TSA通过组蛋白乙酰化影响TLR2基因表达

目的组蛋白去乙酰化酶抑制剂(Trichostatin A,TSA)处理人THP-1细胞,干预组蛋白H3乙酰化水平,探讨组蛋白H3乙酰化对人THP-1细胞中TLR2基因表达水平的影响。方法构建THP-1巨噬细胞模型,分别利用不同浓度TSA处理细胞,Real-time PCR和Western blot方法检测m RNA和蛋白的表达;染色质免疫共沉淀技术(Chromatin immunoprecipitation,Ch IP)比较TSA处理前后启动子区H3乙酰化水平。结果TSA以浓度依赖方式上调表达水平。TSA上调组蛋白H3乙酰化水平,使启动子区H3乙酰化水平明显上升。结论 TSA通过组蛋白H3乙酰化影响基因表达,这是乙酰化调控TLR2基因表达的一种可能机制。     

The role of the SMN gene in proximal spinal muscular atrophy

Childhood spinal muscular atrophy (SMA) is a common recessive autosomal disorder that results in degeneration of lower motor neurons. The identification of the disease gene, Survival of Motor Neuron (SMN), was a major advance in understanding the molecular basis underlying this devastating neuromuscular disease. This finding has greatly improved the genetic counselling of SMA families. Recently, biochemical studies demonstrated its involvement in the biogenesis of spliceosomal snRNPs, suggesting a critical role of SMN in RNA processing. Surprisingly, other studies showed a putative role of SMN in an anti-apoptotic pathway involving Bcl-2. The function of SMN protein is not fully understood. These observations emphasized the difficulty in elucidating the function of any novel protein. Therefore, multidisciplinary approaches are required to understand the pathogenesis of SMA.      

Correlation between histone acetylation and expression of the MYO18B gene in human lung cancer cells

Recently, we isolated a candidate tumor-suppressor gene, MYO18B, which was inactivated in approximately 50% of human lung cancers by deletion, mutation, and promoter methylation. However, more frequent reduction or loss of MYO18B expression and restoration of MYO18B expression by trichostatin A (TSA) treatment suggested the contribution of other mechanisms, especially histone deacetylation, for epigenetic inactivation of the MYO18B gene. In this study, we examined histone modification of the promoter region of the MYO18B gene in 8 human lung cancer cell lines by a chromatin immunoprecipitation assay. In 6 of 7 cell lines with reduced or silenced MYO18B expression, the levels of histones H3 and H4 acetylation surrounding the MYO18B promoter region were lower than those in a cell line with MYO18B expression. By treatment with TSA, the levels of histone H3 and H4 acetylation were increased in all 6 cell lines whose MYO18B expression was restored by TSA, whereas neither H3 nor H4 acetylation was increased in cells whose MYO18B expression was not restored by TSA. Significant correlations were observed between the levels of histone H3/H4 acetylation and MYO18B expression. These results suggest that acetylation of both histones H3 and H4 contributes to regulation of MYO18B expression in lung cancer cells and that histone deacetylation surrounding the promoter region plays an important role in MYO18B silencing and is involved in lung carcinogenesis.     

Phenylbutyrate increases SMN gene expression in spinal muscular atrophy patients

Spinal muscular atrophy (SMA) is caused by insufficient levels of survival motor neuron (SMN) protein. Recently, we found that sodium 4-phenylbutyrate (PB), a well-tolerated FDA approved drug, enhances SMN gene expression in vitro. We provide here the first evidence that oral administration of PB (triButyrate significantly increases SMN expression in leukocytes of SMA patients. This finding provides a strong rationale to further investigate the effects of PB as also supported by preliminary clinical data.     

Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance

Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications.     

Refined characterization of the expression and stability of the SMN gene products

Spinal muscular atrophy (SMA) is characterized by degeneration of lower motor neurons and caused by mutations of the SMN1 gene. SMN1 is duplicated in a homologous gene called SMN2, which remains present in patients. SMN has an essential role in RNA metabolism, but its role in SMA pathogenesis remains unknown. Previous studies suggested that in neurons the protein lacking the C terminus (SMN(Delta7)), the major product of the SMN2 gene, had a dominant-negative effect. We generated antibodies specific to SMN(FL) or SMN(Delta7). In transfected cells, the stability of the SMN(Delta7) protein was regulated in a cell-dependent manner. Importantly, whatever the human tissues examined, SMN(Delta7) protein was undetectable because of the instability of the protein, thus excluding a dominant effect of SMN(Delta7) in SMA. A similar decreased level of SMN(FL) was observed in brain and spinal cord samples from human SMA, suggesting that SMN(FL) may have specific targets in motor neurons. Moreover, these data indicate that the vulnerability of motor neurons cannot simply be ascribed to the differential expression or a more dramatic reduction of SMN(FL) in spinal cord when compared with brain tissue. Improving the stability of SMN(Delta7) protein might be envisaged as a new therapeutic strategy in SMA.     

H3K9Ac在卵巢上皮性肿瘤中的表达及其临床意义

目的 研究组蛋白H3赖氨酸残基9位乙酰化（H3KgAc）在卵巢上皮性肿瘤中的表达及其与卵巢癌组织学分级、临床分期之间的关系。方法 采用免疫组织化学ABC法检测20例良性、16例交界性、40例恶性卵巢上皮性肿瘤中H3K9Ac的表达情况，并分析其与临床病理指标间的关系。结果 ①H3K9Ac在良性、交界性、恶性卵巢上皮性肿瘤中的表达逐渐降低，差异有显著统计学意义（P〈0．01）；②与粘液性囊腺癌相比，H3KgAc在浆液性囊腺癌中表达较低，差异有统计学意义（P〈0．05）；③H3KgAc的表达与卵巢上皮性癌的组织分化程度及临床分期有关，在Ⅲ、Ⅳ期卵巢癌中H3KgAc的表达明显低于Ⅰ、Ⅱ期卵巢癌（P〈0．01）；随着组织分化程度降低，H3KgAc表达也逐渐降低，两两比较差异有统计学意义（P〈0．05）。结论 H3KgAc在良性、交界性、恶性卵巢上皮性肿瘤中的表达差异显著，且随着卵巢上皮性癌恶性程度的增高，表达逐渐降低，H3K9Ac可能为卵巢上皮性肿瘤的良恶性及其预后判断提供一个新的生物学指标。     

Survival motor neuron SMN1 and SMN2 gene promoters: identical sequences and differential expression in neurons and non-neuronal cells

Spinal muscular atrophy (SMA) is a recessive disorder involving the loss of motor neurons from the spinal cord. Homozygous absence of the survival of motor neuron 1 gene (SMN1) is the main cause of SMA, but disease severity depends primarily on the number of SMN2 gene copies. SMN protein levels are high in normal spinal cord and much lower in the spinal cord of SMA patients, suggesting neuron-specific regulation for this ubiquitously expressed gene. We isolated genomic DNA from individuals with SMN1 or SMN2 deletions and sequenced 4.6 kb of the 5' upstream regions of the these. We found that these upstream regions, one of which is telomeric and the other centromeric, were identical. We investigated the early regulation of SMN expression by transiently transfecting mouse embryonic spinal cord and fibroblast primary cultures with three transgenes containing 1.8, 3.2 and 4.6, respectively, of the SMN promoter driving beta-galactosidase gene expression. The 4.6 kb construct gave reporter gene expression levels five times higher in neurons than in fibroblasts, due to the combined effects of a general enhancer and a non-neuronal cell silencer. The differential expression observed in neurons and fibroblasts suggests that the SMN genes play a neuron-specific role during development. An understanding of the mechanisms regulating SMN promoter activity may provide new avenues for the treatment of SMA.     

P38MAPK的激活上调SH-SY5Y细胞APP表达及组蛋白H3乙酰化水平

目的研究P38MAPK信号通路的激活对淀粉前体蛋白(APP)表达的影响及其相关表观遗传学机制。方法体外培养神经母细胞瘤细胞(SH-SY5Y),Western blot法检测SH-SY5Y的APP蛋白表达及组蛋白乙酰化酶(HAT)和组蛋白去乙酰化酶(HDAC)的表达;吸光度值法检测组蛋白3(H3)和组蛋白4(H4)整体乙酰化水平。结果 P38MAPK信号通路经特异性激动剂激活SH-SY5Y 72 h后,APP表达明显增高至对照组的1.5倍(0.41±0.09vs0.28±0.08,P<0.01);同时组蛋白H3整体乙酰化水平增高(0.20±0.04vs0.06±0.03,P<0.01),但H4乙酰化水平无明显改变;组蛋白乙酰化酶CBP表达增高至对照组的2.5倍(0.30±0.03vs0.11±0.05,P<0.01),而组蛋白去乙酰化酶HDAC3表达下降至对照组的40%(0.19±0.05vs0.49±0.03,P<0.01)。结论 P38MAPK信号通路可能通过上调组蛋白乙酰化水平增加SH-SY5Y的APP蛋白表达。     

Inverse correlation between SMN1 and SMN2 copy numbers: evidence for gene conversion from SMN2 to SMN1

Most carriers of autosomal recessive spinal muscular atrophy (SMA) have only one copy of SMN1 because of SMN1 gene deletions or gene conversions from SMN1 to SMN2, which has only one base difference in coding sequence from SMN1. Using SMN gene dosage analysis, we determined the copy numbers of SMN1 and SMN2 in the general population as well as in SMA patients and carriers. Increased SMN1 copy number is associated with decreased SMN2 copy number in the general population; that is, SMN2 copy number was decreased to one or zero copies in 11 of 13 individuals with three or four copies of SMN1, whereas only 71 of 164 individuals with two copies of SMN1 had one or zero copies of SMN2 (P<0.01). SMN2 copy number was increased to three or four in a subset of SMN1 deletion/conversion carriers, and in most SMA patients with a milder phenotype. In conclusion, our data provide evidence that gene conversion from SMN2 to SMN1 occurs, and that SMN1 converted from SMN2 is present in the general population.