HEADWATERS: FROM WHERE THE AGING CASCADE BEGINS

ETERNAL YOUTH 

HEADWATERS

From Where The Aging Cascade Begins

One of the greatest mysteries of biology is the source of sequential orchestration of genetic events in the DNA. For example when we are around 6 years old we begin to lose our milk teeth. What tells exactly at that age to do this? What decides the timing for puberty or menopause? Or just after puberty what tells our cells around that age to bring down the protein production support machinery's efficiency by 70% to begin aging? As stated in my earlier posts there are deliberate very harmful changes that occur in a timed manner to promote aging. If we can find out from where all these instructions come may be the detrimental ones could become a therapeutic or gene therapy target. Is it a single part of our brain that gives these instructions at a particular time/age? No. Apparently each of our 30 trillion cells has its own manager that releases time regulated instructions or temporal regulation. So it seems there is no single boss but 30 trillion managers each managing their own cell and coordinating with other managers. There is a postal system enabled by our blood circulation that scurries messages amongst all the managers probably to react to environmental stimuli or to even out the changes in gene expression. The messages in this postal system are carried in envelopes: EVs: extracellular vesicles and their cargo are the messages. In our body something keeps time and releases instructions for developmental changes in the first phase and aging related in the next phase. No one has been able to point out from where. Even the scientists who are computing clocks that measure changes in methylation in our genome, epigenome and rDNA also have not been able pin point the source of these changes. We will try to do some detective work but later in this essay, next we will share some of the mindblowing discoveries and their movies being made showing us for the first time how genes are turned on. It is important for our quest to understand this.

The brilliant scientist Ibrahim Cisse. Bryce Vickmark

Ibrahim Cisse is a great success story. From a background with few resources in Niger, Africa through education and merit he is today a biophysicist at MIT, USA. His tweak to single cell high resolution microscopy allowed him to take films of RNA transcription in action. This has changed the way we thought about how genes were activated. It seems there are lots of tiny floppy proteins that coalesce into phase change droplets at a target gene switch. They form a mesh in which many proteins would flit in and out sometimes within seconds. They would collectively turn on a gene. The length of their stay would determine how many proteins would get transcribed. Once the job is done these floppy proteins would disperse like a flash mob.

It takes a village of proteins to turn on genes. W.K. Cho Science 2018

It is indeed fascinating to see how the required proteins gather at the required address. Then on their own like magic condense into droplets that coalesce together to form a new village of fast moving droplets. In this temporary body other proteins flit in do their job and zip out sometimes within seconds. Where do these transcription proteins come from? What gives each of those proteins the instructions to all of these complex tasks which would seem to need some level of intelligence at such nano scale?! And voila the selected gene is turned on and begins transcribing a copy of itself. These required proteins do not have a Google Map (t) that home them in at the right address in the looong DNA coils. They bump around like manic blind mice till they bind into the lock made for them. It’s so tempting to believe that some central intelligence is guiding and sequencing these trillions of gene controls of activation or silencing or methylation or acetylation, etc. But such a complex central control tower would be unmanageable. So this becomes our clue as to source of all instructions. We will come back to it later. Next let us review our DNA. The proteins as one saw execute almost all our processes. There may be 90,000 types of proteins. Each one being born in their designated cells and doing their given tasks. Where do proteins come from? Our DNA but only 3.5% of our DNA is protein coding. Until recently scientists used to consider the balance 96.5% as junk DNA. But DNA formation is the culmination of hundreds of millions of years of adaptations and optimizations. There is no way so much DNA would be wasted from generation to generation. Recent discoveries have shown that the non coding 96.5% carries out some of the most important functions. Our body is a collection of cells. Each cell is same and born with identical DNA ‘brain’. Right from the time they differentiate into the 200 different type of cells giving our body distinct organs or structure. Till they die they follow instructions. The instructions come either from within its coding DNA or non coding DNA or from other cells. So just as I formed a conclusion about how aging is executed in our body I have also come to believe that the answer to the greatest mystery of biology is that which instructions will reach which cell and when is decided by the cells themselves as per Nature’s program carried in the non coding section of the DNA. Now I want you to imagine that there are 30 trillion offices around the world. Each office has a central computer with pre-installed software and a 3D printer (transcription/translation). Each office gets designated to a department (organs, teeth, bone, etc.) All the offices are connected by internet. Internet is the signalling system. This is a true democracy so no concept of Leaders. Each office is it’s own leader and co-exists and coordinates with other leaders. Now all the offices have a common instruction manual but the pre installed software controls which pages are visible/actionable and which pages can not be opened. The pre-installed software in each office activates tasks which result in the smooth operation of the entire company. Each one does it’s part and collectively the software orchestrates the functioning of a complex working enterprise. Our DNA is the preinstalled software in each cell (office). Only 3.5% of the software can assign printing jobs that result in posting of instructions/actionable items. Rest of the software is about making sure by individually executing orders it collectively runs our entire body and all its systems and functions. It’s like a live jigsaw puzzle of 30 trillion parts and each part has a chip that ensures it goes and locks on its own at its correct location in the puzzle. If all cells carry the same blue print how do cells differentiate into the 200 different types and how do they function in the role assigned and not some other role? It is done by control of gene expression. At any given time only a few genes are active in a cell approximately 3% to 5%. So how is cellular type, it’s function and it’s production of proteins which in turn control various processes in our body controlled or regulated? There are various mechanisms of gene regulation, structural as well as chemical. Like Chromatin accessibility, histone modifications including acetylation and deacetylation, ubiquitination, phosphorylation, etc., DNA methylation, demethylation, binding affinity alterations, repressors, during transcription, during transport of mRNA, stability of mRNA, during translation and postranslational, etc. As one can see the regulation of genes is highly complex but works in concert with regulation of genes in other cells to culminate into the object of such activity both in the cell and collectively in the body. We are built and we operate based on these controls. Overall there are  three basic types of changes that occur due to regulations of gene expression: developmental changes which build us from a single egg to an adult, stimuli adaptative changes and finally aging related changes (post developmental changes). As soon as developmental changes stop just a little after puberty there are deliberate negative or harmful changes that begin our multi decade process of aging. These changes are also highly conserved to ensure that all humans degrade to a point of death. But the question still remains: From where do the instructions come to execute exactly the control action (these changes) needed in each cell at exactly the time it is needed?

Now we have seen above what a fascinating process it is of activating a gene. The activation and repression of genes in each cells DNA culminates in our creation and operation. What scientists have not yet been able identify so far is from where do activation repression instructions come. Not only that the non stimulus related instructions come in a sequential manner. If it didn’t we would turn 20 then suddenly turn old then turn back into 7 year old, randomly. Another aspect of the mystery is how correct set of genes are switched on and off at exactly the correct location. If not eyes would appear on chest and finger could grow on skull. What is incredible is the low error rate over 200+ million years. The fidelity and precision of the system is mind boggling. As mentioned above controlling millions of complex processes in trillions of cells is impossible to achieve from a single source like a part of our brain. Its would be 100000 times more complex than managing all the flights in the world from a single air control tower. So we can infer that the origin of the instructions are also decentralized. In that case the only place in a cell that contains such data is our DNA. Now we have researched quite a bit on the coding part of our DNA. From that we can conclude that coding part needs upstream instructions to execute it’s control over gene expression and protein creation. From the little we have studied the 96.5% non coding part of the DNA we have noticed some interesting functionalities. The majority of the non coding part of the DNA (which does not print proteins) is highly conserved over 200 million + years. From the research papers that I read it hinted at this portion of DNA controlling the part of ‘which’ instruction will be executed ‘where’ and most importantly ‘When’. The when part ensures we move from a baby to an adolescent and later an elderly person in a sequential manner not suddenly becoming old then turning baby then turning middle age or any such random order. This clock that decides when to trigger which change and where lies hidden and protected in depths of our non coding DNA. There was an ongoing debate wherein one side said that almost all the non coding part of DNA is junk accumulated over the years and has no function. Edward Rubin's team at Lawrence Berkeley National Laboratory snipped out 3% of the non coding DNA in mice and did not find any abnormalities. On the other side Martin Sauvageau and colleagues at Harvard University and Broad Institute found that when they created a knockout mice model without 18 Long Non Coding RNAs (LncRNAs) it caused major growth defects including abnormalities in lungs, heart, gastrointestinal tract and neocortex. While a deletion in a small part of non coding DNA not showing any abnormalities does not prove that rest of the non coding DNA has no function. Whereas deleting a part of non coding RNAs causing fatal abnormalities does prove that non coding sections of DNA have critical functions. Hundreds of studies recently have uncovered more and more functions of the non coding elements. Data from ENCODE suggests that more than 75% of the human genome is transcribed into RNAs, whereas only 3% of these RNAs are from protein coding genes (Djebali et al., 2012; Ecker, 2012; Pennisi, 2012). The balance 72% transcribed RNAs will have functions most of which are yet to be discovered.

This is how we visualize DNA

But this is how it functions with layers of regulation

There are about 20,000 genes in the human genome, but as many as 1 million of regulatory elements in the non coding part of DNA.  If we compare the number of protein-encoding genes in worm and human, for example, humans don’t have that many more protein-coding genes than worms. The noncoding genome scales up much better with the developmental and pathological complexity of an organism. The fraction of protein-coding DNA in the genome decreases with increasing organismal complexity. In bacteria, about 90% of the genome codes for proteins. This number drops off to 68% in yeast, to 23-24% in nematodes and to 1.5-2% (or 3.5% as per some studies) in mammals. Using data from comparative genetic studies, the researchers found that the 300,000 functional elements they found made up about 70 per cent of the evolutionarily conserved non-coding DNA shared by mice and humans. The spatial organization is how regulatory elements know where to execute their tasks. How the regulatory elements contribute to activate a gene is not determined by a specific recognition tag, but by where precisely the gene is in the genome says scientist Francois Spitz. The winding and folding of the DNA around histones and nucleosomes which fold again to form a 30nm fiber which forms loops called Chromatin which again regulates transcription by remaining tightly condensed or open for allowing the village of phase change proteins to activate genes. All this folding is not stochastic but precise. The control of regulation occurs due to specific addresses/locations. Any misfolding can result in incorrect genetic actions. The non coding DNA controls our biological cycle by structural and chemical manipulations. The million non coding regulatory elements are interspersed with coding regulatory elements there are co-regulators and there are regulatory elements that control other regulatory elements which sometimes in turn control other regulatory elements. There are hundreds of different mechanisms of manipulating transcriptional activity acting individually or in clusters. Creating a complex web of intricately managed cellular and biological regulation. This complex management leads us to grow from an egg into an adult and from young to old. The non coding part of our DNA starts this complex regulatory cascade.
Some of the important elements of non coding DNA – this is not an exhaustive listing but to show the massive amount of regulation that originates from the non coding part of DNA and also to see how complex is its control over our biological life:

Transposons: I would like to quote from a very good essay by Francesca Tomasi and Olivia Rhoades: Nearly 46% of our DNA is made up of transposons! For millions of years, transposons enjoyed plenty of travel around our genome. They inserted themselves throughout our evolving DNA for as long as they could before these changes started to make the host human less suited for survival in a given environment. When their random insertion provided some sort of life advantage—increased ability to absorb certain nutrients, for instance—or took place with no negative effect, the resulting modifications to the genome were passed on to future generations. Any insertions that caused death or illness, meanwhile, were a lot less likely to make it past a single generation. As such, over millions and millions of years of trial and error, transposons gradually integrated themselves in increasing numbers throughout our genomes. Eventually, their ability to move without negative consequence likely became, for the most part, saturated. And as a result, over 99% of the transposons in the human genome lost their ability to move. But we still have some active transposable elements within us: sometimes they can wreak havoc and cause disease. At a much finer level of resolution, transposons contribute to creating genes, modifying them, and programming and reprogramming them. Many transposons and retroelements contain captured gene fragments and can be part of gene regulatory regions. The bottom line for genomes is that the cleavage and resection of DNA by transposases virtually guarantees sequence variation, genome scrambling, and the appearance of transposons at rearrangement breakpoints. Simply put, transposases drive genome evolution.

