The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

In this study we initially examined the interaction between CD44v3 (a hyaluronan (HA) receptor) and Vav2 (a guanine nucleotide exchange factor) in human ovarian tumor cells (SK-OV-3.ipl cell line). Immunological data indicate that both CD44v3 and Vav2 are expressed in SK-OV-3.ipl cells and that these two proteins are physically linked as a complex in vivo. By using recombinant fragments of Vav2 and in vitro binding assays, we have detected a specific binding interaction between the SH3-SH2-SH3 domain of Vav2 and the cytoplasmic domain of CD44. In addition, we have observed that the binding of HA to CD44v3 activates Vav2-mediated Rac1 signaling leading to ovarian tumor cell migration. Further analyses indicate that the adaptor molecule, growth factor receptor-bound protein 2 (Grb2) that is bound to p185 HER2 (an oncogene product), is also associated with the CD44v3-Vav2 complex. HA binding to SK-OV-3.ipl cells promotes recruitment of both Grb2 and p185 HER2 to the CD44v3-Vav2 complex leading to Ras activation and ovarian tumor cell growth. In order to determine the role of Grb2 in CD44v3 signaling, we have transfected SK-OV-3.ipl cells with Grb2 mutant cDNAs (e.g. ⌬N-Grb2 that has a deletion in the amino-terminal SH3 domain or ⌬C-Grb2 that has a deletion in the carboxyl-terminal SH3 domain). Our results clearly indicate that the SH3 domain deletion mutants of Grb2 (i.e. the ⌬N-Grb2 (and to a lesser extent the ⌬C-Grb2) mutant) not only block their association with p185 HER2 but also significantly impair their binding to the CD44v3-Vav2 complex and inhibit HA/CD44v3-induced ovarian tumor cell behaviors. Taken together, these findings strongly suggest that the interaction of CD44v3-Vav2 with Grb2-p185 HER2 plays an important role in the coactivation of both Rac1 and Ras signaling that is required for HA-mediated human ovarian tumor progression.The cell adhesion molecule, CD44, is a product of a single gene that undergoes alternative splicing of 12 possible exons to generate variant isoforms (1). Nucleotide sequence analyses reveal that the CD44 isoforms are variants of the standard form, CD44s (1). CD44s (molecular mass Ϸ85 kDa) is the most common isoform of CD44 found in many cell types including human ovarian carcinoma cells (2). CD44 can be further modified by extensive N-and O-glycosylations and glycosaminoglycan additions (3-5). Apparently, both post-translational modifications and/or alternative splicing within the CD44 structure determine the functional outcome of this molecule. CD44 is a transmembrane glycoprotein that is one of the major hyaluronan (HA) 1 receptors (6). CD44 binds to extracellular matrix components (i.e. HA) at its amino terminus of the extracellular domain (7,8). CD44 also contains specific binding sites for the cytoskeleton (9 -14) and various signaling molecules (15-19) within the 70-amino acid carboxyl terminus in the cytoplasmic domain.Both CD44 and HA appear to be overexpressed at sites of tumor attachment and are known to be involved in cell aggregation, proliferation, migration, and...

In this study we have examined the interaction between CD44 (a hyaluronan (HA) receptor) and the trans-

miR-9 mediates the downregulation of BRCA1 and impedes DNA damage repair in ovarian cancer. miR-9 may improve chemotherapeutic efficacy by increasing the sensitivity of cancer cells to DNA damage and may impact ovarian cancer therapy.

Ovarian carcinoma is caused by multiple factors, but its etiology associated with microbes and infection is unknown. Using 16S rRNA high-throughput sequencing methods, the diversity and composition of the microbiota from ovarian cancer tissues (25 samples) and normal distal fallopian tube tissues (25 samples) were analyzed. High-throughput sequencing showed that the diversity and richness indexes were significantly decreased in ovarian cancer tissues compared to tissues from normal distal fallopian tubes. The ratio of the two phyla for Proteobacteria/Firmicutes was notably increased in ovarian cancer, which revealed that microbial composition change might be associated with the process of ovarian cancer development. In addition, transcriptome-sequencing (RNA-seq) analyses suggested that the transcriptional profiles were statistically different between ovarian carcinoma and normal distal fallopian tubes. Moreover, a set of genes including 84 different inflammation-associated or immune-associated genes, which had been named as the human antibacterial-response genes were also modulated expression. Therefore, we hypothesize that the microbial composition change, as a novel risk factor, may be involving the initiation and progression of ovarian cancer via influencing and regulating the local immune microenvironment of fallopian tubes except for regular pathways.

