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Biomedical subjects

H R Schöler

Publications and source records attributed to H R Schöler.

At least 19 recordsLinked to original sources
PubMed2006

Derivation of germ cells from embryonic stem cells.

Embryonic stem cells (ESCs), derivatives of cells of early mammalian embryos, have proven to be one of the most powerful tools in developmental and stem cell biology. When injected into embryos, ESCs can contribute to tissues derived from all three germ layers and to the germline. Prior studies have successfully shown that ESCs can recapitulate features of embryonic development by spontaneously forming somatic lineages in culture. Amazingly, recently it has been shown that mouse ESCs can also give rise to primordial germ cells (PGCs) in culture that are capable of undergoing meiosis and forming both male and female gametes. While the full potential of these ES-derived germ cells and gametes remains to be demonstrated, these discoveries provide a new approach for studying reproductive biology and medicine.

Animals↗
PubMed2006

Cell-cell fusion as a means to establish pluripotency.

Embryonic stem cells (ESCs), embryonic germ cells (EGCs), and embryonic carcinoma cells (ECCs) are three types of pluripotent cells derived from mammalian embryos. The three cell types are capable not only of self-renewal, but also of having the potential to give rise to cells of all tissue types in the fetal and adult body. In several reports, ESCs, ECCs, and EGCs have been described to reprogram somatic cells in vitro. After reprogramming caused by fusion, somatic cells exhibit various features of pluripotent cells: expression of pluripotency markers (e.g., Oct4, nanog, and Rex-1), absence of tissue-specific gene expression, reactivation of inactive X chromosome of female somatic cells, demethylation, as well as histone modification. An activity in pluripotent stem cells appears to be capable of inducing the global changes inherent in the reprogramming of somatic cells. Investigations involving pluripotent stem cells will yield substantial insight into various fundamental biological processes, such as cellular differentiation and de-differentiation. Most importantly for the public, however, is that such studies might lead into cell-based therapies and as such have the potential to change regenerative medicine.

Animals↗
PubMed2006

Recombinant human albumin supports development of somatic cell nuclear transfer embryos in mice: toward the establishment of a chemically defined cloning protocol.

Culturing embryos in different media is a useful approach to characterize their nature in regard to "memory" of the donor nucleus and its "reprogramming" after somatic cell nuclear transfer (SCNT). However, efforts to elucidate the mechanisms of reprogramming are seriously undermined when embryo culture conditions are not completely defined. Using recombinant human albumin (rHA) is a step toward establishing defined culture conditions for mouse cloning. Recombinant HA supports blastocyst formation of cumulus cell-derived clones at a rate comparable with two types of bovine serum albumin (BSA); following transfer of blastocysts to the genital tract, rates of development to midgestation (10.5 dpc) were indistinguishable. rHA also supports the derivation of germline competent embryonic stem (ES) cells from SCNT blastocysts at a substantial rate compared with BSA counterparts and with zygotic blastocysts. Unlike the developmental parameters, the gene expression patterns of clones cultured in rHA or BSA were not superimposed; identical patterns were observed for zygotic blastocysts in the two albumins. In summary, the present study demonstrates that (1) rHA can replace BSA, proving a defined protein source for SCNT in mice; (2) although using rHA is similar to BSA, it is not equal (rHA leaves a mark on gene expression of clones but not zygotes). Future studies that investigate reprogramming after SCNT will need to consider not only the implications of culture media for cloning but also the supplement choice.

Animals↗
PubMed2001

Crystallization of redox-insensitive Oct1 POU domain with different DNA-response elements.

The POU domain of the human Oct1 transcription factor has been crystallized with two different POU-dimer-binding DNA elements. Protein-DNA cocrystals suitable for structural analysis could be obtained only with a redox-insensitive version of the POU domain. The recombinant protein expression in a prokaryotic host was adjusted for fast purification. Optimized crystals were obtained by systematically varying the length of the oligonucleotide and by modifying cryofreezing procedures. These steps are generally applicable to the preparation of protein-DNA complexes for structural studies.

Crystallization↗
PubMed2001

Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences.

The Oct-4 gene encodes a transcription factor that is specifically expressed in embryonic stem cells and germ cells of the mouse embryo. Cells that differentiate into somatic tissues lose Oct-4 expression. Regulation of Oct-4 gene transcription involves a TATA-less minimal promoter and two upstream elements: the proximal (PE) and distal enhancers (DE). We report here the nucleotide sequence of the 5' upstream regulatory regions of the human and murine Oct-4 genes. A comparative alignment analysis between these regions and those of the bovine Oct-4 ortholog reveals four conserved regions of homology (CR 1 to 4) between these species (66-94% conservation). The 1A sequence within the mouse PE is located approximately half-way between CR 2 and CR 3. A putative Sp1/Sp3 binding site and the overlapping hormone responsive element (HRE) in CR1 are identical in all three species. A high number of CCC(A/T)CCC motifs exhibit various levels of homology in these upstream regions. We discuss the importance of these and other sequences and present candidate factors that may bind and regulate Oct-4 gene expression.

Animals↗
PubMed2001

Differential dimer activities of the transcription factor Oct-1 by DNA-induced interface swapping.

Two crystal structures of Oct-1 POU domain bound to DNA provide a rationale for differential, conformation-dependent recruitment of transcription cofactors. The POU-homeo and POU-specific subdomains of Oct-1 contain two different nonoverlapping pairs of surface patches that are capable of forming unrelated protein-protein interfaces. Members of the POU factor family contain one or two conserved sequence motifs in the interface that are known to be phosphorylated, as noted for Oct-1 and Pit-1. Modeling of Oct-4 reveals the unique case where the same conserved sequence is located in both interfaces. Our studies provide the basis for two distinct dimeric POU factor arrangements that are dictated by the architecture of each DNA response element. We suggest interface swapping in dimers could be a general mechanism of modulating the activity of transcription factors.

Amino Acid Sequence↗
PubMed2001

Mouse germline restriction of Oct4 expression by germ cell nuclear factor.

The POU-domain transcription factor Oct4 is essential for the maintenance of the mammalian germline. In this study, we show that the germ cell nuclear factor (GCNF), an orphan nuclear receptor, represses Oct4 gene activity by specifically binding within the proximal promoter. GCNF expression inversely correlates with Oct4 expression in differentiating embryonal cells. GCNF overexpression in embryonal cells represses Oct4 gene and transgene activities, and we establish a link to transcriptional corepressors mediating repression by GCNF. In GCNF-deficient mouse embryos, Oct4 expression is no longer restricted to the germ cell lineage after gastrulation. Our studies suggest that GCNF is critical in repressing Oct4 gene activity as pluripotent stem cells differentiate and in confining Oct4 expression to the germline.

Animals↗
PubMed2001

Reduced fertility and spermatogenesis defects in mice lacking chromosomal protein Hmgb2.

High mobility group 2 protein (Hmgb2) is a member of the HMGB protein family, which includes the ubiquitous Hmgb1 and the embryo-specific Hmgb3. The three proteins are more than 80% identical at the amino acid level and their biochemical properties are indistinguishable. Hmgb1 is an abundant component of all mammalian nuclei and acts as an architectural factor that bends DNA and promotes protein assembly on specific DNA targets. Cells that lack Hmgb1 can survive, although mutant mice die shortly after birth. As Hmgb2 is present in all cultured cells and is abundant in thymus, the preferred source for HMGB proteins, it was considered a ubiquitous variant of Hmgb1. We show that in adult mice Hmgb2 is restricted mainly to lymphoid organs and testes, although it is widely expressed during embryogenesis. Mice that lack Hmgb2 are viable. However, male Hmgb2(-/-) mice have reduced fertility, that correlates with Sertoli and germ cell degeneration in seminiferous tubules and immotile spermatozoa. Significantly, Hmgb2 is expressed at very high levels in primary spermatocytes, while it is barely detectable in spermatogonia and elongated spermatids. This peculiar pattern of expression and the phenotype of mutants indicate that Hmgb2 has a specialised role in germ cell differentiation.

Amino Acid Sequence↗
PubMed2001

Oct-4: gatekeeper in the beginnings of mammalian development.

The Oct-4 POU transcription factor is expressed in mouse totipotent embryonic stem and germ cells. Differentiation of totipotent cells to somatic lineages occurs at the blastocyst stage and during gastrulation, simultaneously with Oct-4 downregulation. Stem cell lines derived from the inner cell mass and the epiblast of the mouse embryo express Oct-4 only if undifferentiated. When embryonic stem cells are triggered to differentiate, Oct-4 is downregulated thus providing a model for the early events linked to somatic differentiation in the developing embryo. In vivo mutagenesis has shown that loss of Oct-4 at the blastocyst stage causes the cells of the inner cell mass to differentiate into trophectoderm cells. Recent experiments indicate that an Oct-4 expression level of roughly 50%-150% of the endogenous amount in embryonic stem cells is permissive for self-renewal and maintenance of totipotency. However, upregulation above these levels causes stem cells to express genes involved in the lineage differentiation of primitive endoderm. These novel advances along with latest findings on Oct-4-associated factors, target genes, and dimerization ability, provide new insights into the understanding of the early steps regulating mammalian embryogenesis.

Animals↗
PubMed2000

Phage display screening reveals an association between germline-specific transcription factor Oct-4 and multiple cellular proteins.

Oct-4 is a transcription factor that is specifically expressed in mouse embryonic stem cells and in cell lines derived thereof. In these cells, Oct-4 activates transcription from remote binding sites due to as of yet unknown co-activators. Expression of Oct-4 in differentiated cells is not sufficient to activate transcription from a distance, rather it requires the co-expression of co-activators such as the adenoviral oncoprotein E1A. In this paper, we used phage display to identify Oct-4-interacting proteins. We first analyzed the interaction between Oct-4 and E1A in order to optimize the biochemical conditions that enable Oct-4-specific interactions with other interacting proteins. A panning approach was used to enrich Oct-4 interacting phages from a pool of excess unspecific phages. The biochemical conditions established in our interaction assays were then used to screen a P19 EC cell cDNA expression library in M13 filamentous phage. A number of phage clones displaying portions of unknown and known transcription factors were obtained, from which the HMG-1 transcription factor was identified. HMG-1, and the closely related factor HMG-2, interact with Oct-4 when co-expressed in mammalian cells. In addition, HMG-1 was found to cooperate with Oct-4 in P19 EC cells. These results provide the first evidence of a non-viral factor that enhances Oct-4 distance-dependent transactivation in stem cells.

Adenovirus E1A Proteins↗
PubMed2000

Synergism with the coactivator OBF-1 (OCA-B, BOB-1) is mediated by a specific POU dimer configuration.

POU domain proteins contain a bipartite DNA binding domain divided by a flexible linker that enables them to adopt various monomer configurations on DNA. The versatility of POU protein operation is additionally conferred at the dimerization level. The POU dimer formed on the PORE (ATTTGAAATGCAAAT) can recruit the transcriptional coactivator OBF-1, whereas POU dimers formed on the consensus MORE (ATGCATATGCAT) or on MOREs from immunoglobulin heavy chain promoters (AT[G/A][C/A]ATATGCAA) fail to interact. An interaction with OBF-1 is precluded since the same Oct-1 residues that form the MORE dimerization interface are also used for OBF-1/Oct-1 interactions on the PORE. Our findings provide a paradigm of how specific POU dimer assemblies can differentially recruit a coregulatory activity with distinct transcriptional readouts.

Amino Acid Motifs↗
PubMed2000

Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer.

Fibroblast growth factor (FGF)-4 gene expression in the inner cell mass of the blastocyst and in EC cells requires the combined activity of two transcriptional regulators, Sox2 and Oct-3, which bind to adjacent sites on the FGF-4 enhancer DNA and synergistically activate transcription. Sox2 and Oct-3 bind cooperatively to the enhancer DNA through their DNA-binding, high mobility group and POU domains, respectively. These two domains, however, are not sufficient to activate transcription. We have analyzed a number of Sox2 and Oct-3 deletion mutants to identify the domains within each protein that contribute to the activity of the Sox2 x Oct-3 complex. Within Oct-3, we have identified two activation domains, the N-terminal AD1 and the C-terminal AD2, that play a role in the activity of the Sox2 x Oct-3 complex. AD1 also displays transcriptional activation functions in the absence of Sox2 while AD2 function was only detected within the Sox2 x Oct-3 complex. In Sox2, we have identified three activation domains within its C terminus: R1, R2, and R3. R1 and R2 can potentiate weak activation by Sox2 in the absence of Oct-3 but their deletion has no effect on the Sox2 x Oct-3 complex. In contrast, R3 function is only observed when Sox2 is complexed with Oct-3. In addition, analysis of Oct-1/Oct-3 chimeras indicates that the Oct-3 homeodomain also plays a critical role in the formation of a functional Sox2 x Oct-3 complex. Our results are consistent with a model in which the synergistic action of Sox2 and Oct-3 results from two major processes. Cooperative binding of the factors to the enhancer DNA, mediated by their binding domains, stably tethers each factor to DNA and increases the activity of intrinsic activation domains within each protein. Protein-protein and protein-DNA interactions then may lead to reciprocal conformational changes that expose latent activation domains within each protein. These findings define a mechanism that may also be utilized by other Sox x POU protein complexes in gene activation.

