Separated at birth? The functional and molecular divergence of OLIG1 and OLIG2.

The basic helix-loop-helix transcription factors oligodendrocyte transcription factor 1 (OLIG1) and OLIG2 are structurally similar and, to a first approximation, coordinately expressed in the developing CNS and postnatal brain. Despite these similarities, it was apparent from early on after their discovery that OLIG1 and OLIG2 have non-overlapping developmental functions in patterning, neuron subtype specification and the formation of oligodendrocytes. Here, we summarize more recent insights into the separate roles of these transcription factors in the postnatal brain during repair processes and in neurological disease states, including multiple sclerosis and malignant glioma. We discuss how the unique functions of OLIG1 and OLIG2 may reflect their distinct genetic targets, co-regulator proteins and/or post-translational modifications.

An amino terminal phosphorylation motif regulates intranuclear compartmentalization of Olig2 in neural progenitor cells.

The bHLH transcription factor Olig2 is expressed in cycling neural progenitor cells but also in terminally differentiated, myelinating oligodendrocytes. Sustained expression of Olig2 is counterintuitive because all known functions of the protein in expansion of neural progenitors and specification of oligodendrocyte progenitors are completed with the formation of mature white matter. How are the biological functions of Olig2 suppressed in terminally differentiated oligodendrocytes? In previous studies, we have shown that a triple serine motif in the amino terminus of Olig2 is phosphorylated in cycling neural progenitors but not in their differentiated progeny. We now show that phosphorylation of the triple serine motif regulates intranuclear compartmentalization of murine Olig2. Phosphorylated Olig2 is preferentially localized to a transcriptionally active "open" chromatin compartment together with coregulator proteins essential for regulation of gene expression. Unphosphorylated Olig2, as seen in mature white matter, is localized mainly within a transcriptionally inactive, chromatin fraction characterized by condensed and inaccessible DNA. Of special note is the observation that the p53 tumor suppressor protein is confined to the open chromatin fraction. Proximity ligation assays show that phosphorylation brings Olig2 within 30 nm of p53 within the open chromatin compartment. The data thus shed light on previously noted promitogenic functions of phosphorylated Olig2, which reflect, at least in part, an oppositional relationship with p53 functions.

Mutation in Rpa1 results in defective DNA double-strand break repair, chromosomal instability and cancer in mice.

Most cancers have multiple chromosomal rearrangements; the molecular mechanisms that generate them remain largely unknown. Mice carrying a heterozygous missense change in one of the DNA-binding domains of Rpa1 develop lymphoid tumors, and their homozygous littermates succumb to early embryonic lethality. Array comparative genomic hybridization of the tumors identified large-scale chromosomal changes as well as segmental gains and losses. The Rpa1 mutation resulted in defects in DNA double-strand break repair and precipitated chromosomal breaks as well as aneuploidy in primary heterozygous mutant mouse embryonic fibroblasts. The equivalent mutation in yeast is hypomorphic and semidominant and enhanced the formation of gross chromosomal rearrangements in multiple genetic backgrounds. These results indicate that Rpa1 functions in DNA metabolism are essential for the maintenance of chromosomal stability and tumor suppression.

The central nervous system-restricted transcription factor Olig2 opposes p53 responses to genotoxic damage in neural progenitors and malignant glioma.

High-grade gliomas are notoriously insensitive to radiation and genotoxic drugs. Paradoxically, the p53 gene is structurally intact in the majority of these tumors. Resistance to genotoxic modalities in p53-positive gliomas is generally attributed to attenuation of p53 functions by mutations of other components within the p53 signaling axis, such as p14(Arf), MDM2, and ATM, but this explanation is not entirely satisfactory. We show here that the central nervous system (CNS)-restricted transcription factor Olig2 affects a key posttranslational modification of p53 in both normal and malignant neural progenitors and thereby antagonizes the interaction of p53 with promoter elements of multiple target genes. In the absence of Olig2 function, even attenuated levels of p53 are adequate for biological responses to genotoxic damage.

