epigenetics and epistasis

Insulators

A class of DNA sequence elements that possess a common ability to protect genes from inappropriate signals emanating from their surrounding environment Two activities define an insulator : Enhancer blocking Barrier to the spread of chromosomal silencing

summary

A gene is a nucleotide sequence in a DNA molecule that acts as a functional unit for the production of a protein, a structural RNA, or a catalytic RNA molecule. In eucaryotes, protein-coding genes are usually composed of a string of alternating introns and exons. A chromosome is formed from a single, enormously long DNA molecule that contains a linear array of many genes The human genome contains 3.2 × 1022 different autosomes and 2 sex chromosomes. Only a small percentage of this DNA codes for proteins or structural and catalytic RNAs. A chromosomal DNA molecule also contains three other types of functionally important nucleotide sequences: replication origins and telomeres allow the DNA molecule to be completely replicated, while a centromere attaches the daughter DNA molecules to the mitotic spindle, ensuring their accurate segregation to daughter cells during the M phase of the cell cycle. The DNA in eucaryotes is tightly bound to an equal mass of histones, which form a repeating array of DNA-protein particles called nucleosomes. The nucleosome is composed of an octameric core of histone proteins around which the DNA double helix is wrapped. Despite irregularities in the positioning of nucleosomes along DNA, nucleosomes are usually packed together (with the aid of histone H1 molecules) into quasi-regular arrays to form a 30-nm fiber. Despite the high degree of compaction in chromatin, its structure must be highly dynamic to allow the cell access to the DNA. Two general strategies for reversibly changing local chromatin structures are important for this purpose: ATP-driven chromatin remodeling complexes, and an enzymatically catalyzed covalent modification of the N-terminal tails of the four core histones

initiation of the PIC eg of different histone modifiers and chromatin remodelling depending on promoter

A real example: Steroid hormones ( lipid soluble so they can pass the lipid membrane without having to bind to a domain ) bind to their receptors inside the cell. e.g. thyroid hormone, oestrogen, testosterone Class I receptors are normally located in the cytoplasm. Upon hormone binding, they translocate to the nucleus to bind to DNA that'll recruit. co-activators and activate transcription

TADs

A topologically associating domain (TAD) is a self-interacting genomic region, meaning that DNA sequenceswithin a TAD physically interact with each other more frequently than with sequences outside the TAD.[1]The median size of TAD in mouse cells is 880 kb which is found to be similar in non-mammalian species.[2]Boundaries at both side of the these domains are conserved between different mammalian cell types and even across species[2] and are highly enriched with CCCTC-binding factor (CTCF) and cohesin binding sites.[1] In addition, some types of genes (such as transfer RNA genes and housekeeping genes) appear near TAD boundaries more often than would be expected by chance.he functions of TADs are not fully understood and still is a matter of debate. Most of the studies indicate TADs regulate gene expression by limiting the enhancer-promoter interaction to each TAD,[5] however, a recent study uncouples TAD organization and gene expression.[6] Disruption of TAD boundaries are found to be associated with wide range of diseases such as cancer,[7][8][9] variety of limb malformations such as synpolydactyly, Cooks syndrome, and F-syndrome,[10] and number of brain disorders like Hypoplastic corpus callosum and Adult-onset demyelinating leukodystrophy.[10] The mechanisms underlying TAD formation are also complex and not yet fully elucidated, though a number of protein complexes and DNA elements are associated with TAD boundaries. However, the handcuff model and the loop extrusion model are described to describe the TAD formation by the aid of CTCF and cohesin proteins.[11] Furthermore, it has been proposed that the stiffness of TAD boundaries itself could cause the domain insulation and TAD formation

EPISTASIS

A type of gene interaction in which one gene alters the phenotypic effects of another gene that is independently inherited.

concluding

ATP-dependent chromatin remodelers regulate chromatin structure during multiple stages of tran- scription. We report that RSC, an essential chromatin remodeler, is recruited to the open reading frames (ORFs) of actively transcribed genes genome wide, suggesting a role for RSC in regulating transcription elongation. Consistent with such a role, Pol II occu- pancy in the ORFs of weakly transcribed genes is drastically reduced upon depletion of the RSC cata- lytic subunit Sth1. RSC inactivation also reduced histone H3 occupancy across transcribed regions. Remarkably, the strongest effects on Pol II and H3 occupancy were confined to the genes displaying the greatest RSC ORF enrichment. Additionally, RSC recruitment to the ORF requires the activities of the SAGA and NuA4 HAT complexes and is aided by the activities of the Pol II CTD Ser2 kinases Bur1 and Ctk1. Overall, our findings strongly implicate ORF-associated RSC in governing Pol II function and in maintaining chromatin structure over tran- scribed regions Hmgn3a stimulates Glyt1 expression and promotes acetylation of H3K14ac across the Glyt1 locus in hepa cells. H3K14ac is known to recruit the RSC complex, which promotes Pol II elongation.

