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M L DePamphilis

Publications and source records attributed to M L DePamphilis.

At least 37 records · Page 2Linked to original sources
PubMed1997

Transcription factor mTEAD-2 is selectively expressed at the beginning of zygotic gene expression in the mouse.

mTEF-1 is the prototype of a family of mouse transcription factors that share the same TEA DNA binding domain (mTEAD genes) and are widely expressed in adult tissues. At least one member of this family is expressed at the beginning of mouse development, because mTEAD transcription factor activity was not detected in oocytes, but first appeared at the 2-cell stage in development, concomitant with the onset of zygotic gene expression. Since embryos survive until day 11 in the absence of mTEAD-1 (TEF-1), another family member likely accounts for this activity. Screening an EC cell cDNA library yielded mTEAD-1, 2 and 3 genes. RT-PCR detected RNA from all three of these genes in oocytes, but upon fertilization, mTEAD-1 and 3 mRNAs disappeared. mTEAD-2 mRNA, initially present at approx. 5,000 copies per egg, decreased to approx. 2,000 copies in 2-cell embryos before accumulating to approx. 100,000 copies in blastocysts, consistent with degradation of maternal mTEAD mRNAs followed by selective transcription of mTEAD-2 from the zygotic genome. In situ hybridization did not detect mTEAD RNA in oocytes, and only mTEAD-2 was detected in day-7 embryos. Northern analysis detected all three RNAs at varying levels in day-9 embryos and in various adult tissues. A fourth mTEAD gene, recently cloned from a myotube cDNA library, was not detected by RT-PCR in either oocytes or preimplantation embryos. Together, these results reveal that mTEAD-2 is selectively expressed for the first 7 days of embryonic development, and is therefore most likely responsible for the mTEAD transcription factor activity that appears upon zygotic gene activation.

Animals↗
PubMed1997

Changes in histone synthesis and modification at the beginning of mouse development correlate with the establishment of chromatin mediated repression of transcription.

The transition from a late 1-cell mouse embryo to a 4-cell embryo, the period when zygotic gene expression begins, is accompanied by an increasing ability to repress the activities of promoters and replication origins. Since this repression can be relieved by either butyrate or enhancers, it appears to be mediated through chromatin structure. Here we identify changes in the synthesis and modification of chromatin bound histones that are consistent with this hypothesis. Oocytes, which can repress promoter activity, synthesized a full complement of histones, and histone synthesis up to the early 2-cell stage originated from mRNA inherited from the oocyte. However, while histones H3 and H4 continued to be synthesized in early 1-cell embryos, synthesis of histones H2A, H2B and H1 (proteins required for chromatin condensation) was delayed until the late 1-cell stage, reaching their maximum rate in early 2-cell embryos. Moreover, histone H4 in both 1-cell and 2-cell embryos was predominantly diacetylated (a modification that facilitates transcription). Deacetylation towards the unacetylated and monoacetylated H4 population in fibroblasts began at the late 2-cell to 4-cell stage. Arresting development at the beginning of S-phase in 1-cell embryos prevented both the appearance of chromatin-mediated repression of transcription in paternal pronuclei and synthesis of new histones. These changes correlated with the establishment of chromatin-mediated repression during formation of a 2-cell embryo, and the increase in repression from the 2-cell to 4-cell stage as linker histone H1 accumulates and core histones are deacetylated.

Acetylation↗
PubMed1996

Uncoupling of transcription and translation during zygotic gene activation in the mouse.

Zygotic gene expression in mice is delayed by a time-dependent mechanism until the two-cell stage in development. To investigate the basis of this 'zygotic clock', the firefly luciferase gene was injected into mouse embryos, and quantitative assays were used to monitor luciferase gene transcription and translation in individual embryos from single mothers. These studies confirmed, at the mRNA level, previous conclusions about the relative capacities of paternal and maternal pronuclei to transcribe genes, and the requirements for promoters and enhancers during zygotic gene activation. Furthermore, these studies revealed that fertilized mouse eggs can delay expression of zygotic genes by uncoupling translation from transcription. An RNA polymerase II-dependent gene could be translated until zygotic gene expression began (a delay of up to 15 h after injection). The time course for nascent mRNA accumulation was biphasic, with the second phase occurring during zygotic gene expression. If the luciferase gene was injected after zygotic gene expression had begun, then translation was tightly linked to transcription. If the second phase of mRNA accumulation was repressed, then luciferase was not produced. Therefore, translation was linked to the accumulation of mRNA during the onset of zygotic gene expression. Similar biphasic time courses also were observed for RNA polymerase I- and III-dependent transcription. These and other results reveal that the zygotic clock regulates the onset of both transcription and translation of zygotic genes.

