed 0. 197 +/- 0.005; MI-Sed 0.247 +/- 0.006; MI-HIST 0.195 +/- 0.006 s) toward normal. Compared with Sham-Sed myocytes, SR Ca(2+)-ATPase expression significantly (P < 0.001) decreased by 44% in MI-Sed myocytes. Surprisingly, expression of SR Ca(2+)-ATPase was further reduced in MI-HIST myocytes to 26% of that measured in Sham-Sed myocytes. There were no differences in calsequestrin expression among the three groups. Expression of phospholamban was not different between Sham-Sed and MI-Sed myocytes but was significantly (P < 0.01) reduced in MI-HIST myocytes by 25%. Our results indicate that HIST instituted shortly after MI improves [Ca(2+)](i) dynamics in surviving myocytes. Improvement in SR function by HIST is mediated not by increased SR Ca(2+)-ATPase expression, but by modulating phospholamban regulation of SR Ca(2+)-ATPase activity.

Animals↗

PubMed1999

Identification of the erythropoietin receptor domain required for calcium channel activation.

Erythropoietin (Epo) activates a voltage-independent Ca2+ channel that is dependent on tyrosine phosphorylation. To identify the domain(s) of the Epo receptor (Epo-R) required for Epo-induced Ca2+ influx, Chinese hamster ovary (CHO) cells were transfected with wild-type or mutant Epo receptors subcloned into pTracer-cytomegalovirus vector. This vector contains an SV40 early promoter, which drives expression of the green fluorescent protein (GFP) gene, and a cytomegalovirus immediate-early promoter driving expression of the Epo-R. Successful transfection was verified in single cells by detection of GFP, and intracellular Ca2+ ([Ca]i) changes were simultaneously monitored with rhod-2. Transfection of CHO cells with pTracer encoding wild-type Epo-R, but not pTracer alone, resulted in an Epo-induced [Ca]i increase that was abolished in cells transfected with Epo-R F8 (all eight cytoplasmic tyrosines substituted). Transfection with carboxyl-terminal deletion mutants indicated that removal of the terminal four tyrosine phosphorylation sites, but not the tyrosine at position 479, abolished Epo-induced [Ca]i increase, suggesting that tyrosines at positions 443, 460, and/or 464 are important. In CHO cells transfected with mutant Epo-R in which phenylalanine was substituted for individual tyrosines, a significant increase in [Ca]i was observed with mutants Epo-R Y443F and Epo-R Y464F. The rise in [Ca]i was abolished in cells transfected with Epo-R Y460F. Results were confirmed with CHO cells transfected with plasmids expressing Epo-R mutants in which individual tyrosines were added back to Epo-R F8 and in stably transfected Ba/F3 cells. These results demonstrate a critical role for the Epo-R cytoplasmic tyrosine 460 in Epo-stimulated Ca2+ influx.

Animals↗

PubMed1998

Transcription by RNA polymerase I.

The genes that code for 45S rRNA, the precursor of 18S, 5.8S and 28S rRNA, are transcribed by RNA polymerase I. In many eukaryotes the genes are arranged as tandem repeats in discrete chromosomal clusters. rDNA transcription and rRNA processing occur in the nucleolus. In vertebrates, at least two factors, SL-1 and UBF, specific for transcription by RNA polymerase I cooperate in the formation of the initiation complex. Interestingly, there are proteins analogous to SL-1 in unicellular eukaryotes, but the requirement for a UBF-like factor appears to vary. Recent advances in our understanding of the rDNA transcription system and its regulation have demonstrated overlap with the other nuclear transcription systems (RNA polymerase II and III). This is exemplified by the utilization of TBP as a component of SL-1 and the role of Rb in regulatory rDNA transcription.

Animals↗

PubMed1998

Topology of recombinant rat upstream binding factor.

Transmission electron microscopy and single particle electron crystallography were employed to reconstruct high-quality projection images of a recombinant, acidic tail deficient form of rat upstream binding factor. The upstream binding factor was found to be dimeric and approximately 10 nm in diameter with a central region of low density. Distinct nodes were observable, of size and spacing consistent with being HMG boxes 3 and 4. The dimerisation domain seemed most probably to be located in the internal region of the structure.

Animals↗

PubMed1998

Regulation of ribosomal DNA transcription by insulin.

