The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing an essential role in many biological processes. The importance of alternative splicing is further illustrated by the increasing number of human diseases that have been attributed to mis-splicing events. Appropriate spatial and temporal generation of splicing variants demands that alternative splicing be subjected to extensive regulation, similar to transcriptional control. The Clk (Cdc2-like kinase) family has been implicated in splicing control and consists of at least four members. Through extensive screening of a chemical library, we found that a benzothiazole compound, TG003, had a potent inhibitory effect on the activity of Clk1/Sty. TG003 inhibited SF2/ASFdependent splicing of ␤-globin pre-mRNA in vitro by suppression of Clk-mediated phosphorylation. This drug also suppressed serine/arginine-rich protein phosphorylation, dissociation of nuclear speckles, and Clk1/ Sty-dependent alternative splicing in mammalian cells. Consistently, administration of TG003 rescued the embryonic defects induced by excessive Clk activity in Xenopus. Thus, TG003, a novel inhibitor of Clk family will be a valuable tool to dissect the regulatory mechanisms involving serine/arginine-rich protein phosphorylation signaling pathways in vivo, and may be applicable for the therapeutic manipulation of abnormal splicing.Recent whole genome sequence analyses revealed that a high degree of proteomic complexity is achieved with a limited number of genes. This surprising finding underscores the importance of alternative splicing, through which a single gene can generate multiple structurally and functionally distinct protein isoforms (1). Based on genome-wide analysis, 35-60% of human genes are thought to encode at least two alternatively spliced isoforms (2). The regulation of splice site usage provides a versatile mechanism for controlling gene expression and for the generation of proteome diversity, playing essential roles in many biological processes, such as embryonic development, cell growth, and apoptosis. Splicing mutations located in either intronic or exonic regions frequently cause hereditary diseases (reviewed in Refs. 3-5). More than 15% of mutations that cause genetic disease affect pre-mRNA splicing (6). Pre-mRNA splicing is also regulated in a tissue-specific or developmental stagespecific manner. Indeed, the selection of splice site can be altered by numerous extracellular stimuli, including growth factors, cytokines, hormones, depolarization, osmotic shock, and UVC irradiation through synthesis, phosphorylation, and a change in localization of serine/arginine-rich (SR) 1 proteins (7). SR proteins are a family of essential factors required for constitutive splicing of pre-mRNA (8) and play an important role in modulating alternative splicing (9). They are highly conserved in eukaryotes and are characterized by having one or two RNA-recognition motifs at the amino terminus a...

The heteroduplex mobility assay (HMA) is widely used to characterize strain variants of human viruses. To determine whether it can detect small sequence differences in homologous templates, we constructed a series of deletion constructs (1–10 bp deletions) in the multiple cloning site (MCS) of pBluescript II. After PCR amplification of the MCS using a mixture of wild-type and one of the deletion constructs, the resulting PCR amplicons were electrophoresed using 15% polyacrylamide gels. Two types of heteroduplexes exhibited retarded electrophoretic migration compared with individual homoduplexes. Therefore, we applied this HMA to detect transcription activator-like effector nucleases (TALEN)-induced insertion and/or deletion (indel) mutations at an endogenous locus. We found that TALEN in vivo activity was easily estimated by the degree of multiple HMA profiles derived from TALEN-injected F0 embryos. Furthermore, TALEN-injected F0 founder fish produced several unique HMA profiles in F1 embryos. Sequence analysis confirmed that the different HMA profiles contained distinct indel mutations. Thus, HMA is a rapid and sensitive analytical method for the detection of the TALEN-mediated genome modifications.

Post-translational histone modifications play key roles in gene regulation, development, and differentiation, but their dynamics in living organisms remain almost completely unknown. To address this problem, we developed a genetically encoded system for tracking histone modifications by generating fluorescent modification-specific intracellular antibodies (mintbodies) that can be expressed in vivo. To demonstrate, an H3 lysine 9 acetylation specific mintbody (H3K9ac-mintbody) was engineered and stably expressed in human cells. In good agreement with the localization of its target acetylation, H3K9ac-mintbody was enriched in euchromatin, and its kinetics measurably changed upon treatment with a histone deacetylase inhibitor. We also generated transgenic fruit fly and zebrafish stably expressing H3K9ac-mintbody for in vivo tracking. Dramatic changes in H3K9ac-mintbody localization during Drosophila embryogenesis could highlight enhanced acetylation at the start of zygotic transcription around mitotic cycle 7. Together, this work demonstrates the broad potential of mintbody and lays the foundation for epigenetic analysis in vivo.

In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A–H2B, we identified protein phosphatase (PP) 2C γ subtype (PP2Cγ/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A–H2B. The disruption of PP2Cγ in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cγ-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes.