Distinct Stimulatory Mechanisms Regulate the Catalytic Activity of Polycomb Repressive Complex 2.

The maintenance of gene expression patterns during metazoan development is achieved, in part, by the actions of polycomb repressive complex 2 (PRC2). PRC2 catalyzes mono-, di-, and trimethylation of histone H3 at lysine 27 (H3K27), with H3K27me2/3 being strongly associated with silenced genes. We demonstrate that EZH1 and EZH2, the two mutually exclusive catalytic subunits of PRC2, are differentially activated by various mechanisms. Whereas both PRC2-EZH1 and PRC2-EZH2 are able to catalyze mono- and dimethylation, only PRC2-EZH2 is strongly activated by allosteric modulators and specific chromatin substrates to catalyze trimethylation of H3K27 in mouse embryonic stem cells (mESCs). However, we also show that a PRC2-associated protein, AEBP2, can stimulate the activity of both complexes through a mechanism independent of and additive to allosteric activation. These results have strong implications regarding the cellular requirements for and the accompanying adjustments in PRC2 activity, given the differential expression of EZH1 and EZH2 upon cellular differentiation.

Smyd2 controls cytoplasmic lysine methylation of Hsp90 and myofilament organization.

Protein lysine methylation is one of the most widespread post-translational modifications in the nuclei of eukaryotic cells. Methylated lysines on histones and nonhistone proteins promote the formation of protein complexes that control gene expression and DNA replication and repair. In the cytoplasm, however, the role of lysine methylation in protein complex formation is not well established. Here we report that the cytoplasmic protein chaperone Hsp90 is methylated by the lysine methyltransferase Smyd2 in various cell types. In muscle, Hsp90 methylation contributes to the formation of a protein complex containing Smyd2, Hsp90, and the sarcomeric protein titin. Deficiency in Smyd2 results in the loss of Hsp90 methylation, impaired titin stability, and altered muscle function. Collectively, our data reveal a cytoplasmic protein network that employs lysine methylation for the maintenance and function of skeletal muscle.

CD19 and BAFF-R can signal to promote B-cell survival in the absence of Syk.

The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B-cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B-cell-specific deletion of the Syk gene and found that a considerable fraction of mature Syk-negative B cells can survive in the periphery for an extended time. Syk-negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL-4, anti-CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk-deficient B cells require BAFF receptor and CD19/PI3K signaling for their long-term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B-cell pool.

Ezh2-mediated repression of a transcriptional pathway upstream of Mmp9 maintains integrity of the developing vasculature.

Maintenance of vascular integrity is required for embryogenesis and organ homeostasis. However, the gene expression programs that stabilize blood vessels are poorly understood. Here, we show that the histone methyltransferase Ezh2 maintains integrity of the developing vasculature by repressing a transcriptional program that activates expression of Mmp9. Inactivation of Ezh2 in developing mouse endothelium caused embryonic lethality with compromised vascular integrity and increased extracellular matrix degradation. Genome-wide approaches showed that Ezh2 targets Mmp9 and its activators Fosl1 and Klf5. In addition, we uncovered Creb3l1 as an Ezh2 target that directly activates Mmp9 gene expression in the endothelium. Furthermore, genetic inactivation of Mmp9 rescued vascular integrity defects in Ezh2-deficient embryos. Thus, epigenetic repression of Creb3l1, Fosl1, Klf5 and Mmp9 by Ezh2 in endothelial cells maintains the integrity of the developing vasculature, potentially linking this transcriptional network to diseases with compromised vascular integrity.

The lipid raft-anchored adaptor protein Cbp controls the oncogenic potential of c-Src.

The tyrosine kinase c-Src is upregulated in various human cancers irrespective of its negative regulator Csk, but the regulatory mechanisms remain unclear. Here, we show that a lipid raft-anchored Csk adaptor, Cbp/PAG, is directly involved in controlling the oncogenicity of c-Src. Using Csk-deficient cells that can be transformed by c-Src overexpression, we found that Cbp expression is markedly downregulated by c-Src activation and re-expression of Cbp efficiently suppresses c-Src transformation as well as tumorigenesis. Cbp-deficient cells are more susceptible to v-Src transformation than their parental cells. Upon phosphorylation, Cbp specifically binds to activated c-Src and sequesters it in lipid rafts, resulting in an efficient suppression of c-Src function independent of Csk. In some human cancer cells and tumors, Cbp is downregulated and the introduction of Cbp significantly suppresses tumorigenesis. These findings indicate a potential role for Cbp as a suppressor of c-Src-mediated tumor progression.

Identification of a new interaction mode between the Src homology 2 domain of C-terminal Src kinase (Csk) and Csk-binding protein/phosphoprotein associated with glycosphingolipid microdomains.

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the ßB/ßC loop of the SH2 domain.

Ezh2, the histone methyltransferase of PRC2, regulates the balance between self-renewal and differentiation in the cerebral cortex.

