Acetylation of sox2 induces its nuclear export in embryonic stem cells.

Embryonic stem (ES) cells require a coordinated network of transcription factors to maintain pluripotency or trigger lineage specific differentiation. Central to these processes are the proteins Oct4, Nanog, and Sox2. Although the transcriptional targets of these factors have been extensively studied, very little is known about how the proteins themselves are regulated, especially at the post-translational level. Post-translational modifications are well documented to have broad effects on protein stability, activity, and cellular distribution. Here, we identify a key lysine residue in the nuclear export signal of Sox2 that is acetylated, and demonstrate that blocking acetylation at this site retains Sox2 in the nucleus and sustains expression of its target genes under hyperacetylation or differentiation conditions. Mimicking acetylation at this site promotes association of Sox2 with the nuclear export machinery. In addition, increased cellular acetylation leads to reduction in Sox2 levels by ubiquitination and proteasomal degradation, thus abrogating its ability to drive transcription of its target genes. Acetylation-mediated nuclear export may be a commonly used regulatory mechanism for many Sox family members, as this lysine is conserved across species and in orthologous proteins.

A positive regulatory role for the mSin3A-HDAC complex in pluripotency through Nanog and Sox2.

Large networks of proteins govern embryonic stem (ES) cell pluripotency. Recent analysis of the critical pluripotency factors Oct4 and Nanog has identified their interaction with multiple transcriptional repression complexes, including members of the mSin3A-HDAC complex, suggesting that these factors could be involved in the regulation of Oct4/Nanog function. mSin3A is critical for embryonic development, but the mechanism by which the mSin3A-HDAC complex is able to regulate ES cell pluripotency is undefined. Herein we show that the mSin3A-HDAC complex positively regulates Nanog expression in ES cells through Sox2, a critical ES cell transcription factor and regulator of Nanog. We have identified the mSin3A-HDAC complex to be present at the Nanog promoter only under proliferating conditions concurrent with histone acetylation. We find that Sox2 associates with mSin3A-HDAC complex members both in vitro and in vivo, similar to the interactions found between Oct4/Nanog and the mSin3A-HDAC complex. Knockdown of mSin3A-HDAC complex members or HDAC inhibitor treatment reduces Nanog expression, and overexpression of mSin3A-HDAC complex subunits stimulates Nanog expression. Our data demonstrate that the mSin3A-HDAC complex can positively regulate Nanog expression under proliferating conditions and that this activity is complementary to mSin3A-mediated p53-dependent silencing of Nanog during differentiation.

Role for Med12 in regulation of Nanog and Nanog target genes.

Nanog, Oct4, and Sox2 form the core of a transcription factor network that maintains embryonic stem cells in the pluripotent state in both humans and mice. These critical factors have been implicated as both positive and negative regulators of transcription, varying by promoter and differentiation state of the cell. The Mediator complex, a ubiquitous conserved complex of approximately 30 subunits, facilitates transcription by coordinating RNA polymerase II binding to target promoters via gene-specific activators and can be divided into several functional subcomplexes. Med12 is part of a subcomplex of four proteins associated with the core Mediator complex and has been found to function both in repressing and activating transcription when recruited to target promoters. We identified an interaction between Med12 and Nanog and present evidence of involvement of Med12 in regulation of Nanog function. Gene expression analysis of embryonic stem cells knocked down for Med12 showed a similarity to Nanog knockdown, with increased expression of Nanog-repressed targets and decreased expression of Nanog-activated targets. Using chromatin immunoprecipitation, we found that Med12 and Nanog co-occupied Nanog target promoters in embryonic stem cells and that Med12 dissociated from target promoters upon differentiation with kinetics similar to Nanog. Our results indicate that Nanog and Med12 function in concert to regulate Nanog target genes and identify a novel role for Med12 in embryonic stem cell regulation.

Domain architecture and biochemical characterization of vertebrate Mcm10.

Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed N-terminal-, internal (ID)-, and C-terminal (CTD)-structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the N-terminal domain forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single- and double-stranded DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the N-terminal 323 residues of the p180 subunit of DNA polymerase alpha-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase alpha-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.

Chromosomal DNA replication in a soluble cell-free system derived from Xenopus eggs.

