Recognition of modified histone species by distinct structural domains within “reader” proteins plays a critical role in the regulation of gene expression. Readers that simultaneously recognize histones with multiple marks allow transduction of complex chromatin modification patterns into specific biological outcomes. Here, we report that chromatin regulator TRIM24 functions as a reader of dual histone marks via tandem Plant Homeodomain (PHD) and Bromodomain (Bromo). The three-dimensional structure of TRIM24 PHD-Bromo revealed a single functional unit for combinatorial recognition of unmodified H3K4 (H3K4me0) and acetylated H3K23 (H3K23ac) within the same histone tail. TRIM24 binds chromatin and estrogen receptor to activate estrogen-dependent genes associated with cellular proliferation and tumor development. Aberrant expression of TRIM24 negatively correlates with survival of breast cancer patients. The PHD-Bromo of TRIM24 provides a structural rationale for chromatin activation via a noncanonical histone signature, establishing a new paradigm by which chromatin readers may influence cancer pathogenesis.

Glioblastoma multiforme (GBM) is a particularly aggressive brain tumor and remains a clinically devastating disease. Despite innovative therapies for the treatment of GBM, there has been no significant increase in patient survival over the past decade. Enzymes that control epigenetic alterations are of considerable interest as targets for cancer therapy because of their critical roles in cellular processes that lead to oncogenesis. Several inhibitors of histone deacetylases (HDACs) have been developed and tested in GBM with moderate success. We found that treatment of GBM cells with HDAC inhibitors caused the accumulation of histone methylation, a modification removed by the lysine specific demethylase 1 (LSD1). This led us to examine the effects of simultaneously inhibiting HDACs and LSD1 as a potential combination therapy. We evaluated induction of apoptosis in GBM cell lines after combined inhibition of LSD1 and HDACs. LSD1 was inhibited by targeted short hairpin RNA or pharmacological means and inhibition of HDACs was achieved by treatment with either vorinostat or PCI-24781. Caspase-dependent apoptosis was significantly increased (>2-fold) in LSD1-knockdown GBM cells treated with HDAC inhibitors. Moreover, pharmacologically inhibiting LSD1 with the monoamine oxidase inhibitor tranylcypromine, in combination with HDAC inhibitors, led to synergistic apoptotic cell death in GBM cells; this did not occur in normal human astrocytes. Taken together, these results indicate that LSD1 and HDACs cooperate to regulate key pathways of cell death in GBM cell lines but not in normal counterparts, and they validate the combined use of LSD1 and HDAC inhibitors as a therapeutic approach for GBM.

Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein-protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.transcription regulation | cellular signaling | protein-protein interaction

Despite years of study focused on the tumor suppressor p53, little is understood about its functions in normal, differentiated cells. We found that p53 directly interacts with lysine-specific demethylase 1 (LSD1) to alter chromatin structure and confer developmental repression of the tumor marker alpha-fetoprotein (AFP). Chromatin immunoprecipitation (ChIP) and sequential ChIP of developmentally staged liver showed that p53 and LSD1 cooccupy a p53 response element, concomitant with dimethylated histone H3 lysine 4 (H3K4me2) demethylation and postnatal repression of AFP transcription. In p53-null mice, LSD1 binding is depleted, H3K4me2 is increased, and H3K9me2 remains unchanged compared to those of the wild type, underscoring the specificity of p53-LSD1 complexes in demethylation of H3K4me2. We performed partial hepatectomy of wild-type mouse liver and induced a regenerative response, which led to a loss of p53, increased H3K4me2, and decreased LSD1 interaction at AFP chromatin, in parallel with reactivation of AFP expression. In contrast, nuclear translocation of p53 in mouse embryonic fibroblasts led to p53 interaction with p21/CIP1 chromatin, without recruitment of LSD1, and to activation of p21/CIP1. These findings reveal that LSD1 is targeted to chromatin by p53, likely in a gene-specific manner, and define a molecular mechanism by which p53 mediates transcription repression in vivo during differentiation.Numerous analyses of p53 functions, in response to cellular stress, underscore its important, pleiotropic roles in arrest of the cell cycle and promotion of apoptosis (5,24,33). Less understood are mechanisms of p53-mediated regulation during differentiation and in maintenance of cellular homeostasis. Previous work from our laboratory established p53 as a major repressor of AFP transcription during hepatic development (60). Developmental repression of AFP, an onco-fetal tumor marker gene, is delayed more than 2 months after birth in p53-null mice, compared to cessation within 2 to 3 weeks in wild-type (WT) mice (45). Transactivating p73 (TA-p73), but not TA-p63, partially compensates for an absence of p53 in p53-null mice, characterized by elevated methylation of histone H3 lysine 4 (H3K4) at the p53/p73 response element of alpha-fetoprotein (AFP) chromatin (12). Therefore, we hypothesized that an H3K4 demethylase is recruited by p53 during hepatic differentiation and that combinatorial alterations in chromatin structure during development dictate the developmental timing of transcription regulation.Enzymatic removal of methyl groups from lysine residues of histone N-terminal tails correlates with either repression or activation of transcription, as determined by the specific histone substrate, amino acid position, and level of methylation. Loss of methylated H3K4 (H3K4me) is generally associated with repression or cessation of active transcription (7,14,55).The first enzyme with the capacity to remove methyl groups from histone lysine residues was identified as BHC110 or histone lysine-specific demethylas...

BACKGROUND Katanin p60 is a microtubule-severing protein and is involved in microtubule cytoskeleton organization in both mitotic and non-mitotic processes. Its role in cancer metastasis is unknown. METHODS Differential protein profiles of bone marrow aspirates were analyzed by chromatography, electropheresis and mass spectrometry. Expression of katanin p60 in primary and metastatic prostate cancer was examined by immunohistochemistry. Cellular function of katanin p60 was further examined in prostate cell lines. RESULTS In a proteomic profiling of bone marrow aspirates from men with prostate cancer, we found that katanin p60 was one of the proteins differentially expressed in bone metastasis samples. Immunohistochemical staining showed that katanin p60 was expressed in the basal cells in normal human prostate glands. In prostatic adenocarcinomas, in which the basal cells were absent, katanin p60 was expressed in the prostate cancer cells. In the specimens from bone metastasis, katanin p60 was detectable in the metastatic cancer cells. Strikingly, some of the metastatic cancer cells also co-expressed basal cell biomarkers including the tumor suppressor p53-homologous protein p63 and the high molecular weight cytokeratins, suggesting that the metastatic prostate cancer cells may have a basal cell-like phenotype. Moreover, overexpression of katanin p60 inhibited prostate cancer cell proliferation but enhanced cell migration activity. CONCLUSIONS Katanin p60 was aberrantly expressed during prostate cancer progression. Its expression in the metastatic cells in bone was associated with the re-emergence of a basal cell-like phenotype. The elevated katanin p60 expression may contribute to cancer cell metastasis via a stimulatory effect on cell motility.