Nucleosomes Are Usually Packed Together into a Compact chromatin fibre

Although long strings of nucleosomes form on most chromosomal DNA, chromatin in a living cell probably rarely adopts the extended "beads on a string" form. Instead, the nucleosomes are packed on top of one another, generating regular arrays in which the DNA is even more highly condensed. Thus, when nuclei are very gently lysed onto an electron microscope grid, most of the chromatin is seen to be in the form of a fiber with a diameter of about 30 nm, which is considerably wider than chromatin in the "beads on a string" form (see Figure 4-23). Several models have been proposed to explain how nucleosomes are packed in the 30-nm chromatin fiber; the one most consistent with the available data is a series of structural variations known collectively as the Zigzag model (Figure 4-29). In reality, the 30-nm structure found in chromosomes is probably a fluid mosaic of the different zigzag variations. We saw earlier that the linker DNA that connects adjacent nucleosomes can vary in length; these differences in linker length probably introduce further local perturbations into the zigzag structure. Finally, the presence of other DNA-binding proteins and DNA sequence that are difficult to fold into nucleosomes punctuate the 30-nm fiber with irregular features (Figure 4-30). Several mechanisms probably act together to form the 30-nm fiber from a linear string of nucleosomes. First, an additional histone, called histone H1, is involved in this process. H1 is larger than the core histones and is considerably less well conserved. In fact, the cells of most eucaryotic organisms make several histone H1 proteins of related but quite distinct amino acid sequences. A single histone H1 molecule binds to each nucleosome, contacting both DNA and protein, and changing the path of the DNA as it exits from the nucleosome. Although it is not understood in detail how H1 pulls nucleosomes together into the 30-nm fiber, a change in the exit path in DNA seems crucial for compacting nucleosomal DNA so that it interlocks to form the 30-nm fiber (Figure 4-31). A second mechanism for forming the 30-nm fiber probably involves the tails of the core histones, which, as we saw above, extend from the nucleosome. It is thought that these tails may help attach one nucleosome to another thereby allowing a string of them, with the aid of histone H1, to condense into the 30- nm fiber (Figure 4-32).

The zig-zag model for nucleosome packing into a 30nm fibre

Based on X-ray crystallography of 4 nucleosomes on positioning sequences

nucleosome

Bead-like structure in eukaryotic chromatin, composed of a short length of DNA wrapped around a core of histone proteins A nucleosome is a section of DNA that is wrapped around a core of proteins. Inside the nucleus, DNA forms a complex with proteins called chromatin, which allows the DNA to be condensed into a smaller volume. When the chromatin is extended and viewed under a microscope, the structure resembles beads on a string. Each of these tiny beads is a called a nucleosome and has a diameter of approximately 11 nm. The nucleosome is the fundamental subunit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a histone octamer. Each histone octamer is composed of two copies each of the histone proteins H2A, H2B, H3, and H4. The chain of nucleosomes is then compacted further and forms a highly organized complex of DNA and protein called a chromosome.

TAD organisation generally correlates very well with:chromatin accessibility, replication timing, epigenetic profiles and transcriptional status.

But: identification of a TAD can depend on sequencing depth and the algorithm used ( more sequence analysis, more depths .. affects interpretation of results as they will be more accurate ( ?) , there are many similar but different models of chromatin organisation. The mechanistic link between transcription and TAD structure appears complex and possibly indirect: Global TAD disruption has mild effects on the transcriptome Specific TAD deletion doesn't always have the predicted effect on phenotype. within a tad all those chromatin loops can interact with each other

Enhancer blocking elements flank the chicken b-globin locus

CTCF is the only enhancer blocking protein identified in vertebrates CTCF sites interact with each other in cells to form chromosomal loops MODEL: Enhancer blocking may be a result of separating enhancers and promoters onto different loops CTCF binds to HS4 and 3'HS and this eventually prevents the FOLR1 receptor from a captivating the B-globin cluster genes in their own cell type and prevents the enhancer cluster from activating folr1

glycine transporter 1 expression

ChIP followed by quantitative PCR at reveals enrichment of histone modifications across a gene locus. investigate where the diff histone modifications were found across this gene so they did crosslinking chip followed by immunoprecepitation for couple of diff modifications ( H3K4ME3/H3K9ac) and compared it with plain h3 that isn't modified which gives similar level of enrichment across the gene then real time PCR with diff primer set were used ( points are graphs) strong peaks can be found at the end of genes aswell

The b-globin genes site within an open chromatin domain

Chromatin immunoprecipitation of active histone modifications in RBC Graph shows results of 28 PCR assays Assay measures the enrichment of specific genomic sequences

the DNA in one cell is about 1.5m long.how is it folded and organised inside the nucleus

Chromatin is a complex of DNA and protein found in eukaryotic cells.[1] Its primary function is packaging long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in reinforcing the DNA during cell division, preventing DNA damage, and regulating gene expression and DNA replication. During mitosis and meiosis, chromatin facilitates proper segregation of the chromosomes in anaphase; the characteristic shapes of chromosomes visible during this stage are the result of DNA being coiled into highly condensed chromatin. To fit our genomes into a tiny cell, the DNA of each chromosome is coiled, compacted, and coiled up some more. At the primary level of compaction, the DNA is wrapped around a group of special proteins called histones. When DNA wraps around a group of histones, it forms a nucleosome. You can think of the system as DNA "thread" wound around a protein "spool". The first scientists who saw nucleosomes with an electron microscope remarked that they looked like "beads on a string," though we now know that nucleosomes are more like "string wrapped around beads." Each nucleosome is made of four different histones -- H2A, H2B, H3, and H4. Two molecules of each histone come together to form an octamer. (Note the prefix "octa" -- an octamer is just a complex made of eight proteins.) The DNA string wraps around the histone octamer bead to create the nucleosome. So the process of fitting all of that DNA into a tiny cell nucleus begins with wrapping the DNA around histones into a nucleosome. But it doesn't end there -- the chain of nucleosomes coils around a central axis to get even more compact. As depicted in the diagram at the right, the packaging actually takes place at a number of scales: The DNA wraps around histone octamers to form a "beads on string" fiber approximately 10 nanometers (nm) in width. The beads-on-string structure in turn coils into a 30-nm-diameter fiber that packs the nucleosomes more closely together. During cellular interphase -- the period in which the cell is not actively dividing -- "scaffold" proteins fold the 30-nm fibers into a somewhat more compact structure to fit within the nucleus. During cell division, the chromatin, through the action of additional scaffold proteins, is radically packed and condensed to form the metaphase chromosome that divides and passes the DNA carrying the genetic code to the two daughter cells.

histone modifications mark chromatin stress

Chromatin profiling has emerged as a powerful means of genome annotation and detection of regulatory activity. The approach is especially well suited to the characterization of non-coding portions of the genome, which critically contribute to cellular phenotypes yet remain largely uncharted. WLS is poised in ES cells, repressed in GM12878 cells, and transcribed in HUVEC and NHLF. Its TSS switches accordingly between poised (purple), repressed (grey) and active (red) promoter states; enhancer regions within the gene body become strongly activated (orange, yellow); and its gene body changes from low signal (white) to transcribed (green). These chromatin state changes summarize coordinated changes in many chromatin marks; for example, H3K27me3, H3K4me3 and H3K4me2 jointly mark a poised promoter, while loss of H3K27me3 and gain of H3K27ac and H3K9ac mark promoter activation. Bottom: Nine chromatin state tracks, one per cell type, in a 900kb region centered at WLS summarize 90 chromatin tracks in directly-interpretable dynamic annotations, showing activation and repression patterns for 6 genes and hundreds of regulatory regions, including enhancer states. b, Chromatin states learned jointly across cell types by a multivariate HMM. Table shows emission parameters learned de novo based on genome-wide recurrent combinations of chromatin marks. Each entry denotes the frequency with which a given mark is found at genomic positions corresponding to the chromatin state

3C

Chromosome conformation capture techniques (often abbreviated to 3C technologies or 3C-based methods[1]) are a set of molecular biology methods used to analyze the spatial organization of chromatin in a cell. These methods quantify the number of interactions between genomic loci that are nearby in 3-D space, but may be separated by many nucleotides in the linear genome.[2] Such interactions may result from biological functions, such as promoter-enhancer interactions, Interaction frequencies may be analyzed directly,[4] or they may be converted to distances and used to reconstruct 3-D structures.[5] The chief difference between 3C-based methods is their scope. For example, when using PCR to detect interaction in a 3C experiment, the interactions between two specific fragments are quantified. In contrast, Hi-C quantifies interactions between all possible pairs of fragments simultaneously. Deep sequencing of material produced by 3C also produces genome-wide interactions maps. detects contacts between different regions of the chromosome such as enhancers and promoters - find an area where the enhancer and promoter are in physical contact where they loop ( crosslinking fixes loop interactions) , formaldehyde - the preparation is then digested with a restriction enzyme ( 6bp) that removes the rest of the loop and you're left with the structure - ligase then ligates the two shorter bits together - reverse crossing by treatment with salt/protease and heat - new species of DNA which is essentially a ligation between those two short bits that were bound at the loop - q-rt-pcr with particular primers to detect this new junction if those two regions were physically interacting you'll be able to detect PCR between this 2 region if they're ligated together .. if they didn't interact you won't get. pcr product since they didn't ligate together 3C you decide where your start point is .. you have a forward primer that's always there and a several different reverse primers and see if any of these interact with the forward primer .. if the enhancer was interacting with the promoter you would see a per product between blue and green PCR but not between the black one the increased signal tells us there's looping between the enhancer and the promoter Many variants of the 3C offer higher throughput (4C, 5C, HiC) • Now possible to study chromosomal interactions on a whole genome scale • Possible to infer chromosome folding from HiC data

Steroid hormone receptors manipulate chromatin structure to regulate transcription

Class I receptors only bind DNA when bound by their ligand. They recruit co-activators, including histone acetyl transferases, to stimulate gene expression. eg testosterone and glucocorticoid receptors. Class II receptors always bind DNA, but in the absence of ligand ( hormone) they recruit co-repressors, and in the presence of ligand (agonist) they recruit co-activators. eg retinoic acid and thyroid hormone receptors

DHS mapping

DHS mapping reveals long range gene regulatory elements Mapping is performed in different cell types Elements shown in red are found only in red blood cells These are enhancers of the β-globin genes HS4/3'HS - always there constitutive gives an indication where the regulatory elements are

CpG islands

DNA regions rich in C residues adjacent to G residues. Especially abundant in promoters, these regions are where non-methylation of cytosine usually occurs. Many CpGs are found in dense clusters called "CpG islands" which are mostly unmethylated regardless of cell type. Approximately 60% of human gene promoters are CpG islands. Many of these genes are tissue specific. However, their promoters remain unmethylated even when genes are not expressed. some regions where there's no methylation ( cpG islands ). the density is higher than the rest o the genome but because they're not methylated they don't help promote chromatin silencing

DNase1 hypersensitive sites

DNase I hypersensitive sites (DHSs) are regions of chromatin that are sensitive to cleavage by the DNase I enzyme. In these specific regions of the genome, chromatin has lost its condensed structure, exposing the DNA and making it accessible. This raises the availability of DNA to degradation by enzymes, such as DNase I. These accessible chromatin zones are functionally related to transcriptional activity, since this remodeled state is necessary for the binding of proteins such as transcription factors.

