hes1-protein--human and Fanconi-Anemia

The Hairy Enhancer of Split 1 (HES1) is a transcriptional repressor that regulates cellular proliferation and differentiation during development. We previously found an interaction between HES1 and Fanconi anemia (FA) proteins. FA is a hematological and developmental disorder caused by mutations in more than 20 different genes. Eight FA gene products form a nuclear core complex containing E3 ligase activity required for mono-ubiquitination of FANCD2 and FANCI, both of which are FA proteins. Given that HES1 interacts with members of the FA core complex, the aim of this study was to determine whether HES1 is mono-ubiquitinated via the FA core complex.. We show that HES1 is mono-ubiquitinated on a highly-conserved lysine residue that is located within a FA-like recognition motif. HES1 modification is dependent on a functional FA complex. Absence of HES1 mono-ubiquitination affects transcriptional repression of its own promoter. This study uncovers a novel post-translational modification of HES1 that regulates its transcriptional activity and suggests that ubiquitination of HES1 occurs in a FA core complex-dependent manner.

Topics: Cell Line; Fanconi Anemia; Fanconi Anemia Complementation Group Proteins; Fibroblasts; HEK293 Cells; HeLa Cells; Humans; Multiprotein Complexes; Transcription Factor HES-1; Ubiquitination

Mutations in one of the 13 Fanconi anemia (FA) genes cause a progressive bone marrow failure disorder associated with developmental abnormalities and a predisposition to cancer. Although FA has been defined as a DNA repair disease based on the hypersensitivity of patient cells to DNA cross-linking agents, FA patients develop various developmental defects such as skeletal abnormalities, microphthalmia, and endocrine abnormalities that may be linked to transcriptional defects. Recently, we reported that the FA core complex interacts with the transcriptional repressor Hairy Enhancer of Split 1 (HES1) suggesting that the core complex plays a role in transcription. Here we show that the FA core complex contributes to transcriptional regulation of HES1-responsive genes, including HES1 and the cyclin-dependent kinase inhibitor p21(cip1/waf1). Chromatin immunoprecipitation studies show that the FA core complex interacts with the HES1 promoter but not the p21(cip1/waf1) promoter. Furthermore, we show that the FA core complex interferes with HES1 binding to the co-repressor transducin-like-Enhancer of Split, suggesting that the core complex affects transcription both directly and indirectly. Taken together these data suggest a novel function of the FA core complex in transcriptional regulation.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Chlorocebus aethiops; COS Cells; Cross-Linking Reagents; Cyclin-Dependent Kinase Inhibitor p21; DNA Repair; Fanconi Anemia; Fanconi Anemia Complementation Group Proteins; Genetic Predisposition to Disease; Homeodomain Proteins; Humans; Multiprotein Complexes; Mutation; Neoplasms; Signal Transduction; Transcription Factor HES-1; Transcription, Genetic

Fanconi anemia (FA) proteins are thought to play a role in chromosome stability and repair of DNA cross-links; however, these functions may not fully explain the developmental abnormalities and bone marrow failure that are characteristic of FA individuals. Here we associate the FA proteins with the Notch1 developmental pathway through a direct protein-protein interaction between the FA core complex and the hairy enhancer of split 1 (HES1). HES1 interaction with FA core complex members is dependent on a functional FA pathway. Cells depleted of HES1 exhibit an FA-like phenotype that includes cellular hypersensitivity to mitomycin C (MMC) and lack of FANCD2 monoubiquitination and foci formation. HES1 is also required for proper nuclear localization or stability of some members of the core complex. Our results suggest that HES1 is a novel interacting protein of the FA core complex.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Line; Cell Line, Transformed; Drug Resistance; Fanconi Anemia; Fanconi Anemia Complementation Group C Protein; Fanconi Anemia Complementation Group Proteins; HeLa Cells; Homeodomain Proteins; Humans; Mice; Mice, Knockout; Mitomycin; Multiprotein Complexes; Protein Binding; Receptor, Notch1; Recombinant Proteins; RNA, Small Interfering; Signal Transduction; Transcription Factor HES-1; Two-Hybrid System Techniques; Ubiquitination