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We think of DNA as the vitally important molecules that carry genetic instructions for most living things, including ourselves. But not all DNA actually codes proteins; now, we're finding more and more functions involving the non-coding DNA scientists used to think of as 'junk'.

A new study suggests thatsatellite DNA a type of non-coding DNA arranged in long, repetitive, apparently nonsensical strings of genetic material may be the reason why different species can't successfully breed with each other.

It appears that satellite DNA plays an essential role in keeping all of a cell's individual chromosomes together in a single nucleus, through the work of cellular proteins.

According to biologists Madhav Jagannathan and Yukiko Yamashita who authored the new study, that important role is managed differently in each species, leading to genetic incompatibility. The clash of the different strategies between species may be what causes chromosomes to scatter outside of the nucleus, at least in part, preventing reproduction.

"We propose a unifying framework that explains how the widely observed satellite DNA divergence between closely related species can cause reproductive isolation," they write intheir paper.

This "satellite DNA divergence" has been well established in previous research, leading to suspicions about its role in speciation. In the case of the chimpanzee genome and the human genome, for example, the protein-coding DNA is almost identical, while the 'junk' DNA is almost entirely different.

In this new study, experimenting on the fruit fly Drosophila melanogaster, the researchers noticed that deleting agene which produces a protein called Prod which binds to a specific bit of satellite DNA caused the flies to die, as their chromosomes scattered outside the cell nucleus.However, that crucial bit of satellite DNA is missing altogether in the flies' nearest relatives, which survive just fine without it.

That suggests these important non-coding sequences of DNA material have evolved differently between species. To take a closer look, the team examined hybrid offspring bred from a D. melanogaster female and a male from the closely related D. simulans species.

Flies bred in this way usually die very quickly or end up sterile. In this case, an examination of the tissue of the hybrid offspring confirmed what the researchers had suspected that the chromosomes (the DNA packages necessary for reproduction) were being disrupted here as well.

"When we looked at those hybrid tissues, it was very clear that their phenotype was exactly the same as if you had disrupted the satellite DNA-mediated chromosomal organization of a pure species," says Yamashita, who works at the Massachusetts Institute of Technology (MIT).

"The chromosomes were scattered, and not encapsulated in a single nucleus."

Digging even further, the researchers produced healthy hybrid flies by removing the genes known to damage hybrid offspring (called 'hybrid incompatibility genes') from their parent flies.These incompatibility genes are known to localize to satellite DNA in the pure species.

Satellite DNA mutates fairly regularly, and the researchers think that the proteins that bind to satellite DNA to keep chromosomes together have to evolve to keep up. This then gives each species its own different strategy when it comes to satellite DNA operations.

Next, the team wants to try and design a protein that successfully binds across the satellite DNA of two species, keeping the chromosomes where they should be. That could enable viable offspring between those species, but it will take years to realize.

"Our study lays the foundation for understanding hybrid incompatibility at a cellular level in Drosophila as well as other eukaryotes," write the researchers.

The research has been published in Molecular Biology and Evolution.

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Biologists Just Got Closer to The DNA Secrets That Stop Species From Interbreeding - ScienceAlert

Have you ever wondered where the honey you add to your morning tea and drizzle on your desserts or oatmeal comes from (besides bees)? The easy answer would be to check the label, which typically offers the country of origin along with all those wonderful nutritional benefits. Unfortunately, as the Department of Homeland SecuritysScience and Technology Directorate(S&T) knows all too well, sometimes labels can be misleading, especially when it comes to honey imported into the U.S.

Honey imports have nearly doubled in the last decadefrom 251 million pounds in 2010 to 416 million pounds in 2019which is great news for consumers who now have more access to some of the sweetest stuff on earth. However, this tremendous growth in demand also has a dark side that many might not know aboutadulteration and mislabeling of honey to hide its true origin have become a global issue.

What does this mean for shoppers and our economy? Well, illicit importers, who are economically motivated to evade tariffs or sanctions, have made it a practice to affix fake labels onto jars, indicating the honey is from a different country of origin or disguising cheaper honey as sought-after expensive types. Some illicit actors even dilute honey with ingredients like syrups and sugar. New Zealand manuka honey, for example, commands a high price on the market, up to 100 times higher than other honey types, as it is very in-demand for its putative health benefits. It is also one of the most adulterated types of honey. In a recent lawsuit, U.S. beekeepers claimed adulterated honey from Asia caused prices to plummet and forced them into financial ruin.

Adulterated honey is a tremendous problem for the U.S. honey industry, because it drives the market price down, and U.S. producers cant compete with the lower market value of imported honey, said Stephen Cassata, a senior science officer and acting lab director of the U.S. Customs and Border Protection (CBP) INTERDICT Science Center. Dealing with this issue is a whole-of-government approach, and we are currently collaborating with other federal agencies (including the Food and Drug Administration and U.S. Department of Agriculture) on joint operations targeting honey enforcement.

CBP is tasked with enforcing hundreds of U.S. trade laws, including the proper classification of goods under the Harmonized Tariff Schedule of the United States, and assessing applicable tariffs to ensure that importers pay the appropriate duties on entered goods. To help CBP determine the true sources of honey, S&T enlisted the expertise of theBorders, Trade, and Immigration Institute Center of Excellence(BTI), led by the University of Houston, for a project calledHoney DNA. S&T invested in cutting-edge forensic science that can improve the speed and efficiency in verifying the country of origin of commercially available honey and its path to the supermarket shelvesspecifically, S&T has been looking at how the unique makeups of products coming to the U.S. match how they are represented on the packaging.

This project developed a means to identify honey countries of origin using the DNA in pollen and DNA dissolved in filtered honey, said BTIexecutive director Kurt Berens.

Honey is filtered for a variety of reasons, including the attempt to hide its source plant by making plant identification by pollen very challenging.

BTIs testing method could potentially provide another capability for CBP to determine country or region of origin for Antidumping and Countervailing Duty enforcement, said CBPdeputy director Patricia Hawes. It complements testing capabilities we already employ to determine country of origin of honey.

BTI conducts research to enhance U.S. border security, facilitate legitimate trade and travel, and ensure immigration system integrity, and for several years this S&T Center of Excellence has been working on detecting the source country of honey by identifying the plant species via DNA from pollen. The Honey DNA project started in early 2020.

Thesize of the availabledatabase of knownDNA sequences from particular plants has exploded recently, making it more likely that any DNA sequence we find can be associated with a particular plant species or small group of plants, said BTI Honey DNA principal investigator Dr. Richard Willson, who is also Huffington-Woestemeyer Professor of Chemical and Biomolecular Engineering. Over the past 15 to 20 years, the cost of DNA sequencing hascollapsed by10,000-fold, making DNA-sequencing-based technologies much more attractive for a wide variety of applications.

Through the Honey DNA project, BTI was able to leverage these technologies to establish techniques that will help mitigate fraud and provide authentic and safe food for consumers. BTI scientists developed methods for sequencing DNA not only from whole pollen grains in unfiltered honey but also from the small amounts of DNA leaked from broken pollen grains in filtered honey.

To study the true origin of honey, BTI scientists collected samples from a variety of places, including directly from apiaries in multiple countries, from small specialty providers and farmers markets, from friends traveling overseas, and even from online specialty stores during the pandemic when travel wasnt possible.