Enhancer transcription and enhancer-promoter communication

Enhancers may use different but not mutually exclusive ways to communicate with their corresponding promoters depending upon the physical distances between these cis-elements. Possible models of communication include a linking model, a tracking/facilitated tracking or scanning model, and a looping model.n the linking model, a number of TFs are recruited sequentially following the binding of a first activator protein (such as pioneer TFs) that induces an open chromatin state at a promoter-proximal sequence during differentiation A chain of TFs then progressively extends along the chromatin fiber from the enhancer to the transcribed gene, and recruits the PIC to the core promoter for transcription initiation. This linking model may only apply to gene regulation between the proximal and core promoter, because this cascade of recruitment may not occur across very long distances.he looping model has been proposed to allow for direct contact of promoters and enhancers over long distances. In this model, the enhancer and promoter make contact by looping out the intervening chromatin (Fig. 2, B and C). The resulting chromatin loops are stabilized by protein-protein interactions. A number of large proteins and protein complexes have been proposed to bridge and direct physical contact between enhancers and promoters, to facilitate both chromatin looping and promoter-proximal pausing of RNAPII. These complexes and proteins include chromatin-remodeling complexes, Mediator, CCCTC-binding factor (CTCF), Cohesin, and many lineage-determining transcription factor

RNAi-mediated centromeric heterochromatin assembly in fission yeast (S. pombe)

Epigenetic mechanisms regulate genome structure and expression profiles in eukaryotes. RNA interference (RNAi) and other small RNA-based chromatin-modifying activities can act to reset the epigenetic landscape at defined chromatin domains. Centromeric heterochromatin assembly is a RNAi-dependent process in the fission yeast Schizosaccharomyces pombe, and provides a paradigm for detailed examination of such epigenetic processes. Here we review recent progress in understanding the mechanisms that underpin RNAi-mediated heterochromatin formation in S. pombe. We discuss recent analyses of the events that trigger RNAi and manipulations which uncouple RNAi and chromatin modification. Finally we provide an overview of similar molecular machineries across species where related small RNA pathways appear to drive the epigenetic reprogramming in germ cells and/or during early development in metazoans. RNA interference (RNAi) is a conserved silencing mechanism whereby double-strand RNA induces specific down-regulation of homologous sequences. In the fission yeast Schizosaccharomyces pombe, centromeric heterochromatin assembly is an RNAi-dependent process. Noncoding RNAs transcribed from pericentromeric repeat sequences are processed into short interfering RNAs (siRNAs) that direct the Argonaute-containing RNA-induced transcriptional silencing (RITS) effector complex to homologous nascent transcripts. RITS is required for H3K9 methylation by the histone methyltransferase (HMT) Clr4; conversely, H3K9 methylation can attract RITS to chromatin via binding of the chromodomain protein Chp1. This codependency has hampered dissection of the order of events and mechanisms of cross talk between the RNAi and chromatin modification machineries. To tackle this problem, we have developed systems that reconstitute heterochromatin at a euchromatic locus, using either hairpin triggers or DNA-tethered chromatin-modifying complexes. These systems reveal that RNAi is sufficient to promote heterochromatin assembly in cis and that direct recruitment of the HMT Clr4 can bypass the role of RNAi in heterochromatin assembly. We have also characterized a new pathway component, Stc1, that translates the RNAi signal into chromatin marks. We discuss the implications of these findings for our understanding of the mechanism and function of RNAi-directed heterochromatin assembly at centromeres. Figure 1. Fission yeast RNAi-mediated centromeric heterochromatin assembly model. (a) Long double stranded RNA (dsRNAs) transcripts are synthesised by RNA polymerase II (RNAPII) activity on the repetitive centromeric outer repeats (otr), which are processed by the ribonuclease Dcr1 into small interfering RNAs (siRNAs). The Argonaute protein Ago1 binds the siRNAs, which guide the effector complex RITS (Ago1, Chp1 and Tas3) to homologous nascent transcripts via a base-pairing mechanism. The RNA-dependent RNA polymerase activity within the RDRC complex that associates with RITS generates more dsRNAs, ultimately increasing the pool of siRNAs. The CLRC complex is tethered to sites of active RNA interference via interaction with RITS. This is mediated mainly through a LIM-domain protein, Stc1 that bridges the two complexes. In the wake of RNAPII passage multiple histone deacetylation activities render the chromatin permissive to CLRC-mediated histone H3 methylation on lysine 9 (H3K9me). Specific chromodomain factors such as Swi6 or Chp1 bind H3K9me thereby assembling the heterochromatin platform on the otr repeats, where more RNAi and heterochomatin factors dock to perpetuate the assembly process. (b) General overview of the different protein complexes and the relevant factors involved in RNAi and heterochromatin formation. RNAPII, RNA polymerase II; HDACs, histone deacetylases; dsRNA, double stranded RNA; siRNAs, short interfering RNAs; Ac, acetylation; Me, methylation; blue balls, histone octamers; S. pombe

epistasis

Epistasis is a phenomenon in genetics in which the effect of a gene mutation is dependent on the presence or absence of mutations in one or more other genes, respectively termed modifier genes. In other words, the effect of the mutation is dependent on the genetic background in which it appears. Epistatic mutations therefore have different effects on their own than when they occur together. Originally, the term epistasis specifically meant that the effect of a gene variant is masked by that of a different gene

