Báo cáo khoa học: Dynamic association of MLL1, H3K4 trimethylation with chromatin and Hox gene expression during the cell cycle

Dynamic Association Of Mll1, H3k4 Trimethylation With Chromatin And Hox Gene Expression During The Cell Cycle

Tác giả: Bibhu P. Mishra, Khairul I. Ansari and Subhrangsu S. Mandal

Lĩnh vực: Chemistry and Biochemistry

Nội dung tài liệu: Nghiên cứu này khám phá mối liên hệ động giữa MLL1, sự trimethyl hóa H3K4 và biểu hiện gen Hox trong chu kỳ tế bào. Kết quả cho thấy MLL1 và dấu ấn H3K4 trimethyl hóa có động lực khác nhau trong chu kỳ tế bào. MLL1 liên kết với chromatin hoạt động và tách ra khỏi chromatin ngưng tụ trong quá trình phân bào, trong khi H3K4 trimethyl hóa vẫn gắn với chromatin trong suốt chu kỳ tế bào. Sự biểu hiện của các gen Hox mục tiêu của MLL bị ảnh hưởng khác nhau trong chu kỳ tế bào, và sự gắn kết của MLL1 cùng với H3K4 trimethyl hóa tại vùng điều hòa của các gen Hox này thay đổi tùy thuộc vào mức độ biểu hiện của chúng. Giảm MLL1 dẫn đến ngừng chu kỳ tế bào ở pha G2/M.

Mục lục chi tiết:

• Dynamic association of MLL1, H3K4 trimethylation with chromatin and Hox gene expression during the cell cycle
• Keywords
• cell cycle; H3K4 methylation; histone methyltransferase; Hox genes; mixed lineage leukemia
• Correspondence
• Abbreviations
• Mixed lineage leukemias (MLLs) are histone H3 at lysine 4 (H3K4)-specific methylases that play a critical role in regulating gene expression in humans. As chromatin condensation, relaxation and differential gene expression are keys to correct cell cycle progression, we analyzed the dynamic association of MLL and H3K4 trimethylation at different stages of the cell cycle. Interestingly, MLL1, which is normally associated with transcriptionally active chromatins (G1 phase), dissociates from condensed mitotic chromatin and returns at the end of telophase when the nucleus starts to relax. In contrast, H3K4 trimethylation mark, which is also normally associated with euchromatins (in G1), remains associated, even with condensed chromatin, throughout the cell cycle. The global levels of MLL1 and H3K4 trimethylation are not affected during the cell cycle, and H3Ser28 phosphorylation is only observed during mitosis. Interestingly, MLL target homeobox-containing (Hox) genes (HoxA5, HoxA7 and HoxA10) are differentially expressed during the cell cycle, and the recruitment of MLL1 and H3K4 trimethylation levels are modulated in the promoter of these Hox genes as a function of their expression. In addition, down-regulation of MLL1 results in cell cycle arrest at the G2/M phase. The fluctuation of H3K4 trimethylation marks at specific promoters, but not at the global level, indicates that H3K4 trimethylation marks that are present in the Gl phase may not be the same as the marks in other phases of the cell cycle; rather, old marks are removed and new marks are introduced. In conclusion, our studies demonstrate that MLL1 and H3K4 methylation have distinct dynamics during the cell cycle and play critical roles in the differential expression of Hox genes associated with cell cycle regulation.
• Histone methyltransferases (HMTs) are key enzymes that post-translationally methylate histones and play critical roles in gene expression, epigenetics and cancer [1-11]. Mixed lineage leukemias (MLLs) are human HMTs that specifically methylate histone H3 at lysine 4 (H3K4) and are linked with gene activation [12-20]. Notably, Set1 is the sole H3K4-specific HMT present in yeast [21-23]. Humans encode six Set1 homologs: MLL1, MLL2, MLL3, MLL4, Set1A and Set1B [12,13,16,19,24-27]. Each of these proteins exists as multiprotein complexes sharing several common subunits, including Ash2, Wdr5, Rbbp5, human CpG-binding protein (CGBP) and Dpy30 [12-14,16,19, 24-31]. MLLs are well known as the master regulators
• Results and Discussion
• Dynamics of MLL1 and its interacting proteins during the cell cycle
• Prior to the analysis of the dynamics of MLL and histone methylation, we synchronized HeLa cells at different phases of the cell cycle using double thymidine treatment, as described previously [38]. Briefly, cells were treated with 10 mm thymidine (18 h), released into fresh medium (9 h), blocked again by the addition of 10 mm thymidine (17h) and finally released into fresh medium at the G₁/S boundary. Cyclins B and E were used as markers for cell cycle synchronization. In agreement with previous studies, cyclin B was expressed prominently in the G2/M phase, whereas cyclin E expression was high in S and G1 phase, but low in G2/M phase (Fig. 1) [39].
• In order to understand the dynamics of MLL1, we performed immunofluorescence staining of the synchronized HeLa cells with anti-MLL1 serum, and visualized its localization using fluorescence microscopy at different stages of the cell cycle. In agreement with our previous studies, we found that MLL1 was localized inside the euchromatic region [less intense 4′,6-diamidino-2-phenylindole (DAPI)-stained region] of the nucleus at the G1 phase of the cells (G1 phase, panels 1-3, Fig. 2) [12]. However, as the cell entered into mitosis and chromatin was condensed, most of the MLL1 protein was dissociated from the chromatin and spread into the cytoplasm, generating a distinct footstep (gap) for condensed chromatin (see metaphase, anaphase and early telophase stages, panels 1-3, Fig. 2). Notably, the spreading of MLL1 protein into the cytoplasm coincided with the disappearance of the nuclear membrane at
• H3K4 trimethylation marks are associated with mitotic chromatins
• In contrast with MLL1 and its interacting proteins, H3K4 trimethylation marks behave differently during the cell cycle. Notably, like MLL1, H3K4 trimethylation is well known to be associated with transcriptionally active euchromatin [12,41]. Therefore, MLL1 and H3K4 trimethylation have been shown (by our laboratory and others) to be colocalized in the euchromatic regions of the nucleus, and this is probably because of their involvement in active gene expression [12,41]. Herein, in order to understand the dynamic association of H3K4 trimethylation with chromatin during the cell cycle, we performed immunofluorescence staining of HeLa cells with anti-H3K4 trimethyl serum at different stages of the cell cycle. The cell nucleus was counter-stained and visualized using DAPI staining. As
• MLL1 and H3K4 trimethylation levels remain unaffected whereas Hox genes are differentially expressed during the cell cycle
• As MLL1 and H3K4 trimethylation show distinct dynamics during cell cycle progression, we analyzed the expression profiles of MLL1, CGBP, Ash2 and Rbbp5, together with cyclins E and B, as a function of the cell cycle. Western blot analysis of the whole-cell extract and histones from different stages of the cell cycle demonstrated that the overall levels of MLL1 and H3K4 trimethylation were unaffected throughout the cell cycle (Fig. 5). Similarly, MLL-interacting proteins, such as CGBP, Ash2 and Rbbp5, were unaffected during the cell cycle (data not shown). Notably, again, our observations showing the unaffected global level of MLL1 (protein level) during the cell cycle contradict the observations by Liu et al. [40], who demonstrated that MLL1 proteins were degraded during late M (mitosis) and S phases. However, in agreement with Liu et al. [40], using RT-PCR analysis, we observed that the expression of MLL1 at the mRNA level was increased from G1/S towards G2/M (Fig. S2, see Supporting information). Furthermore, to confirm cell synchronization, we analyzed the changes in phosphorylation level of H3Ser28, which is considered to be a marker for mitotic cells. Indeed, in agreement with
• MLL1 and H3K4 methylation are critical for Hox gene regulation during the cell cycle
• In order to understand the molecular mechanism of the differential regulation of Hox gene expression, we analyzed the changes in H3K4 methylation and recruitment of MLL1 and RNA polymerase II (RNAP II) at the Hox gene promoters at different phases of the cell cycle using chromatin immunoprecipitation (ChIP) assay [12]. We performed ChIP analysis using anti-RNAP II, anti-MLL1 and anti-H3K4 trimethyl sera at three different phases of the cell cycle [0 h (S), 10 h (G2/M) and 20 h (G1)] after synchronized cells were released at the S phase. In the case of HoxA5, recruitment of RNAP II and MLL1, and the level of H3K4 trimethylation in the promoter, were low at S phase (0 h), increased by 1.7-fold at G2/M (10 h) and decreased again at G1 (20 h) (Fig. 6C,D). Notably, the enrichment of RNAP II, MLL1 and H3K4 trimethylation at the HoxA5 gene promoter at the G2/M phase was correlated with its expression profile (as shown in Fig. 6A,B), indicating the importance of MLL1 and H3K4 trimethylation in HoxA5 gene regulation during cell cycle progression. The association of a certain amount of RNAP II with the HoxA5 gene promoter at 20 h (although much lower in comparison with that at 10h) indicates that a certain amount of basal transcription still continues at this stage of the cell cycle. Similar to HoxA5, the occupancy of RNAP II, MLL1 and H3K4 trimethylation
• Experimental procedures
• Cell culture and synchronization
• Preparation of whole-cell extract, histones and western blotting
• RNA purification and RT-PCR
• Immunofluorescence studies
• Antisense-mediated knockdown of MLL1 and ChIP assay
• Flow cytometry analysis
• Acknowledgements
• References