We analyzed 300 honey samples from which we assembled country-specific plant DNA sequences, said Willson. We also successfully isolated soluble trace DNA from pollen-free, ultra-filtered honey and sequenced it.

To extract DNA from pollen, Willson and his team diluted small samples of honey with water, centrifuged them to help the pollen sink to the bottom of the test tubes, and extracted the DNA. To isolate trace DNA from filtered honey, the BTI scientists again diluted a small sample of pollen-free honey with water, and then extracted and purified the DNA. The extracted DNA was then amplified and sequenced. These findings were recently released in a report. (Read the final report,PDF, 83pgs., 3.71 MB).

DNA sequencing is performed to find the sequence in which the building blocks of DNA, the nucleotides, are arranged in a given DNA strand, which can help identify a plant species.

When the BTI scientists tested the expensive New Zealand manuka honey, which is derived from the manuka plant, they found that many jar labels claiming to contain manuka honey were not accurate.

The BTI team believes that the Honey DNA tracing methods could find broad applications in other types of forensic cases, including identifying the species of other natural products, and even tracing the origins of imported goods and narcotics. Also, the DNA sequences obtained from this project will enrich the public DNA database and help link occurrences of source plants across the world for a more precise identification of honey origin, said Willson.

This new technology could potentially be one of the tools in CBPs toolbox to intercept illegal imports, said Hawes. We are constantly looking at new ways to do our mission.

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DHS S&T Using DNA to Search for the True Origins of Imported Honey | Hstoday - HSToday

By Michael Marshall

Human brains have been shaped by DNA that evolves quickly

comotion_design/Getty Images

Many of the fastest-evolving sections of the human genome are involved in brain development. These rapidly changing segments of DNA may have played key roles in the evolution of the human brain and in our cognitive abilities.

Chris Walsh at Boston Childrens Hospital in Massachusetts and his colleagues studied sections of the human genome dubbed human accelerated regions (HARs). These stretches of DNA are virtually identical in many other mammals that have been studied, suggesting they have important functions but they differ in humans, implying our evolution has changed them.

Previous studies have identified 3171 possible HARs, but Walsh says it is unlikely that they are all important. Probably hundreds of them are, but probably not thousands, he says. His team set out to identify HARs that have played important roles in the evolution of our brains.

The researchers placed copies of each HAR, as well as their chimpanzee equivalents, into developing brain cells from mice and humans. In each cell line, they tracked how much each gene in the genome was expressed. This allowed them to determine whether each HAR enhanced the activity of genes, compared with the equivalent sequence from a chimp.

Using this and other methods, the team identified 210 HARs that significantly enhanced gene activity in the neural cells. These HARs probably affect human brain development.

The researchers then zeroed in on a gene called PPP1R17, which is expressed in some of the cells of the developing brain and regulated by several HARs, so it therefore behaves differently in humans than in other mammals. They compared the expression of PPP1R17 in the developing brains of mice, ferrets, rhesus macaques and humans. In the macaques and humans, the gene was expressed in the cerebral cortex, but it wasnt in the mice and ferrets.

This gives an example of how dynamic these enhancers are over the course of evolution, says Walsh.

It isnt clear why PPP1R17 came to be activated differently in humans, but it may be related to our unusually large brains. Big brains need lots of cells, each of which is likely to contain harmful mutations that need to be fixed. These repairs take time, and PPP1R17 is known to make cells take longer to grow and divide.

Journal reference: Neuron, DOI: 10.1016/j.neuron.2021.08.005

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Rapidly evolving bits of DNA helped develop the human brain - New Scientist

Study on yeast cells reveals how gene affects speed of damage caused by UV radiation

WSU researchers discovered a gene involved in repairing genetic pathways that can lead to cancerous conditions, neurological defects and cause aging.

The gene named ELOF1 was discovered 20 years ago in humans. It was solely recognized for its role in transcribing DNA. The WSU research team is the first to recognize its role in repairing damaged DNA from UV radUnaiation, said Kathir Selvam, postdoctoral researcher with the WSU School of Molecular Biosciences.

Selvam said he and his team cannot test ELOF1 because it is only found in humans, so they are studying its genetic counterpart, Elf1, which can be found in yeast cells. By exposing the cells to UV light, which can cause cancerous conditions or premature aging, the researchers are testing Elf1s role in DNA repair.

In the yeast cell trials, Elf1 is knocked out of the yeast. The cells, which no longer have Elf1, are exposed to UV light in a controlled environment for about one week. When Elf1 is reintroduced to the cell, it is repaired in about one day.

To analyze the process, John Wyrick, WSU School of Molecular Biosciences associate professor, said a genetic sequencing method was developed. The sequence maps where the damage, which can be randomly distributed, forms across a genome.

Genome maps are then compared to analyze the repairing properties of the Elf1 gene, Selvam said.

So far, results show that cells die more readily when Elf1 is removed following UV irradiation, Wyrick said.

Because Elf1 and ELOF1 are counterparts, Elf1s effect in yeast cells DNA repair pathway would be similar to ELOF1s effect in human DNA repair, Selvam said.

Wyrick said he and Selvams group are collaborating with researchers at other universities. This includes the Erasmus University Medical Center in the Netherlands, which focuses on ELOF1s effect on human cells.

Wyrick said the research began in summer 2019 and is centered around studying how DNA is damaged, resulting in possible mutations in genes. The researchs goal was to study a specific type of DNA damage resulting from exposure to UV light.

Nucleotide excision repair is a critical repair pathway that helps repair cell damage, Wyrick said. When there are genetic defects in a DNA pathway, it significantly increases the likelihood an individual will have cancer.

NER repairs damage to DNA lesions caused by UV exposure, Wyrick said. Normally, NER goes along the pathway, searching for distorted DNA lesions and tries to repair them.

Its like finding a needle in a haystack, he said.

Postdoctoral student Kathir Selvam, left, stands with Professor John Wyrick, right.

Typically, DNA is transcribed into RNA by a protein called RNA polymerase. But when the DNA is damaged, the RNA polymerase stalls and the damage has to be repaired.

Failure in this repair can cause a number of human diseases, including Cockayne syndrome a rapidly aging disease that can cause neurological defects and prevent individuals from surviving past their teen years and make an individual more susceptible to cancerous conditions, Selvam said.

Essentially its like a car going along and if a cow gets in your way, then you stop the car, Wyrick said.

If something goes wrong when DNA is transcribed into RNA, a NER subpathway is initiated to repair the damage so transcription can continue.

The cow then gets moved out of the way and the car keeps driving, Wyrick said.

Wyrick said ELOF1 likely plays a role in the pathway because it binds to the RNA polymerase and travels with it, helping to repair and bypass the damage.

Basically, we want to understand what genes are playing a role in that [repair] pathway, and what role they play, Wyrick said.

ELOF1 is not exclusive to repairing damage from UV exposure. It is equally essential in repair pathways found in human genetic diseases or mutations, Wyrick said.

In previous research, Wyrick said mouse embryos were not able to develop without the presence of the ELOF1 gene, indicating it is likely needed for survival.

The research is just focusing on understanding the mechanisms, understanding how the damages are bad, Selvam said. And long term, understanding how mechanisms can, of course, lead to improving treatment.

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Researchers redefine gene involved with DNA repair The Daily Evergreen - The Daily Evergreen

Can we recreate dinosaurs from their DNA? A paleontologist answers the question. Image via Deb Dowd/ Unsplash.

