Control cells are shown in indicates chromosome loss/chromatin breakage. also altered in the absence of HP1. Using chromatin immunoprecipitation analysis, we further demonstrate that this promoters of a number of cell-cycle regulator genes are bound to HP1, supporting a direct role for HP1 in their active transcription. Overall, our data suggest that HP1 is essential for the maintenance of cell-cycle progression and the transcription of cell-cycle regulatory genes. The results also support the view that HP1 is usually a positive regulator of transcription in euchromatin. INTRODUCTION Chromatin in higher eukaryotes is usually subdivided into different functional compartments termed heterochromatin and euchromatin (1). Heterochromatin differs from euchromatin in its DNA composition, replication timing, condensation throughout the cell cycle, and its ability to silence euchromatic genes placed adjacent to or within its territory, often described as position-effect-variegation (PEV) (2). Heterochromatin protein 1 (HP1) was the first protein identified in as a heterochromatin-associated protein (3); the corresponding gene has been cloned from a number of organisms and is highly conserved from yeast to human (4). Polytene chromosome staining showed that, in result in late larval lethality, chromosome breakages/loss, telomere fusion and a high frequency of cells with abnormal anaphase (8,27). Null alleles of the Dynarrestin HP1 functional partner in mice (embryonic Kc cells and an RNA interference (RNAi)-based approach to demonstrate that HP1 plays an important role at S phase and G2/M phases during the cell cycle. We further show that nearly one-third of known/predicted cell-cycle regulators require HP1 to maintain their active transcription. These genes include and a few other cell-cycle regulators. ChIP analysis suggests that HP1 plays a direct role in their transcription. Therefore, the results of this study provide an option explanation for the specific role of HP1 in the regulation of chromatin dynamics and in cell-cycle progression. MATERIALS AND METHODS RNAi in Kc cells Kc cells were routinely cultured at 25C in Schneider medium (GIBCO) supplemented with 10% P2RY5 fetal calf serum, 160 g/ml penicillin, 250 g/ml streptomycin, and 4 mM l-glutamine. Double-stranded RNA (dsRNA) of HP1 was generated by Dynarrestin incubation of single-stranded RNA in annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 3 min at 95C and then placed in a beaker with water at 75C and allowed to cool slowly to room temperature. The detailed process of RNAi was carried out according to the established protocols (http://dixonlab.biochem.med.umich.edu). Briefly, Kc cells were seeded in a six-well dish using serum-free medium at 1 106 cells/ml. HP1 dsRNA (5 g/ml) was added to the Dynarrestin cultured Kc cells. After 60 min at room heat, 2 ml of medium made up of 10% serum was added to each well and the plates transferred to 25C for up to 8 days. Western blotting and RTCPCR were carried out using Dynarrestin the extract/total RNA isolated from control and dsRNA-treated cells on days 2, 6 and 8. Cell-cycle and apoptosis analysis The procedure for circulation cytometric analysis of Kc cells followed that in the manual provided with the BrdU circulation kit (BD PharMingen). The cells were fed with BrdU for 4 h, then scraped and collected. Fluorescence was measured using a FACSCalibur (Becton Dickinson). Data collection Dynarrestin and analysis were performed using CellQuest software. Electrophoresis and immunoblotting Cell extracts (15 g) were fractionated by 10% SDSCPAGE, then transferred to Hybond-P PVDF membranes (Amersham) and probed with main antibodies (CIA9), and secondary antibodies (anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG), obtained from Jackson Immunoresearch Laboratories. Enhanced chemiluminescence reagents (Amersham Pharmacia Biotech) were utilized for transmission detection. For the analysis of H3 ser10 phosphorylation, we used whole-cell extracts from 700?000 Kc cells (control and RNAi at day 8). Western blotting was performed using polyclonal antibodies against ser10-phosphorylated histone H3 at a dilution of 1 1:1000 (Upstate). Kc control cells arrested in mitosis by incubation in 25 M colchicine (Sigma).
