The trithorax and the polycomb group proteins are chromatin modifiers, which play a key role in the epigenetic regulation of development, differentiation and maintenance of cell fates. The polycomb repressive complex 2 (PRC2) mediates transcriptional repression by catalysing the di- and tri-methylation of Lys 27 on histone H3 (H3K27me2/me3). Owing to the essential role of the PRC2 complex in repressing a large number of genes involved in somatic processes, the H3K27me3 mark is associated with the unique epigenetic state of stem cells. The rapid decrease of the H3K27me3 mark during specific stages of embryogenesis and stem-cell differentiation indicates that histone demethylases specific for H3K27me3 may exist. Here we show that the human JmjC-domain-containing proteins UTX and JMJD3 demethylate tri-methylated Lys 27 on histone H3. Furthermore, we demonstrate that ectopic expression of JMJD3 leads to a strong decrease of H3K27me3 levels and causes delocalization of polycomb proteins in vivo. Consistent with the strong decrease in H3K27me3 levels associated with HOX genes during differentiation, we show that UTX directly binds to the HOXB1 locus and is required for its activation. Finally mutation of F18E9.5, a Caenorhabditis elegans JMJD3 orthologue, or inhibition of its expression, results in abnormal gonad development. Taken together, these results suggest that H3K27me3 demethylation regulated by UTX/JMJD3 proteins is essential for proper development. Moreover, the recent demonstration that UTX associates with the H3K4me3 histone methyltransferase MLL2 (ref. 8) supports a model in which the coordinated removal of repressive marks, polycomb group displacement, and deposition of activating marks are important for the stringent regulation of transcription during cellular differentiation.

The recent discovery of a large number of histone demethylases suggests a central role for these enzymes in regulating histone methylation dynamics. Histone H3K27 trimethylation (H3K27me3) has been linked to polycomb-group-protein-mediated suppression of Hox genes and animal body patterning, X-chromosome inactivation and possibly maintenance of embryonic stem cell (ESC) identity. An imbalance of H3K27 methylation owing to overexpression of the methylase EZH2 has been implicated in metastatic prostate and aggressive breast cancers. Here we show that the JmjC-domain-containing related proteins UTX and JMJD3 catalyse demethylation of H3K27me3/2. UTX is enriched around the transcription start sites of many HOX genes in primary human fibroblasts, in which HOX genes are differentially expressed, but is selectively excluded from the HOX loci in ESCs, in which HOX genes are largely silent. Consistently, RNA interference inhibition of UTX led to increased H3K27me3 levels at some HOX gene promoters. Importantly, morpholino oligonucleotide inhibition of a zebrafish UTX homologue resulted in mis-regulation of hox genes and a striking posterior developmental defect, which was partially rescued by wild-type, but not by catalytically inactive, human UTX. Taken together, these findings identify a small family of H3K27 demethylases with important, evolutionarily conserved roles in H3K27 methylation regulation and in animal anterior-posterior development.

Why do seemingly identical cells respond differently to a drug? To address this, we studied the dynamics and variability of the protein response of human cancer cells to a chemotherapy drug, camptothecin. We present a dynamic-proteomics approach that measures the levels and locations of nearly 1000 different endogenously tagged proteins in individual living cells at high temporal resolution. All cells show rapid translocation of proteins specific to the drug mechanism, including the drug target (topoisomerase-1), and slower, wide-ranging temporal waves of protein degradation and accumulation. However, the cells differ in the behavior of a subset of proteins. We identify proteins whose dynamics differ widely between cells, in a way that corresponds to the outcomes-cell death or survival. This opens the way to understanding molecular responses to drugs in individual cells.

ALR (MLL2) is a member of the human MLL family, which belongs to a larger SET1 family of histone methyltransferases. We found that ALR is present within a stable multiprotein complex containing a cohort of proteins shared with other SET1 family complexes and several unique components, such as PTIP and the jumonji family member UTX. Like other complexes formed by SET1 family members, the ALR complex exhibited strong H3K4 methyltransferase activity, conferred by the ALR SET domain. By generating ALR knockdown cell lines and comparing their expression profiles to that of control cells, we identified a set of genes whose expression is activated by ALR. Some of these genes were identified by chromatin immunoprecipitation as direct ALR targets. The ALR complex was found to associate in an ALR-dependent fashion with promoters and transcription initiation sites of target genes and to induce H3K4 trimethylation. The most characteristic features of the ALR knockdown cells were changes in the dynamics and mode of cell spreading/polarization, reduced migration capacity, impaired anchorage-dependent and -independent growth, and decreased tumorigenicity in mice. Taken together, our results suggest that ALR is a transcriptional activator that induces the transcription of target genes by covalent histone modification. ALR appears to be involved in the regulation of adhesion-related cytoskeletal events, which might affect cell growth and survival.

Cells remove proteins by two processes: degradation and dilution due to cell growth. The balance between these basic processes is poorly understood. We addressed this by developing an accurate and noninvasive method for measuring protein half-lives, called "bleach-chase," that is applicable to fluorescently tagged proteins. Assaying 100 proteins in living human cancer cells showed half-lives that ranged between 45 minutes and 22.5 hours. A variety of stresses that stop cell division showed the same general effect: Long-lived proteins became longer-lived, whereas short-lived proteins remained largely unaffected. This effect is due to the relative strengths of degradation and dilution and suggests a mechanism for differential killing of rapidly growing cells by growth-arresting drugs. This approach opens a way to understand proteome half-life dynamics in living cells.