Amanitins↗
PubMed1995

Regulation of gene expression at the beginning of mammalian development.

The maternal to zygotic transition can be viewed as a cascade of events that begins when fertilization triggers the zygotic clock that delays early ZGA until formation of a 2-cell embryo. Early ZGA, in turn, appears to be required for expression of late ZGA, and late ZGA is required to form a 4-cell embryo. ZGA in mammals is a time-dependent mechanism rather than a cell cycle-dependent mechanism that delays both transcription and translation of nascent transcripts. Thus, zygotic gene transcripts appear to be handled differently than maternal mRNA, a phenomenon also observed in Xenopus (55). The length of this delay is species-dependent, occurring at the 2-cell stage in mice, the 4-8-cell stage in cows and humans, and the 8-16-cell stage in sheep and rabbits (4). However, concurrent with formation of a 2-cell embryo in the mouse and rabbit (47,56), perhaps in all mammals, a general chromatin-mediated repression of promoter activity appears. Repression factors are inherited by the maternal pronucleus from the oocyte but are absent in the paternal pronucleus and not available until sometime during the transition from a late 1-cell to a 2-cell embryo. This means that paternally inherited genes are exposed to a different environment in fertilized eggs than are maternally inherited genes, a situation that could contribute to genomic imprinting. Chromatin-mediated repression of promoter activity prior to ZGA is similar to what is observed during Xenopus embryogenesis (31,32) and ensures that genes are not expressed until the appropriate time in development when positive acting factors, such as enhancers, can relieve this repression. The ability to use enhancers appears to depend on the acquisition of specific co-activators at the 2-cell stage in mice and perhaps later in other mammals (47,56), concurrent with ZGA. Even then, the mechanism by which enhancers communicate with promoters changes during development (Fig. 2), providing an opportunity for enhancer-mediated stimulating of TATA-less promoters (e.g. housekeeping genes) early during development while eliminating this mechanism later during development.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Mimosine arrests DNA synthesis at replication forks by inhibiting deoxyribonucleotide metabolism.

Mimosine has been reported to specifically prevent initiation of DNA replication in the chromosomes of mammalian nuclei. To test this hypothesis, the effects of mimosine were examined in several DNA replication systems and compared with the effects of aphidicolin, a specific inhibitor of replicative DNA polymerases. Our results demonstrated that mimosine inhibits DNA synthesis in mitochondrial, nuclear, and simian virus 40 (SV40) genomes to a similar extent. Furthermore, mimosine and aphidicolin were indistinguishable in their ability to arrest SV40 replication forks and mammalian nuclear chromosomal replication forks. In contrast to aphidicolin, mimosine did not inhibit DNA replication in lysates of mammalian cells supplied with exogenous deoxyribonucleotide triphosphate precursors for DNA synthesis. Mimosine also had no effect on initiation or elongation of DNA replication in Xenopus eggs or egg extracts containing high levels of deoxyribonucleotide triphosphates. In parallel with its inhibitory effect on DNA synthesis in mammalian cells, mimosine altered deoxyribonucleotide triphosphate pools in a manner similar to that reported for another DNA replication inhibitor that affects deoxyribonucleotide metabolism, hydroxyurea. Taken together, these results show that mimosine inhibits DNA synthesis at the level of elongation of nascent chains by altering deoxyribonucleotide metabolism.

Animals↗
PubMed1995

A unique role for enhancers is revealed during early mouse development.

