Regulation of p53 activity through lysine methylation

p53 is a tumour suppressor that regulates the cellular response to genotoxic stresses. p53 is a short-lived protein and its activity is regulated mostly by stabilization via different post-translational modifications. Here we report a novel mechanism of p53 regulation through lysine methylation by Set9 methyltransferase. Set9 specifically methylates p53 at one residue within the carboxyl-terminus regulatory region. Methylated p53 is restricted to the nucleus and the modification positively affects its stability. Set9 regulates the expression of p53 target genes in a manner dependent on the p53-methylation site. The crystal structure of a ternary complex of Set9 with a p53 peptide and the cofactor product S-adenosyl-l-homocysteine (AdoHcy) provides the molecular basis for recognition of p53 by this lysine methyltransferase.     

Gene silencing: trans-histone regulatory pathway in chromatin

The fundamental unit of eukaryotic chromatin, the nucleosome, consists of genomic DNA wrapped around the conserved histone proteins H3, H2B, H2A and H4, all of which are variously modified at their amino- and carboxy-terminal tails to influence the dynamics of chromatin structure and function -- for example, conjugation of histone H2B with ubiquitin controls the outcome of methylation at a specific lysine residue (Lys 4) on histone H3, which regulates gene silencing in the yeast Saccharomyces cerevisiae. Here we show that ubiquitination of H2B is also necessary for the methylation of Lys 79 in H3, the only modification known to occur away from the histone tails, but that not all methylated lysines in H3 are regulated by this 'trans-histone' pathway because the methylation of Lys 36 in H3 is unaffected. Given that gene silencing is regulated by the methylation of Lys 4 and Lys 79 in histone H3, we suggest that H2B ubiquitination acts as a master switch that controls the site-selective histone methylation patterns responsible for this silencing.     

MMSET regulates histone H4K20 methylation and 53BP1 accumulation at DNA damage sites

p53-binding protein 1 (53BP1) is known to be an important mediator of the DNA damage response, with dimethylation of histone H4 lysine 20 (H4K20me2) critical to the recruitment of 53BP1 to double-strand breaks (DSBs). However, it is not clear how 53BP1 is specifically targeted to the sites of DNA damage, as the overall level of H4K20me2 does not seem to increase following DNA damage. It has been proposed that DNA breaks may cause exposure of methylated H4K20 previously buried within the chromosome; however, experimental evidence for such a model is lacking. Here we found that H4K20 methylation actually increases locally upon the induction of DSBs and that methylation of H4K20 at DSBs is mediated by the histone methyltransferase MMSET (also known as NSD2 or WHSC1) in mammals. Downregulation of MMSET significantly decreases H4K20 methylation at DSBs and the subsequent accumulation of 53BP1. Furthermore, we found that the recruitment of MMSET to DSBs requires the γH2AX-MDC1 pathway; specifically, the interaction between the MDC1 BRCT domain and phosphorylated Ser?102 of MMSET. Thus, we propose that a pathway involving γH2AX-MDC1-MMSET regulates the induction of H4K20 methylation on histones around DSBs, which, in turn, facilitates 53BP1 recruitment.     

北京棒杆菌天冬氨酸激酶M372I-T379S双突变体的构建及其酶学性质

为构建高酶活力天冬氨酸激酶(aspartokinase, AK), 并削弱或解除Lys(lysine)反馈抑制作用突变体, 通过定点突变和高通量筛选技术构建突变体M372I,T379S和M372I-T379S, 对野生型（WT）和突变体分别进行诱导表达、 纯化及酶学性质表征. 结果表明： 突变体M372I,T379S和M372I-T379S AK与WTAK相比, V_max分别提高了13.77,15.02,15.60倍, K_m和n值均降低； 最适pH值分别升高为8.0,8.5,8.5, 且半衰期分别延长了1.0,0.9,2.3 h； M372I-T379S AK最适温度为30 ℃, 比WT AK高2 ℃； 当浓度为1~10 mmol/L时, 突变体均削弱或部分解除了抑制剂Lys的反馈抑制作用.     

北京棒杆菌天冬氨酸激酶M372I-T379S双突变体的构建及其酶学性质

为构建高酶活力天冬氨酸激酶(aspartokinase, AK), 并削弱或解除Lys(lysine)反馈抑制作用突变体, 通过定点突变和高通量筛选技术构建突变体M372I,T379S和M372I-T379S, 对野生型（WT）和突变体分别进行诱导表达、 纯化及酶学性质表征. 结果表明： 突变体M372I,T379S和M372I-T379S AK与WTAK相比, V_max分别提高了13.77,15.02,15.60倍, K_m和n值均降低； 最适pH值分别升高为8.0,8.5,8.5, 且半衰期分别延长了1.0,0.9,2.3 h； M372I-T379S AK最适温度为30 ℃, 比WT AK高2 ℃； 当浓度为1~10 mmol/L时, 突变体均削弱或部分解除了抑制剂Lys的反馈抑制作用.     

Structure and catalytic mechanism of the human histone methyltransferase SET7/9

Acetylation, phosphorylation and methylation of the amino-terminal tails of histones are thought to be involved in the regulation of chromatin structure and function. With just one exception, the enzymes identified in the methylation of specific lysine residues on histones (histone methyltransferases) belong to the SET family. The high-resolution crystal structure of a ternary complex of human SET7/9 with a histone peptide and cofactor reveals that the peptide substrate and cofactor bind on opposite surfaces of the enzyme. The target lysine accesses the active site of the enzyme and the S-adenosyl-l-methionine (AdoMet) cofactor by inserting its side chain into a narrow channel that runs through the enzyme, connecting the two surfaces. Here we show from the structure and from solution studies that SET7/9, unlike most other SET proteins, is exclusively a mono-methylase. The structure indicates the molecular basis of the specificity of the enzyme for the histone target, and allows us to propose a model for the methylation reaction that accounts for the role of many of the residues that are invariant across the SET family.     

Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization

The p53 tumour suppressor is a short-lived protein that is maintained at low levels in normal cells by Mdm2-mediated ubiquitination and subsequent proteolysis. Stabilization of p53 is crucial for its tumour suppressor function. However, the precise mechanism by which ubiquitinated p53 levels are regulated in vivo is not completely understood. By mass spectrometry of affinity-purified p53-associated factors, we have identified herpesvirus-associated ubiquitin-specific protease (HAUSP) as a novel p53-interacting protein. HAUSP strongly stabilizes p53 even in the presence of excess Mdm2, and also induces p53-dependent cell growth repression and apoptosis. Significantly, HAUSP has an intrinsic enzymatic activity that specifically deubiquitinates p53 both in vitro and in vivo. In contrast, expression of a catalytically inactive point mutant of HAUSP in cells increases the levels of p53 ubiquitination and destabilizes p53. These findings reveal an important mechanism by which p53 can be stabilized by direct deubiquitination and also imply that HAUSP might function as a tumour suppressor in vivo through the stabilization of p53.     

NUMB controls p53 tumour suppressor activity

NUMB is a cell fate determinant, which, by asymmetrically partitioning at mitosis, controls cell fate choices by antagonising the activity of the plasma membrane receptor of the NOTCH family. NUMB is also an endocytic protein, and the NOTCH-NUMB counteraction has been linked to this function. There might be, however, additional functions of NUMB, as witnessed by its proposed role as a tumour suppressor in breast cancer. Here we describe a previously unknown function for human NUMB as a regulator of tumour protein p53 (also known as TP53). NUMB enters in a tricomplex with p53 and the E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing ubiquitination and degradation of p53. This results in increased p53 protein levels and activity, and in regulation of p53-dependent phenotypes. In breast cancers there is frequent loss of NUMB expression. We show that, in primary breast tumour cells, this event causes decreased p53 levels and increased chemoresistance. In breast cancers, loss of NUMB expression causes increased activity of the receptor NOTCH. Thus, in these cancers, a single event-loss of NUMB expression-determines activation of an oncogene (NOTCH) and attenuation of the p53 tumour suppressor pathway. Biologically, this results in an aggressive tumour phenotype, as witnessed by findings that NUMB-defective breast tumours display poor prognosis. Our results uncover a previously unknown tumour suppressor circuitry.     