nucleosome remodelling enzymes

Eukaryotic chromatin is kept flexible and dynamic to respond to environmental, metabolic, and developmental cues through the action of a family of so-called "nucleosome remodeling" ATPases. Consistent with their helicase ancestry, these enzymes experience conformation changes as they bind and hydrolyze ATP. At the same time they interact with DNA and histones, which alters histone-DNA interactions in target nucleosomes. Their action may lead to complete or partial disassembly of nucleosomes, the exchange of histones for variants, the assembly of nucleosomes, or the movement of histone octamers on DNA. "Remodeling" may render DNA sequences accessible to interacting proteins or, conversely, promote packing into tightly folded structures. Remodeling processes participate in every aspect of genome function. Remodeling activities are commonly integrated with other mechanisms such as histone modifications or RNA metabolism to assemble stable, epigenetic states. Very large (~2 MDa) multi-subunit complexes • SWI/SNF and ISWI families. Active subunits are Brg1 or Brm1. • Hydrolyse ATP to remodel nucleosomes • Increase access to the DNA by nucleases and transcription factors • Need to be recruited to chromatin (e.g. by transcription factors) • Can also mediate exchange of histone variants changes the positions of the blue DNA making it accessible for tf's // they don't directly bind to DNA.

The HS4 insulator reduces variability in transgenic animals

Experiment: Microinjection of tyrosinase transgene into albino mouse embryos to regenerate agouti pigment no insulators : variable results if it is flanked : tyrosinase is expressed most consistent all offsprings express it

histone folding

Figure 4-27. The assembly of a histone octamer. The histone H3-H4 dimer and the H2A H2B dimer are formed from the handshake interaction. An H3-H4 tetramer forms the scaffold of the octamer onto which two H2A-H2B dimers are added, to complete the assembly. The histones are colored as in Figure 4-26. Note that all eight N-terminal tails of the histones protrude from the disc-shaped core structure. In the x-ray crystal (Figure 4-25), most of the histone tails were unstructured (and therefore not visible in the structure), suggesting that their conformations are highly flexible A histone fold is a structurally conserved motif found near the C-terminus in every core histone sequence in a histone octamer responsible for the binding of histones into heterodimers. The histone fold averages about 70 amino acids and consists of three alpha helices connected by two short, unstructured loops.[1] When not in the presence of DNA, the core histones assemble into head-to-tail intermediates (H3 and H4 first assemble into heterodimers then fuse two heterodimers to form a tetramer, while H2A and H2B form heterodimers[2]) via extensive hydrophobic interactions between each histone fold domain in a "handshake motif".[3] Also the histone fold was first found in TATA box-binding protein-associated factors, which is a main component in transcription Histone folds play a role in the necleosomal core particle by conserving histone interactions in the nucleosomal core particle when looking at interface surfaces. These contained more than one histone fold. The structure of the nucleosome core particle has two modes that have the largest interaction surfaces with are in groups H3-H4 and H2A-H2B heterotypic dimer interactions. When looking at the H2A-H2A structure it has a modification of the loop at the interface that excludes it from clustering with the same interface of other structures. Which makes it have a different function in the transcriptional activation. Also the two modes are distinct due to having the longest helix chains. These use the handshake interactions between the two histone folds, while they also use it to make themselves unique comparted to the rest of the modes. Similarly modes 5 and 7 of the core nucleosome particle use two types of histone fold dimers which show that all histone domains share a similar structural motif to be able to be able to interact with one another and to interact in different ways. Showing how flexible and adaptive the structure of histones are. H4 and H2A can form an internucleosomal contacts that can be acetylated to be able to perform ionic interactions between two peptides, which in turn could change the surrounding internucleosomal contacts that can make a way to opening the chromatin histone tails contact linker DNA and other nucleosomes and are important for chromatin folding and co-factor recruitment ( proteins to chromatin ) shows end terminal tails that stick out between the DNA

ATP-driven Chromatin Remodeling Machines Change Nucleosome Structure

For many years biologists thought that, once formed in a particular position on DNA, a nucleosome remained fixed in place because of the tight association between the core histones and DNA. But it has recently been discovered that eucaryotic cells contain chromatin remodeling complexes, protein machines that use the energy of ATP hydrolysis to change the structure of nucleosomes temporarily so that DNA becomes less tightly bound to the histone core. The remodeled state may result from movement of the H2A-H2B dimers in the nucleosome core; the H3 H4 tetramer is particularly stable and would be difficult to rearrange (see Figure 4-27). The remodeling of nucleosome structure has two important consequences. First, it permits ready access to nucleosomal DNA by other proteins in the cell, particularly those involved in gene expression, DNA replication, and repair. Even after the remodeling complex has dissociated, the nucleosome can remain in a "remodeled state" that contains DNA and the full complement of histones but one in which the DNA histone contacts have been loosened only gradually does this remodeled state revert to that of a standard nucleosome. Second, remodeling complexes can catalyze changes in the positions of nucleosomes along DNA (Figure 4-33); some can even transfer a histone core from one DNA molecule to another. Cells have several different chromatin remodeling complexes that differ subtly in their properties. Most are large protein complexes that can contain more than ten subunits. It is likely that they are used whenever a eucaryotic cell needs direct access to nucleosome DNA for gene expression, DNA replication, or DNA repair. Different remodeling complexes may have features specialized for each of these roles. It is thought that the primary role of some remodeling complexes is to allow access to nucleosomal DNA, whereas that of others is to re-form nucleosomes when access to DNA is no longer required (Figure 4-34). Chromatin remodeling complexes are carefully controlled by the cell. We shall see in Chapter 7 that, when genes are turned on and off, these complexes can be brought to specific regions of DNA where they act locally to influence chromatin structure. During mitosis, at least some of the chromatin-remodeling complexes are inactivated by phosphorylation. This may help the tightly packaged mitotic chromosomes maintain their structure