By William Ausich, Ohio State University

Would it really be possible to get the DNA of dinosaurs and then recreate them? Lucie R., age 5, Atlanta, Georgia

As a paleontologist thats a scientist who studies ancient life Im asked this question all the time. After all, the scientists in Jurassic Park (and later, Jurassic World) used DNA to recreate dozens of dinosaurs: Triceratops, Velociraptor and T. rex. And if you saw any of those movies, you had to wonder: Could real scientists do that today?

DNA which stands for deoxyribonucleic acid is something in every cell of every organism that ever lived on Earth including dinosaurs. Think of DNA as molecules that carry the genetic code, a set of instructions that helps bodies and minds grow and thrive.

Your DNA is different from everyone elses. It determines many of the characteristics that define you, like the color of your eyes or whether your hair is straight or curly.

DNA is much easier to find in the soft parts of an animal their organs, blood vessels, nerves, muscle and fat. But a dinosaurs soft parts are long gone. They either decomposed or were eaten by another dinosaur.

Dinosaur fossils are all thats left of those prehistoric animals. Immersed for tens of millions of years in ancient mud, minerals and water, the fossils come from the dinosaurs so-called hard parts its bones, teeth and skull.

We find dinosaur fossils in the ground, in riverbeds and lakes, and on the sides of cliffs and mountains. Every now and then, someone finds one in their backyard. Often, theyre quite near the surface, and usually, theyre embedded in sedimentary rock.

With enough fossils, scientists can build a dinosaur skeleton: what you see when you go to the museum.

But scientists have a big problem when trying to find DNA in dinosaur fossils. DNA molecules eventually decay. Recent studies show DNA deteriorates and ultimately disintegrates after about 7 million years. That sounds like a long time, but the last dinosaur died at the end of the Cretaceous Period. Thats more than 65 million years ago.

Dig up a fossil today, and any dino-DNA within would have long since fallen apart. That means, as far as scientists know, and even using the best technology available today, its not possible to make a dinosaur from its DNA.

Although its too late to find dino-DNA, scientists recently found something almost as intriguing. They discovered DNA fragments in the fossils of Neanderthals and other ancient mammals, such as woolly mammoths.

Now that makes sense. Those fragments are less than 2 million years old, well before all of the DNA would decay.

Just for fun, lets imagine that somehow, sometime in the future, researchers came up with fragments of dinosaur DNA. With only fragments, scientists still could not make a complete dinosaur. Instead, they would have to combine the fragments with the DNA of a modern-day animal to create a living organism.

That creature, however, could not be called an actual dinosaur. Rather, it would be a hybrid, a blend of dinosaur and, most likely, a bird or reptile. Think thats a good idea? After all, the scientists in the Jurassic movies tried that. And you know what happened there.

William Ausich, Professor Emeritus of Paleontology, Ohio State University

This article is republished from The Conversation under a Creative Commons license. Read the original article.

Bottom line: We cant recreate dinosaurs from their DNA because the DNA no longer exists. DNA disintegrates in about 7 million years, and dinosaurs lived 65 million years ago.

Members of the EarthSky community - including scientists, as well as science and nature writers from across the globe - weigh in on what's important to them. Photo by Robert Spurlock.

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Can we recreate dinosaurs from their DNA? - EarthSky

ABOVE: Artist rendering of the male found deceased on Aug. 1, 1978 at Poole Knobs Recreation area in La Vergne.

Relatives of missing man James Sanders waited 43 years before learning last week their brothers remains were found in 1981 in Immokalee, Fla., based on DNA samples submitted by Rutherford County Sheriffs detectives.

32-Year-old Sandersof Portland, Tenn., was lastseen Jan. 1, 1978 at a bus stop in Tennessee en route to North Carolina where he planned to work at an aunts tobacco farm. Sanders never arrived at his aunts farm and was never heard from again.

Brother Eddie Sanders contacted Rutherford County Sheriffs Detective Sgt. Dan Goodwin and Steve Kohler in 2014, who were trying to find the identity of a man whose burned body was found Aug. 1, 1978 at Poole Knobs Recreation area in La Vergne.

Sanders wondered if the man found in La Vergne might be his brother. He, his sister and his brothers children gave DNA samples to the detectives who submitted the samples in January 2015 to the North Texas University DNA lab for testing.

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Lab employees notified the detectives six months later the man found in La Vergne was not Sanders (above is a photo of James Sanders as he appeared prior to the last time he was seenon Jan. 1, 1978).

The DNA samples submitted by the Sanders family remained at the lab and now those samples have a match.

Sheriff Kevin Rambosk of Collier County, Florida, announced Friday the lab matched bone from the body found in Immokalee, Florida to Sanders DNA submitted by Rutherford County detectives.

This important development was made possible thanks to DNA technology and the dedication of everyone involved in this investigation, said Rambosk.

Without the combined efforts of multiple agencies, Mr. Sanders would still be unidentified after 40 years, Rambosk said. Now that our victim has a name, the homicide investigation can continue to be actively worked.

The case for the identity of the missing man found in La Vergne is being investigated by Detective Richard Brinkley of the Rutherford County Sheriffs Office.

Brinkley is still trying to identify the suspects who killed the unidentified man while focusing on the identity of the victim.

An autopsy report described the unidentified man as being in his late 30s who was 5-feet-10 tall. He had long, brown hair with a receding hairline and a reddish brown and gray beard. He had a scar on his stomach, no teeth but an upper denture and a quarter-sized mole near his waistline.

Detectives worked with Dr. Lee Meadows Jantz from the state Forensic Anthropology Center who said the mans DNA was submitted to the FBIs Combined DNA Index System to search for a match. No results have been found.

A profile of the unidentified man was added to the National Missing and Unidentified Persons System.

People may view the mans profile athttps://www.namus.gov/UnidentifiedPersons/Case#/1585?nav.

The sheriffs office is now partnering with Othram, a private DNA lab who recovers human DNA to solve murder cases with hopes for an identification. The team is actively working on matches.

Were pursuing the same thing that solved the Florida case, DNA evidence, Brinkley said.

People who may have information on the man may contact Brinkley atrbrinkley@rcsotn.orgor at 615-904-3045.

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DNA from Rutherford County Leads to Identification of Body Found in Florida 40-Years-Ago - Wgnsradio

Significance

During the aging process, senescent cells secrete inflammatory factors, causing various age-related pathologies. Thus, controlling the senescence-associated secretory phenotype (SASP) can tremendously benefit human health. Although SASP seems to be induced by the alteration of chromosomal organization, its underlying mechanism remains unclear. Here, it has been revealed that noncoding RNA (ncRNA) transcribed from pericentromeric repetitive elements impairs the DNA binding of CCCTC-binding factor, resulting in the alteration of chromosomal accessibility and the activation of SASP-like inflammatory genes. Notably, the ncRNA was transferred into surrounding cells via small extracellular vesicles, acting as a tumorigenic SASP factor. Our study highlights a novel mechanism regulating chromatin interaction and inflammatory gene expression in senescence and cancer.

Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and noncell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.

Cellular senescence is a state of essentially irreversible cell cycle arrest induced by several stressors under various physiological and pathological conditions (1). Senescent cells that accumulate in vivo over the course of aging communicate with surrounding tissues through the production of proinflammatory proteins, termed the senescence-associated secretory phenotype (SASP), which are thought to promote multiple age-related diseases, including some cancers, such as breast and colon cancer (27). Therefore, elucidating the regulatory mechanism of the SASP is essential for developing new preventive and therapeutic strategies against age-related cancer.

Recent studies have reported that abnormal nuclear morphologies, observed as micronuclei or nuclear buds, induce SASP gene expression via the activation of the DNA-sensing pathway during cellular senescence (812). In addition, cellular senescence causes a dramatic alteration of chromatin organization, characterized by an increase in short-range chromatin contacts and genome-wide shrinkage of chromosome arms (13). Chromatin organization and global gene expression are coordinately maintained by the CCCTC-binding factor (CTCF), a zinc-finger (ZF) nucleic acidbinding protein, and the cohesin complex; together, these factors orchestrate higher-order chromatin conformation through the formation of intrachromosomal and interchromosomal loops (1416). Given that the nuclear localization and RNA-binding capacity of CTCF dynamically change due to cellular stress (17) and the alteration of CTCF distribution and/or followed by chromatin reorganization occur during cellular senescence (13, 18), postulating that CTCF distribution is associated with SASP gene expression in senescent cells is reasonable. However, the molecular mechanism underlying the connection between CTCF regulation and its association with SASP gene expression remains elusive.

In this study, we demonstrate that noncoding RNA (ncRNA) transcribed from pericentromeric repetitive satellite sequences changes the distribution of CTCF binding on the genome, thereby inducing SASP-like inflammatory gene expression via the functional impairment of CTCF in senescent cells. Furthermore, pericentromeric satellite RNA provokes tumorigenesis in a cell-autonomous or noncell-autonomous manner via a pathway involving exosomes, a type of small extracellular vesicle (EV). Our findings reveal a mechanism of CTCF regulation by pericentromeric satellite RNA during cellular senescence, which may contribute to the risk of tumorigenesis.

To elucidate the molecular mechanism underlying the alteration of chromatin organization and gene expression during cellular senescence, we first compared genome-wide chromatin accessibility between X-rayinduced senescent and proliferating IMR-90 cells, which are normal human diploid fibroblasts. Assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis revealed that the peak intensities in 16,325 regions were dramatically altered (false discovery rate [FDR] < 0.05) during cellular senescence (Fig. 1 A and B), and a high incidence of distal intergenic regions and introns was identified (SI Appendix, Fig. S1A). From ATAC-seq analysis, 14,356 peaks were identified as higher chromatin accessibility (red) in X-rayinduced senescent cells versus proliferating IMR-90 cells; 1,969 peaks were identified as lower chromatin accessibility (blue; Fig. 1B). The 16,325 ATAC-seq peaks that showed differential chromatin accessibility values in X-rayinduced senescent cells compared to those in proliferating cells (Fig. 1B) were annotated to 652 transcripts using databases, including GRCh37/hg19 (coding genes and some noncoding regions) and RepeatMasker (repetitive elements). Next, we focused on these 652 transcripts and reanalyzed their expression level using published RNA-sequencing (RNA-seq) data of proliferating and X-rayinduced senescent IMR-90 cells (GSE130727; Fig. 1C) (19). Thus, loci containing pericentromeric repetitive sequences called human satellite II (hSATII), which are epigenetically silenced in normal somatic cells, were highly accessible (yellow; Fig. 1B), and hSATII ncRNA expression was markedly up-regulated in X-rayinduced senescent IMR-90 cells compared to proliferating cells (log10 fold change = 2.8) among the transcripts showing an FDR < 1010 (Fig. 1C). When we integrated our ATAC-seq data with published RNA-seq data of proliferating and X-rayinduced senescent IMR-90 cells (GSE130727) (19), a comparative analysis of senescent and proliferating cells represented both higher chromatin accessibility and increased transcription at hSATII loci in senescent cells versus proliferating cells (Fig. 1D). In accordance with previous studies of senescent cells and many types of cancer (2023), we detected hSATII RNA expression by RT-qPCR and Northern blot analysis in H-RasV12 and serial passageinduced senescent cells (SI Appendix, Fig. S1 BF). To interpret the biological effects of hSATII RNA expression, we overexpressed hSATII RNA in SVts8 cells, a conditionally immortalized human fibroblast cell line suitable for transfection analysis (24). The ectopic expression of hSATII RNA, but not centromeric human satellite alpha RNA (hSAT), induced SASP-like inflammatory gene expression, which was shown as the enrichment of signatures related to the inflammatory response and SASP by gene set enrichment analysis and altered the chromatin accessibility of the loci of SASP genes (Fig. 1 EG and SI Appendix, Fig. S2 AD) (25). Importantly, the knockdown of hSATII RNA diminished the expression of SASP genes in X-rayinduced senescent SVts8 cells (Fig. 1H) or X-ray and serial passageinduced senescent IMR-90 cells (SI Appendix, Fig. S2 E and F). These data suggest that hSATII RNA regulates SASP-like inflammatory gene expression by altering chromatin accessibility during cellular senescence.

Pericentromeric hSATII RNA regulates SASP factor gene expression during cellular senescence. (AC) Screening of unique transcripts showing increased chromatin accessibility and active transcription during X-rayinduced senescence in IMR-90 cells. (A) A scheme of the screening steps. (B) Volcano plot of ATAC-seq signals showing fold change (FC) (x-axis) and FDR (y-axis) of chromatin accessibility between proliferating and X-rayinduced senescent IMR-90 cells. Red peaks show significantly increased chromatin accessibility in X-rayinduced senescent cells. Blue peaks showing significantly increased chromatin accessibility in proliferating cells. Black peaks show no significant changes. Yellow peaks containing hSATII loci show significantly increased chromatin accessibility. (C) Volcano plot of RNA-seq data (GSE130727) showing FC (x-axis) and FDR (y-axis) concerning 652 transcripts involved in an increased chromatin accessibility region between proliferating and X-rayinduced senescent IMR-90 cells from ATAC-seq analysis in B. The 47 transcripts showing FDR < 1010 are shown as red (up-regulated) or blue (down-regulated) dots. (D) Peaks of uniquely mapped reads by ATAC-seq and RNA-seq (GSE130727) in hSATII loci in proliferating or X-rayinduced senescent IMR-90 cells. Two biological replicates are shown. (EG) RNA-seq analysis of hSAT or hSATII-overexpressed and X-rayinduced senescent SVts8 cells. (E) Heatmap regarding SASP-related gene expression in hSAT or hSATII-overexpressed and X-rayinduced senescent SVts8 cells. (F) Scatterplot showing FC in hSAT (x-axis) or hSATII (y-axis) RNA-overexpressed SVts8 cells compared to empty vectorexpressed cells. Red dots indicate genes up-regulated (FC > 10) in vicinity of specific chromatin accessible peaks in hSATII RNAoverexpressed cells. (G) Gene set enrichment analysis of signatures associated with senescence (Upper) and inflammatory response (Lower) in hSAT or hSATII RNAoverexpressed SVts8 cells. NES, normalized enrichment score. (H) The effect of hSATII RNA knockdown on hSATII RNA and SASP gene expression in proliferating or X-rayinduced senescent SVts8 cells by RT-qPCR. The relative expression is shown as the FC from control small-interfering RNAtreated proliferating cells. Each bar represents mean SD of three biological replicates. ***P < 0.001 by one-way ANOVA, followed by the Tukeys multiple comparisons post hoc test.

To understand how hSATII RNA promotes SASP-like inflammatory gene expression, we attempted to identify hSATII RNAbinding proteins. Several studies have reported the association of centromeric hSAT RNA with specific proteins (26); however, thus far, none have reported such an association for pericentromeric hSATII RNA. RNA pull-down and mass spectrometry analysis identified 280 hSATII RNAbinding proteins (Fig. 2A and SI Appendix, Fig. S3A and Table S1). Among these proteins, we identified 26 chromatin-binding proteins by Gene Ontology (GO) analysis (GO: 0003682) and focused on CTCF because of both its high intensity score (unique peptides) and its relevance to chromatin organization (15, 16, 27) (Fig. 2A). Unlike hSAT RNA, hSATII RNA bound to CTCF, whereas both ncRNAs bound to lamin B1 (Fig. 2B) (26). Because CTCF binding to genomic DNA is important for the maintenance of genomic integrity and CTCF is an RNA-binding protein (2831), we performed RNA immunoprecipitation (RIP) analysis, demonstrating that the ZF DNA- and RNA-binding domains of CTCF are important for their binding to not only an exogenous hSATII RNA in human embryonic kidney (HEK)-293T cells (Fig. 2 C and D and SI Appendix, Fig. S3 B and C) but also an endogenous hSATII RNA in X-rayinduced senescent IMR-90 cells (SI Appendix, Fig. S3D). Of the 11 ZF domains of CTCF, the binding of ZF1 or ZF10 of CTCF to RNA is important for CTCF to form chromatin loops and regulate gene expression (31); however, we found that hSATII RNA bound to neither ZF1 nor ZF10 (SI Appendix, Fig. S3 B and C). Further analysis revealed that ZF3-ZF6 of CTCF-, known as DNA-binding domain (32), deficient mutant (CTCF ZF3-6) could not bind to hSATII RNA (Fig. 2 C and D), indicating that ZF3-ZF6 of CTCF is important for binding to hSATII RNA. Note that CTCF also binds to RNA through ZF domains; therefore, ZF3-6 might be an unfolding protein and not function appropriately. Further analysis will be needed to determine the interaction between CTCF and hSATII RNA. Importantly, the up-regulation of SASP-like inflammatory gene expression caused by hSATII RNA was canceled in the presence of excessive CTCF in SVts8 cells (Fig. 2E and SI Appendix, Fig. S3E). In contrast, CTCF depletion by RNA interference resulted in SASP-like inflammatory gene expression in proliferating cells (Fig. 2F and SI Appendix, Fig. S3F). Based on these results, we considered it likely that SASP-like inflammatory gene expression induced by hSATII RNA depends on the functional impairment of CTCF. Unexpectedly, we found that hSATII RNA expression was also up-regulated by CTCF depletion (Fig. 2F). Moreover, CTCF expression decreased during cellular senescence (SI Appendix, Fig. S3 G and H) (33). Together, these findings imply that CTCF regulates hSATII RNA expression during cellular senescence.

Pericentromeric hSATII RNA binds to CTCF. (A) GO analysis of 280 hSATII RNAbinding proteins (Left). Among these proteins, 26 genes were categorized as chromatin-binding (GO: 0003682), and the top 10 ranked genes and unique peptides are listed (Right). (B) RNA pull-down assay using SVts8 cell lysate followed by Western blotting confirmed hSATII RNA but not hSAT RNA bound to CTCF. (C) Western blot analysis of FLAG-tagged CTCF (WT: wild type) or CTCF ZF (deletion of ZF domain) in HEK-293T cells. (D) RIP followed by qPCR confirmed the binding of FLAG-tagged CTCF WT, but not CTCF ZF1-11 or ZF3-6, to hSATII RNA in HEK-293T cells. (E) RT-qPCR analysis of SASP-like inflammatory genes in hSATII RNAoverexpressed SVts8 cells with excess CTCF. The relative expression shows the value normalized from empty vectorexpressed cells. (F) RT-qPCR analysis of SASP-like inflammatory genes in CTCF-depleted SVts8 cells. The relative expression shows the value normalized from small-interfering control (siControl)treated cells. Each bar represents mean SD of three technical replicates repeated in two independent experiments (D, E, and F). *P < 0.05, **P < 0.01, ***P < 0.001, or N.S. (not significant) by one-way ANOVA, followed by the Tukeys (D and E) or Dunnetts (F) multiple comparisons post hoc test.

Furthermore, we investigated the expression of mouse major satellite (MajSAT) RNA, which is located at the pericentromeric locus of chromosomes, as well as human hSATII RNA. In mouse embryonic fibroblasts (MEFs), DNA damage induced by doxorubicin increased MajSAT RNA expression along with some canonical markers of cellular senescence (SI Appendix, Fig. S3I). As expected, the induction of MajSAT RNA was negatively correlated with the expression of CTCF (SI Appendix, Fig. S3I), and CTCF bound to pericentromeric MajSAT RNA but not mouse centromeric minor satellite (MinSAT) RNA, resulting in the up-regulation of SASP-like inflammatory genes (SI Appendix, Fig. S3 J and K). Taken together, these findings indicate that CTCF is crucial for the regulation of both pericentromeric satellite RNA and the expression of SASP-like inflammatory gene during cellular senescence (SI Appendix, Fig. S3M).

Because the ZF3-ZF6 DNA binding domain of CTCF was relevant to its binding to hSATII RNA (Fig. 2D), we hypothesized that hSATII RNA changes the DNA-binding capacity of CTCF via direct binding to its ZF domains. As expected, the ectopic expression of hSATII RNA altered the distribution of CTCF at its binding sites (Fig. 3 A and B). Remarkably, both chromatin immunoprecipitation (ChIP)-qPCR and electrophoretic mobility shift assay (EMSA) revealed that hSATII RNA inhibited the DNA-binding capacity of CTCF to an imprinting control region (ICR) positioned between IGF2 and H19, a well-known representative CTCF binding site, in a dose-dependent manner (Fig. 3 C and D) (15). In accordance with this data, pericentromeric MajSAT RNA bound to CTCF and diminished the binding of CTCF to ICR (SI Appendix, Fig. S3L). These notions raised the possibility that pericentromeric satellite RNA could change chromatin interaction as the binding of CTCF to DNA is important to maintain genomic integrity. To validate this assumption, we performed a chromosome conformation capture (3C) assay of the SASP genes in the vicinity of the CXCL10/CXCL11 locus because a robust interaction was noted in proliferating fibroblasts and various cell lines (SI Appendix, Fig. S4 AF) (34). We discovered that the ectopic expression of hSATII RNA significantly weakened interactions in the T2 and T22 regions, as revealed by 3C-qPCR assay, and increased chromatin accessibility within these loci, as revealed by ATAC-seq analysis, followed by the up-regulation of SASP-like inflammatory gene expression, as also observed in X-rayinduced senescent cells (Fig. 3 E and F). Together, these data indicate that the up-regulation of hSATII RNA in senescent cells causes a conformational change of chromatin structure in some SASP gene loci. Chromatin organization and global gene expression are coordinately regulated by CTCF during a variety of physiological and pathological events, such as embryonic development and carcinogenesis (27). However, the molecular mechanism underlying the connection between CTCF regulation and its association with SASP gene expression during cellular senescence has not been elucidated. Our findings demonstrated that pericentromeric satellite RNA influences chromatin interaction by interfering with CTCF function, resulting in changes in SASP-like inflammatory gene expression (SI Appendix, Fig. S4G).

Pericentromeric hSATII RNA changes chromosomal interaction via CTCF disturbance. (A and B) Venn diagram showing overlap of CTCF binding sites from ChIP-seq analysis. (B) Enrichment of peaks from ChIP-seq analysis whose signals on peak summit 2 kb region are shown as profile plot (Left) and heatmaps split into two clusters using the k-means algorithm (Right) over sets of genomic regions in SVts8 cells. Wilcoxon rank-sum test P values are shown. (C) ChIP-qPCR for CTCF binding to an ICR positioned between IGF2 and H19. (D) EMSA showing the effect of hSAT or hSATII RNA on CTCF binding to ICR. (E and F) RNA-seq, CTCF ChIP-seq, and ATAC-seq profiles of SVts8 cells in representative loci of the SASP factor genes, CXCL10 and CXCL11 (E), and chromatin conformation by 3C-qPCR assay (F). The interaction of a constant primer (C) with each target primer (T) is shown. Each bar represents mean SD of three technical replicates repeated in two independent experiments (C and F). *P < 0.05, ***P < 0.001, or N.S. (not significant) by one-way ANOVA, followed by the Tukeys multiple comparisons post hoc test (C) or unpaired two-sided t test (F).

Human and murine satellite RNAs have the potential to induce chromosomal instability (CIN), leading to tumorigenesis (26, 35, 36). Hence, we explored the possibility that the loss of CTCF could contribute to satellite RNAinduced CIN. In accordance with previous reports, we confirmed that the ectopic expression of hSATII RNA provoked multipolarity and chromosomal bridge formation (SI Appendix, Fig. S5 AC), which are typical characteristics of CIN (37). Furthermore, hSATII RNAoverexpressing cells exhibited obvious phenotypes of tumor cells, such as an abnormal chromosomal number and anchorage-independent growth (SI Appendix, Fig. S5 D and E). Notably, we found that excessive CTCF expression abolished CIN induced by hSATII RNA (SI Appendix, Fig. S5 F and G), a finding suggesting that CTCF plays a role in hSATII RNAinduced CIN, which is a risk factor for tumor development. Similarly, the ectopic expression of MajSAT RNA in MEFs also provoked multipolarity and chromosomal bridge formation in mitosis (SI Appendix, Fig. S6 AC), causing the formation of transformed foci and polyploidy transition (SI Appendix, Fig. S6 D and E). Surprisingly, these cells exhibited the ability to form tumors in immunodeficient mice, although control cells did not (SI Appendix, Fig. S6F). Collectively, we concluded that pericentromeric satellite RNA may promote susceptibility to carcinogenesis.

Furthermore, to gain insight into the biological significance of our findings, we focused on the function of hSATII RNA in the tumor microenvironment. We and others recently reported that small EVs secreted from cancer cells and/or senescent stromal cells dynamically contribute to tumor incidence and progression in a noncell-autonomous manner in the tumor microenvironment (3841). Intriguingly, the amounts of hSATII RNA, but not those of hSAT, were higher in small EVs derived from senescent cells than in those derived from proliferating cells (Fig. 4A). From an analysis of recently published RNA-seq data (42), we discovered that hSATII RNA could be detected in exosomes, a type of EV, secreted from cells of different human colon cancer cell lines (SI Appendix, Fig. S7A). Based on these observations, we speculated that hSATII RNA derived from senescent stromal cells would be transferred into surrounding cells through small EVs and function as a SASP-like inflammatory factor. Supporting this hypothesis, small EVs derived from senescent cells promoted anchorage-independent growth and CIN in normal cells (SI Appendix, Fig. S7 B and C). To assess the involvement of hSATII RNA in these phenotypes, we used EXOsomal transfer into cells (EXOtic) synthetic biology (43) to establish a designer exosome that contains hSATII RNA and found that these designer exosomes have tumorigenic activity similar to those of small EVs derived from senescent cells (SI Appendix, Fig. S7 DF). Importantly, these designer exosomes promoted SASP-like inflammatory gene expression (Fig. 4B). Together, these findings show that pericentromeric hSATII RNA in small EVs secreted from senescent cells promote SASP-like inflammatory gene expression and CIN in neighboring cells in the tumor microenvironment.

Pericentromeric hSATII RNA promotes tumor development in a cell-autonomous and noncell-autonomous manner. (A) RT-qPCR analysis of hSATII and hSAT RNA in the same number of small EVs derived from RPE-1/hTERT cells. Each value represents three biological replicates. *P < 0.05 or ***P < 0.001 by one-way ANOVA followed by the Dunnetts multiple comparisons post hoc test. (B) An effect of the designer exosome produced by the EXOtic devices on SASP-like inflammatory gene expression in SVts8 cells was evaluated by RT-qPCR. Each value was normalized from EXOtic-Nluc-treated cells. (CE) Representative and magnified (100) images (C) or quantified data (D and E) of RNA-ISH with hSATII RNA probe in colon cancer specimens. Black and red arrows indicate normal epithelial and tumor cells, respectively. Black and red arrowheads indicate fibroblasts and cancer-associated fibroblasts, respectively. (Scale bar, 200 m.) In the boxplot, the bottom and top hinges indicate the first and third quartile, respectively. The horizontal lines into the boxes indicate the median. The upper and lower whiskers define the highest and lowest value within 1.5 times of the interquartile range, respectively. n = 20 for each sample. **P < 0.01 or ***P < 0.001 by the Wilcoxon rank-sum test. Statistical analysis was performed using all samples and included outliers. (F) Schematic representation of this study. The up-regulation of pericentromeric satellite RNA during cellular senescence or aging provokes the expression of aberrant SASP-like inflammatory gene by interfering with the function of CTCF. In the tumor microenvironment, inflammatory proteins and hSATII RNA in small EVs are secreted from senescent stromal cells into surrounding tissue, where they act as SASP factors, thereby increasing the risk of carcinogenesis.

Finally, we checked the expression of pericentromeric ncRNA in the tumor microenvironment. Because the expression of murine satellite RNA is higher in malignant organoids derived from colon cancer (Apc716 Trp53R270H/R270H) than in nonmalignant organoids (Apc716) and accompanied by the reduction of CTCF and the up-regulation of SASP-like inflammatory genes (44) (SI Appendix, Fig. S7G), we evaluated the expression of hSATII RNA in surgical resection specimens from patients with primary colon carcinoma. We found that colon cancer cells expressing hSATII RNA were highly abundant in specimens compared with normal epithelial cells by RNA in situ hybridization (RNA-ISH) analysis (Fig. 4 C and D). Strikingly, we also observed that the population of hSATII RNApositive cells was significantly higher among cancer-associated fibroblasts than among fibroblasts in normal stromal tissues (Fig. 4 C and E). In summary, our findings suggest that senescent stromal cells expressing hSATII RNA support tumor development in a noncell-autonomous manner in the tumor microenvironment via the secretion of SASP-like inflammatory factors and small EVs containing hSATII RNA (Fig. 4F).

Cellular senescence causes a dramatic alteration of chromatin organization (13, 18); however, its effect on gene expression and implication for senescent cells are not fully understood. We identified ncRNA derived from pericentromeric repetitive elements as a novel inducer of SASP-like inflammatory gene expression that acts by altering chromatin interaction. Importantly, pericentromeric satellite RNA is up-regulated during cellular senescence and aging in vivo (Fig. 1 AD and SI Appendix, Fig. S1 BF), which decreases CTCF binding to genomic DNA and alters both chromatin interaction and transcription in SASP-like inflammatory gene loci, thereby increasing the risk of tumor development (Fig. 4F). To verify the physiological role of pericentromeric satellite RNA, we confirmed that the expression level of ectopic ncRNA was equivalent to that found in senescent cells. Furthermore, the knockdown of endogenous pericentromeric satellite RNA diminished the expression of SASP-like inflammatory genes in senescent cells despite having no effect on the induction of senescence, clearly indicating that endogenous pericentromeric satellite RNA plays a role in the expression of SASP-like inflammatory genes.

CTCF and the cohesin complex are essential for stabilizing chromatin organization, which has divergent effects on gene regulation, embryonic development, and tumorigenesis (27), through their binding ability to specific sequences in genomic DNA (15, 16, 45). Recently, some groups have reported that CTCF shows a high affinity for specific RNA and depends on binding with RNA to form chromatin loops and genome organization in mouse embryonic stem cells (30, 31). Another group also has shown that the interaction of CTCF with long ncRNA, such as Tsix and Xite, mediates long-range chromosomal interactions, inducing homologous X chromosome pairing in mouse embryonic stem cells (29). In contrast, in senescent cells (in pathological conditions), we have revealed a novel molecular mechanism in which pericentromeric satellite RNA regulates chromatin interaction and gene expression by CTCF disturbance. Because pericentromeric satellite RNA expression is at an extremely low level in normal cells (SI Appendix, Fig. S1 BF) (20), we considered that it is insufficient for the RNA to disturb CTCF function in physiological conditions but not in senescent and tumor cells that aberrantly express pericentromeric satellite RNA. In the previous study, Zirkel et al. revealed that, upon senescence entry, the high-mobility group B protein (HMGB2) nuclear depletion provokes the alteration of CTCF distribution and CTCF spatial clustering (18). Moreover, Lehman et al. showed that stressors, such as acute oxidative stress, cause CTCF reduction from nuclear speckles and changes in CTCF RNA interaction (17). These reports support our findings that pericentromeric satellite RNA up-regulated during cellular senescence directly binds to CTCF and disturbs CTCF function.

Furthermore, our observations also showed that CTCF maintains pericentromeric satellite RNA expression at extremely low levels in normal cells by directly binding the pericentromeric hSATII locus (46); however, CTCF expression significantly decreased during cellular senescence (SI Appendix, Fig. S3 G and H). Therefore, once pericentromeric satellite RNA is predominantly expressed by CTCF depression, satellite RNA alters chromatin interaction and induces CIN and SASP-like inflammatory gene expression via CTCF disturbance (SI Appendix, Fig. S3M). Previous studies have revealed that CTCF binds to pericentromeric/centromeric regions and recruits the centromeric protein CENP-E to these regions in mitotic chromosomes (46, 47). In addition to these findings, we have identified a regulatory machinery involving CTCF that controls pericentromeric satellite RNA expression.

In patients with gastrointestinal cancer, driver mutations were detected in the ZF domains of CTCF that provoke CIN and aberrant gene expression (48). Although CTCF-knockout mouse embryos die at early implantation stages (49), CTCF haploinsufficient (Ctcf+/) mice are markedly susceptible to cancer, and transformed foci are observed in Ctcf+/ mousederived MEFs (35). These reports strongly support our conclusion that the disturbance of CTCF function caused by pericentromeric satellite RNA results in aberrant gene expression, CIN, and tumorigenesis. Centromeric satellite RNA (hSAT and MinSAT RNA) is associated with CIN in some cell lines (26), but we believe that there must be another mechanism, as these centromeric satellite RNAs neither bind to CTCF nor contribute to SASP-like inflammatory gene expression in normal fibroblasts (Figs. 1 E and F and 2B and SI Appendix, Figs. S2 A and B and S3K).

We conclude that pericentromeric satellite RNA plays a prominent role in tumorigenesis by cell-autonomous and noncell-autonomous pathways in vivo: 1) DNA damage caused by various oncogenic stresses induces cellular senescence of normal epithelial cells, thereby acting as a tumor-suppressor mechanism. In these senescent cells in benign tumors, pericentromeric satellite RNA is up-regulated and leads to SASP-like inflammatory induction (1, 2, 4, 50). If these senescent cells override their cell cycle arrest, pericentromeric satellite RNA may contribute to transformation from a benign to a malignant tumor through CIN (51). 2) Pericentromeric satellite RNA could be transferred into cancer cells via small EVs from senescent stromal cells, provoking CIN and SASP-like inflammatory gene expression, resulting in tumor progression. Our data suggest that secreted pericentromeric satellite RNA also functions as a tumorigenic SASP factor via small EVs in the tumor microenvironment. Furthermore, the down-regulation of CTCF expression with age may trigger the up-regulation of pericentromeric satellite RNA expression and diminish CTCF function via a positive-feedback loop, subsequently promoting SASP-related inflammation and tumorigenesis during aging (Fig. 4F). Our findings clearly indicate that pericentromeric satellite RNA represents a therapeutic target for age-related pathologies.

A full description of the following methods is described in the SI Appendix, Supplementary Information Methods: cell culture, plasmid construction, RNA interference, RT-PCR, RT-qPCR, Northern blot, RNA pull-down assay, Western blotting, mass spectrometric analysis, ChIP followed by ChIP sequencing (ChIP-seq), 3C-qPCR, ATAC-seq, RIP, EMSA, immunofluorescence imaging, karyotype analysis, focus formation assay, anchorage-independent soft agar colony formation assay, RNA-seq, extraction and application of exosome-like EVs, RNA-ISH, organoid culture experiments, in vivo allograft assays, bioinformatical analysis, and statistical analysis.

TIG-3 cells (11, 38, 52) and IMR-90 cells were obtained from the Japanese Cancer Research Resources Bank and American Type Culture Collection, respectively. TIG-3 cells, IMR-90, and IMR-90/ER:H-RasV12 cells (52) were cultured in Dulbeccos Modified Eagles (DME) medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Sigma-Aldrich) at physiological oxygen conditions (92% N2, 5% CO2, and 3% O2) at 37 C. RPE-1/hTERT cells (39) and HEK-293T cells (52) were cultured in DME medium (Nacalai Tesque) supplemented with 10% FBS and penicillin/streptomycin (Sigma-Aldrich) in a 5% CO2 incubator at 37 C. SVts8 cells (24) were cultured in DME medium (Nacalai Tesque) supplemented with 10% FBS and penicillin/streptomycin (Sigma-Aldrich) in a 5% CO2 incubator at 34 C. MEFs were generated from CD-1 mice as previously described (53) and then cultured in DME medium (Nacalai Tesque) supplemented with 10% FBS and penicillin/streptomycin (Sigma-Aldrich) at physiological oxygen conditions (92% N2, 5% CO2, and 3% O2) at 37 C. All cell lines used were negative for mycoplasma.

Cell pellets were lysed in lysis buffer (0.1 M TrisHCl pH 7.5, 10% glycerol, and 1% sodium dodecyl sulfate [SDS]), boiled for 5 min, and then centrifuged for 10 min at 15,000 rpm. All protein concentrations were determined by BCA Protein Assay Reagent (Pierce). Each cell lysate was electrophoresed by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking with 5% skim milk (Megumilk) or 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membrane was treated with primary antibodies to p16 (IBL, #11104, 1:250 dilution), lamin B1 (Abcam, #ab16048, 1:1,000 dilution), GAPDH (Proteintech, #60004-1-lg, 1:10,000 dilution), vinculin (Sigma-Aldrich, #V9131, 1:1,000), CTCF (Cell Signaling Technology, #3418, 1:1,000 dilution), DDDK-tag (MBL, #M185-3L, 1:5,000), and ras (Oncogene, #OP41, 1:1,000 dilution) overnight at 4 C in blocking buffer. Membranes were then washed three times in TBST and incubated with an enhanced chemiluminescence (ECL) anti-mouse IgG, horseradish peroxidaselinked whole antibody (GE Healthcare, NA931V) or ECL anti-rabbit IgG, horseradish peroxidaselinked whole antibody (GE Healthcare, NA934V) for 1 h at room temperature. After washing the membrane three times in TBST, the signal was resolved with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and imaged on a FUSION imaging system (Vilber Lourmat).

hSATII RNA was detected on formalin-fixed paraffin-embedded (FFPE) sections in primary colon cancer specimens using an Advanced Cell Diagnostics (ACD) RNAscope 2.5 HD Reagent Kit-BROWN (ACD, #322300) and the RNAscope Target Probe Hs-HSATII (ACD, #504071) according to the manufacturers instructions. For each sample (n = 10), two images (100) of normal mucosa, submucosa, and tumor were randomly selected. The areas of hSATII RNA positivity and total cells were analyzed using the ImageJ software (https://imagej.nih.gov/ij/docs/faqs.html). The hSATII RNApositive area per field (percent) of each type of cell was calculated as the proportion of the total positive area to the total area of cells.

The FFPE sections in primary colon cancer specimens were collected from patients who provided informed consent for genetic and cell biological analyses. All methods were performed in accordance with protocols approved by the Institutional Review Board (approval number: 2013-1090) of the Japanese Foundation for Cancer Research (JFCR).

MEF/Vector or MEF/MajSAT RNA (5 106 cells) in Hanks Balanced Salt Solution (Gibco/Thermo Fisher Scientific) were subcutaneously injected with an equal volume of Matrigel (BD Pharmingen) into 4- or 5-wk-old female BALB/c-nu/nu mice (Charles River Laboratories). After 20 or 30 d of cell injection, the mice were euthanized and tumor weight was measured. All animal procedures were performed using protocols approved by the JFCR Animal Care and Use Committee in accordance with the relevant guidelines and regulations (approval number: 1804-05).

Parametric statistical analyses were performed using the unpaired two-tailed Students t test (Fig. 3F and SI Appendix, Figs. S1C, S3 G, I, J, and L, S5 BE, S6 BE, and S7 EG) or one-way ANOVA, followed by the Dunnetts (Figs. 2F and 4A and SI Appendix, Figs. S2A and S7C) or Tukeys (Figs. 1H, 2 D and E, and 3C and SI Appendix, Figs. S2 E and F, S3 C and D, and S5G) multiple comparisons post hoc test using the R software for statistical computing (64-bit version 3.6.1). Nonparametric statistical analyses were performed using the Wilcoxon rank-sum test (Figs. 3B and 4 D and E) or the KruskalWallis H test (one-way ANOVA on ranks) followed by the Steels multiple comparisons post hoc test (SI Appendix, Fig. S6F) using the R software for statistical computing. P < 0.05 was considered statistically significant. All experiments, except for mass spectrometric analysis, were repeated at least twice.

The sequence and processing data have been deposited in the DNA Data Bank of Japan with the accession numbers DRA009771 for RNA-seq, DRA010750 for ChIP-seq, and DRA010749 for ATAC-seq. All other data supporting the findings of this study are available within the article and SI Appendix.

We thank K. Nagasaka for valuable suggestions; T. Yamamoto and N. Saitoh for technical assistance for chromatin conformation analysis; K. Matsumoto for technical assistance for northern blot; S. Kuraku, C. Obuse, S. Adachi, and T. Natsume for mass spectrometry; N. Tanaka for bioinformatics analysis; G. Hannon and D. Beach for providing the MaRX vector; R. Asaka, K. Baba, and K. Takeuchi for technical advice for in situ experiments; H. Siomi for technical assistance for the RNAprotein interaction study; and members of the A.T. laboratory for helpful discussions during the preparation of this manuscript. This work was supported in part by the Japan Science and Technology Agency (JST)-Precursory Research for Embryonic Science and Technology (PRESTO) under grant number JPMJPR17H7; JST-Moonshot R and D under grant number JPMJPS2022; the Japan Agency of Medical Research and DevelopmentAdvanced Research and Development Programs for Medical Innovation (PRIME) under Grant 19gm6110023h0001; the Japan Society for the Promotion of Science (JSPS) under Grants 19H03507, 18K15254, and 20K16344; the Princess Takamatsu Cancer Research Fund; the Mitsubishi Foundation; and the Takeda Science Foundation. This research was also supported by the Research Fellowships for Young Scientists from JSPS under Grant 19J00796.

Author contributions: K.M. and A.T. designed research; K.M., Y.I., S.H., M. Nishio, T.M.L., R.O., R.F., L.J., H.Z., T.S., and A.T. performed research; L.Y., T.N., R.M., R.K., M. Nakayama, M.O., S.N., H. Seimiya, T.H., and H. Saya contributed new reagents/analytic tools; K.M., K.U., S.T., K.S., E.H., and A.T. analyzed data; and K.M. and A.T. wrote the paper.

The authors declare no competing interest.

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We found a handful of new Jurassic World items today. Our finds include a Mr. DNA reversible bucket hat, a Dinosaur Observation Committee crossbody bag, a Camp Cretaceous lunch bag, and a pin set. Lets dig in to the new merchandise.

Both sides of this bucket hat are bright and colorful.

The blue side has the Jurassic World logo, surrounded by DNA strands and Mr. DNA himself.

Universal Studios text and a mosquito are embroidered on the other side.

Reversing the hat reveals the yellow side.

The yellow side is all about Mr. DNA.

A Universal Studios tag is stitched to one side.

The Jurassic World logo hangs from the bag.

Two zippered pockets are on the opposite side.

A Dinosaur Observation Committee badge is attached in the corner. The artwork has been used on other Jurassic World merchandise.

Inside, we find a Jurassic World tag and an additional zippered pocket.

This lunch box is perfect for day trips to Camp Cretaceous.

We dont think anyone will steal our lunch with this dino protecting it. Three unique patches round out the design.

The opposite side has additional artwork and a Universal Studios text logo. A single mesh pouch is provided to hold a bottle.

The zipper pulls are dinosaur teeth, complete with the Jurassic World logo!

This Jurassic World pin set includes three separate pins.

The largest pin is a blue Jurassic World sign pin. It is surrounded by DNA.

The Mr. DNA pin is the most colorful in the set.

An atom rounds out the set. We found all four items in Jurassic Outfitters. The reversible hat and lunch bag are also available at Dinostore.

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Original post:
PHOTOS: New Jurassic World Pin Set, Crossbody, Lunch Bag, and Mr. DNA Reversible Bucket Hat Available at Universal's Islands of Adventure - wdwnt.com