Ageing

Question 1

What is heterochromatin?

Answer 1

Heterochromatin is a functionally distinct genomic compartment that is characterized by its relatively low gene density, enrichment for repetitive sequences and transposable elements, highly compact chromatin architecture, and late onset of DNA replication.

Question 2

Why use Hi-C?

Answer 2

Hi-C is a very powerful extension of 3C, and it offers the advantage of measuring long-range interactions between different loci genome-wide

Question 3

What is the Hi-C workflow?

Answer 3

The typical workflow of a Hi-C assay comprises crosslinking chromatin, digesting the DNA with a restriction enzyme, and incorporating a biotinylated nucleotide into the restriction fragment ends. Next, the restriction fragments are ligated. At this step two fragments which are in proximity 3-dimensionally are joined together by the ligase enzyme, even though they may be kilobases away in an unfolded genome. The biotinylated ligation products are pulled down with streptavidin coated beads and are processed for sequencing. The sequencing data, along with the direct relationship between ligation frequency and distance in 3D, allows for the 3-dimensional organization of chromatin to be inferred computationally [8]. Typically, the event where two restriction fragments are ligated to one another is referred to as an interaction, and the genome wide interaction profiles generated by Hi-C are used to identify different features of chromatin organization.

Question 4

What is the looping exclusion model?

Answer 4

Very high resolution in-situ Hi-C datasets have demonstrated that smaller TADs tend to contain recognition sequences for the CCCTC-binding factor (CTCF) protein. In what has become known as the “looping extrusion model”, the DNA in between these recognition sequences is passed through a CTCF cohesion protein complex. Once the CTCF binding sites reach this complex, the extrusion is stopped, and the result is a chromatin loop.

Question 5

What are frequently interacting regions (FIREs) and where are they found?

Answer 5

A large study comparing Hi-C data from different human tissues and cell types found that many chromatin loops and FIREs are tissue and cell type specific.

Question 6

What is Senescence Associated Secretory Phenotype (SASP) and how may it be used to increase lifespan?

Answer 6

The cumulative non-repaired DNA damage which each cell in an organism endures throughout its life leads to the accumulation of senescent cells [20], as well as an increase in their inflammatory products, a collection of proinflammatory signaling molecules and cytokines which are collectively referred to as the Senescence Associated Secretory Phenotype (SASP) [21]. Attenuating SASP in mice results into a marked increase in both healthspan and lifespan. For example, eliminating SASP at its source by selectively inducing the apoptosis in senescent cells extends the lifespan of mice

Question 7

What does telomere shortening cause?

Answer 7

Fibroblasts that were passaged to replicative exhaustion. In this form of senescence, referred to as replicative senescence, the shortening of telomeres which occurs during cell division leads to constitutive activation of the DNA damage response pathway, causing the cell to permanently exit the cell cycle.

Question 8

What is Oncogene Induced Senescent (OIS)?

Answer 8

OIS cells have extensive changes in their nuclear architecture. For example, Chandra et al. used Hi-C on WI-38 human lung fibroblasts undergoing OIS to demonstrate that regions of the genome which are typically near the nuclear periphery dissociate and localize to a more centralized area. This discovery helped to shed light on the formation of senescence-associated heterochromatic foci (SAHF), regions with high amounts of heterochromatic chromatin.

Question 9

What are LADs?

Answer 9

Lamina Associated Domains (LADs). Typically, the LADs are heterochromatic regions which attach to the nuclear envelope via the structural proteins Lamin A/C and Lamin B.

Question 10

What happens to LADs during ageing?

Answer 10

In replicative senescence these regions become detached due to a decrease in Lamin B1 proteins, resulting in LADs becoming more euchromatic. This observation is especially interesting since structural alterations in LADs are known to drive other age-related pathologies such as Gilford’s Hutchinson Progeria Syndrome.

Question 11

What happens to LADs in cancer cells?

Answer 11

Cell hyperproliferation is linked to many other epigenetic changes. Similar to what is seen in cellular senescence, the LADs of cancer cells detach from the nuclear lamina and become more open. This type of alteration can lead to expression of the many transposable elements which are within LADs, and the subsequent duplication of these repetitive sequences can further perturb the organization of the chromatin. Moreover, it has been found that cancer cells have unusually high mutation rates found at CTCF sites. It is expected that these mutagenic events contribute to the altered genomic architecture and gene regulation which characterizes cancer.

Question 12

What effect does progeria have on LADs?

Answer 12

Progeria express Progerin. This defunct translational product prevents LADs from attaching to the nuclear envelope, causing a perturbed 3-dimensional architecture, as well as premature aging phenotypes and frailty.

Question 13

What is the telomere position effect?

Answer 13

Perturbations to the 3-dimensional architecture of telomere sequences can also drive disease states. As previously mentioned, continual cell division leads to telomere attrition, and this has been found to affect gene expression via a phenomenon known as the Telomere Position Effect (TPE). In young cells telomeres are able to form loops with loci megabases away, and as telomeres shorten the structure of these loops are altered

Question 14

What is cytosolic DNA and how does it occur?

Answer 14

parts of the genome are completely expelled from the nucleus. This type of nuclear breakdown has been described in cancer and more recently in cellular senescence. Significantly, a growing body of evidence has shown the resulting cytosolic DNA to initiate various inflammatory pathways. The breakdown of the 3-dimensional nuclear architecture and can lead to inflammation and tissue dysfunction.

Question 15

Name 2 types of cytosolic DNA?

Answer 15

cancer-associated cytosolic DNA (CACD), and senescence-associated cytosolic DNA (SACD)

Question 16

What are micronuclei and what cause them?

Answer 16

Nuclear dysmorphism and cytosolic DNA in cancer and senescent cells are precursors to inflammation. Nuclei from cancer and senescent cells share common aberrant nuclear structural properties such as nuclear envelope distensions (blebs) and altered lamin expression. In the cancer nucleus, nuclear blebbing and micronuclei are formed due to genotoxic stresses, lamin A/C (red) or lamin B (blue) dysregulation, and excessive force of perinuclear structural proteins (neon blue). In senescent cells the 3D genome changes represented by dissociation of chromatin (grey) from regions of the nuclear lamina. Lamin B1 (blue) is targeted for degradation with its associated DNA (grey) and is extruded into the cytosol via LC3II (yellow). Detection of aberrant DNA species in the cytosol of cancer and senescent cells by cGAS-STING, propagates a pro-inflammatory type I interferon response.

Question 17

How are laminators involved in cancer?

Answer 17

Lamins are nuclear envelope structural proteins that form the backbone of nucleus morphology. Dysregulation of lamin proteins have been implicated in multiple forms of cancers, as these proteins are directly or indirectly involved in the regulation of gene expression, DNA repair, and apoptosis

Question 18

Altered expression of either or both lamin A/C or lamin B in cancers have been associated to the acute release of nuclear genetic material to the cytosol. How does this occur?

Answer 18

One possible mechanism of DNA exit through the movements of metastatic cells, where the nuclear envelope is morphed by mechanotransduction in and out of tight spaces [53]. Because of the dramatic changes in lamin structure and organization, the shape of the nucleus is compromised, often forming outward distensions leading to nuclear envelope ruptures and release of genetic material into the cytoplasm. Another similar mechanism has been hypothesized where extrusion of DNA from the nucleus to the cytoplasm is mediated by “pinching” of sites on the nuclear envelope that are lamin-depleted. In this case, the mechanical forces produced by perinuclear structural proteins (tubulin, actin) exceeds the anchoring and physical strength capabilities of the nuclear lamina. The pinching of the nuclear envelope eventually reaches a critical point causing nuclear envelope rupture events and subsequent leakage of chromatin into the cytoplasm.

Question 19

What are CACDs and how do they differ?

Answer 19

cancer-associated cytoplasmic DNA (CACD). Interestingly, CACDs display remarkable molecular conformation heterogeneity as double stranded DNA, single stranded DNA, as well as DNA:RNA hybrids

Question 20

Do cancer and cell senescence have any similarities?

Answer 20

Yes - striking similarities between the physiology of cancers and cellular senescence. Just as it has been observed in cancer cells, senescent cells are riddled with altered proteostasis, genomic instabilities, and compromised nuclear envelope. Many groups have reported on genomic dysfunction of repetitive elements and the appearance of nuclear distensions (nuclear blebbing) in senescent cells. These features preceded the observation of senescence-associated cytosolic DNA (SACD), which has been recently described by multiple groups. Though the similarities between cancer and senescence cannot be denied, the unique features in DNA conformation that define the senescent state and the genesis of disease are not yet well understood.

Question 21

Why is irreversible cell cycle arrest needed?

Answer 21

the irreversible cell cycle arrested state fulfills essential roles in embryonic development, tissue repair and remodeling, and in tumor suppression.

Question 22

What happens to lamin B1 during cell stress?

Answer 22

proposed a mechanism of nuclear DNA expulsion from the nucleus that showed SACD is selected based on its association with the nuclear envelope structural protein, lamin B1. Because nuclear lamins are designated for degradation in moments of cellular stress, it was hypothesized that senescent cells actively degrade lamin B1 through an autophagy-mediated process. To accomplish this, it was shown that the autophagic factor, microtubule-associated proteins 1A/1B light chain 3B II (LC3II) escorts lamin B1 with its coupled chromatin to the outside of the nucleus into autophagosomes. Autophagosome-containing SACD subsequently fuse to lysosomes, a process that typically leads to the degradation of the contained DNA.

Question 23

What may cytosolic DNA be linked too?

Answer 23

Two of the most significant findings are that the formation of cytosolic DNA in senescence and in cancer have been linked to increased cell metastasis potential, and that cytoplasmic DNA results in cell intrinsic and extrinsic pro-inflammatory responses [43,49,65]. How can this be? Anomalous DNA in the cytoplasm is sensed by the cytosolic DNA-sensing factor, cyclic GMP-AMP synthase (cGAS). Upon cGAS binding to the aberrant DNA, the stimulator of interferon genes (STING) initiates a signaling cascade that triggers the expression of type I interferons as well as other canonical pro-inflammatory factors of the innate immune system. The result is that the affected cell responds in the same manner as if it were infected by a virus, promoting the release of factors and cytokines with the intent of warning neighboring cells, including cells of the immune system. Because of the strong overlap of cytoplasmic DNA detection and immune response between cancer and senescence, this may mean that the widespread effects of cytoplasmic DNA are not dependent on cellular state or disease context.