Good morning. Can you hear me? Thank you very much for that wonderful introduction, and thank you very much for joining me here today to talk about regulatory and epigenetic landscapes. I'll start by saying I have no commercial affiliations to report. I want to start with this quote by Carl Sagan, where he says, the nitrogen in our DNA, the calcium in our teeth, the iron in our blood, the carbon in our apple pies were made in the interiors of collapsing stars. We are star stuff. Just as the universe is the focal point of astronomy, the human genome is the focal point of biology. It represents a universe to biologists. The physical universe is vast and unexplored. By comparison, the genome is vast, yet invisible, and after 60 years of exploration and full sequencing, it retains secrets that haven't been explained. In 2003, the Human Genome Project laid the foundation for the future of medicine. As scientists directly involved in disease research, you and I are likely to use genomic sequence data every day. And yet, how likely are we to see a piece of genomic sequence and know its function? Or if it contains variants that contribute to disease? These are the questions facing biomedical research today. Now that the genomic blueprint has been generated, biologists need to characterize genome composition to understand the inner workings of the genome. They need to assess the impact of sequence alterations and address the biological and environmental causes of disease. We can begin to illuminate the relevant components of the genome by discussing functional elements, histone modifications, DNA methylation, as this outline enumerates. And the question of what's next belongs to each and every one of you. As you pursue important biological questions with relevance to health and human disease. Think about it this way. If Facebook can get 1 billion hits in 24 hours, how far off can the estimate of 1 billion genomes be? Age of omics. Entire collections of functional elements can be examined comprehensively, through computational and experimental means. You've heard the catchphrases genomics, proteomics, transcriptomics, epigenomics. What this slide lacks is emphasis on the critical need for integration across datasets, cell types, and time points. Otherwise, these snapshots of the cell only give a static view of the dynamic system. In fact, much power can be gained by comparing changes in large scale data to derive the action and response system of the cell. So let's start with genome composition. Winston Churchill has a quote where he says, now this is not the end. It is not even the beginning of the end, but it is perhaps the end of the beginning. This is particularly appropriate for studies of the human genome. We have the sequence of 3.2 billion haploid base pairs, 22,000 protein coding genes, and we know that the genome is 98% non-coding sequence. Clearly, we cannot ignore the 98%. Repetitive elements comprise 48%, and another 50% is unknown. Determining what these unknown sequences do and how they contribute to disease is what makes our outlook as biologists so fascinating. It's no secret that regulatory regions outnumber protein coding regions. And in this talk, I will show you that regulatory sequence can outnumber coding sequence by four or five times. But predicting regulatory content and finding regulatory elements are two different challenges. These mysterious sequences have been referred to as the dark matter of the genome, analogous to the unseen matter and energy that inspired Carl Sagan in his study of the universe. Explain what I mean. We like to think that the human species is the most advanced species on the planet. This suggests that genomic complexity has been critical to our success. Yet the origin of that complexity is not obvious from studying the human genome itself. For example, if you thought that the number of bases in the genome was significant, then I'm sorry to tell you that by that measure of complexity, the person sitting next to you falls between a turkey and a lungfish. And in terms of the number of genes in humans, we fall somewhere between a chicken and a grape. So while the complexity isn't controlled by total sequence or gene number alone, it is now up to genomic research to determine how the dark matter makes us special. One place to see complexity is in disease studies. For example, diseases are monogenic or multigenic. Somewhat amazingly, we know the mutations behind 4100 Mendelian or single gene diseases. These diseases have a devastatingly simple origin whereby a single genetic misspelling cripples a protein. Finding the faulty gene means that you've instantly revealed the biological story of the disease. But very few of these disease mutations are found outside of coding regions. A few examples are starting to appear in enhancers and other non-coding regions in small numbers. In contrast, the genetics of complex diseases caused by the effects of multiple genes, lifestyle and environmental factors currently evades description. And with contributions from hundreds of genes, the risk for disease is only slightly elevated by any given variant. Therefore, understanding what these variants do and how they may interact to cause disease is the goal of current research. Since transcription of protein coding genes is required for their function, you might hypothesize that any transcribed region could carry function. A study of transcription across the genome, comprehensively covering over 100 cell types, indicated that the total amount of the genome with evidence of transcription was 70 to 80 percent. So while we don't know what the function of that transcription might be or whether it has any function, it sheds new light that the dark matter of the genome is largely transcribed. This means that even the darkest recesses are shuttled through the dynamically active system. This suggests that the complexity of the human genome could easily be hidden in the recesses of the dark matter that occupies each cell type. Yet it's not totally, it's not the totality of transcription that's important, but the diversity of products that are produced, diverse cell types, diverse regulatory elements, alternative splicing, alternative promoters and non-coding RNA. So perhaps the adage that one man's junk is another man's treasure applies to individual cells, where human complexity could be born. Is it possible that one cell's junk DNA is another cell's dark matter? We know that mutations cannot be tolerated because they interrupt functional elements, and we know that sequence variants can be tolerated because they are found in one out of every 100 to 300 bases. Interpreting the functional impact of sequence changes depends on your perspective. It requires you to think about the genome in a cellular context. You are familiar with the linear genomic sequence illustrated in every textbook on molecular biology. This consecutive nucleotide display conveys the content of the genomic sequence, but overlooks the necessary packaging. We often describe DNA folding as a convenience to cram nearly six and a half feet of linear DNA into a small sphere. This feat requires tremendous compaction. But we don't often as here, we don't as often hear about how the architecture of the folding facilitates interactions along the DNA. These interactions can join regulatory elements and gene targets that are one million bases apart. Like strands of a tied shoelace, loops bring distant genes and gene control switches into close proximity. Consider then DNA wrapped up into a folded structure in order to allow inter chromosomal contacts. Suddenly the positioning of chromosomes is relevant to their functional instructions. Sequences at the periphery of the nucleus are transcriptionally silent and sequences near the interior are transcriptionally active. What we fervently want to know is how does this cellular topology affect diseases and how do diseases change this topology? So as you think about the genome and you explore the impact of a single nucleotide variant, ask yourself, is it affecting the linear flow of information, the looping interactions of gene regulation or the 3D spatial positioning and dynamic responsiveness of the chromosome? Since the 1950s, when Francis Crick described the central dogma, a laser like focus has been placed on finding mutations that affect protein function. Now with whole exome sequencing, these are rapidly being hunted to completion. And in the last decade, the recognition of the vast amount of non-coding RNA started to expand the scope of functional elements in the genome. We know now that as much as 50 to 90 percent of the transcriptome has no protein coding potential, but rather represents an important class of regulatory molecules responsible for orchestrating gene expression. This opens up a new search for disease causing mutations that are present in the non-coding sequences, a much larger target that requires whole genome sequencing to address. So returning to the issue of complexity in the human genome, it has been argued that the extent of long non-coding RNA is correlated with organismal complexity, especially in processes related to brain function. Then it is no surprise that link RNAs have roles in cancer, diabetes, hypertension, as well as psychiatric disorders, including schizophrenia, bipolar disorder, depression, and autism. Let's begin with the linear sequence. One way to narrow the search for meaningful pieces of the non-coding genome is to align genomic sequences from species that are very different. For example, of the 46 existing genomes from species ranging from humans to fish, they span an evolutionary time span of 450 million years. Clues to function are found in regions that stay the same between species, as well as those that change. For example, relationships can be depicted using a phylogenetic tree. Written on the tree are important questions that can be addressed by particular comparisons, such as the origin of therian sex chromosomes X and Y. To address this question, you would look at differences in placental mammals and marsupials versus monotremes. The question of changes accompanying pig domestication is focused on comparisons of wild and domestic pigs. And human language evolution involves comparison of Neanderthals, Denisovans, and modern humans. Transposable element exceptation or co-option into functional regulatory elements started with comparisons of chickens and lizards and expanded to show that 10% of transcription factor binding sites in the human genome were deposited by mobile elements. So for each of these questions, the answer requires having sequences separated by the appropriate phylogenetic time span. Now looking directly at sequence alignments in mammals provides clues to regulatory elements. Here are five species aligned to show that regulatory elements of the beta globin enhancer region. Boxed areas represent known or predicted transcription factor binding sites. Dots represent identical sequences across species and dashes represent gaps. This computational exercise of finding regulatory conservation is called a phylogenetic footprint. The similarity is caused by negative selection, otherwise known as purifying selection. This is the pressure to prevent or remove deleterious changes from occurring within a sequence. It is a strong signal of regions whose sequence has been maintained over time because it is critical for a biological function. This image illustrates how sequence conservation represents functional regions. Now at closer distances like in primates, comparative