The Drosophila Dbf4 Ortholog Chiffon Forms A Complex with Gcn5 That Is Necessary for Histone Acetylation and Viability

Metazoans contain two homologs of the Gcn5-binding protein Ada2, Ada2a and Ada2b, which nucleate formation of the ATAC and SAGA complexes respectively. In Drosophila melanogaster, there are two splice isoforms of Ada2b: Ada2b-PA and Ada2b-PB. Here we show only the Ada2b-PB isoform is in SAGA; in contrast, Ada2b-PA associates with Gcn5, Ada3, Sgf29 and Chiffon forming the Chiffon Histone Acetyltransferase (CHAT) complex. Chiffon is theDrosophila ortholog of Dbf4, which binds and activates the cell cycle kinase Cdc7 to initiate DNA replication. In flies, Chiffon and Cdc7 are required in ovary follicle cells for gene amplification, a specialized form of DNA re-replication. Although chiffon was previously reported to be dispensable for viability, here we find that Chiffon is required for both histone acetylation and viability in flies. Surprisingly, we show that chiffon is a dicistronic gene that encodes distinct Cdc7- and CHAT-binding polypeptides. Although the Cdc7-binding domain of Chiffon is not required for viability in flies, Chiffon’s CHAT-binding domain is essential for viability but is not required for gene amplification, arguing against a role in DNA replication

At the Crossroad of Gene Regulation and Genome Organization: Potential Roles for ATP-Dependent Chromatin Remodelers in the Regulation of CTCF-Mediated 3D Architecture

In higher order organisms, the genome is assembled into a protein-dense structure called chromatin. Chromatin is spatially organized in the nucleus through hierarchical folding, which is tightly regulated both in cycling cells and quiescent cells. Assembly and folding are not one-time events in a cell’s lifetime; rather, they are subject to dynamic shifts to allow changes in transcription, DNA replication, or DNA damage repair. Chromatin is regulated at many levels, and recent tools have permitted the elucidation of specific factors involved in the maintenance and regulation of the three-dimensional (3D) genome organization. In this review/perspective, we aim to cover the potential, but relatively unelucidated, crosstalk between 3D genome architecture and the ATP-dependent chromatin remodelers with a specific focus on how the architectural proteins CTCF and cohesin are regulated by chromatin remodeling

PBRM1 Regulates the Expression of Genes Involved in Metabolism and Cell Adhesion in Renal Clear Cell Carcinoma

<div><p>Polybromo-1 (PBRM1) is a component of the PBAF (Polybromo-associated-BRG1- or BRM-associated factors) chromatin remodeling complex and is the second most frequently mutated gene in clear-cell renal cell Carcinoma (ccRCC). Mutation of PBRM1 is believed to be an early event in carcinogenesis, however its function as a tumor suppressor is not understood. In this study, we have employed Next Generation Sequencing to profile the differentially expressed genes upon PBRM1 re-expression in a cellular model of ccRCC. PBRM1 re-expression led to upregulation of genes involved in cellular adhesion, carbohydrate metabolism, apoptotic process and response to hypoxia, and a downregulation of genes involved in different stages of cell division. The decrease in cellular proliferation upon PBRM1 re-expression was confirmed, validating the functional role of PBRM1 as a tumor suppressor in a cell-based model. In addition, we identified a role for PBRM1 in regulating metabolic pathways known to be important for driving ccRCC, including the regulation of hypoxia response genes, PI3K signaling, glucose uptake, and cholesterol homeostasis. Of particular novelty is the identification of cell adhesion as a major downstream process uniquely regulated by PBRM1 expression. Cytoskeletal reorganization was induced upon PBRM1 reexpression as evidenced from the increase in the number of cells displaying cortical actin, a hallmark of epithelial cells. Genes involved in cell adhesion featured prominently in our transcriptional dataset and overlapped with genes uniquely regulated by PBRM1 in clinical specimens of ccRCC. Genes involved in cell adhesion serve as tumor suppressor and maybe involved in inhibiting cell migration. Here we report for the first time genes linked to cell adhesion serve as downstream targets of PBRM1, and hope to lay the foundation of future studies focusing on the role of chromatin remodelers in bringing about these alterations during malignancies.</p></div

Presence of PBRM1 resulted in decreased proliferation rate of ccRCC cells.

<p>Cellular proliferation rate was monitored in (A) Caki1 and (B) A498 with either control knockdown or PBRM1 knockdown, as well as (C) Caki2 and (D) A704 with either control vectors or PBRM1 re-expression vectors. Both cell lines were treated with ethanol or doxycycline to control for cell line variation or doxycycline effects. n = 8 independent biological replicate experiments. P < 0.0001 (****) (two-way ANOVA test) between PBRM1 re-expression cell lines treated with ethanol (EtOH) or Doxycycline (Dox) at day 11. Error bars represent s.e.m.</p

Downregulation of genes involved in cell proliferation upon PBRM1 re-expression.

<p>(A) Validating relative expression of genes regulating cell cycle (identified in RNA_seq data) in Caki2+Vectorand Caki2+PBRM1 cells by qRTPCR. A designation of * = P < 0.05 (paired Student <i>t</i>-test). n = 3 independent biological replicate experiments. Error bars represent s.e.m. (B) Percentage of cells in different stages of cell cycle (G1, S and G2) determined by flow cytometric analysis. A designation of * = P < 0.05 (paired Student <i>t</i>-test). n = 3 independent biological replicate experiments. Error bars represent s.e.m.</p