Cytoplasmic egg extracts from the frog Xenopus laevis represent a powerful cell-free system to study eukaryotic chromosomal DNA replication. In the classical approach, sperm chromatin is added to unfractionated egg cytoplasm, leading to the assembly of transport-competent nuclei that undergo a single, complete round of DNA replication. The need for nuclei in this system has been circumvented. Sperm chromatin or plasmid DNA is first incubated with clarified egg cytoplasm to form chromatin-bound prereplication complexes. Subsequently, a highly concentrated nucleoplasmic extract is added that stimulates initiation from these prereplication complexes, and a single complete round of chromosomal DNA replication ensues. This review describes the preparation of the cytosolic and nucleoplasmic extracts, as well as their use in DNA replication, origin unwinding, and chromatin isolation assays.

Embryonic stem cells: a great hope for a new era of medicine.

Current stem-cell research has the potential to lead to new approaches for the treatment of cardiovascular, neurodegenerative and musculoskeletal diseases, as well as diabetes and cancer. Stem-cell-based approaches could be employed in cell-replacement therapy or in drug treatments that encourage adult stem cells to migrate and activate at a site of injury or disease. For such therapeutic approaches to be successful, a greater understanding of the signaling pathways that determine the diverse developmental fates of these cells is needed. From a drug-discovery perspective, efforts are being deployed in developing cell-based assays to screen for small molecules that can modulate stem-cell fate. Such compounds will provide new insights into stem-cell biology, and may ultimately contribute to effective disease treatments.

Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication.

Little is known about the architecture and biochemical composition of the eukaryotic DNA replication fork. To study this problem, we used biotin-streptavidin-modified plasmids to induce sequence-specific replication fork pausing in Xenopus egg extracts. Chromatin immunoprecipitation was employed to identify factors associated with the paused fork. This approach identifies DNA pol alpha, DNA pol delta, DNA pol epsilon, MCM2-7, Cdc45, GINS, and Mcm10 as components of the vertebrate replisome. In the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45, GINS, and MCM2-7 are enriched at the pause site. The data suggest the existence of a large molecular machine, the "unwindosome," which separates DNA strands at the replication fork and contains Cdc45, GINS, and the MCM2-7 holocomplex.

The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication.

The initiation of eukaryotic DNA replication involves origin recruitment and activation of the MCM2-7 complex, the putative replicative helicase. Mini-chromosome maintenance (MCM)2-7 recruitment to origins in G1 requires origin recognition complex (ORC), Cdt1, and Cdc6, and activation at G1/S requires MCM10 and the protein kinases Cdc7 and S-Cdk, which together recruit Cdc45, a putative MCM2-7 cofactor required for origin unwinding. Here, we show that the Xenopus BRCA1 COOH terminus repeat-containing Xmus101 protein is required for loading of Cdc45 onto the origin. Xmus101 chromatin association is dependent on ORC, and independent of S-Cdk and MCM2-7. These results define a new factor that is required for Cdc45 loading. Additionally, these findings indicate that the initiation complex assembly pathway bifurcates early, after ORC association with the origin, and that two parallel pathways, one controlled by MCM2-7, and the other by Xmus101, cooperate to load Cdc45 onto the origin.

MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts.

The MCM2-7 complex is believed to function as the eukaryotic replicative DNA helicase. It is recruited to chromatin by the origin recognition complex (ORC), Cdc6, and Cdt1, and it is activated at the G(1)/S transition by Cdc45 and the protein kinases Cdc7 and Cdk2. Paradoxically, the number of chromatin-bound MCM complexes greatly exceeds the number of bound ORC complexes. To understand how the high MCM2-7:ORC ratio comes about, we examined the binding of these proteins to immobilized linear DNA fragments in Xenopus egg extracts. The minimum length of DNA required to recruit ORC and MCM2-7 was approximately 80 bp, and the MCM2-7:ORC ratio on this fragment was approximately 1:1. With longer DNA fragments, the MCM2-7:ORC ratio increased dramatically, indicating that MCM complexes normally become distributed over a large region of DNA surrounding ORC. Only a small subset of the chromatin-bound MCM2-7 complexes recruited Cdc45 at the onset of DNA replication, and unlike Cdc45, MCM2-7 was not limiting for DNA replication. However, all the chromatin-bound MCM complexes may be functional, because they were phosphorylated in a Cdc7-dependent fashion, and because they could be induced to support Cdk2-dependent Cdc45 loading. The data suggest that in Xenopus egg extracts, origins of replication contain multiple, distributed, initiation-competent MCM2-7 complexes.