approaches might reveal that functional and neutrally evolving sequences remain too similar to be distinguished. But functional words can be found in regions where no changes occur, as shown here by the black lines, as long as enough primate species are included. These words often reveal primate specific regulatory elements. You'll note that the boxes show regions where the sequences have changed. This approach is known as phylogenetic shadowing, and it can be used to study any differences within any lineage, as long as enough sequences are available. Now in the early days of genome sequencing, only human and mouse genomes were available. They were used to calculate the total amount of the human genome under purifying selection. This could be used as a proxy for how much of the genome was functionally important. In total, 40% of the human genome aligned with the mouse genome, as shown by the blue curve. The vast majority of this was neutrally evolving, as shown by the red curve, and carried low sequence similarity. The statistical estimate of the sequence conserved above the neutral rate was 5%, as shown by the gray curve. Now keep in mind, this is at least two times larger than the estimate of coding sequence in the genome. And as conserved regions became the focus of function, this approach could say that there were regions in the genome that appear to be functional, but it wasn't proof of function and it certainly couldn't say what those functions were. Now some very extreme examples of negative selection exist in the genome. For example, ultra conserved elements are longer than 200 base pairs and have 100% identity with no insertions or deletions between human, mouse, and rat genomes. They were originally thought to be traces of contamination by human DNA in these other genomes. It was later shown that these sequences actually belong in these genomes. This extreme level of conservation vehemently predicts functional importance. And today we know that ultra conserved elements throughout the genome can serve as enhancers, regulators of splicing, as well as axons of protein coding genes. What's really interesting about ultra conserved elements is that some of them carry a dual regulatory purpose. They are axons in protein coding genes and they are enhancers for neighboring genes. So the search for functional elements required new exploration of the genome beyond the coding regions. Donald Rumsfeld has a quote. There are known knowns. These are the things we know. There are known unknowns. These are the things we do not know. There are also unknown unknowns. These are the ones we don't know. We don't know. It is the latter category that tend to be the difficult ones. This is not a picture of Donald Rumsfeld. This is a picture of Captain obvious. But the question of what fraction of the human genome is under purifying selection has remained contentious ever since those first estimates in 2002. At that time it was not well appreciated that the amount of human constrained sequence that is also constrained in mouse is a minority of all human constrained sequence. This is because there is relatively rapid gain and loss of functional sequences in both lineages since their last common ancestor. In other words at that time it was an unknown unknown. By examining genomes of multiple species the constrained sequence could more easily be seen despite losses in some species. And that's what's being shown here. The saturated colors show us the fraction of constrained sequence that has been retained whereas the pastel colors show how much has turned over. Based on this study the amount of the genome under negative selection to preserve function is estimated at 8.2 percent. This is nearly four times the amount of protein coding sequence. The opposite end of the spectrum some regions are under selection to increase the number of nucleotide changes in a particular species. This is known as positive natural selection the force that drives the increase in advantageous substitutions. A well-known example although you might not consider this advantageous is the sickle cell mutation in the beta-globin gene. In its heterozygous form it actually protects against malaria. And that's because infected blood cells are greatly weakened by the presence of the sickle allele causing them to be rapidly cleared from the body. This connection explains why the mutation has been retained and the incidence of sickle cell corresponds to regions endemic with malaria. Another example is a mutation in a regulatory region near the gene for lactase. For around 10 percent of Americans 10 percent of Africa's Tutsi tribe 50 percent of Spanish and French people and 99 percent of Chinese. A tall cold glass of milk means an upset stomach. Most of the adults in the world are lactose intolerant and cannot digest lactose the primary sugar in milk. And yet regardless of our ancestry most of us begin our lives happily drinking milk. So what happened in that intervening time. In people who become lactose intolerant adults the lactase gene is switched off after weaning. Recent evidence suggests as African and European populations began hurting cattle lactose tolerance became an advantageous trait. These mutations were favored by natural selection and quickly spread through dairy dependent populations.  Now rapidly changing regions in the human genome provide a window on acquired mutations that provide a favorable trait. In 2006 a large scale study was undertaken to identify all transcripts with accelerated divergence called human accelerated regions or horrors for short up to 1000 regions have been identified. They are conserved across species and some have accelerated changes in human relative even to chimp. One example of an accelerated change occurs in a special enhancer sequence that targets expression to the opposable thumb. It may also be responsible for modifications in the ankle or foot that correspond to walking upright. What this figure shows is that when the human sequences were placed in a transgenic mouse they could drive expression that supported the hypothesis of the opposable thumb. When the mouse sequences were used they could not support those expression patterns. So given all the non-coding sequence under selection perhaps it is even more impressive that many years ago in 1975 Mary Claire King published a paper that stated the evolutionary changes in human anatomy and way of life are more often based on changes in the mechanisms controlling the expression of genes than on sequence changes in proteins. How prophetic that statement would turn out to be. And with it we continue exploring the non-coding dark matter of the genome. So Winston Churchill has another quote out of intense complexities, intense simplicities emerge. I've just told you how complex the genome can be. Could it be possible to describe it in simplistic terms? That's what we would all like right? I guess my answer is it depends on how you define simplistic. In other words the rules and patterns define ways to reduce complex items into straightforward examples. Following the completion of the genome project the linear sequence could be used to identify these rules and define regulatory elements. From their experimental techniques and computational approaches have been used to predict functional regions. The basic descriptions of regulatory elements and the mapping of proteins that bind to them helps us to define these rules. We can define enhancers as clusters of transcription factor binding sites that are each 6 to 20 base pairs long. Evolutionary sequence conservation is typically present and enhancers are being found just about anywhere. They are adjacent to genes embedded within coding exons of genes, in introns of unrelated genes, megabases away from their targets and even in gene deserts. Their modular construction being made up of transcription factors could allow the accumulation of mutations or even variants that carry a modest effect of disease risk. Explaining why the changes persist in the genome and that they contribute to type 2 diabetes, colorectal cancers and even coronary artery disease are very fascinating questions. Promoters of the regulatory switches controlling gene expression. They are bound by the RNA Paltu transcription machinery. They can be described in terms of having canonical and non-canonical elements. All active promoters have nucleosome free regions to provide clear access for the transcriptional machinery. Not all of these elements are present in all vertebrate promoters. Some protein coding and micro RNA promoters have these canonical core promoter elements. But a large majority of human promoters or mammalian promoters lack these classical core promoter elements. But instead they contain broad regions such as CPG islands or ATG deserts or transcription initiation platforms. One important protein that is central to most regulatory processes is CTCF. It is an 11 zinc finger protein that functions as an insulator or boundary element. To fulfill these functions there are up to 40,000 CTCF binding sites in the human genome. CTCF partitions the genome into distinct regions of open and closed chromatin domains like fences in a neighborhood. It has recently been shown to bind in tandem to mark the boundaries of chromatin domains and it also functions to insulate enhancers from spurious interactions with unintended promoters. Variants that disrupt CTCF binding alter its normal function in the cell but usually at only one of the two alleles leading to variable consequences. And you can begin to see what changes around the genome in CTCF binding locations could have complex outcomes. Now there are also regulatory regions that affect splicing. A portion of these disrupt events that happen directly in the splice junctions and we all know about these. They change the splice boundary information. Others reveal hidden regulatory elements and what I mean by hidden is that they are embedded within coding sequences. Splicing instructions in central positions of axons contain specialized elements known as exonic splicing enhancers and exonic splicing silencers. These are the ESE and ESS positions in the image. These are binding sites for RNA proteins. So in this case even synonymous substitutions in the DNA can affect regulatory function in the RNA. A similar set of regulatory sites falls in introns and acts as intron splicing enhancers and intron splicing silencers. So I've just told you that introns carry functional elements. Luckily some of these elements look very much like transcription factor binding sites and that and they can be conserved and that gives us some clues to how to predict them. So what have we learned about enhancers? Recently two groups published work on enhancers that are exquisitely long. They call them super enhancers or stretch enhancers. They're so long they look like landing strips at an airport. They're only found upstream with genes that are critical for forming a cell's identity. We find that genetic variations associated with common disease are highly enriched in stretch enhancers. For example, stretch enhancers that are specific to pancreatic islets, harbor variants linked to type 2 diabetes. Also notably this work was published from our own Francis Collins. We also recently learned about enhancer RNA. These are RNA that are expressed as very short pieces of non-coding RNA whose transcription is coupled to a partner protein coding gene. If transcription is lost at the enhancer it is reduced or lost at the protein coding gene. This actually provides a way to predict enhancers using small RNA sequencing. Now while the linear sequence of the genome is very important it gives the impression that disconnected pieces of the genome function in isolation. When in fact genomic function is much more like a game of twister where many distant pieces show connections. So consider the linear enhancer view that I showed you in a previous slide. This enhancer actually functions by looping interactions mediated by the protein CTCF and an accessory protein cohesin. Enhancer A interacts with the promoter to give specialized limb expression and enhancer B interacts with the same promoter to give specialized brain expression. And it's the combination of both enhancers that confer the brain and limb expression patterns seen in the gene expression profile at the top of the image. Enhancer variants contribute to gene expression variability by precluding the proper interactions. In this way only one of the productive interactions could occur in a cell creating an allele specific imbalance. This variability can be very important to the generation of complex diseases. For example Crohn's disease or cardiovascular disease where fine tuning of the amount of products may be important. Now a very strong effect is seen in mutations that affect the sonic hedgehog gene enhancer. This is a gene involved in the formation of digits and mammals and thins in fish. Mutations occur in an enhancer located one megabase away from the gene. These mutations allow unregulated activity of the enhancer during development which creates extra digits. The polydactyl phenotype occurs in humans. It can be reproduced in laboratory mice and it also has seen in Hemingway's cats which are selectively bred at the Hemingway House in Key West Florida if you'd like to go see them. Now in addition to cute cats genomic looping plays an important role in the function of non-coding RNA. The exist non-coding RNA is expressed from one chromosome in every female cell and it coats that chromosome to repress expression in a process called X chromosome inactivation. This image shows two superimposed plots where the distance from the exist expression site shown in the black box is drawn along the X axis. The peaks in red are locations of the exist non-coding RNA along the chromosome. The peaks in blue co-localize and represent the frequency of interaction of those positions of the chromosome with the exist expression site. So in this way exist is delivered to its target sites without ever diffusing through the nucleus. Many other non-coding RNA are delivered through strategies that use this type of targeted proximity rather than diffusion. This allows for very low levels of non-coding RNA transcription to be effective as regulatory molecules. I think this is a very important point because people say well RNA expressed at such a low level probably is noise. This is one example where it's not noise and in fact exist is needed at very high copies but many other non-coding RNA probably are effective at very low expression levels. So if genes have non-random location then a corollary is that chromosomes facilitate interactions through their nuclear organization as well. Chromosome territories are the spatial locations defining the positions of each chromosome. Regions with transcriptionally silent heterochromatin contain gene poor sequences and low overall transcriptional activity. They are pushed to the nuclear periphery. Gene rich regions are in highly transcribed DNA situated in the center of the nucleus. When a gene loses function it often changes its location in the nucleus. Now in newer views of chromosome packaging chromatin from different chromosomes is allowed to expand into the surrounding territories. These are known as inter-chromosomal networks. You can think of this as an explanation for translocations that occur between chromosomes. Double-strand breaks formed in regions of intermingling are more likely to produce inter-chromosomal rearrangements whereas those deep within a territory are more likely to produce intra-chromosomal rearrangements. At the edges of the nuclear periphery are the regulators of structure. In this silenced region important proteins include the lamin proteins. They form an intra-nuclear scaffold known as the lamina around the edges of the nucleus. This lamina connects the chromatin or the heterochromatin to the nuclear envelope. These proteins support the nuclear architecture by separating regions of silenced chromatin from active chromatin. Hutchinson-Gilford-Prageria syndrome is a premature aging syndrome with mutations in the protein lamin A. Symptoms include accelerated aging represented by early cessation of growth, baldness at the age of two, degeneration of the skin, muscle and bone and often fatal heart conditions in childhood. In this disease the attachment of chromatin with the nuclear envelope becomes altered. The nuclear organization is lost along with transcription and transcription-coupled repair that is necessary to correct DNA damage. So you can see how the proper architecture within the nucleus is very important for many of the functions that go on. The reduced ability to address DNA damage triggers cell death and senescence promoting accelerated aging. So what you might all relate to is the fact that the cellular alterations in the lamin A processing seen in Prageria play a role in the aging process in everyone. It just happens at a slower pace. So even in cancer regions of heterochromatin at the nuclear periphery become repositioned in a tumor cell. These regions have special names. They're called LOX and LADS which stand for characteristics of lamina associated domains. These changes indicate regulatory reprogramming is going on in the tumor cells. And rather than conferring ages, aging, the changes confer immortalization. So within the last 10 years, we've all lived through a monumental shift from the gene centric paradigm to the genome scale perspective. And along with this shift came new technologies to study epigenetics defined as changes in gene expression that cannot be explained by changes in DNA sequence. These changes are due to modifications of the histones that wrap the DNA or to modifications of the DNA itself. So I will cover these in the next two sections. At the root of DNA packaging is the nucleosome. This creates the fundamental unit of chromatin, approximately 147 bases of DNA are wrapped around an octamer of histone proteins, including two copies of H2A, H2B, H3 and H4. In nuclei of mammals, the most obvious form of structural genome organization is the compartmentalization into U-chromatin and heterochromatin. Multiple modifications occur on these histone tails as they wag outside of the nucleosome. There are over 150 different modifications that are described both by their position and their type. These include activating marks like acetylation and phosphorylation or methylation which can be activating or repressing. In its simplest form, closed chromatin provides an accessibility barrier and is repressive to gene expression. Open chromatin is a hallmark of an enhancer region and assays such as DNA hypersensitivity can find these locations experimentally. Histone modifications correspond to the accessibility oops. Histone modifications correspond to the accessibility of the chromatin. For example, active enhancers are marked by active histone modifications such as H3K27 acetylation and H3K4 monomethalation. In contrast, closed or inactive enhancers have specific identifying marks as well. These include repressive marks such as H3K27 trimethylation but typically the active mark of H3K4 monomethalation is present. So what does it all mean? It means that we can read the functional state of the genome by histone modifications and just as the Rosetta Stone converted in ancient language to a modern one, the computer program Chrome HMM converts the histone code to types of function in the genome. Yellow enhancers, red promoters and green gene bodies differentially color the genome in every cell type. To illustrate the role of histone modifications, kabuki syndrome is a congenital disease with an intellectual disability. Mutations occur in two proteins KMT2D and KDM6A. These two proteins collaborate in a larger complex to turn repressed chromatin into active chromatin through exchanging the histone marks. Without the full function of these proteins occurring in all cells, you can see the emergence of this disease. Without these functions, in other words, they're not just passive marks but they're required for opening up the chromatin. Now, another aspect of the epigenetic code is conferred by DNA methylation patterns which are specific for each tissue type. For example, DNA methylation plays a role in aging. And as Groucho Marx says, age is not a particularly interesting subject. Anyone can get old. All you have to do is live long enough. But it is an interesting subject because it's a biological phenomenon. And in fact, the longer you live, the more likely the total DNA methylation levels will drop across your genome. This could manifest as more transposable element movement, less rigorous control of gene expression, and select positions might acquire methylation somewhat sporadically. Some of these events occur at tumor suppressor genes which might create a predisposition to cancer. DNA methylation stably alters gene expression frequently by gene silencing. It suppresses the expression of viral genes and it prevents genomic rearrangements between repetitive elements. Therefore, it is crucial for maintaining genomic stability. Now, Waddington coined the term epigenetics in 1942. He described the cellular programming as a dependency on epigenetics and he drew a parallel between the path a cell takes towards terminal differentiation to that of a ball traveling downwards along branching valleys. Once a ball entered into a canal, it could not easily cross the mountains and enter into neighboring valleys. In other words, differentiation into a tissue is set by a series of events that cannot be changed into another type. We know now that the reversal of differentiated states can occur in induced pluripotent cells that occurs through the introduction of multiple transcription factors including oct4, nanog and SOX2. And one can imagine through their interactions with other proteins in the cell, a series of changes occur that culminate in repositioning of the chromatin within the cell to induce a pluripotent state. It is in this state that the repressive DNA methylation marks that gave the cell its differentiated identity are removed. So DNA methylation also takes center stage in behavioral studies. In rats, the normal nurturing behavior of mothers is frequent grooming and licking of their offspring. This behavior is transmissible across generations, but it's not transmitted through the germline. It is a context-dependent effect. As a result of the grooming and the licking, the glucocorticoid receptor in the brains of the pups becomes demethylated. This allows high levels of gene expression in their brains. The result is that there are low levels of the circulating ligand, the glucocorticoid stress hormone. These offspring grow up to be adults who are calm and easygoing and they also exhibit the nurturing behavior towards their own pups. Offspring raised by mothers who exhibit low nurturing grow up to be mothers who are low nurturing. Their glucocorticoid receptors carry DNA methylation and decreased expression. As adults, these pups are highly anxious and stressed and perpetuate the low nurturing behavior. So this phenotype is reversible and there are two ways that can be reversed. Pups of low nurturing mothers can be adopted by high nurturing mothers and they become high nurturing mothers themselves. Pups of low nurturing mothers can have compounds injected into their brains that erase the methylation patterns. When that happens, they grow up to be high nurturing mothers. This tells you that the effects of the environment are mediated through epigenetics, including DNA methylation. Now, methylation is very important in cancer and we can see a reversal of the normal methylation processes where the epigenetic landscape changes dramatically compared to a normal cell. For example, repetitive sequences in intergenic regions that are normally repressed, lose their methylation and become transcriptionally active in cancer. CPG islands, which are normally un-methylated, acquire methylation leading to transcriptional repression. The shores and shelves of regions surrounding CPG islands also gain methylation and block transcription of important genes. And it is through the process of these epigenetic changes that the interaction sites of DNA change within tumor cells. This recently published report of brain tumors demonstrates this phenomenon where two genes that are normally confined by good fences to separate loop domains and rarely interact become closely associated in brain tumors. In this large-scale study, the authors identified roughly 10,000 of these shoelace configurations showing how the genome harnesses form to regulate function. The process includes gene mutations leading to altered positions of DNA methylation which causes interference in CTCF binding sites, the loss of neighborhoods and inappropriate gene expression. So in this final section, I'd like to recap some important points. The ENCODE consortium reported that a surprisingly large amount of the human genome, 80.4 percent is covered by at least one ENCODE-identified element when examined in over 100 different cell types. These elements include transcribed regions, biochemical marks like histone modifications and while it doesn't prove function of 80 percent of the genome, the biochemical evidence complements the genetic evidence, the sequence conservation and the protein coding information. Note that none of these indicators of function are completely overlapping. We can now tally the number of functional elements in the genome. The 20,000 protein coding genes are being rivaled by 13,000 long non-coding RNA and more are appearing every day. 70,000 promoters, a company nearly 400,000 enhancers and over 600,000 transcription factor binding sites. And it's still just the end of the beginning of human genome research because the functional roles of most of these factors are still not known. So regarding annotating the functional content of the genome, let me show you another astute quote by Carl Sagan that the absence of evidence is not the evidence of absence. This idea is illustrated by an experiment conducted many years ago when the deletion of an ultra-conserved region sent shock waves through the community because its loss should no functional effects. The laboratory mice were healthy and they produced healthy offspring and many experiments later, we know of numerous ultra-conserved elements that have a phenotype when they are deleted. But this result provided a unique unpredictable insight on the occurrence of the absence of experimental evidence despite this region having the strongest prediction of function. Now one explanation is that the genome carries elements known as shadow enhancers. They provide redundant function for other enhancers and when one is lost the other can compensate. The number and extent of the duplicity in the genome is a known unknown. Other regions in the genome seem to contradict the rules for function by not showing sequence conservation across species. Link RNA or long non-coding RNA provide an example of functional elements that lack high levels of sequence similarity. However, they might not require very much nucleotide sequence conservation to maintain their functionality. So whereas protein coding genes require a correct linear amino acid code to produce a functional open reading frame, RNA molecules utilize secondary structure as the mode of function. This doesn't require strict linear sequence conservation. If an important nucleotide changes in one location a compensatory change somewhere else can restore the molecule to full function. So as we end I would like to finish up with just some remarks about seemingly non-functional regions. There are regions in the genome that are parasitic. The majority of transposable elements don't add any function to the genome. In fact, they can cause disease when they're not dealt with properly. The genome has specific approaches to fold these regions into segments of repeat pairing structures known as repeat assemblies. Thus as I've told you, TEs are methylated and repressed and they are non-functional in the majority of cases. But their relevance to disease implicates that they should be sequenced and studied across the genome. Finally, let's return to this idea of the vast amounts of non-coding RNA in the genome. We are left with the question is it random? Does the cell use it for functional purposes? Or is it in or is the act of transcription itself enough? A recent OMIC study using a new technique called CHIP-XO cleaves the DNA adjacent to the RNA-POL2 molecule giving base pair resolution to its location. This is shown in the red CHIP-XO peak compared to the CHIP-Seq peak or the older style CHIP-CHIP data each of which have less resolution. This new approach revealed 160,000 positioned RNA-POL2 molecules in the genome where 150,000 of them were not located at protein coding genes. They were located in the non-coding regions of the genome. So right now the debate rages on whether this is spurious binding or regulated binding and whether it's producing functional or non-functional product. But with this new data the tapestry of the genome annotations grow ever more refined. So I will close with this because it's a final quote from Carl Sagan. He says, at the heart of science is an essential balance between two seemingly contradictory attitudes an openness to new ideas no matter how bizarre or counterintuitive they may be and the most ruthless skeptical scrutiny of all ideas old and new. This is how deep truths are winnowed from deep nonsense. I think that's appropriate to studying the genome and I'd like to add this is how function is winnowed from the dark matter of the genome. Thank you very much for your attention.


Coming to you from our GIS Studios, I am Anisia Antoine and this is your media news brief. The Curriculum and Material Development Unit, CAMDU, within the Department of Education, Innovation, Gender Relations and Sustainable Development in collaboration with the Division of Human Services, hosted a discipline development seminar to sensitize the heads of the Department of Education as it relates to positive discipline. CAMDU has been implementing effective and child-friendly schools across the island throughout the years. This is the 30th Convention hosted by CAMDU with the aim of enhancing the education system in St. Lucia. Bevelian Poyot, Acting Director of the Division of Human Services, presented on the work being done with the Justice Tribunal Project. She stated that working with the communities and an environment in which young people engage plays an important role in enhancing the abilities and mannerisms of young people. In observation of World Press Freedom Day and Reading Month, the National Association of Librarians and Information Professionals and the Public Library will be hosting a writing and publishing workshop on Friday, May 3rd, 2019 from 9.30am to 4.30pm. This workshop encapsulates the basic fundamentals of writing and publishing and will be facilitated by locally renowned writers, journalists, authors, poets and publishers. Topics include, but are not limited to, general principles of writing, the art of journalism, the basic principles of publishing to name a few. The group earmarked to participate in this activity include writers of short stories, students of English language, English literature and communication studies, budding novelists, poets, playwrights, persons pursuing careers in journalism, writing, publishing both seasoned and novices. The management of the Victoria Hospital would like to inform the public that outpatient pharmacy services will be unavailable from Monday, April 29th, 2019 to Friday, May 3rd, 2019. Thereafter, service will resume at the Owen King EU Hospital from Monday, May 6th, 2019. During this period, the public is heard by advised to access pharmacy services at the wellness centres in their respective communities. This has been your media news brief. Thank you for watching.


Let's see the breakfast in plus TV Africa. It's time for us to talk sports football to be precise. The flamingos after a narrow defeat against Germany in their FIFA under 17 women's world cup opener. The girls have been urged to put their defeat to Germany behind them and go all out for a three point when they take the pitch against New Zealand counterparts today the under 17 women's world cup in India. And so it is a makeover for the Nigerians flamingos and the New Zealand today as a win for one would see the exit of the other from the game. We have Monday Thomas joining the conversation this morning. Monday it's good to have you join us. Good morning Mercy. Hope you're having a fantastic time. Definitely. Let's get straight to the crux of the matter. What would you make of the opener for the flamingos? Do you think it's a case of lack of confidence or as an issue of defense? How would you describe that game? The first 45 minutes was quite impressive. I didn't expect that from the girls but they were in all departments. They were very strong and very solid and they even went ahead to score the Germans first. A brilliant strike from the free kick by Miracle Osani. That was world class and I think they had a great time in the first 45 minutes. But you know football is a game of two halves. It can favor you in the first half and favor the opponent in the second half and that was the case right there for the German side. The second 45 minutes whatever the coach told them in the lockout room certainly worked out in the second 45 minutes. The Germans were able to score two quick goals. One in the 59 minutes and the other in the 61st minute to see them bid the flamingos who had a brilliant game even when they went down by two goals to one. They were still putting in the fight hoping that they could equalize some tactical substitutions there by the coach Bankole Oluo-Kere who I'm very confident in. I trust him because of how he qualified the ladies to this particular World Cup. I mean having a great run of form in the qualifiers dominating African football and also having a very fair outing in Turkey for the 10-day training. So a lot is expected from them and like today's game, yes a lot is expected from them. And I actually spoke with one of the number-nines Alvin Dasu and she's saying that she's expecting to just let the past be the past. Let that defeat just get behind them and they'll be looking to get a win today. Just like you said, it's going to be a make-or-mall for the flamingos. And I must let you know that the flamingos have not won any of their last five games at the under-17 World Cup. A streak that is stretching back to a three-nil defeat in the quarter-finals in the