Phosphorylation state of Olig2 regulates proliferation of neural progenitors.

The bHLH transcription factors that regulate early development of the central nervous system can generally be classified as either antineural or proneural. Initial expression of antineural factors prevents cell cycle exit and thereby expands the pool of neural progenitors. Subsequent (and typically transient) expression of proneural factors promotes cell cycle exit, subtype specification, and differentiation. Against this backdrop, the bHLH transcription factor Olig2 in the oligodendrocyte lineage is unorthodox, showing antineural functions in multipotent CNS progenitor cells but also sustained expression and proneural functions in the formation of oligodendrocytes. We show here that the proliferative function of Olig2 is controlled by developmentally regulated phosphorylation of a conserved triple serine motif within the amino-terminal domain. In the phosphorylated state, Olig2 maintains antineural (i.e., promitotic) functions that are reflected in human glioma cells and in a genetically defined murine model of primary glioma.

Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae.

The availability of an annotated genome sequence for the yeast Saccharomyces cerevisiae has made possible the proteome-scale study of protein function and protein-protein interactions. These studies rely on availability of cloned open reading frame (ORF) collections that can be used for cell-free or cell-based protein expression. Several yeast ORF collections are available, but their use and data interpretation can be hindered by reliance on now out-of-date annotations, the inflexible presence of N- or C-terminal tags, and/or the unknown presence of mutations introduced during the cloning process. High-throughput biochemical and genetic analyses would benefit from a "gold standard" (fully sequence-verified, high-quality) ORF collection, which allows for high confidence in and reproducibility of experimental results. Here, we describe Yeast FLEXGene, a S. cerevisiae protein-coding clone collection that covers over 5000 predicted protein-coding sequences. The clone set covers 87% of the current S. cerevisiae genome annotation and includes full sequencing of each ORF insert. Availability of this collection makes possible a wide variety of studies from purified proteins to mutation suppression analysis, which should contribute to a global understanding of yeast protein function.

Haploinsufficiency of Flap endonuclease (Fen1) leads to rapid tumor progression.

Flap endonuclease (Fen1) is required for DNA replication and repair, and defects in the gene encoding Fen1 cause increased accumulation of mutations and genome rearrangements. Because mutations in some genes involved in these processes cause cancer predisposition, we investigated the possibility that Fen1 may function in tumorigenesis of the gastrointestinal tract. Using gene knockout approaches, we introduced a null mutation into murine Fen1. Mice homozygous for the Fen1 mutation were not obtained, suggesting absence of Fen1 expression leads to embryonic lethality. Most Fen1 heterozygous animals appear normal. However, when combined with a mutation in the adenomatous polyposis coli (Apc) gene, double heterozygous animals have increased numbers of adenocarcinomas and decreased survival. The tumors from these mice show microsatellite instability. Because one copy of the Fen1 gene remained intact in tumors, Fen1 haploinsufficiency appears to lead to rapid progression of cancer.

Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility.

Exonuclease 1 (Exo1) is a 5'-3' exonuclease that interacts with MutS and MutL homologs and has been implicated in the excision step of DNA mismatch repair. To investigate the role of Exo1 in mammalian mismatch repair and assess its importance for tumorigenesis and meiosis, we generated an Exo1 mutant mouse line. Analysis of Exo1(-/-) cells for mismatch repair activity in vitro showed that Exo1 is required for the repair of base:base and single-base insertion/deletion mismatches in both 5' and 3' nick-directed repair. The repair defect in Exo1(-/-) cells also caused elevated microsatellite instability at a mononucleotide repeat marker and a significant increase in mutation rate at the Hprt locus. Exo1(-/-) animals displayed reduced survival and increased susceptibility to the development of lymphomas. In addition, Exo1(-/-) male and female mice were sterile because of a meiotic defect. Meiosis in Exo1(-/-) animals proceeded through prophase I; however, the chromosomes exhibited dynamic loss of chiasmata during metaphase I, resulting in meiotic failure and apoptosis. Our results show that mammalian Exo1 functions in mutation avoidance and is essential for male and female meiosis.