Transcription and replication of genes in mammalian cells always requires a promoter or replication origin, respectively, but the ability of enhancers to stimulate these regulatory elements and the interactions that mediate this stimulation are developmentally acquired. The primary function of enhancers is to prevent repression, which appears to result from particular components of chromatin structure. Factors responsible for this repression are present in the maternal nucleus of oocytes and its descendant, the maternal pronucleus of mouse 1-cell embryos and in mouse 2-cell embryos, but are absent in the paternal pronucleus. Thus, enhancers are not needed to achieve efficient transcription and replication in paternal pronuclei. However, enhancers, even in the presence of their specific activation protein, are inactive prior to formation of a 2-cell embryo, suggesting that a coactivator essential for enhancer function is not available until zygotic gene expression begins. Furthermore, enhancer stimulation of transcription appears to be mediated through a promoter transcription factor, but this interaction can change as cells undergo differentiation, switching from a TATA-box independent to a TATA-box dependent mode.

Animals↗
PubMed1995

Repression of gene expression at the beginning of mouse development.

The transition from maternal to zygotic gene expression in the mouse occurs in the 2-cell embryo. Previous studies in which DNA was injected into 2-cell embryos revealed that transcription promoters and origins of DNA replication are strongly repressed in cleavage stage embryos unless linked to an embryo-responsive enhancer. Repression also occurs when DNA is injected into the paternal pronucleus of a 1-cell embryo and the injected embryo subsequently undergoes mitosis, except that repression is no longer relieved by enhancers. Here we extend this observation to maternal pronuclei in 1-cell embryos and show that this repression could not be relieved either by linking the promoter to an embryo-responsive enhancer or by inducing hyperacetylation of chromatin inorder to increase its accessibility to transcription factors. However, repression could be relieved by transplanting the injected pronucleus to a 2-cell embryo, even when the recipient cell subsequently underwent mitosis. Both the extent of promoter repression and the ability of enhancers to stimulate promoter activity increased as development proceeded from the early 2-cell stage to the 4-cell stage. Once repression was established in late 2-cell embryos, transplanting an injected 2-cell embryo nucleus back to an early 1-cell embryo failed to restore activity to the injected promoter, even when it was linked to an enhancer. These and other data demonstrate that cytoplasmic factors appear during formation of a 2-cell embryo that can repress promoter activity and activate enhancer activity. These factors are absent from the paternal pronucleus and cytoplasm of early (S-phase arrested) 1-cell embryos. Moreover, the cytoplasm of early 1-cell embryos appears to lack the ability to reprogram expression of genes once they have progressed to the late 2-cell stage in mouse development.

Animals↗
PubMed1995

Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.

Animals↗
PubMed1994

Requirements for DNA transcription and replication at the beginning of mouse development.

In mice, the first round of DNA replication occurs in fertilized eggs (1-cell embryos), while the onset of zygotic gene transcription begins approximately 20 hours after fertilization, a time that normally coincides with formation of a 2-cell embryo. One approach to investigating the mechanisms that control these developmentally regulated events has been to microinject plasmid DNA into the nuclei of mouse oocytes and embryos in order to determine the requirements for unique DNA sequences that regulate transcription and replication. The results from these and other studies have revealed two important mechanisms that regulate the beginning of animal development. The first is a time dependent "zygotic clock" of unknown detail that delays the onset of transcription, regardless of whether or not a 2-cell embryo is formed. The second is a mechanism that represses the activity of promoters and origins of replication specifically in maternal pronuclei of oocytes and 1-cell embryos, and in all nuclei of 2-cell embryos, regardless of their parental origin or ploidy. This repression is linked to chromatin, but the striking ability to relieve this repression with specific embryo-responsive enhancers first appears with formation of a 2-cell embryo. The need for a TATA-box to mediate enhancer stimulation of promoter activity appears even later when cell differentiation becomes evident. Thus, a biological clock delays transcription until both paternal and maternal genomes are replicated and remodeled from a post-meiotic state to one in which transcription is repressed by chromatin structure in a manner that can be relieved by cell-specific enhancers at appropriate times during development.

Animals↗
PubMed1994

Papillomavirus contains cis-acting sequences that can suppress but not regulate origins of DNA replication.

