Age-associated increase in heterochromatic marks in murine and primate tissues

Chromatin is highly dynamic and subject to extensive remodeling under many physiologic conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin-associated proteins. We found increased levels of the heterochromatin-associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1β) in human fibroblasts during replicative senescence in culture, and for the first time, an age-associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver, we observed increased age-associated localization of mH2A to regions of pericentromeric heterochromatin. In the skeletal muscle, we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogene-induced cellular senescence, and are prominently found in terminally differentiated, postmitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to investigate further the progressive epigenetic changes that occur during aging.     

Maternal Depletion of Piwi,a Component of the RNAi System,Impacts Heterochromatin Formation in Drosophila

A persistent question in epigenetics is how heterochromatin is targeted for assembly at specific domains, and how that chromatin state is faithfully transmitted. Stable heterochromatin is necessary to silence transposable elements (TEs) and maintain genome integrity. Both the RNAi system and heterochromatin components HP1 (Swi6) and H3K9me2/3 are required for initial establishment of heterochromatin structures in S. pombe. Here we utilize both loss of function alleles and the newly developed Drosophila melanogaster transgenic shRNA lines to deplete proteins of interest at specific development stages to dissect their roles in heterochromatin assembly in early zygotes and in maintenance of the silencing chromatin state during development. Using reporters subject to Position Effect Variegation (PEV), we find that depletion of key proteins in the early embryo can lead to loss of silencing assayed at adult stages. The piRNA component Piwi is required in the early embryo for reporter silencing in non-gonadal somatic cells, but knock-down during larval stages has no impact. This implies that Piwi is involved in targeting HP1a when heterochromatin is established at the late blastoderm stage and possibly also during embryogenesis, but that the silent chromatin state created is transmitted through cell division independent of the piRNA system. In contrast, heterochromatin structural protein HP1a is required for both initial heterochromatin assembly and the following mitotic inheritance. HP1a profiles in piwi mutant animals confirm that Piwi depletion leads to decreased HP1a levels in pericentric heterochromatin, particularly in TEs. The results suggest that the major role of the piRNA system in assembly of heterochromatin in non-gonadal somatic cells occurs in the early embryo during heterochromatin formation, and further demonstrate that failure of heterochromatin formation in the early embryo impacts the phenotype of the adult.     

染色质可及性与植物基因表达调控

真核生物基因组上的核小体呈现不均匀分布, 转录活跃区域的染色质结构相对松散且易被调节蛋白结合, 这些区域的可接近程度称为染色质可及性。随着测序技术的发展, DNase-seq、ATAC-seq、MNase-seq和NOMe-seq等组学技术的应用, 全基因组范围内染色质可及性检测变得简便且高效。该文主要介绍了真核生物染色质可及性的4种基本检测方法的技术原理, 总结了核小体定位、组蛋白修饰以及转录因子结合与染色质可及性的关系, 并综述了染色质可及性参与植物生长发育和环境响应研究进展, 以期为植物领域全基因组水平染色质可及性研究、顺式调控元件挖掘及发育和环境响应过程中基因表达调控网络的解析提供借鉴。     

Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior

Examining the role of chromatin modifications and gene expression in neurons is critical for understanding how the potential for behaviors are established and maintained. We investigate this question by examining Drosophila melanogaster fru P1 neurons that underlie reproductive behaviors in both sexes. We developed a method to purify cell-type-specific chromatin (Chromatag), using a tagged histone H2B variant that is expressed using the versatile Gal4/UAS gene expression system. Here, we use Chromatag to evaluate five chromatin modifications, at three life stages in both sexes. We find substantial changes in chromatin modification profiles across development and fewer differences between males and females. Additionally, we find chromatin modifications that persist in different sets of genes from pupal to adult stages, which may point to genes important for cell fate determination in fru P1 neurons. We generated cell-type-specific RNA-seq data sets, using translating ribosome affinity purification (TRAP). We identify actively translated genes in fru P1 neurons, revealing novel stage- and sex-differences in gene expression. We also find chromatin modification enrichment patterns that are associated with gene expression. Next, we use the chromatin modification data to identify cell-type-specific super-enhancer-containing genes. We show that genes with super-enhancers in fru P1 neurons differ across development and between the sexes. We validated that a set of genes are expressed in fru P1 neurons, which were chosen based on having a super-enhancer and TRAP-enriched expression in fru P1 neurons.     

