| BackgroundWith the in-depth study of tumorigenesis in recent years, people recognize that cancer is not only dependent on the gene itself, but is also closely related to disorders of epigenetic regulations, such as histone modifications. The dynamic balance of histone modifications is dependent on two opposing enzymes, like histone methyltransferases and demethylases, histone acetyltransferases and deacetylases. Many studies have addressed the role of histone modificating enzymes in diverse human diseases and tumors. Two histone deacetylase inhibitors, Zolinza and Istodax, have been approved by the US FDA for the treatment of cutaneous T-cell lymphoma. In the coming years, researchers will focus on the better understanding of the function of histone modificating enzymes responsible for gene regulation, with the final goal to obtain new drug candidates for the treatment of cancer.Mammalian KMT2 enzyme, as an important histone modificating enzyme, has become one of the research hotspots recently. Sequence analyses of human tumors documented in The Cancer Genome Atlas (TCGA) have revealed many tumors like breast cancers are closely related to various disease-specific mutations in the KMT2 family. Researches about KMT2 enzymes could be divided to two main aspects. On one hand, the function analyses of KMT2 enzymes have been achieved by knocking out different subtypes of K.MT2 family members. Germline homozygous deletion of Mill results in the death of the embryos, while Mill heterozygous mice show retarded growth with hematopoietic abnormalities. It has been well-documented that Mill is relevant with some specific forms of leukemia in humans, suggesting its critical role in hematopoietic stem and progenitor cells. Loss of M112 slows embryonic growth, increases apoptosis and leads to the death of mice around embryonic day 10.5 (E10.5). Mll2 deletion in oocytes leads to anovulation and oocyte death. Conditional knockout of Mll2 leads to male mice infertility, indicating its important role in spermatogenesis. Setla is required at the earliest stage of embryonic development and its deletion leads to embryonic death at E5.5 with gastrulation failure. Setlb deficient embryos could survive to E11.5 without major morphological defects, but are grossly retarded. On the other hand, the functional analyses of KMT2 enzymes could be studied by knocking out the core subunits including WDR5, RbBP5, ASH2L and DPY30. Loss of ASH2L reduces the enzymatic activity of KMT2, thus disturbing H3K4me3 which is found primarily at promoters that possess open chromatin, which apparently cannot be compensated.Researchers have already made progress in the studies about structural and functional analyses of ASH2L. ASH2L gene was cloned in 1999 by Ikegawa and colleagues. The human ASH2L is located on chromosome 8p11.2, spans more than 34 kb of genomic DNA and its transcript is 2,368 bp long. Until now, three known different alternative splice isoforms of ASH2L have been described. Isoform 1 is considered as the classical isoform and consists of 628 amino acids and contains 16 exons. Structural analyses have found ASH2L consists of five identified domains, including an N-terminus containing a plant homeodomain (PHD) finger and a helix-wing-helix (HWH) motif, a central nuclear localization signal (NLS) domain and a C-terminus containing an SPla and Ryanodine receptor (SPRY) domain and an Sdcl Dpy-30 interacting (SDI) domain. These structural characteristics determine its localization, interaction partners and functions in regulating the biological behavior in cells.The mouse Ash2l shows 79.2% identities and similar structure to its human counterpart. Considering the medical ethics and the high similarity between human ASH2L and mouse Ash21, Ash21 has been analyzed widely for its functional role. Ash21 has been found to be essential for mouse development while at embryonic age E3.5 Ash2l-/-embryos could be found, no embryos could survive beyond E8.5. Ash21 has also been found to be crucial in maintaining the pluripotency of embryonic stem cells (ES), open chromatin and X chromosome inactivation. Moreover Ash21 might be involved in the pathogenesis of inflammation, cardiac hypertrophy and fibrosis, and hypoxia induced pulmonary hypertension. Some previous findings have proposed the potential involvement of ASH2L in tumorogenesis. Firstly, ASH2L has been identified as an interaction partner of the oncoprotein MYC. Secondly, ASH2L is overexpressed in the majority of human tumors. In addition a potential role of ASH2L in cell transformation is also implied by the observation that it cooperates with activated Ha-RAS to transform primary rat embryonic fibroblasts (REFs). In the later experiments, ASH2L is exogenously expressed in liver specifically, but it seems that ASH2L cannot induce or promote hepatic carcinomas in mice. Overexpression of ASH2L in mouse hepatocytes has no significant influence on the growth of hepatocytes, the function and the histology of mouse liver.To sum up, the characteristics of ASH2L sequence and its protein structure have been studied adequately. ASH2L exerts its function mainly in the context of KMT2 enzymes, regulating gene expression through histone posttranslational modifications. The functional role of Ash21 has been addressed in maintaining the pluripotency of ES cells and embryonic development. However the potential role of ASH2L in tumorigenesis is largely unknown. The role of ASH2L in mouse hepatic carcinoma has been studied by overexpression of ASH2L in hepatocytes, but no positive findings could be acquired. Thus the functional role of ASH2L is still worthy of discussion and research in depth.Objective1. The hepatocyte specific Ash21 KO mouse model was established to investigate the functional role of Ash21 in liver and mice postnatal development.2. The inducible Ash2l KO MEFs were generated to investigate the functional role of Ash21 in cell physiology and explore its potential mechanism.Methods For the hepatocyte-specific disruption of Ash2l, Ash2lfl/fl animals were crossed with mice harboring a transgene for the expression of Cre recombinase driven by the hepatocyte-specific serum albumin (Alb) promoter. Littermates were crossed to obtain the homozygous KO genotype Ash2lfl/fl Tg(AlbCre) and various control animals(Ash2lfl/fl,Ash2lfl/fl,Ash2lfl/fl Tg(AlbCre), Ash2l+/+ Ash2l-/- Tg(AlbCre)).1. The body weight, general conditions, behaviors and clinical parameters were observed everyday routinely and scored. The mice had to be sacrificed once the score was more than 20.2. Blood was taken by penetrating the retro-orbital sinus with a glass capillary in Serum Gel microcentrifuge tubes. The separated serum was used to measure some clinical hepatic parameters like ALP, ALT, AST and TBIL.3. The Ash2l deletion efficiency was checked on both DNA and RNA levels using PCR with specific primer pairs.4. The loss of Ash2l expression on protein level was analyzed by Western blot.5. The histologic changes were evaluated by hematoxylin and eosin (HE), periodic acid-schiff (PAS) and modified Gomori’s stainings.6. Immunohistochemistry (IHC) stainings were performed in liver sections using antibody against Ash2l, H3K4me3, Ki67 and yH2A.X.7. IHC staining of cleaved caspase-3 and TUNEL assay was conducted to detect apoptotic cells. Ash2lfl/fl mice were crossed with mice harboring a transgene for the expression of CreERT2 driven by the cytomegalvirus immediate early enhancer-chicken β-actin hybrid (CAG) promoter. Littermates were further crossed to obtain embryos with the genotype of Ash2l+/+ Tg(CAGCreERT2) and Ash2lfl/fl Tg(CAGCreERT2). The primary MEFs (pMEFs) were generated from pregnant mice at E13.5 post coitum. Plat-E cells were transiently transfected with pRetroSuperBlasti-shp-19ARF using calcium phosphate to produce retroviruses. Immortalized MEFs (iMEFs) were generated by retroviruses infection of pMEFs with continuous blasticidin selection for 2 weeks.1. The deletion efficiency of Ash2l was analyzed on the DNA level by PCR with specific primer pairs and on the protein level by Western blot.2. Cell growth curves were described and methylene blue stainings were used to monitor the cell proliferation.3. DNA contents in cells were tested with PI staining to determine the cell cycle distribution by flow cytometry.4. MEFs were stained with AnnexinV-FITC and PI to check apoptotic cells by flow cytometry.5. β-Galactosidase stainings were used to observe senescent cells whose galactosidase activity were high at the pH of 6.0.6. The expression of Ash21 in whole cell lysates and in the single cell was analyzed by Western blot and immunofluorescence staining respectively. The impact of H3K4me3 and yH2A.X was also determined once loss of Ash21.7. Western blot analyses were performed to find the potential changes of histone modifications due to the loss of Ash21 in iMEFs.Results(1) Ash21 is essential for liver function.The hepatocyte-specific Ash2l KO mouse model was established and the KO efficiency was confirmed on DNA, RNA and protein levels. As early as 11 days, the Ash2l KO mice looked smaller and lacked physical activity compared to the control mice of the same gender from the same cote. The scores of all Ash21 KO mice were higher than 20 and had to be sacrificed. However,6 Ash2l KO mice died before we stopped the experiments, with an average age of 26.17 ±9.19 days. The body weight of control (C) and KO animals was compared. Liver specific deletion of Ash21 caused growth retardation apparently (female:7.45± 2.36g(KO)vs.11.19±4.61 g (C), P< 0.05; male:6.56±1.05 g (KO) vs.8.06± 1.84 g(C),P< 0.05).Several liver enzymes as marker of liver damage were tested. ALP, ALT, AST increased significantly in Ash21 KO mice compared to the control mice (ALP: 2022.20 ± 195.70 U/L (KO) vs.467.05±52.01 U/L (C), P< 0.001; ALT:110.68 ±17.77 U/L (KO) vs.49.96±6.70 U/L (C), P< 0.001; AST:248.34±37.75 U/L (KO) vs.104.31±17.52 U/L (C), P< 0.001). Albeit increased TBIL levels were measured, this change did not reach statistical significance compared to control mice (48.26±12.33 μmol/L (KO) vs.17.90±10.42 μmol/L (C), P= 0.070).In IHC stainings of Ash2l KO mice, Ash21 proteins were absent in hepatocytes but still present in other cell types, including Kupffer cells and endothelial cells. Meanwhile, the hepatocytes without Ash21 showed a concomitant strong reduction in histone H3K4me3. HE stainings revealed a normal liver morphology of control mouse. In the KO animals, the liver sections exhibited ballooning and feathery degenerations of the hepatocytes. With increasing age, an enhanced ductular reaction could be observed and PAS staining revealed a gain of Ceroid-storing macrophages. In addition with advancing age, modified Gomori’s stain showed small areas of hepatocyte collaps and progressive portal-portal bridging fibrosis in KO animals. There was no difference between the control sections and the sections from KO mice in cleaved caspase-3 and TUNEL staining. Increased yH2A.X stainings were found in the KO vs. control sections of littermates in some but not all age-groups.IHC detecting the Ki-67 revealed a consistent albeit moderate increase in the amount of stained cells with larger nuclei and intensive coloring in mice older than 11 days.(2)Ash21 is essential for cell proliferation.The inducible Ash21 KO MEFs were generated and the deletion efficiency was confirmed on both DNA and protein levels.Growth curves and methylene blue staining revealed a reduced prolif-eration after the loss of Ash21.Cell cycle analyses found Ash2l KO MEFs were mainly arrested in G0/G1 phase accompanied by a decrease of cells in S phase of pMEFs.Annexin V-FITC/PI stainings showed that loss of Ash21 in the iAsh2lfl/fl MEFs made the distribution Of the cells in the FSC and SSC axes more widely.The f.raction of apoptotic and early apoptotic cells in iAsh2lfl/fl MEFs(+HOT) increased significantly when compared with the control cells(apoptotic cells: 18.19士1.15％(+HoT)vs.7.55±1.49%(-HOT),P<0.05；early apoptotic cells:10.38±0.49%(+HOT)vs.0.92±0.07%(-HOT),P<0.001).Treatment with HOT did not affect the apoptOSis in the iAsh2l+/+ MEFs(apoptotic cells: 11.01±0.27%(+HOT)vs.11.92±1.33%(-HOT),P=0.359；early apoptotic cells:2.14±0.17%(+HOT)vs.2.25±0.49%(-HOT),P<0.739).Similarly, the rate of apoptotic and early apoptotic cells in pAsh2lfl/fl MEFs(+HOT) increased significantly(apoptotic cells:34.13%±0.85%(+HOT)vs.22.93%± 0.37%(-HOT),P<0.001；early apoptotic cells:22.30±0.95%(+HOT)vs. 13.00±0.50%(-HOT),P<0.001).There was no significant difference between the pAsh2l+/+ MEFs after HOT treatment(apoptotic cells:24.62±1.72%(+HOT) vs.26.87±1.42%(-HOT),P=0.157；early apoptotic cells:13.60±0.46%(+ HOT)vs.14.10±1.21%(-HOT),P=0.541).In addition, about 80% of floating cells in iAsh2lfl/fl MEFs(+HOT)group underwent apoptosis and 63.90% were in the early stage of apoptosis.However the floating cells in the iAsh2l+/+ MEFs and iAsh2lfl/fl MEFs without HOT induction were so limited that we could not use these cells for further FACs analyses.β-Galactosidase staining displayed the blue color mainly in the cytoplasm of the irregularly enlarged and flattened Ash2lfl/fl MEFs with HOT treatment. Compared with untreated iAsh2lfl/fl MEFs and pAsh2lfl/fl MEFs, the P-gal positive cells increased significantly in both treated iAsh2lfl/fl MEFs and pAsh2lfl/fl EFs (iAsh2lfl/fl:65.04 ± 4.35%(+HOT) vs.1.24±0.62%(-HOT), P< 0.001; pAsh2lfl/fl:72.19 ± 3.17%(+HOT) vs.2.58±0.49%(-HOT), P< 0.001). Although HOT induction also increased β-gal positive Ash2l+/+ MEFs, the total fraction of β-gal positive cells was less than 3%(iAsh2l+/+:2.59 Π 1.01%(+ HOT) vs.0.95 ± 0.05%(-HOT), P= 0.048;pAsh2l+/+:1.88 ± 0.19%(+HOT) vs. 0.86 ± 0.11%(-HOT), P= 0.001).Western blot anlyses showed an increase of yH2A.X in the whole cell lysates from Ash2l KO iMEFs. Immunifluorescence staining pointed out the increased yH2A.X in some Ash2l KO cells.Many histone modifications were analyzed and H3K4mel/me2/me3 decreased after the loss of Ash21. There was no influence on other histone modifications, such as H3K9ac, H3K9me3, H3K27ac and H3K27me3. Immunifluorescence staining showed a concomitant strong reduction of H3K4me3 in Ash2l KO cells.Conclusion1. The established hepatocyte specific Ash21 KO animal model reveals that loss of Ash21 results in retarded growth, liver damage and shortened life expectancy.2. The histologic abnormalities in the liver from Ash2lfl/fl Tg(AlbCre) mouse are probably caused by the loss of Ash21 through the strong reduction of H3K4me3.3. Inducible Ash2l KO MEFs were generated and loss of Ash21 inhibits cell proliferation with cell cycle arrest.4. The retardance of cell growth by Ash2l KO is probably arisen by promoting cellular apoptosis in a small degree and cellular senescence.Significance1. Firstly studied and proved the essential role of Ash21 in liver using the hepatocyte specific Ash2l KO mouse model.2. Further explored and proved the role of Ash21 in cell proliferation.3. Ash21 was proposed as a potential therapeutic target of cancer. |
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