Williams Syndrome Transcription Factor is critical for neural crest cell function in Xenopus laevis.

Williams Syndrome Transcription Factor (WSTF) is one of ∼25 haplodeficient genes in patients with the complex developmental disorder Williams Syndrome (WS). WS results in visual/spatial processing defects, cognitive impairment, unique behavioral phenotypes, characteristic "elfin" facial features, low muscle tone and heart defects. WSTF exists in several chromatin remodeling complexes and has roles in transcription, replication, and repair. Chromatin remodeling is essential during embryogenesis, but WSTF's role in vertebrate development is poorly characterized. To investigate the developmental role of WSTF, we knocked down WSTF in Xenopus laevis embryos using a morpholino that targets WSTF mRNA. BMP4 shows markedly increased and spatially aberrant expression in WSTF-deficient embryos, while SHH, MRF4, PAX2, EPHA4 and SOX2 expression are severely reduced, coupled with defects in a number of developing embryonic structures and organs. WSTF-deficient embryos display defects in anterior neural development. Induction of the neural crest, measured by expression of the neural crest-specific genes SNAIL and SLUG, is unaffected by WSTF depletion. However, at subsequent stages WSTF knockdown results in a severe defect in neural crest migration and/or maintenance. Consistent with a maintenance defect, WSTF knockdowns display a specific pattern of increased apoptosis at the tailbud stage in regions corresponding to the path of cranial neural crest migration. Our work is the first to describe a role for WSTF in proper neural crest function, and suggests that neural crest defects resulting from WSTF haploinsufficiency may be a major contributor to the pathoembryology of WS.

Mitochondrial and nuclear genomic integrity after oxidative damage in Saccharomyces cerevisiae.

All cells have the ability to adjust their metabolism to their changing environment to be able to survive. This adaptation is coordinated by various systems in the cell and mitochondria seem to play a unique and important role. Most endogenous oxidative damage to cells is actually generated as a byproduct of the mitochondrial function, which in turn damages mitochondrial structures more extensively due to their proximity to the source. Excessive damage to mitochondria leads to loss of parts or all of mtDNA, but unlike other organisms, S. cerevisiae cells are able to survive without mtDNA or respiration when grown on fermentative carbon sources. This allows studies of the role of mitochondria in the maintenance of cellular integrity, since lack of mitochondrial DNA frequently leads to genomic instability. Mitochondria are known for their role in respiration, ATP production and apoptosis, but it is now becoming clear that their function is intimately connected to diverse processes such as calcium and iron homeostasis and amino acid metabolism, and thus their dysfunction is not well tolerated. In this review, we discuss the mechanisms by which mitochondrial dysfunction can lead to genomic instability and the effect of the carbon source on this process.

Life without RNAi: noncoding RNAs and their functions in Saccharomyces cerevisiae.

There are a number of well-characterized and fundamental roles for noncoding RNAs (ncRNAs) in gene regulation in all kingdoms of life. ncRNAs, such as ribosomal RNAs, transfer RNAs, small nuclear RNAs, small nucleolar RNAs, and small interfering RNAs, can serve catalytic and scaffolding functions in transcription, messenger RNA processing, translation, and RNA degradation. Recently, our understanding of gene expression has been dramatically challenged by the identification of large and diverse populations of novel ncRNAs in the eukaryotic genomes surveyed thus far. Studies carried out using the budding yeast Saccharomyces cerevisiae indicate that at least some coding genes are regulated by these novel ncRNAs. S. cerevisiae lacks RNA interference (RNAi) and, thus, provides an ideal system for studying the RNAi-independent mechanisms of ncRNA-based gene regulation. The current picture of gene regulation is one of great unknowns, in which the transcriptional environment surrounding a given locus may have as much to do with its regulation as its DNA sequence or local chromatin structure. Drawing on the recent research in S. cerevisiae and other organisms, this review will discuss the identification of ncRNAs, their origins and processing, and several models that incorporate ncRNAs into the regulation of gene expression and chromatin structure.

Noncoding but nonexpendable: transcriptional regulation by large noncoding RNA in eukaryotes.

Genome sequencing and annotation has advanced our understanding of genome organization and gene structure but initially only allowed predictions of how many genes might be present. Mechanisms such as alternative splicing reveal that these predictions only scratch the surface of the true nature of the transcriptome. Several thousand expressed partial gene fragments have been cloned but were considered transcriptional noise or cloning artifacts. We now know that genomes are indeed expressed at much higher levels than was previously predicted, and much of the additional transcription maps to intergenic regions, intron sequences, and untranslated regions of mRNAs. These transcripts are expressed from either the sense or the antisense strand and can be confirmed by conventional techniques. In addition to the already established roles for small RNAs in gene regulation, large noncoding RNAs (ncRNAs) are also emerging as potent regulators of gene expression. In this review, we summarize several illustrative examples of gene regulatory mechanisms that involve large ncRNAs. We describe several distinct regulatory mechanisms that involve large ncRNAs, such as transcriptional interference and promoter inactivation, as well as indirect effects on transcription regulatory proteins and in genomic imprinting. These diverse functions for large ncRNAs are likely to be only the first of many novel regulatory mechanisms emerging from this growing field.

Diverse roles for histone H2A modifications in DNA damage response pathways in yeast.

There are many types of DNA damage that are repaired by a multiplicity of different repair pathways. All damage and repair occur in the context of chromatin, and histone modifications are involved in many repair processes. We have analyzed the roles of H2A and its modifications in repair by mutagenizing modifiable residues in the N- and C-terminal tails of yeast H2A and by testing strains containing these mutations in multiple DNA repair assays. We show that residues in both tails are important for homologous recombination and nonhomologous end-joining pathways of double-strand break repair, as well as for survival of UV irradiation and oxidative damage. We show that H2A serine 122 is important for repair and/or survival in each of these assays. We also observe a complex pattern of H2A phosphorylation at residues S122, T126, and S129 in response to different damage conditions. We find that overlapping but nonidentical groups of H2A residues in both tails are involved in different pathways of repair. These data suggest the presence of a set of H2A "damage codes" in which distinct patterns of modifications on both tails of H2A may be used to identify specific types of damage or to promote specific repair pathways.

Regulation of two JunD isoforms by Jun N-terminal kinases.

The JunD transcription factor is one member of the Jun family of proteins that also includes c-Jun and JunB. Although c-Jun can function to promote cell proliferation and can cooperate with other oncogenes to transform cells, JunD slows proliferation of fibroblasts and antagonizes transformation by activated ras. Two isoforms of JunD, a full-length isoform containing 341 amino acids (JunD-FL) and a truncated isoform lacking 48 amino acids at the N terminus (Delta JunD), are generated through utilization of two translation start sites within a single mRNA. Here we show that both isoforms of JunD are phosphorylated by Jun N-terminal kinases (JNKs) at three identical residues and that both contain a docking domain that specifically binds JNKs. The JunD-FL isoform binds to and is phosphorylated by JNK more efficiently than Delta JunD in vitro; correspondingly, JunD-FL is a more potent transcriptional activator than Delta JunD. Although increased JNK signaling can activate both JunD isoforms, mutating either the JNK docking domain or the target JNK phosphorylation sites blocks this activation. These results identify two distinct isoforms of JunD with differential responses to JNK signaling pathways.