Position effect variegation (PEV)

Genes positioned adjacent to a repressive chromatin domain can become silenced The extent of heterochromatin spreading will vary depending on- the position of the gene with respect to the source of repression- the relative abundance of repressive versus active chromatin factors This can result in variegated gene expression between cells in a tissue ( sometimes the red box will be silenced and some times it won't ) inherited during mitosis

HS4 lies at the boundary between the b-globin domain and a 16kb region of heterochromatin

H3K9me3

summary

HAT (GCN5) writer - adds acetyl group Bromodomain protein ( CBP) is a reader binds acetyl lysine HDAC is an eraser that removes acetylation ( HDAC1)

Competition between chromatin states

HS4 is acting as a barrier its blocking the spread of blue to the active region

TADs identified using Hi-C are represented like this:

Hi-C used to identify TADs in the human HOXA locus, with a CTCF insulator site between them. TADS only become apparent when assaying many cells, so represent an average of interactions over time. Some TADs are delineated by CTCF binding sites, but not all CTCF sites are TAD boundaries

HOX Genes as an Example of TAD-Switching During Controlled Developmental Gene Expression

Hox gene clusters are a well-studied system of chromatin architecture. Gene expression inside a cluster is coordinated in space and time during embryonic development. Notably, these genes are arranged in the genome in an order that corresponds to their expression along the body axis, a phenomenon termed colinearity (reviewed in [30]). The HoxD cluster is involved in many stages of vertebrate limb development, but early development requires two successive waves of expression. In early limb formation, sequential activation of anterior and central HoxD genes (13 genes in total) occurs, which drives the development of the proximal limb (forearm and arm). Later, a second, partially overlapping, sub-group of HoxD genes (Hoxd8 to Hoxd13) in the more distal part of the gene cluster are expressed, defining what will become the extremities of the limb (hand and digits). The mechanism that regulates gene transcription in early and late phases of HoxD cluster expression involves a switch in contact between promoters and regulatory elements, and these define two distinct TADs, called here the telomere-proximal and centromere-proximal TADs (T-TAD and C-TAD, respectively; Figure 2A). The T-TAD and C-TAD are on opposite sides of the HoxD cluster [28]. Their spatial separation argues for a 180 degrees switch in long-range folding of the locus during limb development. Using a mouse model, gene expression and histone marks were monitored at two different temporal and spatial stages of arm/forearm limb development. The transcriptional induction of Hoxd11 to Hoxd8, key targets for early limb development, correlates with the spread of the active H3K27ac mark towards the C-TAD. Coincident with this, Hoxd4 and Hoxd3 experience transcriptional repression and a loss of the active histone mark H3K27ac, as H3K27me3 accumulates and spreads towards the T-TAD. Interestingly, the expression of Hoxd11 to Hoxd9 genes in the early phase is a prerequisite to the proper expression of these genes in the distal region later in development. This was not the case for Hoxd13, however, which is expressed only in the distal region, thanks to regulatory elements in the C-TAD. this study suggests that long-range chromatin contacts play a role in the fine-tuning of gene expression during development and that changes in chromatin interactions involve regulatory elements localized in different topological domains

3C studies have shown that CTCF often forms the basis for chromatin loops.

Initial simple model: Interactions between enhancers and promoters within the same loop are favoured, those between loops are blocked. Loop domains bordered by CTCF sites typically associated with cohesin. Interactions between enhancers and promoters within the same loop are favored; those between loops are blocked. At loops bordered by the strongest and most conserved CTCF sites, CTCF is oriented as shown, with the N terminus of each protein facing into the loop enhancer cannot activate a gene on another loop

However, Hi-C experiments have suggested the chromatin is organised into TADs: (Topologically Associated Domains) consisting of multiple loops

Inside TADs, long-range chromatin interactions can be detected in a dynamic manner between promoters and enhancers (bold stars), which leads to the activation or repression of gene transcription. Inside the nucleus one can visualize the segregation of chromatin into different domains which are often defined in relation to landmarks such as the nucleolus, nuclear pore complex (NPC) or nuclear envelope (NE). Chromatin is organized into unit blocks termed topologically associated domains (TADs) (see text), and these can be classified into two types of compartments: A-type, which are active domains (blue, green); or B-type, which are inactive (orange, red, yellow). This reflects the enrichment of certain proteins and the expression of genes within[52_TD$DIFF] a given TAD. Inside TADs, long-range chromatin interactions can be detected in a dynamic manner between promoters and enhancers (bold stars), which leads to the activation or repression of gene transcription. TADs associated with the nuclear lamina have also been defined as LADs. Importantly, one LAD can consist of few TADs or a TAD can contain one or more LADs.

Lamina-associated domains

Lamina-associated domains (LADs) are parts of the chromatin that heavily interact with the lamina, a network-like structure at the inner membrane of the nucleus.LADs consist mostly of transcriptionally silent chromatin, being enriched with trimethylated Lys27 on histone H3, which is a common posttranslational histonemodification of heterochromatin. LADs have CTCF-binding sites at their periphery.

Evidence suggests that the loops are formed by extrusion through cohesin rings. Extrusion may be driven by cohesion itself or transcription

Loop extrusion as a model for TAD formation. Cohesin is continuously loaded and unloaded from interphase chromatin, and bidirectionally extrudes loops while engaged on chromatin. Elements forming a roadblock to extrusion, such as the collision of convergent CTCF-bound sites, become a more stable barrier for the extruded TAD dna is pulled out of cohesion loops ( RNA polymerase)

lysine 4 methylation // writers readers and erasers of H3K4me3

MLL - writer JARID1 - demethylates MLL Is unregulated in cancer chromodomains - bind methyl lysine CHD1 - remodelling enzyme

pre-initiation complex (PIC)

Mediator recruited, General transcription factors released, and RNA polymerase recruited. Occurs at promoter Assembly of the pre-initiation complex allows phosphorylation of the polymerase C-terminal domain (CTD) .CTD phosphorylation allows the polymerase holoenzyme to clear the promoter and initiate transcription

Chromatin Immunoprecipitation (ChIP)

Method of gene expression analysis: o Fixed tissue is sonicated to shear small bits of DNA off then protein bound to DNA is immuno-precipitated with an antibody o Quantify DNA bound to that protein with PCR or microarray analysis chromatin preparation 1) crosslinking method - fixes cells where proteins interact with DNA ( formaldehyde ) chromatin then gets sonicated.. so the chromosomes are broken down since they are very long . short chromatin fragments .. good when looking at where TFs are bound 1) native method - no cross linking method used here, use live cells to work with .. nuclease digestion and purification of chromatin its very look at looking at histone modifications cut chromatins are stable . you get a lot more chromatin too so its more efficient method shown in picture Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly defining cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers.[1] Briefly, the conventional method is as follows: DNA and associated proteins on chromatin in living cells or tissues are crosslinked (this step is omitted in Native ChIP). The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion. Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated from the cell debris using an appropriate protein-specific antibody. The associated DNA fragments are purified and their sequence is determined. Enrichment of specific DNA sequences represents regions on the genome that the protein of interest is associated with in vivo.

Alteration of CTCF binding sites can affect gene expression

Mutation of Isocitrate dehydrogenase leads to increased expression of the oncogene PDGFRA in some types of glioma Effect of methylation of a CTCF site on boundary activity. In certain human gliomas, the product of the mutated isocitrate dehydrogenase (IDH) gene interferes with DNA demethylation at a critical CTCF-binding site, resulting in loss of CTCF binding and insulation and inappropriate activation of the PDGFRA gene, a glioma oncogene, by a distal enhancer (green hexagon) ( idh has additional activity to do with DNA methylation

chromatin structure

Negatively charged DNA loops twice around histone octamer (2 each of the positively charged H2A, H2B, H3, and H4) to form nucleosome bead. H1 ties nucleosomes together in a string. (Think of "beads on a string"; H1 is the only histone that is not in the nucleosome core.) In mitosis, DNA condenses to form mitotic chromosomes.

HS4 marks the 5' boundary of the b-globin domain

Open chromatin domain spans ~30 kb Ends abruptly at HS4 HS4 is found in all cell types and is not an enhancer

A CTCF binding site near EPHA4 is important for proper limb formation

Proper limb formation requires the TAD borders of the EPHA4-containing TAD. Elimination of the TAD border was sufficient for phenotypic limb malformation (e.g., polydactyly) and ectopic interactions with genes of flanking TADs, as well as gain of expression pattern similar to EPHA4 by these misregulated genes deletion of ctcf binding site is consistent with loss of enhancer activity / misregulation of gene expression and a phenotypic effect

CTCF often interacts with the cohesin ( protein complex) at the base of chromatin loops - condenses chromosomes

Proposed mechanisms for generating loop domains terminated by convergently oriented CTCF sites Cohesin bound to chromatin extrudes a loop and continues until it reaches a properly oriented CTCF site on each arm of the loop. It then stops searching; CTCF either comigrates with cohesin or is prebound, but cohesin is deposited only when CTCFs are properly oriented. Two possible configurations of cohesin are shown, corresponding to proposed models of cohesin interaction with chromatin . This process would require an energy source, suggested here to be an as yet unspecified helicase, shown as orange arrows.

linker histone H1

Pulls nucleosomes together into the 30-nm fiber promotes nucleosome compaction bind on the outside of nucleosome near where liker dan enters and leaves the nucleosome and the tails are positively charged they interact with he negatively charger linker DNA and help bind DNA and compact chromatin Histone H1 (green) consists of a globular core and two extended tails. Part of the effect of H1 on the compaction of nucleosome organization may result from charge neutralization: like the core histones, H1 is positively charged (especially its C-terminal tail), and this helps to compact the negatively charged DNA. Unlike the core histones, H1 does not seem to be essential for cell viability; in one ciliated protozoan the nucleus expands nearly twofold in the absence of H1, but the cells otherwise appear normal. with linker Histone H1, the linker DNAs are brought together/ zig-zag

Heterochromatin and barrier elements

Repressive chromatin can spread Repressive chromatin marks promote chromatin condensation (heterochromatin) and dominantly interfere with transcription

