Abstract
BackgroundANKHD1, Ankyrin repeat and KH domain-containing protein 1 is highly expressed in CD138+ cells of patients with multiple myeloma (MM) as well as in MM cell lines (U266, RPMI 8226 ,MM1S and MM1R. Our microarray studies showed modulation of several histone variants after ANKHD1 silencing (not published). Furthermore ANKHD1 silencing in MM cell lines resulted in S phase arrest (1). As genes involved in histone transcription are upregulated in S phase, and ANKHD1 downregulation inhibits cell cycle progression at S phase, we hypothesized that ANKHD1 might be a protein that gets upregulated in S phase and plays a role in histone gene transcription. Hence, in the present study ANKHD1 expression was sought at different phases of cell cycle and a possible interaction of ANKHD1 with histones was also investigated, in addition to the effect of ANKHD1 downregulation on histone expression in a MM cell line.MethodsMM cell line U266 was synchronized at G1 phase by serum starvation (16h), S phase by double thymidine block (2mM) followed by release in 24 μM deoxycytidine (4h) or G2 phase by nocodazole treatment(50ng/ml;16h). Cells were stained with PI and analyzed by flow cytometry for DNA content. Percentage of cells in G1, S, or G2/M was calculated using the ModFit program. Western blot was then carried out for ANKHD1 expression in U266 cells untreated or synchronized at different G1, S and G2 phases of cell cycle. ANKHD1 expression was inhibited by lentiviral mediated ANKHD1shRNA transduction and its effect on expression of histones was studied by qPCR and immunoblot. Further chromatin immunoprecipitation (ChIP) assay was performed to study the interaction between ANKHD1 and histones using EZ-Magna ChIP™ A kit(Millipore) followed by qPCR with primers specific to core histones promoter region. Immunofluorescence was performed to determine the localization of ANKHD1 before and after leptomycin B treatment of U266 cells.Results. In the present study, endogenous ANKHD1 expression showed a clear cell-cycle-dependence, peaking during S phase, when cells were synchronized by double thymidine block followed by deoxycytidine release. Further down-regulation of ANKHD1 expression in U266 cells by lentiviral mediated shRNA against ANKHD1 resulted in a significant reduction of histones (p<0.05) at both mRNA and protein levels. Chromatin immunoprecipitation followed by qPCR with primers specific to core histones promoter region showed that ANKHD1, though not IgG (negative control) coprecipitated with histone gene chromatin, thus confirming the interaction between the core histone promoter regions and ANKHD1. Fold enrichment (mean ± sd) of promoter sequences bound to ANKHD1 were 7.74 ± 0.048, 7.78± 0.129 and 7.06± 0.178 for histones H2B/r, H3/c and H4/e, respectively. Immunofluorescence after leptomycin B treatment (20ng/ml) of U266 wild type cells for 24 hours showed accumulation of ANKHD1 inside nucleus as compared to untreated cells where ANKHD1 was found to be predominantly in cytoplasm. This suggests transport of ANKHD1 between nucleus and cytoplasm.ConclusionANKHD1 expression peaks during S phase of cell cycle and downregulation of ANKHD1 protein by shRNA results in downregulation of histones. ANKHD1 interacts with histone gene promoter sequences and modulates histones transcription. These results suggest that ANKHD1 might be an important component of the machinery required for histone mRNA expression and cell-cycle progression. Furthermore, ANKHD1 protein which was earlier reported to be localized predominantly in cytoplasm (1) is herein suggested to shuttle between cytoplasm and nucleus thereby playing a role in gene regulation. Extensive studies are required to understand the mechanism underlying the regulation of gene transcription by ANKHD1.
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