fourth edition way back in 2014. Their last victory came in the group stages of the 2014 three-nil thumping of Mexico. And since then they have scored just one goal in the last edition which was in 2016. They didn't score a goal, they didn't score a single goal in that particular one and their best result was a neonial draw against England way back in 2016. So what I'm trying to see is that the flamingos that in the World Cup or at the World Cup, they've not had that brilliant form. But by the way they qualified, by the football they played against Germany, who we know that Germany were going to be their toughest opponent. So a two-one finish was not so bad against a German side that many people were expecting that the flamingos will maybe go down by three goals in a year or four goals in a year. But the ladies were able to put up a great performance and saw them still lose that particular game. But the imagine which they lost was still very impressive for me. And against the New Zealand side who lost their first game to Chile by three goals to one. So I think Nigeria, the flamingos will just age past this particular one. I'm seeing a narrow victory for the flamingos. Just before we get to the flamingos with New Zealand because as much as we would say that yes this is Africa's you know best in putting out right there in Nigeria, you can also agree with me that New Zealand is also not a walk in the park just as Germany. They also had not to crack. But like I asked you earlier on what do you think the problem lies? Do you think it's the issue of confidence especially when you talk about you know the exposure and playing with you know a team as Germany and or where the problem is on the defense? I mean you can say that the problem with the flamingos losing to Germany was on our defense. No it was it was just football happening. I mean I think the main issue right there for the flamingos, they were over confident after seeing that in the first 45 minutes the Germans were not what people told them about. So they had a fantastic first half, they scored first and they grew and I think they were over confident to that. They would get all three points against the Germans. But you know the Germans had all the plans. So it's not a case of not having confidence. It's not a case of not having a quality defense because at all departments I think Bankola, Lua, Caris girls were top notch but they just lacked that consistency to see off the game. So they became complacent in the late minutes of that game especially from the 59 minutes where they considered the first goal and you know in a football game when you score a goal you get to build from there. You get to have momentum to go for the order and that's what the Germans did. They scored the first goal, they had momentum and they went ahead for the second one and that's how they were able to beat the flamingos.  A lot of persons have actually queried whether or not you know the flamingos are really a material for the World Cup. And so I'm asking do you see an exit? Do you think that this is just going to be an exit falling out of the game today? There's no exit. The flamingos are still in this. They're still in this. I told you that you may have mentioned of New Zealand who are not clearly working at Park. I agree with you because for you to play at the World Cup it means you did something great in your continent, in the Oceanic and the Asian continent. It means you did well. That's why you qualified to the World Cup. So the New Zealand are not going to be any work over but I think the flamingos have got what they take to each pass them and get their first victory and see how they can of course get a draw or another victory against the Chileans to see them qualify to the knockout stages of the Under 17 World Cup. Well so if you had the chance to be the coach of the flamingos for just you know a day, what would you be looking at? What exactly would you be putting the team through? Monday can you hear me? Yeah yeah Mercy I can dig it out. All right so I'm asking that if you had a chance to be the coach or to coach the flamingos for just a day, what exactly would you be telling them to look at? What would be your strategy? What exactly would you be doing to get us you know to that point not to fall out of the game especially on this day? Well Mercy I can't be a coach. I don't think I'll ever want to be a coach in the future. I'm just saying let's assume. Why? Because I trust Bankole. I trust Bankole coach. Bankole is a world class coach. So I think he's doing exactly what I'll be doing. He's doing exactly what I'll be doing right now with the girls. I mean I was impressed. I mean I wouldn't quickly or I wouldn't completely judge them for losing against the German side. The German side clearly where the top is open and like I said people were expecting the flamingos to lose by maybe a three-goal margin but they lost by two goals at once. So there is a lot of positive we can take out of that game. Bankole or Luocare is a world class manager and whatever he's doing right now is Monday. You're not even getting the question. No one is in doubt about the positive of the game. I'm just saying as much as you'd say that was a fantastic play whether or not we actually lost out narrowly to Germany but you can also say that you're putting your best man out there in the continent and then you're losing out. It doesn't also add up. So I'm saying what are the lacunas? What exactly have you seen? What can be done to improve the game? Let's assume the girls are listening and let's just even say you're in the position to fix one or two as fantastic as the coach may be. All right. So even if I'm saying that I would do better than the coach and this is what the coach is not doing and this is what I'll probably do that is just like saying that the coach is not fit for this particular job and I wouldn't want to say that. So what I'm saying is that I got your question clearly. I got your question clearly. Now I've been with the girls. I've followed them since their qualifying series before the World Cup and before traveling to Turkey. These ladies have been brilliant. The coach is very vocal. The coach always tries to instill confidence in them and this is what I will do as well as a coach and also the tactics on the pitch of play is a top notch with the coach. So I don't see any loophole. That's what I'm trying to say. I don't see any loophole. I don't see anything I would do better with the girls that the coach is not getting it done right now. That's what I was trying to tell you. Oh well. So I think that you're trying to just be politically correct especially when it feels like you're a major fan of you know Bankole. The truth is you can never have a system where you have absolutes. You're not absolutes you're related. So as much as the system might just be perfect and a coach there might just be you know one or two you know to put out there. But however we're wishing the girls you know a great outing today but quickly just before we could sit down it would just be on a lighter note. We saw at Old Trafford Francis Zoho a world-class goalkeeper and we saw the fact that he faced 34 shots conceding one goal making 12 saves and nine of them inside the box and so what happens because over time we see that our players get to do better when they're outside of the Nigerian league but when they come to play at home it's a different thing. What exactly can we do? What magic can we perform for Francis to actually have a performance as this for us? All right first of all Francis needs to work on himself more. I mean that was a brilliant brilliant performance. I mean world-class goalkeeping display at the Old Trafford. It was there for everyone to see that it's got what it takes to be a world-class goalkeeper and great great one for the young star who is going to be 24 in the next 14 14 days from now. He had a brilliant time at the Old Trafford and the question is can he continue this particular form to see how we can also help the Super Eagles goalkeeping problems and now what he needs to do right now is to just be consistent and I think if the coaches right there in Amona Nicosia can have so much faith in him to make him the number one goalkeeper to see him playing week in week out because some people might say you might just have been a stroke of luck for Francis Zoho. You know every footballer every goalkeeper everybody in sport know that there are some days that just come in favor of them and there are some really bad days and for you to get rid of the bad days frequently you need to put in more work. You need to be more consistent. You need to be the number one goalkeeper. You need to be a goalkeeper that is playing week in week out because football is you as good as your last game. So if you keep on playing if he becomes the number one goalkeeper of Amona Nicosia the number one goalkeeper was not fit to stand for the separate side and that's why Francis Zoho was in goal against his boyhood club Manchester United and I think he just needs to play more. He just needs to become the number one goalkeeper for Amona Nicosia because the Super Eagles the stakes are very high and there are high demands right there. I don't know it's quite disappointing that our goalkeepers right now are not number ones in their respective clubs. If we take a look at the Senegalese the Senegalese goalkeeper Edward Mendy is maybe no longer the number one goalkeeper for Chelsea but for last season and for the start of this season he was the number one goalkeeper. For big countries the number one goalkeepers are also number one in their respective clubs. Francis Zoho to be a number one goalkeeper not just a number one goalkeeper but a reliable number one. He needs to be the number one of Amona Nicosia. So first thing first he needs to get to that number one shirt of Amona Nicosia and then we'll see how we can contribute this brilliant performance the same way you did at the Old Trafford for the Super Eagles of Nigeria. Oh it didn't quite really sound like you were proud of what he did. Anyways I think you took it too serious now. Mendy we're just trying to just tease Mendy Francis at this point but that's fine. Just before we go quickly I'd like you to tip forth Flamingos. What score lines are we looking at? It's going to be a narrow victory for the Flamingos. I'm seeing a 1-0 victory or 2-1 victory in favour of Nigeria. Alright then fingers across let's see how that pans out. This game would actually be played this afternoon. I really don't know what time it will be transmitted to the sort of part of the world talking about West Africa. We will be looking at the match and let's see what happens and how the girls who make Nigeria proud were very cheering for them and supporting and we wish them the very best. Thank you so much Mendy Thomas for being part of the show. He is a sports analyst. He joins us all the way from Aquibom State. Thank you. We appreciate your time and we look forward to sharing your thoughts some other time. It's always a pleasure talking to you Mercy. I mean you're always actually really really interesting questions and I try to answer them in the safe possible way. Van Kole is still my number one and I'll stick with him. I understand the bias and that's fine. That's the much we can take at this point on the breakfast. If you missed out on it it's okay follow us on Facebook, Twitter and Instagram and do subscribe to our YouTube channel Plus TV Africa, Plus TV Africa Lifestyle and that's it. My name is Messi Boko. Have a fantastic Friday.