Base Sequence↗
PubMed2000

Oct-4: control of totipotency and germline determination.

The transcription factor Oct-4 is expressed in totipotent embryonic cells and germ cells. As totipotent cells differentiate to form somatic and/or extraembryonic tissues, the Oct-4 gene is downregulated. Primordial germ cells are the only cells in which Oct-4 expression is maintained after postgastrulation. Recent in vivo ablation of the Oct-4 function has shown that the absence of this transcription factor causes early embryonic lethality due to trophectodermal differentiation of cells which normally would give rise to the inner cell mass of the blastocyst. This result strongly suggests that Oct-4 is necessary for the maintenance of the totipotent phenotype of embryonic cells and that this factor likely plays a role as a determinant of the totipotency of germ cells by preventing their differentiation to a somatic cell phenotype during gastrulation. The involvement of Oct-4 in the biology of totipotent and germ cells is here discussed in view of new understanding about Oct-4 function.

Animals↗
PubMed1999

Simplified generation of targeting constructs using ET recombination.

ET recombination is a way to engineer DNA in Escherichia coli using homologous recombination. Here we develop the potential of ET recombination in two ways relevant to complex engineering exercises such as building gene targeting constructs. First, a targeting construct was made in a single step. Second, ET recombination was used to place two unique restriction sites at precise positions in a large genomic clone. Subsequently a complex targeting construct was created by ligation with a multifunctional cassette.

Animals↗
PubMed1999

Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice.

The Pic-1, Oct-1,2, Unc-86 (POU) transcription factor Oct-4 is specifically expressed in the germ cell line, and a previous study has indicated that the expression of the lacZ gene inserted into an 18 kb genomic fragment encompassing the Oct-4 gene can come close to mimicking the endogenous embryonic expression pattern of Oct-4 in transgenic mice. In the present study transgenic mice expressing green fluorescent protein (GFP) in the germ cell line were generated using the same Oct-4 genomic fragments and the expression pattern was analyzed in detail through all stages of germ cell development. The GFP expressing primordial germ cells were first detected as early as 8.0 days post-coitum (d.p.c.; early head fold stage) at the base of the allantois in living embryos. The GFP expression was thereafter found in both male and female germ cells at all developmental stages except in male germ cells after differentiating into type A spermatogonia in the postnatal testis. There was also a lower level of expression in female germ cells in the prophase of the first meiotic division. These transgenic mice therefore proved to be powerful tools for isolating living germ cells at various developmental stages to study their nature and to isolate new genes.

Animals↗
PubMed1999

Synergism with germ line transcription factor Oct-4: viral oncoproteins share the ability to mimic a stem cell-specific activity.

Activation of transcription by Oct-4 from remote binding sites requires a cofactor that is restricted to embryonal stem cells. The adenovirus E1A protein can mimic the activity of this stem cell-specific factor and stimulates Oct-4 activity in differentiated cells. Here we characterize the Oct-4-E1A interaction and show that the E1A 289R protein harbors two independent Oct-4 binding sites, both of which specifically interact with the POU domain of Oct-4. Furthermore, we demonstrate that, like E1A, the human papillomavirus E7 oncoprotein also specifically binds to the Oct-4 POU domain. E7 and Oct-4 can form a complex both in vitro and in vivo. Expression of E7 in differentiated cells stimulates Oct-4-mediated transactivation from distal binding sites. Moreover, Oct-4, but not other Oct factors, is active when expressed in cells transformed by human papillomavirus. Our results suggest that different viruses have evolved oncoproteins that share the ability to target Oct-4 and to mimic a stem cell-specific activity.

Adenovirus E1A Proteins↗
PubMed1999

Oct-4: lessons of totipotency from embryonic stem cells.

The Oct-4 transcription factor is expressed in embryonic stem cells and germ cells of the mouse embryo. Cells that are driven into embryonic and extraembryonic somatic differentiation lose Oct-4 expression. Oct-4 is crucial for the maintenance of the totipotency of embryonic cells at early developmental stages as demonstrated recently by homologous recombination. Since during embryogenesis Oct-4 is restricted to germ cells, it is possible that it plays a key role for the germline lineage by preventing differentiation of these cells during gastrulation. Thus, Oct-4 appears to be a key regulator of totipotency during the life cycle of mammals.

Animals↗