Bovine papillomavirus (BPV) DNA has been reported to restrict its own replication and that of the lytic simian virus 40 (SV40) origin to one initiation event per molecule per S phase, which suggests BPV DNA replication as a model for cellular chromosome replication. Suppression of the SV40 origin required two cis-acting BPV sequences (NCOR-1 and -2) and one trans-acting BPV protein. The results presented in this paper confirm the presence of two NCOR sequences in the BPV genome that can suppress polyomavirus (PyV) as well as SV40 origin-dependent DNA replication as much as 40-fold. However, in contrast to results of previous studies on SV40, most of the suppression of the PyV origin was due to NCOR-1, a 512-bp sequence that functioned independently of distance or orientation with respect to the PyV origin and that was not required for BPV DNA replication. Moreover, NCOR-1 alone or together with NCOR-2 did not restrict the ability of the PyV ori to reinitiate replication within a single S phase and did not require any BPV protein to exert suppression. Furthermore, NCOR-1 did not suppress BPV origin-dependent DNA replication except in the presence of PyV large tumor antigen (T-ag). Since NCOR-1 suppression of PyV origin activity also varied with T-ag concentration, suppression of origins by NCOR sequences appeared to require papovavirus T-ag. Therefore, it is unlikely that NCOR sequences are involved in regulating BPV DNA replication. When these results are taken together with those from other laboratories, BPV appears to be a slowly replicating version of papovaviruses rather than a model for origins of DNA replication in eukaryotic cell chromosomes.

Antigens, Viral, Tumor↗
PubMed1994

TATA-dependent enhancer stimulation of promoter activity in mice is developmentally acquired.

Herpes simplex virus (HSV) thymidine kinase (tk) promoter activity depends on four transcription factor binding sites, one of which is a TATA box sequence, and the presence of either a cis-acting enhancer sequence or a transactivator protein. Studies presented here show that this TATA box was required for promoter activity only after cells began to differentiate and then only when promoter activity was stimulated by either an enhancer or a transactivator. When the HSV tk promoter was utilized by mouse embryos from the one-cell to eight-cell stage of development or by undifferentiated mouse embryonic stem cells, disruption of the HSV tk TATA box by site-specific mutations did not reduce promoter activity. This was true even when HSV tk promoter activity was stimulated strongly by either the embryo-responsive polyomavirus F101 enhancer or its natural transactivator, the HSV ICP4 gene product. However, stimulated expression was dependent on a distal Sp1 DNA binding site. Similarly, disruption of the TATA box did not reduce tk promoter activity in primary mouse embryonic fibroblasts or in immortalized 3T3 mouse fibroblasts; in fact, promoter activity was increased up to 2.6-fold. However, in these differentiated cells, stimulation of the HSV tk promoter by either the F101 enhancer or ICP4 protein required the TATA box. HSV tk promoter activity also was dependent on its TATA box in the mouse oocyte, a terminally differentiated cell with an endogenous transactivating activity. These results reveal that the need for a TATA box is developmentally acquired and depends on at least two parameters: the differentiated state of the cell and stimulation of the promoter by either an enhancer or a transactivator.

3T3 Cells↗
PubMed1993

Origins of DNA replication in metazoan chromosomes.

In metazoan chromosomes, initiation of DNA replication occurs primarily at specific sites (0.5-3 kb; the OBR) using the same replication fork mechanism favored by simple genomes. Nucleosome segregation is distributive. However, initiation events also occur at other sites randomly distributed throughout a larger initiation zone (8-55 kb). These nonspecific initiation events presumably occur at a lower frequency than those at the OBR. Although origins in native chromosomes appear to be genetically determined, ori sequences have been convincingly demonstrated only during programmed gene amplification. It seems likely that metazoan origins include both specific DNA sequences as well as chromatin structure and nuclear organization, something that may be difficult to reproduce with plasmid DNA. Definitive answers will come only with a functional assay for initiation that exhibits the sequence-specific characteristics observed in vivo.

Animals↗
PubMed1993

Analysis of gene expression in mouse preimplantation embryos demonstrates that the primary role of enhancers is to relieve repression of promoters.