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Should you supplement champion the new supplement from Madmong the famous supplement brand for gamers? Well champion is a very interesting supplement that can definitely improve your well-being And support your immune system It does contain some very important entropics and vitamins like vitamin D3, vitamin K, B complex and so on. In addition It does contain some important mineral like zinc and selenium. It does contain some very powerful Antioxidants like lutein, xiaxantin and because of that, this is a very interesting and very potent Supplement for improving your well-being. So if you're a gamer that wants to support your general health, your mood, your well-being and so on If I were you I would definitely consider taking champion because I think champion can help you out


is a presentation of TFNN. The Tom O'Brien Show is produced every business day. Tom takes your phone calls toll-free at 1-877-927-6648 internationally at<PHONE_NUMBER>. Let's go to Eddie and book our time. Hey Eddie, what's going on? Hey Tom, how are you man? I'm doing great man, yourself? Good, good. It is a treasure to have TFNN every hour during the trading day to be there to help you to guide you and even to give you some peace of mind or like did somebody else is there with you while you're trading this crazy market. These are up or down? Well listen, we appreciate you growling and prowling us out here because we wouldn't be out here folks if we didn't have all you guys, gals, tigers and tigers as clients and you know the market teaches you every single day man. Now, Tom O'Brien. Welcome folks, this is Tom O'Brien of TFNN. We go five days a week. We go seven hours a day. We go 24 hours a day on the internet at TFNN.com. Always remember folks, whatever you think about, you bring about whatever. You focus on growth so everyone's having a great day, safe day. September 15th man, you're gonna love it. It's making a great night and a great week. Be impeccable with your word. Replace a fear with love. This is a great card folks. The human mind is like a fertile ground with seeds that continue to be implanted. When you're impeccable with your word, your mind is no longer a fertile ground for the words that come from fear. Your mind is only a fertile for the words that come from love. Knock it off! Let's take a look at it out here. We have the Dow Industrial's up by 218. King Nasdaq up 112. S&P's up 33. Gold. Gold contract down $13.90. Trade at $17.93 an ounce. You get Silver Dow 9 cents, $23.79 an ounce. Late sweet crude up 220. $72.66 a barrel. Notes and bonds. The 10-year note down 7 ticks. Trade in $133.12. 30-year off half a point. 15 ticks at $163.27 and King Dollar. $17 down 66 ticks. Trade in $92.557. Euro 118. Yen 109.43 and the British pound at $138 to one U.S. dollar. iPhone numbers<PHONE_NUMBER>. Give us a call, folks. I know what's going on in your world. In the world of the S&P's, let's take a look at them. What do you have? We take a look at the spy out here. You're up 350. You rejected lower price. We didn't get to the low of yesterday. This is kind of intriguing. But the bottom line is that you got down a price point of 443.44 today. And that baby that's going right into where we went topside on the 23rd. Now we went topside on the 23rd with 54 million folks. We're going to do more than that. So this roll is still on downtown. That's the bottom line. That's how I read this. NDX100, same type of setup inside the NDX. It looks to me like the NDX is going to get the volume today. You got a rejection of lower price thus far, but it looks to me if you get 34 million, then what you actually have is that you got a lower low with volume. That's saying you're going to be right back down there. Now it's a little clearer of what the NDX or the 3Qs have actually done here. They got down to a price point of 373.37. Well, the high of its strength is 373.93. So you get down close to that high, rejected lower price, bottom line. It's going to be the same type of setup though. If we get 34 million or more, that's saying you have a lower low with volume saying it wants lower price. We're going to take a look at the small caps inside the IWM. What do we have with the IWM? The IWM out here, you get a bounce. Now it's 17.9 million. This is a light bounce, man. This is going to be intriguing with the small caps because the small caps, you have the commodity stocks in the small caps and that's when they do catch a bid, they're a big bid. That's what we did have happen when you take a look at this back on September 27th, August 27th. That being said, we're still sticking out like a sore thumb of these two high volume lows. You get a high volume low at 208 as well as 209. So it might take particularly how this is bouncing today. See, the small caps are bouncing with tremendously lighter volume today. That sets up lower prices coming at you. Notes and bonds. We take a look at the note and bond market. You're going to see the note and bond market. They're pulling back. You're pulling back with tremendously lighter volume. We did 1.5 or 1.7 yesterday, let me see. We're down today on 1.2 million contracts. Yeah, 1.553. This one's higher price. We went up at 1.553 and backward at 1.2. We're going to take a look at the 30 year. Now, the 30 year really had some action. The 30 year actually had taken its nose and jumped above the whole consolidation it was in yesterday. And you pulled back today, so today we're coming back with 325, 225,000 contracts. We'll go up yesterday with 493. It wants a higher price. Good old king dollar. What do we have a king dollar out here? This one is going to get really intriguing because today it's just a toss up, man. What you had yesterday is that you rejected lower price at $92,300. You didn't go higher today. You went sideways today. You got a sideways move like this. It's like, okay, you're going to go for the high of Tuesday first, reversal or low. And bottom line, it's not giving us much information. That's the real bottom line. Let's go over to the Euro and take a look at the Euro. So the way the Euro is set up, the Euro looks like it wants a little bit lower price. So that would be saying that it'd be higher for the dollar. We're going to take a look at the German pound. When I say lower price here, let me give you the lower price, too. Because this is interesting, too, because this is what... Now, the dollar should be up a lot more. Oh, I see what's happening. Oh, I had the wrong one up. Interesting. So the Euro is actually up. But that's still sideways. So the Euro is sideways, 118. We take a look at the British pound. The pound's up also. Now, the pound wants higher price. Looking at the pound, that's telling me they want higher price. So they're tossed up on the Euro right now. The yen, we take a look at the yen. Oh, this is good for the gold market. The yen broke. So we broke lower in the end. We went to 109.11. You're at 109.41, but that sets up... That sets up a swing of 108.72. Yeah, this yen broke pretty good last night. Let me go take a look at the Nikkei. So the Nikkei, all of Asia was down last night. Nikkei wasn't down bad, 158. Whoa, that being said, it's wild.  Okay, so let's see. Yeah, that'd be right. That's in real time. So this pattern here, folks, if you're into candlestick pattern, this is a very hard pattern to do. Tristadoji.  And that's a Tristadoji, man. If the Nikkei goes down at all tonight, that is a signal that the Nikkei is going to go all the way back down to the lower end of consolidation, which would be a total mind blow up. That is some serious business inside the Nikkei right now. Dow. Dow industrials are up $251 now, except $119. S&P is up $37. Stay right there, folks. Come right back. Tom's Daily Market Newsletter, Market Insights, is published every morning when the market's open to give you the competitive informational edge you need to succeed. These newsletters are packed full of Tom's advanced technical analysis and are geared to deliver comprehensive strategies for a successful portfolio. Get Tom O'Brien's newsletter, Market Insights, today and try all of our products and newsletters 30 days risk-free with our money back guarantee at TFNN.com. Educating Investors. Commodities. Futures. And Forex. This powerful suite of tools leverages instant trade filtering and strategy formulation to show you emerging trades before they happen.  Level the playing field with the TAS Profile Scanner, which you can find under the services tab at TFNN.com. Sign up today. 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Dow is up 269. Nasdaq is up 127. S&P's are up 39. Let's go inside the end of the X100 and take a look at what we have, the strength versus the weakness out here. Well, the strength out here today is eBay. eBay is up 4%. You get Fox up 3.5 and you get Walgreens boots up 3.4. Taken away from it. Electronic Arts is down 5.4%. You get Starbucks off 3.8. Pindu Odua's off 3 and Qualcomm's off 2.3. Now, if we go over to Electronic Arts, I want to show you something here with Electronic Arts because this is, if you really want to get your head wrapped around price and volume, this is like a classic and it's very subtle. So if you have my book, The Out of Time in the Trade, Your Ultimate Trading and Mastery System, you've seen this before. And this is one of these deals that's harder to teach this than to actually, you got to be through it a few times. So I'm going to go through this a little bit slow so you can really kind of get it. And remember the program's archived in case you're in your car. But this is a classic, man. This is what it is. So I have a six month daily up. What you're going to see is that first Electronic Arts hit a high up here with $348.89. Now when it hit that high of $348.89, you can see the volume had contracted dramatically after you actually went down with some volume about a month before that. So we'll start at that level. You start at the high of the $146.89. Bottom line, it failed on price with light volume. Bottom line ends up going sideways for a bit. Then basically takes a huge deep dive on the 5th of August. On the 5th of August it goes from a price point of $145 to $134. Now what you have there is that you get a high volume low. And when you look at this you're going to see that it grinded all the way back top side again. That's what it did. It grinded, grinded, grinded. You got volume in the equity on $1.5 million, $147.13. Well the high of what it was going after was $146.45. You had $1.6 million there. It got up to that level with $1.5. Now this is where it's subtle here folks and this is what you want to just wrap your head around. So watch what happens here. It's $1.695 that you came down on. You have light, light, light volume. You get up to that level again and you do $1.52. So at that point there it's like oh man okay are you going higher or not. That's a toss up but you do have lighter volume by I think $200,000. Actually more than $200,000. $260,000. So you still have a good what that's good 15%. But that's not where you really get the signal. What happens then is this. Then you get to a higher high with $767,000. And when you do that you're failing on price and failing on volume on the test of that was generated out here on the 31st as well as the high volume downdraft of August 5th. Okay. What ends up happening? And you're going to see this. Bottom line, those sideways gets a little bit lower. Volume expands as it gets lower. Gets higher. Yesterday do nothing and slam. You come down. You come down hard. This baby, my point more