High-yield and high-purity amide bond formation using DMTMM PF6 for DNA-encoded libraries.

In this study, we report on the ability of DMTMM PF6 to improve the amidation reaction. The on-DNA amidation reaction using DMTMM PF6 demonstrates higher conversion rates than those using HATU or DMTMM Cl, particularly with challenging sterically hindered amines and carboxylic acids. The developed method enables the expansion of available building blocks and the efficient synthesis of high-purity DNA-encoded libraries.

Regulation of DNA demethylation during maturation of CD4+ naive T cells by the conserved noncoding sequence 1.

Demethylation of transcriptional regulatory elements and gene coding regions is an important step in the epigenetic regulation of gene expression. Several noncoding conserved regions are required for the efficient transcription of cytokine genes. In this paper, we show that the deletion of one such sequence, conserved noncoding sequence 1 (CNS-1), interferes with the efficient demethylation of Th2 cytokine genes but has little effect on histone modifications in the area. Th2 cells derived from CD4 single-positive (SP) mature thymocytes exhibit more rapid demethylation of CNS-1 and Th2-specific cytokine genes and produce more Th2 cytokines than do Th2 cells derived from CD4-positive peripheral naive T cells. De-repression of the Th1 cytokine IFN-gamma was also detected in Th2-primed CD4 SP thymocytes but not in naive T cells. Our results indicate that susceptibility to demethylation determines the efficiency and kinetics of cytokine gene transcription. The extrathymic maturation step undergone by naive T cells suppresses robust and rapid cytokine expression, whereas mature CD4 SP thymocytes maintain a rapid and less-specific cytokine expression profile. Finally, we detected the methyl cytosine binding protein MBD2 at CNS-1 in mature thymocytes, suggesting that this protein may regulate the demethylation of this region.

Replication initiation from a novel origin identified in the Th2 cytokine cluster locus requires a distant conserved noncoding sequence.

Lineage commitment of Th cells is associated with the establishment of specific transcriptional programs of cytokines. However, how Th cell differentiation affects the program of DNA replication has not been addressed. To gain insight into interplays between differentiation-induced transcription regulation and initiation of DNA replication, we took advantage of an in vitro differentiation system of naive T cells, in which one can manipulate their differentiation into Th1 or Th2 cells. We searched for replication origins in the murine IL-4/IL-13 locus and compared their profiles in the two Th cell lineages which were derived in vitro from the same precursor T cells. We identified a replication origin (ori(IL-13)) downstream from exon 4 of IL-13 and showed that this origin functions in both Th2 and Th1 cells. A distant regulatory element called CNS-1 (conserved noncoding sequence 1) in the IL-4/IL-13 intergenic region coincides with a Th2-specific DNase I-hypersensitive site and is required for efficient, coordinated expression of Th2 cytokines. Replication initiation from ori(IL-13) is significantly reduced in Th1 and Th2 cells derived from CNS-1-deficient mice. However, the replication timing of this locus is consistently early during S phase in both Th1 and Th2 cells under either the wild-type or CNS-1 deletion background. Thus, the conserved noncoding element in the intergenic region regulates replication initiation from a distant replication origin in a manner independent from its effect on lineage-specific transcription but not the replication timing of the segment surrounding this origin.