Hungjiun Liaw

Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks

Chronic stalling of DNA replication forks caused by DNA damage can lead to genomic instability. Cells have evolved lesion bypass pathways such as postreplication repair (PRR) to resolve these arrested forks. In yeast, one branch of PRR involves proliferating cell nuclear antigen (PCNA) polyubiquitination mediated by the Rad5-Ubc13-Mms2 complex that allows bypass of DNA lesion by a template-switching mechanism. Previously, we identified human SHPRH as a functional homologue of yeast Rad5 and revealed the existence of RAD5-like pathway in human cells. Here we report the identification of HLTF as a second RAD5 homologue in human cells. HLTF, like SHPRH, shares a unique domain architecture with Rad5 and promotes lysine 63-linked polyubiquitination of PCNA. Similar to yeast Rad5, HLTF is able to interact with UBC13 and PCNA, as well as SHPRH; and the reduction of either SHPRH or HLTF expression enhances spontaneous mutagenesis. Moreover, Hltf-deficient mouse embryonic fibroblasts show elevated chromosome breaks and fusions after methyl methane sulfonate treatment. Our results suggest that HLTF and SHPRH are functional homologues of yeast Rad5 that cooperatively mediate PCNA polyubiquitination and maintain genomic stability.

The exon junction complex component Magoh controls brain size by regulating neural stem cell division

Brain structure and size require precise division of neural stem cells (NSCs), which self-renew and generate intermediate neural progenitors (INPs) and neurons. The factors that regulate NSCs remain poorly understood, and mechanistic explanations of how aberrant NSC division causes the reduced brain size seen in microcephaly are lacking. Here we show that Magoh, a component of the exon junction complex (EJC) that binds RNA, controls mouse cerebral cortical size by regulating NSC division. Magoh haploinsufficiency causes microcephaly because of INP depletion and neuronal apoptosis. Defective mitosis underlies these phenotypes, as depletion of EJC components disrupts mitotic spindle orientation and integrity, chromosome number and genomic stability. In utero rescue experiments showed that a key function of Magoh is to control levels of the microcephaly-associated protein Lis1 during neurogenesis. Our results uncover requirements for the EJC in brain development, NSC maintenance and mitosis, thereby implicating this complex in the pathogenesis of microcephaly.

DNA-PK-Dependent RPA2 Hyperphosphorylation Facilitates DNA Repair and Suppresses Sister Chromatid Exchange

Hyperphosphorylation of RPA2 at serine 4 and serine 8 (S4, S8) has been used as a marker for activation of the DNA damage response. What types of DNA lesions cause RPA2 hyperphosphorylation, which kinase(s) are responsible for them, and what is the biological outcome of these phosphorylations, however, have not been fully investigated. In this study we demonstrate that RPA2 hyperphosphorylation occurs primarily in response to genotoxic stresses that cause high levels of DNA double-strand breaks (DSBs) and that the DNA-dependent protein kinase complex (DNA-PK) is responsible for the modifications in vivo. Alteration of S4, S8 of RPA2 to alanines, which prevent phosphorylations at these sites, caused increased mitotic entry with concomitant increases in RAD51 foci and homologous recombination. Taken together, our results demonstrate that RPA2 hyperphosphorylation by DNA-PK in response to DSBs blocks unscheduled homologous recombination and delays mitotic entry. This pathway thus permits cells to repair DNA damage properly and increase cell viability.

Mph1p promotes gross chromosomal rearrangement through partial inhibition of homologous recombination.

Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3′–5′ DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Δ mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA.

Sir3 C-terminal domain involvement in the initiation and spreading of heterochromatin

ABSTRACT Heterochromatin is nucleated at a specific site and subsequently spreads into distal sequences through multiple interactions between modified histones and nonhistone proteins. In the yeast Saccharomyces cerevisiae, these nonhistone proteins include Sir2, Sir3, and Sir4. We have previously shown that loss of the C-terminal Rap1 domain containing Sir3 and Sir4 association sites can be overcome by tethering a 144-amino-acid C-terminal domain (CTD) of Sir3 adjacent to the telomere. Here, we explore the substructure and functions of the CTD. We demonstrate that the CTD is the minimum domain for Sir3 homodimerization, a function that is conserved in related yeasts. However, CTD heterodimers associate at only low efficiencies and correspondingly have low levels of tethered silencing, consistent with an essential role for dimerization in tethered silencing. Six missense alleles were generated, with ctd-Y964A producing the most extreme phenotypes when tethered to the LexA binding sites. Although ctd-Y964A is capable of dimerization, telomere silencing is abrogated, indicating that the CTD serves a second essential function in silencing. Chromatin immunoprecipitation analyses of wild-type and ctd-Y964A mutant cells indicate an association of the CTD with the deacetylated histone tails of H3 and H4 that is necessary for the recruitment of Sir3. The efficiency of spreading depends upon the apparent stoichiometry and stability during the initiation event. The predicted Cdc6 domain III winged-helix structure may well be responsible for dimerization.

Genomic organization of the chicken CD8 locus reveals a novel family of immunoreceptor genes.

The genomic organization of the chicken CD8α gene was investigated to determine the basis of its polymorphism. Contiguous to the CD8α gene we identified multiple DNA blocks possessing sequences homologous to CD8α. Gene conversions and recombination over evolutionary time among CD8α and these CD8α homologous genes seem to account for the observed polymorphism. Furthermore, these CD8α-like DNAs encode a distinct multigene family of immunoreceptors that have a charged or polar residue in place of the interspecies-conserved CD8α transmembrane proline residue and a short cytoplasmic tail nonhomologous to CD8α. The identification of this novel multigene family with an organization reminiscent of human killer Ig-like receptors raises compelling questions on their evolutionary relationship among immunoreceptors.

Retinoic acid mediates the expression of glutamate transporter‐1 in rat astrocytes through genomic RXR action and non‐genomic protein kinase C signaling pathway

J. Neurochem. (2012) 121, 537–550.

Ecological genomics in Xanthomonas : the nature of genetic adaptation with homologous recombination and host shifts

BackgroundComparative genomics provides insights into the diversification of bacterial species. Bacterial speciation usually takes place with lasting homologous recombination, which not only acts as a cohering force between diverging lineages but brings advantageous alleles favored by natural selection, and results in ecologically distinct species, e.g., frequent host shift in Xanthomonas pathogenic to various plants.ResultsUsing whole-genome sequences, we examined the genetic divergence in Xanthomonas campestris that infected Brassicaceae, and X. citri, pathogenic to a wider host range. Genetic differentiation between two incipient races of X. citri pv. mangiferaeindicae was attributable to a DNA fragment introduced by phages. In contrast to most portions of the genome that had nearly equivalent levels of genetic divergence between subspecies as a result of the accumulation of point mutations, 10% of the core genome involving with homologous recombination contributed to the diversification in Xanthomonas, as revealed by the correlation between homologous recombination and genomic divergence. Interestingly, 179 genes were under positive selection; 98 (54.7%) of these genes were involved in homologous recombination, indicating that foreign genetic fragments may have caused the adaptive diversification, especially in lineages with nutritional transitions. Homologous recombination may have provided genetic materials for the natural selection, and host shifts likely triggered ecological adaptation in Xanthomonas. To a certain extent, we observed positive selection nevertheless contributed to ecological divergence beyond host shifting.ConclusionAltogether, mediated with lasting gene flow, species formation in Xanthomonas was likely governed by natural selection that played a key role in helping the deviating populations to explore novel niches (hosts) or respond to environmental cues, subsequently triggering species diversification.

SHPRH regulates rRNA transcription by recognizing the histone code in an mTOR-dependent manner

Significance Transcription of ribosomal RNA (rRNA), which composes the ribosome with other proteins, is tightly regulated to maintain the right number of ribosomes. Many DNA repair proteins have functions in addition to their role in DNA repair. We provide evidence that SHPRH functioning in DNA repair at stalled DNA replication forks recognizes epigenetic histone codes of rDNA through its plant homeodomain (PHD) and modulates 47S rRNA transcription. SHPRH bound to rDNA promoters and promoted RNA polymerase I recruitment for rRNA transcription. SHPRH localization to the rDNA promoter was inhibited by trimethylation of histone H3 lysine 4, which is a mark of the rDNA promoter at poised status on starvation. Collectively, we suggest a mechanism controlling 47S rRNA transcription by SHPRH in a histone methylation-dependent manner. Many DNA repair proteins have additional functions other than their roles in DNA repair. In addition to catalyzing PCNA polyubiquitylation in response to the stalling of DNA replication, SHPRH has the additional function of facilitating rRNA transcription by localizing to the ribosomal DNA (rDNA) promoter in the nucleoli. SHPRH was recruited to the rDNA promoter using its plant homeodomain (PHD), which interacts with histone H3 when the fourth lysine of H3 is not trimethylated. SHPRH enrichment at the rDNA promoter was inhibited by cell starvation, by treatment with actinomycin D or rapamycin, or by depletion of CHD4. SHPRH also physically interacted with the RNA polymerase I complex. Taken together, we provide evidence that SHPRH functions in rRNA transcription through its interaction with histone H3 in a mammalian target of rapamycin (mTOR)-dependent manner.

The Yeast Nucleosome Atlas (YNA) database: an integrative gene mining platform for studying chromatin structure and its regulation in yeast

BackgroundHistone modification and remodeling play crucial roles in regulating gene transcription. These post-translational modifications of histones function in a combinatorial fashion and can be recognized by specific histone-binding proteins, thus regulating gene transcription. Therefore, understanding the combinatorial patterns of the histone code is vital to understanding the associated biological processes. However, most of the datasets regarding histone modification and chromatin regulation are scattered across various studies, and no comprehensive search and query tool has yet been made available to retrieve genes bearing specific histone modification patterns and regulatory proteins.DescriptionFor this reason, we developed the Yeast Nucleosome Atlas database, or the YNA database, which integrates the available experimental data on nucleosome occupancy, histone modifications, the binding occupancy of regulatory proteins, and gene expression data, and provides the genome-wide gene miner to retrieve genes with a specific combination of these chromatin-related datasets. Moreover, the biological significance analyzer, which analyzes the enrichments of histone modifications, binding occupancy, transcription rate, and functionality of the retrieved genes, was constructed to help researchers to gain insight into the correlation among chromatin regulation and transcription.ConclusionsCompared to previously established genome browsing databases, YNA provides a powerful gene mining and retrieval interface, and is an investigation tool that can assist users to generate testable hypotheses for studying chromatin regulation during transcription. YNA is available online at http://cosbi3.ee.ncku.edu.tw/yna/.