Non Coding RNA: a good reference is a chapter: Loudu Srijyothi, Saravanaraman Ponne, Talukdar Prathama, Cheemala Ashok and Sudhakar Baluchamy (October 10th 2018). Roles of Non-Coding RNAs in Transcriptional Regulation, Transcriptional and Post-transcriptional Regulation, Kais Ghedira, IntechOpen, DOI: 10.5772/intechopen.76125. Available from: https://www.intechopen.com/books/transcriptional-and-post-transcriptional-regulation/roles-of-non-coding-rnas-in-transcriptional-regulation.
Non coding RNAs are functional RNA molecules from our non coding DNA but do not code proteins. There are many types of non coding RNA: such as small non coding RNAs – sncRNAs: miRNA, piRNA, SiRNA, SnRNA and long non coding RNAs – lncRNA: lincRNA, NAT, eRNA, circRNA, ceRNA, PROMPTS. ncRNAs play critical roles in defining DNA methylation patterns as well as chromatin remodeling this having a substantial effect on epigenetic signaling. ncRNAs play roles in transcriptional and post transcriptional regulation. Methylation patterns change in a linear fashion through out our life and to an extent where they are used by algorithms to predict biological age. ncRNA regulation is tissue specific and also makes changes in a linear fashion following our stages of lifecycle: earlier ensuring developmental changes and just after puberty age related changes.
Find below a diagram encompassing the RNA universe:

Small interfering RNA – siRNA and micro RNA- miRNA: These molecules induce mRNA degradation or translational repression which thereby changes gene expression. Surprisingly about 60% of the translated protein coding genes are negatively regulated by miRNAs! To make it even more complex there are lncRNAs which bind and degrade target miRNAs thereby upregulating that gene's expression. Layer upon layer of regulation. miRNAs also play role in cell proliferation, cell differentiation, development and cell death.
Long non coding RNAs: Their actions can be divided into 4 types. The diagram below will explain them:

LncRNAs have diverse regulatory functions and might regulate gene expression by modulating chromatin remodeling, cis and trans gene expression, gene transcription, post-transcriptional regulation, translation, protein trafficking and cellular signaling. These below are some of the ways in which lncRNAs regulate:
Transcriptional regulation is done by:
Enhancer ncRNAs – eRNAs – as name suggests they upregulate gene expression.
Activating ncRNAs – transcriptional activating function. Although function is similar to eRNAs their mechanisms are different.
lncRNAs that recruit chromatin modifiers- they recruit chromatin remodeling complexes to specific DNA location to activate or repress genes.
ncRNAs involved in genomic imprinting- participate in epigenetic silencing of an allele inherited from either parent. One example is X-chromosome inactivation in females.
Post translational regulation is done by acting as competing endogenous RNAs that regulate microRNA levels which in turn modulate mRNA levels by altering mRNA stability, mRNA decay, and translation. Some of their regulatory actions:
LncRNAs as a source of miRNAs- 50% of the miRNAs are produced from non coding transcripts. LncRNA genes contain embedded miRNA sequences which may be located within an exon or an intron or occur in clusters within the genome. Though the sources are different, the pathways converge at the level of pre-miRNA structure which produce miRNA.
LncRNAs as negative regulator of miRNA- as mentioned above miRNAs act as negative regulator of gene expression lncRNAs competitively bind them and degrade them thereby upregulating target gene expression.
LncRNA mediated mRNA degradation- they do this mRNA degradation directly independent of miRNAs.

Cis Regulatory Elements: Non Coding regulatory elements near a gene. Cis-regulatory events are complex processes that involve chromatin accessibility, transcription factor binding, DNA methylation, histone modifications, and the interactions between them, control of chromosomal replication,  condensation, pairing and segregation. Types:
Promoter: helps in Initiating transcription of a gene.
Enhancers: provide binding site to Transcription Factors to enhance gene expression.
Silencers: repress the gene after binding with Transcription Factors.
Response Elements: provide locational homing for Transcription Factors.
Insulators: Acts as a boundary wall.
Almost 1/3rd of the genome- about 1 billion base pairs – may be involved in cis-regulatory functions.

Trans Regulatory Elements: modify expression of distant genes.

Introns: Introns are non coding elements inside a gene which are spliced out before transcription of the remaining gene (exon). Alternative splicing allows multiple proteins to be generated from the same gene. 90% of our protein coding genes have introns and of those 95% have alternative splicing! Human genome contains an average of 8.4 introns/gene: 139,480 in our entire genome. Accounts for 25% of our genome. Why our genome has so many conserved Introns is still being discovered but some of the regulatory functions that have been found are transcription initiation, transcription termination, time delay during transcription, alternative splicing, recruitment of nuclear export factors, recruitment of shuttle proteins, it increases translation yields, etc.