The experiments reported here used 3T6-Swiss albino mouse fibroblasts and H4-II-E-C3 rat hepatoma cells as model systems to examine the mechanism(s) through which insulin regulates rDNA transcription. Serum starvation of 3T6 cells for 72 h resulted in a marked reduction in rDNA transcription. Treatment of serum-deprived cells with insulin was sufficient to restore rDNA transcription to control values. In addition, treatment of exponentially growing H4-II-E-C3 with insulin stimulated rDNA transcription. However, for both cell types, the stimulation of rDNA transcription in response to insulin was not associated with a change in the cellular content of RNA polymerase I. Thus we conclude that insulin must cause alterations in formation of the active RNA polymerase I initiation complex and/or the activities of auxiliary rDNA transcription factors. In support of this conclusion, insulin treatment of both cell types was found to increase the nuclear content of upstream binding factor (UBF) and RNA polymerase I-associated factor 53. Both of these factors are thought to be involved in recruitment of RNA polymerase I to the rDNA promoter. Nuclear run-on experiments demonstrated that the increase in cellular content of UBF was due to elevated transcription of the UBF gene. In addition, overexpression of UBF was sufficient to directly stimulate rDNA transcription from a reporter construct. The results demonstrate that insulin is capable of stimulating rDNA transcription in both 3T6 and H4-II-E-C3 cells, at least in part by increasing the cellular content of components required for assembly of RNA polymerase I into an active complex.

Animals↗

PubMed1997

cDNA cloning, genomic organization, and in vivo expression of rat N-syndecan.

The amino acid sequence of rat N-syndecan core protein was deduced from the cloned cDNA sequence. The sequence predicts a core protein of 442 amino acids with six structural domains: an NH2-terminal signal peptide, a membrane distal glycosaminoglycan attachment domain, a mucin homology domain, a membrane proximal glycosaminoglycan attachment domain, a single transmembrane domain, and a noncatalytic COOH-terminal cytoplasmic domain. Transfection of human 293 cells resulted in the expression of N-syndecan that was modified by heparan sulfate chain addition. Heparitinase digestion of the expressed proteoglycan produced a core protein that migrated on SDS-polyacrylamide gels at an apparent molecular weight of 120, 000, identical to N-syndecan synthesized by neonatal rat brain or Schwann cells. Rat genomic DNA coding for N-syndecan was isolated by hybridization screening. The rat N-syndecan gene is comprised of five exons. Each exon corresponds to a specific core protein structural domain, with the exception of the fifth exon, which contains the coding information for both the transmembrane and cytoplasmic domains as well as the 3'-untranslated region of the mRNA. The first intron is large, with a length of 22 kilobases. The expression of N-syndecan was investigated in late embryonic, neonatal, and adult rats by immunoblotting and Northern blotting analysis. Among the tissues and developmental stages studied, high levels of N-syndecan expression were restricted to the early postnatal nervous system. N-syndecan was expressed in all regions of the nervous system, including cortex, midbrain, spinal cord, and peripheral nerve. Immunohistochemical staining revealed high levels of N-syndecan expression in all brain regions and fiber tract areas.

Amino Acid Sequence↗

PubMed1996

Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy.

The accelerated protein accumulation characteristic of cardiomyocyte hypertrophy results from increased cellular protein synthetic capacity (elevated ribosome content). The rate limiting step in ribosome accumulation is transcription of the rRNA genes. During neonatal cardiomyocyte hypertrophy induced by norepinephrine or spontaneous contraction, changes in the expression of a ribosomal DNA transcription factor, UBF, correlated with increased rates of ribosome biogenesis. We hypothesized that elevated expression of UBF was part of the mechanism by which these hypertrophic stimuli effected increases in the rate of transcription from the rDNA promoter. In this study, we have examined directly the effect of overexpressing UBF on rDNA transcription in neonatal cardiomyocytes in culture. In control experiments, a novel reporter construct for rDNA transcription (pSMECAT) showed similar increases in activity in response to hypertrophic stimuli (10(-4) M phenylephrine, 10(-7) M endothelin, and spontaneous contraction) as did the endogenous rRNA genes. When contraction-arrested cardiomyocytes were cotransfected with pSMECAT and increasing amounts of a UBF1 expression vector; a dose-dependent (3-5 fold) increase in rDNA transcription was observed. Western blot analysis confirmed that the overexpressed, FLAG-tagged UBF accumulated in the cardiomyocyte nuclei. The observation that overexpression of UBF1 is sufficient to increase rDNA transcription in neonatal cardiomyocytes provides evidence in support of the hypothesis that the regulation of UBF is a key component of the increased ribosome biogenesis and protein accumulation associated with cardiomyocyte hypertrophy.