Aging and Loss of Germination in Rye Seeds Is Accompanied by a Decreased Fragmentation of Nuclear DNA at Loop Domain Boundaries

To investigate the processes that occur in the embryo cell nuclei in the course of natural and accelerated aging of rye seeds, nuclear DNA structural organization into chromatin loop domains was studied. The loss of germination was shown to be accompanied by a decreased excision of chromatin loop domains. The study of chromatin accessibility to DNase I did not reveal any considerable changes in chromatin architecture that would explain the decreased DNA fragmentation at matrix attachment regions. A soluble nuclear protein of ca. 31 kD was found to manifest nuclease activity, which declined with the loss of germination. The study of DNA fragmentation in histone-depleted nuclei (nucleoids) disclosed a nuclease activity resistant to 2 M NaCl extraction and sensitive to the specific inhibitors of DNA topoisomerase II; the latter activity also declined with aging. The authors conclude that the changes in DNA fragmentation patterns in aging seeds were primarily caused by a decreased activity of the enzymes accounting for the excision of chromatin loop domains.     

Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation.     

Discovery of cell-type specific regulatory elements in the human genome using differential chromatin modification analysis

Chromatin modifications have been comprehensively illustrated to play important roles in gene regulation and cell diversity in recent years. Given the rapid accumulation of genome-wide chromatin modification maps across multiple cell types, there is an urgent need for computational methods to analyze multiple maps to reveal combinatorial modification patterns and define functional DNA elements, especially those are specific to cell types or tissues. In this current study, we developed a computational method using differential chromatin modification analysis (dCMA) to identify cell-type-specific genomic regions with distinctive chromatin modifications. We then apply this method to a public data set with modification profiles of nine marks for nine cell types to evaluate its effectiveness. We found cell-type-specific elements unique to each cell type investigated. These unique features show significant cell-type-specific biological relevance and tend to be located within functional regulatory elements. These results demonstrate the power of a differential comparative epigenomic strategy in deciphering the human genome and characterizing cell specificity.     

Age-associated increase of spontaneous mutant frequency and molecular nature of mutation in newborn and old lacZ-transgenic mouse

Accumulation of mutation has long been hypothesized to be a cause of aging and contribute to many of the degenerative diseases, which appear in the senescent phase of life. To test this hypothesis, age-associated changes in spontaneous mutation in different tissues of the body as well as the molecular nature of such changes should be examined. This kind of approach has become feasible only lately with a development of new transgenic mice suitable for mutation assay. Here, using one of these transgenic mice harboring lacZ gene, we have shown that the age-associated increase in spontaneous mutant frequency is common to all tissues examined; spleen, liver, heart, brain, skin and testis, while the rates of increase in mutant frequency differed among the tissues. DNA sequencing of the 496 lacZ mutants recovered from the tissues of newborn and old mice has revealed that spectra of mutations are similar at the two age points with G:C to A:T transition at CpG site being a predominant type of mutation. Furthermore, some mutations in old tissues are complex type and not found in tissues of newborn mice. These results suggest that similar mechanisms may be operating for mutation induction in fetal and postnatal aging process. In addition, the appearance of complex types of mutations in the old tissues suggests a unique cause for these mutations in aging tissues.     