Repeated non coding DNA sequences: repeated noncoding DNA sequences at the ends of chromosomes form telomeres. Telomeres protect the ends of chromosomes from being degraded during the copying of genetic material. Repetitive noncoding DNA sequences also form satellite DNA, which is a part of other structural elements. Satellite DNA is the basis of the centromere, which is the constriction point of the X-shaped chromosome pair. Satellite DNA also forms heterochromatin, which is densely packed DNA that is important for controlling gene activity and maintaining the structure of chromosomes.

As ENCODE data suggests that 75% of the human genome is transcribed to RNAs but only 3% of that is from protein coding genes. So rest of the RNA and other factors have complex functions as mentioned above – most of which are yet to be discovered. We read above how multiple transcription factors help activate a gene. Many of these transcription factors are coded in the non protein coding part of our DNA. Each has a function creating an affinity for co-factors, their shapes locking to the exact location. There are regulatory factors that regulate other regulatory factors. So for example one example of factors would regulate as per their purpose but then another layer of factors emerge due to temporal reasons as during aging to bind and block the first layer from activating genes. Such factors emerging from transcription of non coding DNA seem to regulate at various levels, pre-transcription, post transcription, post translation, etc. Both spatial and temporal organization of genome is incredible but what fascinates me is the temporal organization of genomic activity. All the little information we have discovered about the world of non coding DNA shows incredibly complex and precise management of our body through the management of the intracellular and extracellular environment through the management of gene expression and post gene expression. To go one step ahead one can say that which proteins get printed correctly, are stable and get activated and which proteins are blocked at any given time in our lifecycle determines the homeostatic status of our various systems which culminates into our lifestage at that time. The non coding elements not only regulate gene expression/repression but also ultimately protein production/repression.  Our biological destiny is defined by the proteome which is regulated by the transcriptome. The 90,000 types of proteins that are produced, which help run our bodies, from the 3.5% of the genome are regulated by the factors transcribed from 96.5% of the genome! We can safely deduce that the highly conserved part of the non coding genome holds the ‘clock’ that triggers various transcriptions based on time or life stage. This temporal execution turns us from an egg to an adult and then gradually and deliberately in a calibrated manner destabilizes homeostatic balance and efficiency of important processes like repair to make us grow old and eventually die. Just like the developmental program, the aging program too is global, unfolding in every cell in our body. Change is constant in our lifecycle. I am not talking about the changes that occur for day to day operations. I am talking about macro global changes. If there were no changes we would remain an egg. We would remain a baby if changes stopped after we developed into one. These changes make us an adult and then begin aging till we die. Now here is what is fascinating: If we stop these lifecycle changes from occurring after we become an adult there is no reason why we could not remain young forever. This is not a theoretical speculation. Researcher Richard Dixon from University of North Texas and collaborators discovered that a 1,000 year old Gingko Biloba tree's gene expression was the same as a 20 year old tree with no sign of senescence or deterioration. The tree's ability to photosynthesize, germinate seeds, grow leaves or resist disease/infections remains the same as trees thousand years younger.

1,400 year old Gingko Biloba tree planted by Chinese Emperor of Tang Dynasty (618-907) in full bloom at Zen Monastery in Shaanxi!

Our biological systems are so brilliantly designed that at its homeostatic peak it could maintain optimum operations almost forever. If the Gingko Biloba tree can figure it out why can't we. If we want to remain forever in homeostatic bliss and at our youthful peak we will need to discover the region of non coding DNA that gives birth to elements that trigger the various changes that lead to the aging phenotype. Once discovered we would need to either edit or block those regions or elements. We would need to freeze our gene expression pattern as soon as we become an adult. Reward will be eternal youth. This, whenever does happen, would be a permanent solution to remaining young but what about now? Another strategy is to hack the factors and proteins that cause progressive age related changes in the activation and repression of genes and replace them with factors and proteins from a young environment. This will change the gene expression signature back to what it was in youth. In turn that would make us young again. Only catch is that unlike the permanent change to youth that is possible by discovering the birthplace of elements that make the age related changes in gene expression, the hacking protocol requires regular hacks for life to maintain youth.