Animals↗

PubMed1996

Intracellular distribution of HMG1, HMG2 and UBF change following treatment with cisplatin.

Cisplatin (CDDP) is a widely used cancer chemotherapeutic agent. CDDP forms well characterized intrastrand cross-links between adjacent purines in genomic DNA. In mammalian cells, these lesions are repaired by the nucleotide excision repair system. An early event in the recognition and processing of cis-Pt-DNA adducts may well involve the binding of specific proteins to the sites of damage. Several proteins have been identified, including high mobility group (HMG) proteins 1 and 2 and upstream binding factor (UBF), which recognize CDDP-DNA. However, the physiological significance of this binding has not been established. In this study, we have utilized antibodies to these proteins to examine the effect of CDDP on their intracellular distribution. Marked changes in the immunofluorescent staining pattern of HMG1/HMG2 were noted in cells treated with CDDP. At higher drug concentrations, the distribution of UBF also changed, from a clustered appearance associated with the nucleoli to more diffuse nuclear staining. These results demonstrate that HMG1/HMG2 and UBF respond to drug treatment, presumably by recognizing cis-Pt-DNA adduct formation in intact cells. Hence, these proteins may play an important role in directing the response of tumor cells following exposure to CDDP.

Antineoplastic Agents↗

PubMed1996

Structure of recombinant rat UBF by electron image analysis and homology modelling.

We have studied the structure of recombinant rat UBF (rrUBF), an RNA polymerase I transcription factor, by electron microscopy and image analysis of single particles contrasted with methylamine tungstate. Recombinant rat UBF appeared to be a flat, U-shaped protein with a central region of low density. In the dominant projections, 2-fold mirror symmetry was seen, consistent with the dimerization properties of this molecule, and of dimensions in agreement with the length of DNA that rat UBF protects in footprinting studies. Electron microscopy of various rrUBF-DNA complexes confirmed that our recombinant protein was fully able to bind the 45S rDNA promoter, and that it caused substantial bends in the DNA. Upon extended incubation in a droplet covered by a lipid monolayer at the liquid-air interface, rrUBF formed long filamentous arrays with a railway track appearance. This structure was interpreted to consist of overlapping rrUBF dimers 3.5 nm apart, which value would represent the thickness of the protein. Our results show rrUBF to interact with and bend the promoter DNA into a roughly 10 nm diameter superhelix. Based on all these electron microscopical results, an atomic structure was predicted by homology modelling of the HMG fingers, and connected by energy minimized intervening segments.

Animals↗

PubMed1996

Regulation of ribosomal DNA transcription during contraction-induced hypertrophy of neonatal cardiomyocytes.

Cardiac hypertrophy requires protein accumulation. This results largely from an increased capacity for protein synthesis, which in turn is the result of an elevated rate of ribosome biogenesis. The process of ribosome formation is regulated at the level of transcription of the ribosomal RNA genes. In this study, we examined the amounts and activities of various components of the ribosomal DNA transcription apparatus in contraction-arrested neonatal cardiomyocytes and in spontaneously contracting cardiomyocytes that hypertrophy. Nuclear run-on assays demonstrated that spontaneously contracting cardiomyocytes supported a 2-fold increased rate of ribosomal DNA transcription. However, enzymatic assay of total solubilized RNA polymerase I and Western blots demonstrated that contraction-induced increases in ribosomal RNA synthesis were not accompanied by increased activity or amounts of RNA polymerase I. In contrast, accelerated ribosome biogenesis was accompanied by an increased amount of the ribosomal DNA transcription factor, UBF. Immunoprecipitation of [32P]orthophosphate-labeled UBF from hypertrophying, neonatal cardiomyocytes indicated that the accumulated UBF protein was phosphorylated and, thus, in the active form. UBF mRNA levels began to increase within 3-6 h of the initiation of contraction and preceded the elevation in rDNA transcription. Nuclear run-on assays demonstrated increased rates of transcription of the UBF gene. Transfection of chimeric reporter constructs containing deletions of the 5'-flanking region of the UBF gene revealed the presence of contraction response elements between -1189 and -665 relative to the putative start of transcription. These results are consistent with the hypothesis that UBF is an important factor in the regulation of rDNA transcription during contraction-mediated neonatal cardiomyocyte hypertrophy.

Animals↗