Changes at the nuclear lamina alter binding of pioneer factor Foxa2 in aged liver

Increasing evidence suggests that regulation of heterochromatin at the nuclear envelope underlies metabolic disease susceptibility and age‐dependent metabolic changes, but the mechanism is unknown. Here, we profile lamina‐associated domains (LADs) using lamin B1 ChIP‐Seq in young and old hepatocytes and find that, although lamin B1 resides at a large fraction of domains at both ages, a third of lamin B1‐associated regions are bound exclusively at each age in vivo. Regions occupied by lamin B1 solely in young livers are enriched for the forkhead motif, bound by Foxa pioneer factors. We also show that Foxa2 binds more sites in Zmpste24 mutant mice, a progeroid laminopathy model, similar to increased Foxa2 occupancy in old livers. Aged and Zmpste24‐deficient livers share several features, including nuclear lamina abnormalities, increased Foxa2 binding, de‐repression of PPAR‐ and LXR‐dependent gene expression, and fatty liver. In old livers, additional Foxa2 binding is correlated to loss of lamin B1 and heterochromatin (H3K9me3 occupancy) at these loci. Our observations suggest that changes at the nuclear lamina are linked to altered Foxa2 binding, enabling opening of chromatin and de‐repression of genes encoding lipid synthesis and storage targets that contribute to etiology of hepatic steatosis.     

Age‐associated de‐repression of retrotransposons in the Drosophila fat body,its potential cause and consequence

Eukaryotic genomes contain transposable elements (TE) that can move into new locations upon activation. Since uncontrolled transposition of TEs, including the retrotransposons and DNA transposons, can lead to DNA breaks and genomic instability, multiple mechanisms, including heterochromatin‐mediated repression, have evolved to repress TE activation. Studies in model organisms have shown that TEs become activated upon aging as a result of age‐associated deregulation of heterochromatin. Considering that different organisms or cell types may undergo distinct heterochromatin changes upon aging, it is important to identify pathways that lead to TE activation in specific tissues and cell types. Through deep sequencing of isolated RNAs, we report an increased expression of many retrotransposons in the old Drosophila fat body, an organ equivalent to the mammalian liver and adipose tissue. This de‐repression correlates with an increased number of DNA damage foci and decreased level of Drosophila lamin‐B in the old fat body cells. Depletion of the Drosophila lamin‐B in the young or larval fat body results in a reduction of heterochromatin and a corresponding increase in retrotransposon expression and DNA damage. Further manipulations of lamin‐B and retrotransposon expression suggest a role of the nuclear lamina in maintaining the genome integrity of the Drosophila fat body by repressing retrotransposons.     

Sex Differences in Drosophila melanogaster Heterochromatin Are Regulated by Non-Sex Specific Factors

The eukaryotic genome is assembled into distinct types of chromatin. Gene-rich euchromatin has active chromatin marks, while heterochromatin is gene-poor and enriched for silencing marks. In spite of this, genes native to heterochromatic regions are dependent on their normal environment for full expression. Expression of genes in autosomal heterochromatin is reduced in male flies mutated for the noncoding roX RNAs, but not in females. roX mutations also disrupt silencing of reporter genes in male, but not female, heterochromatin, revealing a sex difference in heterochromatin. We adopted a genetic approach to determine how this difference is regulated, and found no evidence that known X chromosome counting elements, or the sex determination pathway that these control, are involved. This suggested that the sex chromosome karyotype regulates autosomal heterochromatin by a different mechanism. To address this, candidate genes that regulate chromosome organization were examined. In XX flies mutation of Topoisomerase II (Top2), a gene involved in chromatin organization and homolog pairing, made heterochromatic silencing dependent on roX, and thus male-like. Interestingly, Top2 also binds to a large block of pericentromeric satellite repeats (359 bp repeats) that are unique to the X chromosome. Deletion of X heterochromatin also makes autosomal heterochromatin in XX flies dependent on roX and enhances the effect of Top2 mutations, suggesting a combinatorial action. We postulate that Top2 and X heterochromatin in Drosophila comprise a novel karyotype-sensing pathway that determines the sensitivity of autosomal heterochromatin to loss of roX RNA.