Byung Joon Hwang

Xeroderma pigmentosum p48 gene enhances global genomic repair and suppresses UV-induced mutagenesis.

UV-damaged DNA-binding activity (UV-DDB) is deficient in some xeroderma pigmentosum group E individuals due to mutation of the p48 gene, but its role in DNA repair has been obscure. We found that UV-DDB is also deficient in cell lines and primary tissues from rodents. Transfection of p48 conferred UV-DDB to hamster cells, and enhanced removal of cyclobutane pyrimidine dimers (CPDs) from genomic DNA and from the nontranscribed strand of an expressed gene. Expression of p48 suppressed UV-induced mutations arising from the nontranscribed strand, but had no effect on cellular UV sensitivity. These results define the role of p48 in DNA repair, demonstrate the importance of CPDs in mutagenesis, and suggest how rodent models can be improved to better reflect cancer susceptibility in humans.

p48 Activates a UV-Damaged-DNA Binding Factor and Is Defective in Xeroderma Pigmentosum Group E Cells That Lack Binding Activity

ABSTRACT A subset of xeroderma pigmentosum (XP) group E cells lack a factor that binds to DNA damaged by UV radiation. This factor can be purified to homogeneity as p125, a 125-kDa polypeptide. However, when cDNA encoding p125 is translated in vitro, only a small fraction binds to UV-damaged DNA, suggesting that a second factor is required for the activation of p125. We discovered that most hamster cell lines expressed inactive p125, which was activated in somatic cell hybrids containing human chromosome region 11p11.2-11cen. This region excludedp125 but included p48, which encodes a 48-kDa polypeptide known to copurify with p125 under some conditions. Expression of human p48 activated p125 binding in hamster cells and increased p125 binding in human cells. No such effects were observed from expression of p48 containing single amino acid substitutions from XP group E cells that lacked binding activity, demonstrating that the p48 gene is defective in those cells. Activation of p125 occurred by a “hit-and-run” mechanism, since the presence of p48 was not required for subsequent binding. Nevertheless, p48 was capable of forming a complex with p125 either bound to UV-damaged DNA or in free solution. It is notable that hamster cells fail to efficiently repair cyclobutane pyrimidine dimers in nontranscribed DNA and fail to express p48, which contains a WD motif with homology to proteins that reorganize chromatin. We propose that p48 plays a role in repairing lesions that would otherwise remain inaccessible in nontranscribed chromatin.

A cell-specific enhancer that specifies lin-3 expression in the C. elegans anchor cell for vulval development.

During C. elegans vulval development, the anchor cell (AC) in the somatic gonad expresses lin-3, activating the EGF receptor signaling pathway in vulval precursor cells (VPCs) and thereby inducing and patterning VPCs. Previous studies with lin-3 mutants and transgene expression have revealed that the level of LIN-3 in the AC must be precisely regulated for proper vulval development. To understand how lin-3 expression is achieved in the AC, we identified a 59 bp lin-3 enhancer sufficient to activate lin-3 transcription solely in the AC. The enhancer contains two E-box elements, and one FTZ-F1 nuclear hormone receptor (NHR) binding site that is mutated in a vulvaless mutant, lin-3(e1417). Mutagenesis studies show that both E-boxes and the NHR binding site are necessary to express lin-3 in the AC. In vitro DNA-binding studies and in vivo functional assays indicate that distinct trans-acting factors, including the E-protein/Daughterless homolog HLH-2 and unidentified nuclear hormone receptor(s), are necessary for lin-3 transcription in the AC and thus are involved in vulval development.

Isolation of a cDNA encoding a UV-damaged DNA binding factor defective in xeroderma pigmentosum group E cells

XPE binding factor (XPE-BF) is deficient in a subset of patients from xeroderma pigmentosum complementation group E. Binding activity copurifies with a 125 kDa polypeptide (p125) that binds to DNA damaged by ultraviolet (UV) radiation and many other agents. We isolated cDNA encoding a polypeptide with a predicted amino acid sequence that matched the sequences of eleven tryptic peptides derived from digestion of XPE-BF purified from human placenta. In vitro transcription and translation of the cDNA yielded a polypeptide of 125 kDa that bound specifically to UV-damaged DNA. Therefore the cDNA encodes either all or the major component of XPE-BF.

Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress

ABSTRACT Aminoacyl-tRNA synthetases (ARSs) acylate transfer (t)RNAs with amino acids. Charging tRNAs with the right amino acids is the first step in translation; therefore, the accurate and error-free functioning of ARSs is an essential prerequisite for translational fidelity. A recent study found that methionine (Met) can be incorporated into non-Met residues of proteins through methionylation of non-cognate tRNAs under conditions of oxidative stress. However, it was not understood how this mis-methionylation is achieved. Here, we report that methionyl-tRNA synthetase (MRS) is phosphorylated at Ser209 and Ser825 by extracellular signal-related kinase (ERK1/2) under conditions of stress caused by reactive oxygen species (ROS), and that this phosphorylated MRS shows increased affinity for non-cognate tRNAs with lower affinity for tRNAMet, leading to an increase in Met residues in cellular proteins. The expression of a mutant MRS containing the substitutions S209D and S825D, mimicking dual phosphorylation, reduced ROS levels and cell death. This controlled inaccuracy of MRS seems to serve as a defense mechanism against ROS-mediated damage at the cost of translational fidelity.

C. elegans EVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

During C. elegans development, LIN-12 (Notch) signaling specifies the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and then invades into the vulval epithelium. Although these cellular processes are essential for the proper organogenesis of vulva and appear to be temporally regulated, the mechanisms that coordinate the processes are not well understood. We computationally identified egl-43 as a gene likely to be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of an enhancer element similar to the one that transcribes lin-3 in the same cells. Genetic epistasis analyses reveal that egl-43 acts downstream of or parallel to lin-12 in AC/VU cell fate specification at an early developmental stage, and functions downstream of fos-1 as well as upstream of zmp-1 and him-4 to regulate AC invasion at a later developmental stage. Characterization of the egl-43 regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2 (E12/47), which is required for the specification of the VU fate during AC/VU specification. EGL-43 also regulates basement membrane breakdown during AC invasion through a FOS-1-responsive regulatory element that drives EGL-43 expression in the AC and VU cells at the later stage. Thus, egl-43 integrates temporally distinct upstream regulatory events and helps program cell fate specification and cell invasion.

Interaction between UV-damaged DNA binding activity proteins and the c-Abl tyrosine kinase

The c-Abl tyrosine kinase is activated by some forms of DNA damage, including ionizing radiation, but not UV radiation. The functions of this activation in the damage response pathways remain obscure. To identify potential targets of c-Abl kinase, we utilized the yeast two-hybrid system to screen a murine cDNA library. One c-Abl binding protein of particular interest was the large subunit (DDB1) of the heterodimeric complex with UV-damaged DNA binding activity (UV-DDB). This complex binds with high specificity to DNA damaged by UV, is absent in a subset of xeroderma pigmentosum group E cells, and is required for global genomic repair of UV-induced damage. The association of c-Abl with DDB1 required the kinase domain of c-Abl and preserved the interaction between DDB1 and the small subunit (DDB2) of the UV-DDB complex. Significantly, overexpression of c-Abl increased tyrosine phosphorylation of DDB2 and suppressed UV-DDB activity. Conversely, a dominant negative, kinase-deficient allele of c-Abl decreased tyrosine phosphorylation of DDB2 and dramatically stimulated UV-DDB activity. These results suggest that one role of c-Abl may be to negatively regulate UV-DDB activity by phosphorylation of DDB2.

Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins

Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein.

Live image profiling of neural crest lineages in zebrafish transgenic lines

Zebrafish transgenic lines are important experimental tools for lineage tracing and imaging studies. It is crucial to precisely characterize the cell lineages labeled in transgenic lines to understand their limitations and thus properly interpret the data obtained from their use; only then can we confidently select a line appropriate for our particular research objectives. Here we profiled the cell lineages labeled in the closely related neural crest transgenic lines Tg(foxd3:GFP), Tg(sox10:eGFP) and Tg(sox10:mRFP). These fish were crossed to generate embryos, in which foxd3 and sox10 transgenic neural crest labeling could be directly compared at the cellular level using live confocal imaging. We have identified key differences in the cell lineages labeled in each line during early neural crest development and demonstrated that the most anterior cranial neural crest cells initially migrating out of neural tube at the level of forebrain and anterior midbrain express sox10:eGFP and sox10:mRFP, but not foxd3:GFP. This differential profile was robustly maintained in the different-tiating progeny of the neural crest lineages until 3.5dpf. Our data will enable researchers to make an informed choice in selecting transgenic lines for future neural crest research.

Internal sequence analysis of proteins eluted from polyacrylamide gels.

We have developed an elution-digestion-sequencing (EDS) method, which yields the internal amino acid sequence of partially purified proteins. The overall yield for the method was greater than 60%. The method yielded peptide peaks that could be sequenced on HPLC for all tested proteins with masses from 45 to 200.10(3) and yielded internal amino acid sequence information when as little as 10 pmol of partially purified protein was used as the starting material. The EDS method was extremely reliable and gave sequence information for each of 25 proteins tested, including high-molecular-mass proteins (M(r) > 100.10(3)) that were difficult to sequence by other methods.