Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.

Embryonic stem (ES) cells require a coordinated network of transcription factors to maintain pluripotency or trigger lineage specific differentiation. Central to these processes are the proteins Oct4, Nanog, and Sox2. Although the transcriptional targets of these factors have been extensively studied, very little is known about how the proteins themselves are regulated, especially at the post-translational level. Post-translational modifications are well documented to have broad effects on protein stability, activity, and cellular distribution. Here, we identify a key lysine residue in the nuclear export signal of Sox2 that is acetylated, and demonstrate that blocking acetylation at this site retains Sox2 in the nucleus and sustains expression of its target genes under hyperacetylation or differentiation conditions. Mimicking acetylation at this site promotes association of Sox2 with the nuclear export machinery. In addition, increased cellular acetylation leads to reduction in Sox2 levels by ubiquitination and proteasomal degradation, thus abrogating its ability to drive transcription of its target genes. Acetylation-mediated nuclear export may be a commonly used regulatory mechanism for many Sox family members, as this lysine is conserved across species and in orthologous proteins. STEM CELLS

The Ena/vasodilator-stimulated phosphoprotein (VASP) protein family is implicated in the regulation of a number of actin-based cellular processes, including lamellipodial protrusion necessary for whole cell translocation. A growing body of evidence derived largely from in vitro biochemical experiments using purified proteins, cell-free extracts, and pathogen motility has begun to suggest various mechanistic roles for Ena/VASP proteins in the control of actin dynamics. Using complementation of phenotypes in Ena/VASP-deficient cells and overexpression in normal fibroblasts, we have assayed the function of a panel of mutants in one member of this family, Mena, by mutating highly conserved sequence elements found in this protein family. Surprisingly, deletion of sites required for binding of the actin monomer-binding protein profilin, a known ligand of Ena/VASP proteins, has no effect on the ability of Mena to regulate random cell motility. Our analysis revealed two features essential for Ena/VASP function in cell movement, cyclic nucleotide-dependent kinase phosphorylation sites and an F-actin binding motif. Interestingly, expression of the C-terminal EVH2 domain alone is sufficient to complement loss of Ena/VASP function in random cell motility.

Drugging large protein pockets is a challenge due to the need for higher molecular weight ligands, which generally possess undesirable physicochemical properties. In this communication, we highlight a strategy leveraging small molecule active site dimers to inhibit the large symmetric binding pocket in the STING protein. By taking advantage of the 2:1 binding stoichiometry, maximal buried interaction with STING protein can be achieved while maintaining the ligand physicochemical properties necessary for oral exposure. This mode of binding requires unique considerations for potency optimization including simultaneous optimization of protein−ligand as well as ligand−ligand interactions. Successful implementation of this strategy led to the identification of 18, which exhibits good oral exposure, slow binding kinetics, and functional inhibition of STING-mediated cytokine release.