Molecular characterization of transgene integration by next-generation sequencing in transgenic cattle.

As the number of transgenic livestock increases, reliable detection and molecular characterization of transgene integration sites and copy number are crucial not only for interpreting the relationship between the integration site and the specific phenotype but also for commercial and economic demands. However, the ability of conventional PCR techniques to detect incomplete and multiple integration events is limited, making it technically challenging to characterize transgenes. Next-generation sequencing has enabled cost-effective, routine and widespread high-throughput genomic analysis. Here, we demonstrate the use of next-generation sequencing to extensively characterize cattle harboring a 150-kb human lactoferrin transgene that was initially analyzed by chromosome walking without success. Using this approach, the sites upstream and downstream of the target gene integration site in the host genome were identified at the single nucleotide level. The sequencing result was verified by event-specific PCR for the integration sites and FISH for the chromosomal location. Sequencing depth analysis revealed that multiple copies of the incomplete target gene and the vector backbone were present in the host genome. Upon integration, complex recombination was also observed between the target gene and the vector backbone. These findings indicate that next-generation sequencing is a reliable and accurate approach for the molecular characterization of the transgene sequence, integration sites and copy number in transgenic species.

SET8 plays a role in controlling G1/S transition by blocking lysine acetylation in histone through binding to H4 N-terminal tail.

We report evidence suggesting that methyltransferase SET8 plays a novel role in regulating cell cycle by suppressing DNA replication through histone binding. First, the distribution of SET8 is strongly cell cycle-dependent. SET8 was concentrated in the nucleus during G(1) and G(2) phases, and was excluded from the nucleus during S phase. Second, at G(1)/S transition, SET8 was degraded through ubiquitination via SCF/Skp2. Third, it was evident that the SET8 binds to the H4 N-terminal tail (H4NT) and blocks the acetylation of lysine residues K5, K8 and K12 of histone H4 during G(1). Such a blockage can hinder DNA replication. Fourth, SET8 binds to hypoacetylated but not hyperacetylated H4NT. Finally, overexpressing the histone-binding domain of SET8 appeared to suppress DNA replication and arrest the cell cycle before the G(1)/S transition. Taken together, these findings suggest that SET8 can be a negative regulator of DNA replication and the destruction of SET8 is required for the onset of S phase.

MH1 domain of SMAD4 binds N-terminal residues of the homeodomain of Hoxc9.

Smad family proteins mediate signaling initiated by bone morphogenetic proteins (BMPs). Upon BMP stimulation, the Smads such as Smad4 can interact directly with Hox proteins and suppress their DNA-binding activity. Although the interaction between the MAD-homology 1 (MH1) domain of Smad4 and Hox was found to regulate the transcription activity of Hox proteins, the molecular mechanism is not well characterized and direct contact residues remain to be elucidated. In the present study, the interaction between the recombinant homeodomain (HD) of Hoxc9 and MH1 domain of Smad4 was investigated with the use of the GST pull-down assay, surface plasmon resonance (SPR) analysis as well as multidimensional nuclear magnetic resonance (NMR) techniques. The Hoxc9-HD was precipitated with the GST-fused Smad4-MH1 but not with GST alone, demonstrating a direct interaction between Hoxc9-HD and Smad4-MH1 in vitro. SPR measurement further confirmed a moderately strong interaction (K(d) approximately 400 nM) between these two domains. Moreover, NMR titration experiments showed that a strong and specific binding occurred between Smad4-MH1 and Hoxc9-HD. NMR triple-resonance experiments and backbone assignments revealed that the N-terminal arm of Hoxc9-HD, spanning the positive-charged DNA-binding segment of Arg190-Arg196, was intimately involved in the interaction with Smad4-MH1. Ala-substitutions of Arg190-Arg196 led to the loss of interaction between Hoxc9-HD and Smad4-MH1 in both GST-pull down assay and SPR analysis; further provided functional evidence for the critical role of this positive-charged region in binding to Smad4-MH1. This suggested that Smad4-MH1 could occupy one of the DNA binding sites of Hoxc9 and consequently inhibits its transcription activity. The above results are in good agreement and yield the first insight into the interaction between the homeodomain of Hox proteins and the conserved MH1 domain of Smad family proteins.

SET8 recognizes the sequence RHRK20VLRDN within the N terminus of histone H4 and mono-methylates lysine 20.

Methylation of lysine 20 in histone H4 has been proven to play important roles in chromatin structure and gene regulation. SET8 is one of the methyltransferases identified to be specific for this modification. In this study, the minimal active SET domain of SET8 has been mapped to the region of amino acids 195-352. This region completely retains the same methylation activity and substrate specificity as the full-length SET8. The SET domain recognizes a stretch of specific amino acid sequence around lysine 20 of H4 for its methylation activity. Methylation assays with N terminus mutants of H4 that contain deletions and single alanine or glutamine substitutions of charged residues revealed that SET8 requires the sequence RHRK20VLRDN for methylation at lysine 20. The individual mutation of any charged residue in this sequence to alanine or glutamine abolished or greatly decreased levels of methylation of lysine 20 of H4 by SET8. Interestingly, mutation of lysine 16 to alanine, arginine, glutamine, or methionine did not affect methylation of lysine 20 by the SET domain. Mass spectrometric analysis of synthesized H4 N-terminal peptides modified by SET8 showed that SET8 selectively mono-methylates lysine 20 of H4. Taken together, our results suggested that the coordination between the amino acid sequence RHRK20VLRDN and the SET domain of SET8 determines the substrate specificity and multiplicity of methylation of lysine 20 of H4.

Studies on the Separation, Purification of Beijing Duck Apo A-I CNBr Cleavaged Fragments and the Localization of Their Function Domain.

Apo A-I of Beijing duck was first cleavaged by CNBr into 11 peptide fragments which were further separated and purified by preparative SDS-PAGE and HPLC, then their molecular weights and N-terminal amino acid sequences were also determined. According to the molecular weights and N-terminal amino acid sequences of each fragment the positions of peptide fragments 3-10 in apo A-I molecule were localized at the sites of amino acid sequences 64-240, 74-240, 64-206, 1-163, 1-36, 171-206, 207-240 and 137-170 respectively. The studied results of their main functions are as following: (1) All the fragments could combine with lipids to form lipsomes of various sizes. The fragment Longer, the size will be larger. (2) The ability of the fragments 3-10 to activate LCAT equals to 65%, 52%, 60%, 39%, 8%, 7%, 0% and 2% of that of the whole apo A-I molecule respectively, showing that the activating sequence is localized in amino acid residues 64-136. (3) Only the liposomes formed with fragments 3, 4 and 9 could combine with the HDL receptor of liver cell membrane of Beijing duck; the rest of the liposomes demonstrated essentially no binding ability. As peptide fragments 3 and 4 also contained fragment 9, the result indicated that amino acids 207-240 maybe the fragment that combined with HDL receptor.