Multipotent progenitor cells of the cerebral cortex balance self-renewal and differentiation to produce complex neural lineages in a fixed temporal order in a cell-autonomous manner. We studied the role of the polycomb epigenetic system, a chromatin-based repressive mechanism, in controlling cortical progenitor cell self-renewal and differentiation. We found that the histone methyltransferase of polycomb repressive complex 2 (PCR2), enhancer of Zeste homolog 2 (Ezh2), is essential for controlling the rate at which development progresses within cortical progenitor cell lineages. Loss of function of Ezh2 removes the repressive mark of trimethylated histone H3 at lysine 27 (H3K27me3) in cortical progenitor cells and also prevents its establishment in postmitotic neurons. Removal of this repressive chromatin modification results in marked up-regulation in gene expression, the consequence of which is a shift in the balance between self-renewal and differentiation toward differentiation, both directly to neurons and indirectly via basal progenitor cell genesis. Although the temporal order of neurogenesis and gliogenesis are broadly conserved under these conditions, the timing of neurogenesis, the relative numbers of different cell types, and the switch to gliogenesis are all altered, narrowing the neurogenic period for progenitor cells and reducing their neuronal output. As a consequence, the timing of cortical development is altered significantly after loss of PRC2 function.

The catalytic domain of the histone methyltransferase NSD2/MMSET is required for the generation of B1 cells in mice.

Humoral immunity in mammals relies on the function of two developmentally and functionally distinct B-cell subsets-B1 and B2 cells. While B2 cells are responsible for the adaptive response to environmental antigens, B1 cells regulate the production of polyreactive and low-affinity antibodies for innate humoral immunity. The molecular mechanism of B-cell specification into different subsets is understudied. In this study, we identified lysine methyltransferase NSD2 (MMSET/WHSC1) as a critical regulator of B1 cell development. In contrast to its minor impact on B2 cells, deletion of the catalytic domain of NSD2 in primary B cells impairs the generation of B1 lineage. Thus, NSD2, a histone H3 K36 dimethylase, is the first-in-class epigenetic regulator of a B-cell lineage in mice.

The ubiquitously expressed Csk adaptor protein Cbp is dispensable for embryogenesis and T-cell development and function.

Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of "lipid rafts" is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

Signaling function of PRC2 is essential for TCR-driven T cell responses.

Differentiation and activation of T cells require the activity of numerous histone lysine methyltransferases (HMT) that control the transcriptional T cell output. One of the most potent regulators of T cell differentiation is the HMT Ezh2. Ezh2 is a key enzymatic component of polycomb repressive complex 2 (PRC2), which silences gene expression by histone H3 di/tri-methylation at lysine 27. Surprisingly, in many cell types, including T cells, Ezh2 is localized in both the nucleus and the cytosol. Here we show the presence of a nuclear-like PRC2 complex in T cell cytosol and demonstrate a role of cytosolic PRC2 in T cell antigen receptor (TCR)-mediated signaling. We show that short-term suppression of PRC2 precludes TCR-driven T cell activation in vitro. We also demonstrate that pharmacological inhibition of PRC2 in vivo greatly attenuates the severe T cell-driven autoimmunity caused by regulatory T cell depletion. Our data reveal cytoplasmic PRC2 is one of the most potent regulators of T cell activation and point toward the therapeutic potential of PRC2 inhibitors for the treatment of T cell-driven autoimmune diseases.

Coupling of T cell receptor specificity to natural killer T cell development by bivalent histone H3 methylation.

The fidelity of T cell immunity depends greatly on coupling T cell receptor signaling with specific T cell effector functions. Here, we describe a chromatin-based mechanism that enables integration of TCR specificity into definite T cell lineage commitment. Using natural killer T cells (iNKT cell) as a model of a T cell subset that differentiates in response to specific TCR signaling, we identified a key role of histone H3 lysine 27 trimethylation (H3K27me3) in coupling iNKT cell TCR specificity with the generation of iNKT cells. We found that the Zbtb16/PLZF gene promoter that drives iNKT cell differentiation possesses a bivalent chromatin state characterized by the simultaneous presence of negative and positive H3K27me3 and H3K4me3 modifications. Depletion of H3K27me3 at the Zbtb16/PLZF promoter leads to uncoupling of iNKT cell development from TCR specificity and is associated with accumulation of iNKT-like CD4(+) cells that express a non-iNKT cell specific T cell repertoire. In turn, stabilization of H3K27me3 leads to a drastic reduction of the iNKT cell population. Our data suggest that H3K27me3 levels at the bivalent Zbtb16/PLZF gene define a threshold enabling precise coupling of TCR specificity to lineage commitment.

Polycomb group protein ezh2 controls actin polymerization and cell signaling.

Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. Here, we report the existence of the cytosolic Ezh2-containing methyltransferase complex and tie the function of this complex to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes.