Lsh, DNA methylation and agingLsh, DNA methylation and aging

报告人报告人 : : 周瑞周瑞

Normal DNA methylationNormal DNA methylation

• In mammals, DNA methylation patterns:• an initial wave of global demethylation; • the rapid establishment of gene and tissue-specific p

atterns of methylation which are relatively stable afterwards

• In humans, approximately 70% of CpG dinucleotides are methylated in adult cells.

Normal DNA methylationNormal DNA methylation

• In the regulation of gene transcription: • highly expressed genes tend to be hypomethylat

ed ;• silent genes tend to be hypermethylated

• Its direct contribution to gene expression remains somewhat unclear

Normal DNA methylationNormal DNA methylation

• Be implicated in chromatin structure and chromosomal stability.

• Silencing of repetitive sequences and integrated foreign DNA sequences:

• a defense mechanism against the deleterious effects of genomic invasion by parasitic DNA

CpG islandsCpG islands

• CpG islands: dense (one per 10bp) 0.5-3kb located in promoters and 5’ regions unmethylated or hypomethylated

• Promoters: CpG-island promoters CpG-deficient promoters

CpG island promoter methylation and gene expression

Much evidence exists linking promoter methylation and inhibited gene transcription.

• Physiological observation:

Inactive X-chromosome in women

Imprinted genes, including H19, IGF 2R and others• Pathological situation:

the fragile X syndrome

neoplasia

CpG island promoter methylation and gene expression

• Mechanism:

a. involves methylated DNA binding proteins(MeCPs) with transcriptional repression properties b. assembly of a nucleosomal structure involving a protein complex that includes histones, histone deacetylases and others c. the formation of a closed chromatin structure that silences gene expression through exclusion of trans- cription factors

CpG island promoter methylation and gene expression

• Inhibition of gene expression:

• CpG rich promoters:• depending on the density of methylation• Irreversible

• CpG poor promoters:• be regulated in part by DNA methylation• normal mechanisms of regulation of gene expression for some genes• Reversible

DNA methylation changes in aging

• Hypomethylation in aging cells and tissues

• CpG island hypermethylation in aging cells and tissues

DNA methylation changes in aging

• About Age-related methylation :• (a) affects only a small proportion of the human

genome.• (b) affects both genes that are expressed and

unexpressed in the tissues examined.• (c) is tissue-specific.

LshLsh

• belongs to the family of SNF2/helicases which

are frequently involved in chromatin remodeling.

• Lsh regulates DNA methylation levels in mice

• Targeted deletion of Lsh results in perinatal lethality, with abnormal lymphoid development

and renal defects

Lsh ,required for genome-wide methylation

• Object:

Genomic DNA derived Lsh−/− mice

Lsh−/− mice, in which exons 6 and 7 containing helicase domains I, Ia, and part of II were deleted, results in perinatal lethality with a rather normal development.

Results:Results:

(1) Hypomethylation in multiple repetitive sequences: minor satellite sequences major satellite sequences single copy sequences:β-Globin and Pgk-2(2) Global hypomethylation in Lsh−/− mice.(3) Expression of DNA methyltransferases and measurem

ent of Mtase activity in Lsh−/− mice.

Figure 1. Hypomethylation of minor satellite sequences in Lsh−/− mice.

(A) Southern analysis of genomic DNA derived at day 13.5 of gestation. DNA was digested with HpaII or MspI, blotted, and probed for minor satellite sequences using MR150.

(B) Southern analysis of genomic DNA derived from newborn mice within 24 hours after birth. Whole body comprises every tissue with the exception of the examined internal organs. DNA was digested with HpaII or MspI, blotted, and probed for minor satellite sequences using MR150.

Figure2. Global hypomethylation in Lsh−/− mice. (A) Genomic DNA derived fromthymus and MEF of Lsh de-ficient mice was digested with HpaII and MspI, subjected to agarose gel electrophoresis. (B) Methyl acceptance assay.Equal amounts of genomic DNA derived from day 13.5 embryonic body,embryonic liver, or from newborn brain were methylated in vitro by SssI CG methylase using radiolabeled S-adenosyl-methionine as donor.(C) Direct measurementof methyl-cytosine in genomic DNA.

Figure 3. Expression of Dnmts and measurement of Mtase activity in Lsh−/− mice.

(A) RT–PCR analysis.

(B) Western analysis.

(C)Mtase activity.

• Object:

• Mice with disruption of PASG expression by deleting

exons 10,11, and 12 containing helicase domains III, IV,

and part of II using homologous recombination.

Lsh and aging

Results:Results:

1.growth retardation and premature aging

2.Increased expression ofβ-gal

3.Altered gene expression pattern

Figure 4. PASG−/− mice age prematurely.(A) Newborn mice.PASG−/− mice (arrows) appear morphologically normal exceptfor their smaller size.(B) PASG−/− mice (shown at day 17) rapidly develop gray hair and balding. (C) PASG−/− mice at day 15show loss of body mass, muscle atrophy, cachexia, and a profounddecrease in adipose tissue. (D) Surviving PASG−/− mice at15 d of age show kyphosis.

Figure 5. Increased expression of the senescence biomarker,β-gal, in Lsh−/− mice and/or MEFs. (A,B) β-gal positive cells are found in Lsh−/− tubules of the renal cortex (A) and the thymus (B). Tissues from normal littermates did not show increased expression of β-gal . (C) Lsh−/− MEFs show increased β-gal positive cells compared to wild-type MEFs (D) Lsh−/− MEFs showed a flattenedand enlarged morphology compared to the normal morphology of the wild-type MEFs.

Figure 6.

Increased level of p16INK4a, p53, and p21 and decreased level of bmi-1 are observed inthe kidney of Lsh−/− mice.

Figure 7. Increased

level of p16INK4a and p19ARF and decreased level of bmi-1 are observed in PASG−/− MEFs at passage 2 (P2) and passage 5 (P5).

Lsh, genomic methylation, agingLsh, genomic methylation, aging

Lsh impact on the accessibility of impact on the accessibility of

chromatin to DNA methyltransferases chromatin to DNA binding proteins

CpG methylation Aging DNA transcriptional regulation

alteration of DNA structure

impact on the binding of DNA and TFs

谢 谢