Enhancers are generally viewed simply as extensions of promoters, lacking a function of their own. However, previous studies of mouse preimplantation embryos revealed that 1-cell embryos can utilize enhancer-responsive promoters efficiently without an enhancer, whereas 2-cell embryos require an enhancer to achieve the same levels of expression. This suggested that enhancers relieved a repression in 2-cell embryos that is absent in 1-cell embryos. Results presented here demonstrate first that the ability of 1-cell embryos to dispense with enhancers does not result from the absence of specific activation proteins. Under conditions where GAL4-VP16 activated a GAL4-dependent promoter in both embryos, GAL4-VP16 activated a GAL4-dependent enhancer only in 2-cell embryos. Moreover, the role of an enhancer is not to compensate for either changes in promoter requirements, or for reduced levels of promoter-specific transcription factors. Linker-scanning mutations in a natural promoter revealed that both embryos utilized the same promoter elements, and comparison of different promoters revealed that these embryos have equivalent transcriptional capacities. In addition, titration experiments revealed less Sp1 activity in 1-cell embryos where enhancers are dispensable than in 2-cell embryos where enhancers are required. Therefore, we propose that the primary function of enhancers, first evident with formation of a mouse 2-cell embryo, is to prevent repression of weak promoters, probably by altering chromatin structure. Consistent with this hypothesis is the fact that butyrate, an agent that alters chromatin structure, stimulated promoters in 2-cell embryos, but not in 1-cell embryos.

Animals↗
PubMed1993

Transcription enhancer factor-1 (TEF-1) DNA binding sites can specifically enhance gene expression at the beginning of mouse development.

In an effort to identify transcriptional elements that are recognized at different stages of early mouse development, polyomavirus (PyV) enhancer mutations were selected for their ability to support PyV transcription and replication in various mouse undifferentiated embryonal carcinoma (EC) and embryonic stem (ES) cell lines. Several of these enhancer mutations were then isolated, sequenced and tested for their ability to stimulate the PyV early gene promoter in plasmid DNA that was either transfected into EC, ES and fibroblast cell lines, or injected into the nuclei of mouse 1-cell and 2-cell embryos. EC, ES and fibroblast cell lines showed clear preferences for different enhancer configurations, and cleavage-stage embryos (2- to 8-cell stage) strongly preferred the same enhancer configuration favored by ES cells. This 'embryo responsive' (ER) enhancer configuration was characterized by a tandem duplication of the region containing a single point mutation that created a DNA binding site for Transcription Enhancer Factor-1 (TEF-1). ER enhancers stimulated the PyV promoter up to 350-fold in embryos, and were up to 74-fold more active than the wild-type PyV enhancer. Most of the activity from PyER enhancers could be duplicated in 2-cell embryos by synthesizing only the tandemly repeated sequence. Comparison of these synthetic enhancers with ER enhancers confirmed that TEF-1 DNA binding sites were highly preferred in ES cells and cleavage-stage embryos, and suggested that ER enhancer activity resulted primarily from cooperative interaction between either two closely spaced TEF-1 DNA binding sites or two TEF-1 DNA binding sites separated by a third, as yet unidentified, transcription factor binding site. These results provide a prototype of a mammalian embryo responsive enhancer, and suggest that TEF-1 plays an important role in activation of gene expression at the beginning of mammalian development.

Animals↗
PubMed1993

Requirements for promoter activity in mouse oocytes and embryos distinguish paternal pronuclei from maternal and zygotic nuclei.

Fertilization of mouse eggs produces a 1-cell embryo containing both a paternal and maternal pronucleus. These two nuclei combine during the first mitosis to form the zygotic nuclei of 2-cell embryos. This transition is accompanied by the onset of transcription and the decline of maternal mRNA-dependent gene expression. To determine how changes in nuclear composition affect gene expression, plasmid DNA containing a promoter and an enhancer that function throughout a broad host range was injected into nuclei of oocytes and embryos. The requirements for promoter activity in paternal pronuclei of 1-cell embryos were distinct from those in maternal or zygotic nuclei: (1) Paternal pronuclei permitted high levels of promoter activity relative to maternal or zygotic nuclei. (2) Butyrate, an agent that alters chromatin structure, stimulated promoter activity in maternal or zygotic nuclei, but not in paternal pronuclei. (3) The embryo-responsive polyomavirus F101 enhancer also stimulated promoter activity, but only after formation of a 2-cell embryo. Either butyrate or the F101 enhancer stimulated promoter activity in zygotic nuclei to the level observed in paternal pronuclei. Stimulation also was observed with 2-cell embryos containing nuclei of only maternal or paternal origin, but their transcriptional capacity was more limited. These and other results support the hypothesis that the need for enhancers in 2-cell embryos results from repression by chromatin structure, and the role of enhancers is to relieve this repression.

Animals↗