than anything is that if you basically wrap your head around this on the way up and way down and it keeps it keeps you in an aspect that, okay, now does it want to break out the lows? What does it want to do? Does it want to break it? Is there more pressure on the way down? Is there more pressure on the way up? If it wants to go higher what should happen is that there be less pressure on the way down then as it goes up you're going to see expansion of volume as it goes up. That's electronic odds. Now watch this. We'll go to Starbucks. Starbucks is in trouble. Starbucks is going to be an ABC structured down. So Starbucks is down 450 today. What you have with Starbucks, Starbucks bottom line is down off its highs. You can see it doesn't have any volume at highs. Light volume at highs tremendously. It made a high with 4.8 million shares. Failed. Couldn't handle price. Comes back up. Now watch this one. This comes up to ice. Ice is a Wyckoff term that's also in time in Australia but that's where it's basically broken down somewhat on the third of August. Comes up to that level. Couldn't hold price. Now what you've done is that you've come down to the B point. The B point is 113.61. Well you broke the B point today and you broke it with volume folks. So what happens now is this. When you break a B point and you break it with volume but you close above it that just turns into a complex ABC structure on the way down. So my take is that you still have an ABC structure on the way down. Your A point is there as a 126. Your B point is 113. So is it 26? No, I said yes. So you get 13 bucks. Your C point is up here at 120. So we're talking about 107. Now you're talking about 107. There's a swing point there. And let's put this up on a weekly. We put this on a weekly. Bottom of the consolidation is 109. The top of the worst strength is 107. So this looks to me it's going to 107. That being said it can go a lot lower man because Starbucks basically has going forward without volume going all the way up. But there's two of those right there inside of the MDX100 that could give you just a real nice clue as to how markets work. And it blows my mind. There's no doubt man. Let me tell you something.  You can test that high but when you test that high on light of volume and you have a high volume low and you get a failure on price of volume. That's where it wants to go man. It blows my mind about the whole aspect of how technically price and volume go is that they actually can get it up that high with no volume and then it lets loose again on the downside. Let's go with the pendur do and take a look at that. That's down 307 today. And this baby here so this came this had a low that was generated out there at $74 and it gets up to $108 this is backing down. This is going to consolidate for a while. That's what it looks like. We were talking about when in Las Vegas sands yesterday they're having some serious problems out here today. Wins down another $650 so when was trading before they opened yesterday at $104 it's $85 where's Las Vegas sands? When we went to the numbers yesterday it was interesting Las Vegas sands actually had a bigger exposure to Macau but it rejected lower price there at $3615. I wouldn't be buying this. I wouldn't buy anything folks that you have to depend on China for because what you're going to see here what that was all about was when and Macau is that that's all about the aspect. Right now if you do business in Macau the way it worked out is that the local companies had owned 10% of the company. Bottom line they came out with the memo yesterday morning and guess what they're going to own a lot more of it. Number one number two which I think it's the biggest thing picture that you're a big whale in China right do you think you're really going to go to Macau and start gambling millions of dollars when you get every freaking watchdog in the world there and you know that they're clamping down no that ain't going to happen man the big gamble is going to stay away for a bit stay right there folks come right back Are you having fun trading the markets but having trouble finding like-minded individuals to discuss your trading and investment ideas with become an apex predator in the trading markets and join the Tiger's Den trading room only at tfnn.com The Tiger's Den is an exclusive trading room where successful traders from around the world come to exchange trades and ideas join the den and surround yourself with the sharpest minds in the trading world Subscribers to the Tiger's Den are also the first to have their questions answered live on air and can privately chat with our tfnn hosts live 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they're estimates they raised their own estimates and you'll see this at $230 they came out and said they raised their own estimates intraday by 5% to 7% now folks companies in the 90's used to do this all the time to basically rocket their stock well bottom line would end up happening is that yes the stock rocketed up to $59.54 and some fund must have been just waiting there to pounce on it because look at this 10 minute bar $2.5 million bang the next bar $5.4 million shares Cisco just did itself in whatever else they said at the forum they're at right now at the investor day are in general because now with this thing sets up now a run down of $54 when you get such a failure it's pretty incredible too so it's going to be wild watching this basically shake out and you know that's so intriguing when that happens intraday because you don't see it a lot anymore intraday and the 90's it used to happen all the time it goose it right before the close 15 minutes before the close and always you really had to get straight in the 90's folks is that where I say well Sun Micro Microsoft Dell and Cisco were going those four right there and that's when the NDX100 was hot if you got those right you could really push things around it was pretty crazy man but guess what that just backfired on Cisco whatever that was they just either gave a big fund to it a large way let's go take a look at some of the higher volume equities here in this marketplace Apple just doesn't have any strength man Apple's up 93 cents you're dealing at 149 but there's not much there we have Bank of America is up 43 cents you got Uber is down 50 you got Win Reserves off 630 that's a big one we talked about that not a whole lot of action higher volume ones inside the Dow industrials the strength versus the weakness point wise out here you get the Dow up 260 a lead in the Dow is United Health up 37 positive points Microsoft 33 Caterpillar 22 taking away from it Goldman's 5 no big deal if we take a look at the Dow industrials now what you're going to see the Dow has been the end to see lead in us lower folks and what you have out here is that we got let's see you have to get anything yeah 35,000 091 it has to get back above that okay in order to basically even think about going after a tie a once it got below that area folks that's trading a lower zone my take is that the Dow's going down to this 17th of a 18th of June is the one of the numbers the other one's July right is that July 19th yeah July 19th and July 19th would be first but the bottom line the lower end of this consolidation is down to that level TWI let's see what we got here this is Titan I believe this is the wheel people Titan is national yeah so this is the lowest 254 the highs 1182 we next time they come up with numbers is on the 5th of November look at this this hasn't got the traction okay so you rejected lower price let me see 454,000 okay so you did reject lower price with tremendously lighter volume let me put this on a weekly for a second okay so last May of 2020 or $1.16 you go to 1182 you pull back into strength see what this was too okay so this did almost a 50% retracement of its move you came back into some strength with tremendous lighter volume now the strength that you came back in was the strength from January of 2021 okay um you rejected lower price tremendously lighter volume even last week we had well last week is a holiday 1.3 million it was a week before 1.3 so that's still good yeah that's just saying to me you rejected lower price right now now the real question is going to be can you build some cars to get to higher price that's how that baby is set up right now and if we take a look at what they're looking for in November they're going to be looking for $415 million which is slightly lower than the last quarter and uh the next quarter is $399 but then it goes up tremendously again so the bottom line is that they are looking for a couple softer quarters we go and take a look at the oil contract the oil contract this thing uh this wants higher price man yeah you're up to $228 you've done $380,000 contracts that's that we won't do that now see this gets interesting this is almost an ABC structure on the way up but what happens is that even though we have $380,000 you won't get $20,000 contracts even though we got a half hour left because what does happen with the oil market folks most of this contract volume inside of the oil market bottom line comes in very early in the day that being said if I put the oil market on a continuous contract so you can get a little bit more clarity technically on this watch this I'll put this on a weekly and you're going to see that it's like glaring at you that this $76.98 is gained that's how this is kind of set up right now uh so I expect we're going to see it let's go to the XLE uh XLE yeah you're up in volume uh well let's go to the OIH because I know we have a couple traders out there trading this OIH okay so this got volume behind the move today yeah this is a nice move man it's taking it's swing point out oh you look at this you got an ABC up okay hold let's do it $193.97 ooh it's a big one man holy cow it's 29 bucks uh $77 that's $201, $203 so $203 is coming at us and $215 is your next swing point $203 is coming at you that's how this baby is set up right now you get volume coming in we get $1.2 million shares today your B point on this is $603,000 shares B point is $193.97 this one's higher price let's go see what Exxon and Chevron are doing and Exxon right now that's a $0.78 maybe that's paying a 6% dividend man unbelievable Chevron that's paying a 5.4% dividend that's at these prices Exxon looks a little bit stronger than Chevron right now stay right there folks come right back are you in the market for buying or selling real estate in the bay area including the surrounding St. Petersburg Tampa and Clearwater markets Tiger Real Estate LLC is a firm that has extensive experience in the Tampa Bay area whether you're looking to sell your current property for maximum value or you're in the market for a second home or investment property Tiger Realty has the experience across all areas of real estate in the Tampa Bay area to help buyers and sellers make the most informed decisions across all price levels from the price you should be paying per square foot in certain up and coming areas to the type of cash flow investment properties are capable of creating Tiger Real Estate can help you make the best decision when it comes to all areas of the market before you make one of the biggest decisions of your financial future call Tiger Real Estate LLC today at<PHONE_NUMBER> or email us at tiger at tfnn.com that's<PHONE_NUMBER> call us today the technology around us is changing every day with so much happening it can seem impossible to keep up with all the information David White's investment newsletter the technology insider is designed to give