SUMMARY Histone H2B ubiquitination plays an important role in transcription regulation. It has been shown that H2B ubiquitination is regulated by multiple upstream events associated with elongating RNA polymerase. Here we demonstrate that H2B K34 ubiquitylation by the MOF-MSL complex is part of the protein networks involved in early steps of transcription elongation. Knocking down MSL2 in the MOF-MSL complex affects not only global H2BK34ub, but also multiple co-transcriptionally regulated histone modifications. More importantly, we show that the MSL, PAF1 and RNF20/40 complexes are recruited and stabilized at active gene promoters by direct binary interactions. The stabilized complexes serve to regulate chromatin association of pTEFb through a positive feedback loop and facilitate Pol II transition during early transcription elongation. Results from our biochemical studies are underscored by genome-wide analyses that show high RNA Pol II processivity and transcription activity at MSL target genes.

Summary Aberrant expression of HOXA9 is a prominent feature of acute leukemia driven by diverse oncogenes. Here we show that HOXA9 overexpression in myeloid and B progenitor cells leads to significant enhancer reorganizations with prominent emergence of leukemia-specific de novo enhancers. Alterations in the enhancer landscape lead to activation of an ectopic embryonic gene program. We show that HOXA9 functions as a pioneer factor at de novo enhancers and recruits CEBPα and the MLL3/MLL4 complex. Genetic deletion of MLL3/MLL4 blocks histone H3K4 methylation at de novo enhancers and inhibits HOXA9/MEIS1-mediated leukemogenesis in vivo. These results show that therapeutic targeting of HOXA9-dependent enhancer reorganization can be an effective therapeutic strategy in acute leukemia with HOXA9 overexpression.

DNA methylation and histone modification are two major epigenetic pathways that interplay to regulate transcriptional activity and other genome functions. Dnmt3L is a regulatory factor for the de novo DNA methyltransferases Dnmt3a and Dnmt3b. Although recent biochemical studies have revealed that Dnmt3L binds to the tail of histone H3 with unmethylated lysine 4 in vitro, the requirement of chromatin components for DNA methylation has not been examined, and functional evidence for the connection of histone tails to DNA methylation is still lacking. Here, we used the budding yeast Saccharomyces cerevisiae as a model system to investigate the chromatin determinants of DNA methylation through ectopic expression of murine Dnmt3a and Dnmt3L. We found that the N terminus of histone H3 tail is required for de novo methylation, while the central part encompassing lysines 9 and 27, as well as the H4 tail are dispensable. DNA methylation occurs predominantly in heterochromatin regions lacking H3K4 methylation. In mutant strains depleted of H3K4 methylation, the DNA methylation level increased 5-fold. The methylation activity of Dnmt3a largely depends on the Dnmt3L's PHD domain recognizing the histone H3 tail with unmethylated lysine 4. Functional analysis of Dnmt3L in mouse ES cells confirmed that the chromatin-recognition ability of Dnmt3L's PHD domain is indeed required for efficient methylation at the promoter of the endogenous Dnmt3L gene. These findings establish the N terminus of histone H3 tail with an unmethylated lysine 4 as a chromatin determinant for DNA methylation.T he genomic DNA in mammalian cells is not only bound with basic histone proteins to form chromatin, but also covalently methylated at cytosine bases in specific regions. DNA cytosine methylation together with histone modifications in chromatin present profound epigenetic platforms for the regulation of gene expression and genome functions in various developmental and disease processes (1).A fundamental question concerns how DNA methylation occurs in the first place. In mammals, de novo methyltransferases Dnmt3a and 3b function to establish the methylation patterns in the genome, and the maintenance enzyme Dnmt1 acts to replicate the methylation patterns in cell divisions (2). Cross-talk between DNA methylation and histone modification exists in various contexts of transcriptional regulation and chromatin functions (3). There is some evidence that chromatin cues might be a determinant of DNA methylation. In the filamentous fungus Neurospora crassa, either the replacement of histone H3 lysine 9 (H3K9) with other amino acids, or deletion of the sole H3K9 methyltransferase DIM5 results in the loss of DNA methylation (4). In mouse ES cells, deficiency in the histone H3K9 methyltransferases Suv39h1-Suv39h2 is associated with hypomethylation at a subset of repeat elements (5). These correlative observations fail to establish a clear link between histone modifications and DNA methylation, partially because deletion of a histone methyltransferase or loss of ...