PML regulates p53 acetylation and premature senescence induced by oncogenic Ras

The tumour suppressor p53 induces cellular senescence in response to oncogenic signals. p53 activity is modulated by protein stability and post-translational modification, including phosphorylation and acetylation. The mechanism of p53 activation by oncogenes remains largely unknown. Here we report that the tumour suppressor PML regulates the p53 response to oncogenic signals. We found that oncogenic Ras upregulates PML expression, and overexpression of PML induces senescence in a p53-dependent manner. p53 is acetylated at lysine 382 upon Ras expression, an event that is essential for its biological function. Ras induces re-localization of p53 and the CBP acetyltransferase within the PML nuclear bodies and induces the formation of a trimeric p53-PML-CBP complex. Lastly, Ras-induced p53 acetylation, p53-CBP complex stabilization and senescence are lost in PML-/- fibroblasts. Our data establish a link between PML and p53 and indicate that integrity of the PML bodies is required for p53 acetylation and senescence upon oncogene expression.     

The pathological response to DNA damage does not contribute to p53-mediated tumour suppression

The p53 protein has a highly evolutionarily conserved role in metazoans as 'guardian of the genome', mediating cell-cycle arrest and apoptosis in response to genotoxic injury. In large, long-lived animals with substantial somatic regenerative capacity, such as vertebrates, p53 is an important tumour suppressor--an attribute thought to stem directly from its induction of death or arrest in mutant cells with damaged or unstable genomes. Chemotherapy and radiation exposure both induce widespread p53-dependent DNA damage. This triggers potentially lethal pathologies that are generally deemed an unfortunate but unavoidable consequence of the role p53 has in tumour suppression. Here we show, using a mouse model in which p53 status can be reversibly switched in vivo between functional and inactive states, that the p53-mediated pathological response to whole-body irradiation, a prototypical genotoxic carcinogen, is irrelevant for suppression of radiation-induced lymphoma. In contrast, delaying the restoration of p53 function until the acute radiation response has subsided abrogates all of the radiation-induced pathology yet preserves much of the protection from lymphoma. Such protection is absolutely dependent on p19(ARF)--a tumour suppressor induced not by DNA damage, but by oncogenic disruption of the cell cycle.     

An update on histone lysine methylation in plants

Histone methylation plays crucial roles in epigenetic regulation. The SET domain proteins are now recognized as generally having methyltransferase activity targeted to specific lysine residues of histones. The enzymes and their specific histone lysine methylation have enormous impacts on the regulation of chromatin structure and function. In this review, we discuss recent advances made on histone lysine methylations and their diverse functions in plant growth and development.     

Oncogene-induced senescence as an initial barrier in lymphoma development

Acute induction of oncogenic Ras provokes cellular senescence involving the retinoblastoma (Rb) pathway, but the tumour suppressive potential of senescence in vivo remains elusive. Recently, Rb-mediated silencing of growth-promoting genes by heterochromatin formation associated with methylation of histone H3 lysine 9 (H3K9me) was identified as a critical feature of cellular senescence, which may depend on the histone methyltransferase Suv39h1. Here we show that Emicro-N-Ras transgenic mice harbouring targeted heterozygous lesions at the Suv39h1, or the p53 locus for comparison, succumb to invasive T-cell lymphomas that lack expression of Suv39h1 or p53, respectively. By contrast, most N-Ras-transgenic wild-type ('control') animals develop a non-lymphoid neoplasia significantly later. Proliferation of primary lymphocytes is directly stalled by a Suv39h1-dependent, H3K9me-related senescent growth arrest in response to oncogenic Ras, thereby cancelling lymphomagenesis at an initial step. Suv39h1-deficient lymphoma cells grow rapidly but, unlike p53-deficient cells, remain highly susceptible to adriamycin-induced apoptosis. In contrast, only control, but not Suv39h1-deficient or p53-deficient, lymphomas senesce after drug therapy when apoptosis is blocked. These results identify H3K9me-mediated senescence as a novel Suv39h1-dependent tumour suppressor mechanism whose inactivation permits the formation of aggressive but apoptosis-competent lymphomas in response to oncogenic Ras.