you all the information you need to understand the technology that shapes today's markets and tomorrow's future David White has made his living staying on the cutting edge of technology this weekly newsletter will give you specific recommendations for valued tech stocks as well as entry prices, target prices and stops to set for each trade Dave delivers his weekly newsletters every Friday with updates throughout the week you can get the technology insider at tfnn.com for only $0.50 sign up for David's newsletter the technology insider and get an inside look at everything the technology sector has to offer try it risk free today with our 30 day money back guarantee tfnn educating investors biotech is booming but for how long whether you think the biotech bull has room to run or has run its course trade LABU or LABD directions daily S&P biotech bull and bear ETFs visit directioninvestments.com slash biotech today an investor should consider the investment objectives risks charges and expenses of the direction shares carefully before investing the prospectus and summary prospectus contain this and other information about direction shares to obtain a prospectus or summary prospectus please contact direction shares at<PHONE_NUMBER> the prospectus or summary prospectus should be read carefully before investing an investment fund is subject to risk including the possible loss of principal the funds are designed to be utilized only by sophisticated investors such as traders and active investors distributor for side fund services LLC free at 1-877-927-6648 internationally at<PHONE_NUMBER> welcome back folks down investors right now I'm going to check up 130 S&P's up 40 let's go to our man Jose and Lakeland what's going on brother you there okay I don't have a mr. left him but I think he was last time I talked to him he was trading Nike and uh hello what's going on man hey how's it going Tom Tom, uh, a couple weeks ago you made a provocative statement uh you said something about Wells Fargo kind of negative do you care to retract that statement no no I mean Tom they did give me a sunbeam toaster 20 years ago when I opened the checking account that's all the best of the old reason I'm asking that's a beautiful thing man totally yeah it did burn out and burn down it's shorted out and burned down my kitchen I'm not too happy but anyway Tom royal gold uh I'm long royal gold right now against my better judgment okay so I see it pop up two and a half in the morning and then fades away all day yeah and it let's see 109.95 okay this closes all right man it's it's the one it's the 109.01 that you gotta watch okay so where I'm going with that Jose that's the high volume low of I mean uh June 18th I mean June 8th all right no June 18th lower price again this morning at that at that level so that's saying higher price is coming at you man I mean it's it's a close call here but that's the gold pullback with the general market all right listen I know you know they didn't this is what it would end up happening last week when we got that strength that was the first day that we actually got strength with the market was coming down you know so it's we know we've been doing this long enough that most of the time the general market pulls back goes pulled back to you know that's that's we know that that's the bottom line that being said when we got that strength last week gold the gold the gold miners and the oil they're the ones that went higher dramatically and stayed higher so right now the miners have outperformed the metal and that's what I was waiting for so is that what you're seeing the miners seem to be yes there's no doubt about that right today or so and right and these are up right right yeah hey what you do at Nike oh boy I was long I was short now I'm out okay I thought you may have been wrong on that big rally last week at Nike but I think you'll end up being correct again and that gap will get filled to what 155 150 yeah it'll take a while but it got down to 155 67 today and rejected price so you'll get some kind of a bounce but it's building cause to get into that gap man so it's going to be intriguing watching that play out because we know Nike's a great company man I mean if Nike he goes into that gap man everything's going down do you know what I mean it's like okay yeah it'll it'll head down with everything else I think this um I think you'll probably get a rally right through options expiration Friday but I don't know no that AMD also I can see that cause even Dave White was talking about in his show you know this is prior to this market going up here folks okay that you know he watches these option market makers in a big way what they're looking at and they weren't getting nervous at all you know bottom line saying that the S&P should be somewhere around 4500 where 4487 so I mean well he uses technical analysis I use a Ouija board so that's good I like okay man have a great one have a safe one<PHONE_NUMBER> let's go take a look at the XAU and the HUI because I believe what we actually got here yesterday is that we're going to need more volume in both of these let's see so we popped yesterday with 25 million well that's not bad you're going into 22 on the XAU we take a look at the gold bugs index this is up 26 cents today 19 versus 17 okay so hey in both of those this is a little tricky in both of those they both want higher price man meaning that I thought we actually didn't have enough volume this day but we did we take a look at oh look at this thing this is unbelievable okay so PPLT this is the platinum ETF in this baby for the year $77 the highs $122 you're trading $88 let me bring this up okay I gotta bring this up this has been a one way route in the way down PPL I'm gonna bring up actually the platinum active contract first you're at $934 you know what is amazing folks okay and many of you have listened for years you know commodities no doubt can go higher lower all the above and the play on platinum has been one of the most dramatic plays that I've seen in a long period of time meaning that gold's almost $2,000 platinum is always traded higher than gold excluding the last five years and platinum is only $934 so the question that one of the tigers have out here right now let me put this on a generic contract P01 when I go to a generic contract folks by the way what I'm doing is this the reason I go to that is that the future contract only gets back three months so when you go to the generic you can put this on a longer term monthly okay and weekly okay first I'm gonna put on a three year weekly just to see where you came back into okay so you came back into strength that was developed out here going back to December of 2020 you rejected lower price right now yeah listen man if you're looking to take a shot at the PPLT I'd say that yeah you can do it and if you're looking folks you know for physical metal you know the bottom line is that let me compare it right now compare this to there it is okay so I just put this up comparing it to gold and I'm gonna put this on a ten year monthly and where do you see this okay so that doesn't work let's see five year weekly you're gonna see that okay so going back yeah going back to 2017 oh here's the cross right here the cross is right there right yellow yellow it's the gold yeah yellow is the gold right so the cross happened in 17 we're 21 you know and what I mean by the cross folks the cross meaning that platinum went under the price of gold gold keeps going platinum keeps going down yeah this would be a great place to start it so if you do buy platinum folks right what you want to do don't you know you if you if you're gonna keep something for a long period of time yeah you could go get platinum coins but you gotta pay the premiums gonna be huge on them what you can do is you can just get the platinum bounces they are a better investment if you're looking just to make money on the platinum on the metal itself that's that's how that goes okay because the thank you Mike the bars the bars the bars are much better investment and you know tomorrow morning I might buy a bunch of it myself you know it's like it's it's hard to comprehend but guess what the bottom line it is what it is and platinum is out here at nine and change in golds at 1795 say right there folks come right back down down industrials right now up to 16 as except 132 s&p's are up 40 will come right back sharpening your skills as an investor is like getting better at playing a musical instrument you have to practice sure but you also need excellent instruction from experts at tfnn you'll get advice and guidance from the authority and technical market analysis and it's not just dry tedious text either tfnn airs live financial content streamed live on tfnn.com and tfnn's youtube channel with tiger tv live every market day from 8 30 a.m. to 4 p.m. eastern for free each host is an experienced trader and gives their take on the market while taking calls and questions 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Petersburg, Florida your investment can be anywhere from 100,000 to 500,000 do you want to make 1000 per year on 100,000 invested or 7,000 per year on a secured target first mortgage the target first mortgage program may be just the program for you the target first mortgage program pays 7% per year paid monthly for more information you can call or call don't forget you can listen to tfnn live on your mobile device 24 hours per day go to tfnn.com then hit watch tiger tv that's tfnn.com then hit watch tiger tv welcome back folks so the target just turned me on to I know this company but man I haven't seen it for a long time this is a score folks okay the low for the year out here is $10 the highest 24 this pays a 9.4% dividend so if you're looking for dividend stocks simultaneously now this is a very illiquid stock okay so you can see it only did 11,000 shares today what do we have here 17,000 yes oh my god it does no volume but man that is one monster premium and so this is predicated more than likely on the price of platinum and palladium okay when you break this down they do palladium 33 billion rhodium 32 billion platinum 17 billion other gold and silver 6.4 million billion rather they do 137 million a billion rather a billion with a B this is something to look at man if you're in this for a longer period of time now the spread is big you can see the spread is 72 cents just to get out of it so you don't buy this to trade it but you don't see 9.4% dividends and the way that a lot of these work because of South Africans I've held these South Africans sometimes for quite some time and they pay twice a year folks okay big dividends though depending what you well they're not big dividends not like that and thank you because I'm buying this frickling tomorrow for sure I mean I'll take the shot that platinum is close to the lows and I think we can get some action man you know we don't have to go hand over fist but 10% premium man 10% dividend that's we've seen them I've seen them in the pipelines and all that but we know what the aspect there is after I want to look at this with the platinum number is play the platinum number which is out there someone just sold down those NQ's that's for sure always remember folks the bank and claw your heart out the bull can run you over and thank god there's always another trade health habits and prosperity have a great night folks have a safe night come back and visit Tommy tomorrow morning kicks us off 9am great show have a great win folks have a safe building wealth trading in the stock market seems impossible