Mutations have DIFFERENT phenotypes

Seen among mutations at different genes/loci that cause DIFFERENT phenotypes: e.g,:Albinism (recessive) is epistatic to ALL other mutations that affect EYE color (blue, green, brown, beige.....). The Albinism locus "allows" color. Reflects SOME in vivo functional (inter-)dependence of the gene products Tells you that there IS some functional link - but does NOT tell you HOW - that link could be direct or indirect, via protein, RNA, metabolite... Allows a Geneticist to CLUSTER and ORDER genes (products) in in vivo functional pathways and based SOLELY on analyzing phenotype of progeny • CLUSTER to ORDER:If a cluster of genes-can ORDER products by action • ORDER to CLUSTER:If you can robustly order using epistasis,then they MUST act in pathway

Lysine methylation

Similar to HATs, Lysine can be methylated up to 3 times to reduce affinity for DNA backbone. -increase transcription Protein lysine methylation is a critical and dynamic post-translational modification that can regulate protein stability and function. This post-translational modification is regulated by lysine methyltransferases and lysine demethylases. The process of protein lysine methylation consists of enzymes adding or removing methyl groups on particular substrates33,34. The lysine ε-amino group of proteins can accept up to three methyl groups, resulting in either mono-, di-, or trimethyl lysine, (me1, me2, or me3) with the various methylation states of lysines exerting distinct functions35. To date, more than 50 KMTs and 20 lysine demethylases (KDMs) have been reported HMT they use S-adenosyl methionine to take a methyl group and add it to the end of lysine residue .. you can have them added to mono/di/tri those have different structures demethylase removes the methyl

Covalent Modification of the Histone Tails Can Profoundly Affect Chromatin

The N-terminal tails of each of the four core histones are highly conserved in their sequence, and perform crucial functions in regulating chromatin structure. Each tail is subject to several types of covalent modifications, including acetylation of lysines, methylation of lysines, and phosphorylation of serines (Figure 4-35A). Histones are synthesized in the cytosol and then assembled into nucleosomes. Some of the modifications of histone tails occur just after their synthesis, but before their assembly. The modifications that concern us, however, take place once the nucleosome has been assembled. These nucleosome modifications are added and removed by enzymes that reside in the nucleus; for example, acetyl groups are added to the histone tails by histone acetyl transferases (HATs) and taken off by histone deacetylases (HDACs). The various modifications of the histone tails have several important consequences. Although modifications of the tails have little direct effect on the stability of an individual nucleosome, they seem to affect the stability of the 30-nm chromatin fiber and of the higher-order structures discussed below. For example, histone acetylation tends to destabilize chromatin structure, perhaps in part because adding an acetyl group removes the positive charge from the lysine, thereby making it more difficult for histones to neutralize the charges on DNA as chromatin is compacted. However, the most profound effect of modified histone tails is their ability to attract specific proteins to a stretch of chromatin that has been appropriately modified. Depending on the precise tail modifications, these additional proteins can either cause further compaction of the chromatin or can facilitate access to the DNA. If combinations of modifications are taken into account, the number of possible distinct markings for each histone tail is very large. Thus, it has been proposed that, through covalent modification of the histone tails, a given stretch of chromatin can convey a particular meaning to the cell (Figure 4-35B). For example, one type of marking could signal that the stretch of chromatin has been newly replicated, and another could signal that gene expression should not take place. According to this idea, each different marking would attract those proteins that would then execute the appropriate functions. Because the histone tails are extended, and are therefore probably accessible even when chromatin is condensed, they provide an especially apt format for such messages. As with chromatin remodeling complexes, the enzymes that modify (and remove modifications from) histone tails are usually multisubunit proteins, and they are tightly regulated. They are brought to a particular region of chromatin by other cues, particularly by sequence-specific DNA-binding proteins. We can thus imagine how cycles of histone tail modification and demodification can allow chromatin structure to be dynamic locally compacting and decompacting it, and, in addition, attracting other proteins specific for each modification state. It is likely that histone-modifying enzymes and chromatin remodeling complexes work in concert to condense and recondense stretches of chromatin; for example, evidence suggests that a particular modification of the histone tail attracts a particular type of remodeling complex. Moreover, some chromatin remodeling complexes contain histone modification enzymes as subunits, directly connecting the two processes.

Chromosomal position effect on transgene expression

The expression of a randomly integrated transgene (green dots) is dictated by its chromatin environment - chromosomal position effect Major hindrance for• academic research• industrial production of therapeutic proteins from transgenic cells/animals • human genetic therapy low gene expression = low transgene expression chromatin env dependant

the core histone proteins

The high-resolution structure of a nucleosome core particle, solved in 1997, revealed a disc-shaped histone core around which the DNA was tightly wrapped 1.65 turns in a left-handed coil (Figure 4-25). All four of the histones that make up the core of the nucleosome are relatively small proteins (102 135 amino acids), and they share a structural motif, known as the histone fold, formed from three α helices connected by two loops (Figure 4-26). In assembling a nucleosome, the histone folds first bind to each other to formH3 H4 and H2A-H2B dimers, and the H3 H4 dimers combine to form tetramers. An H3 H4 tetramer then further combines with two H2A-H2B dimers to form the compact octamer core, around which the DNA is wound (A) Each of the core histones contains an N-terminal tail, which is subject to several forms of covalent modification, and a histone fold region, as indicated. (B) The structure of the histone fold, which is formed by all four of the core histones. (C) Histones 2A and 2B form a dimer through an interaction known as the "handshake." Histones H3 and H4 form a dimer through the same type of interaction, as

Relationship with promoter-enhancer contacts

The majority of observed interactions between promoters and enhancers do not cross TAD boundaries. Removing a TAD boundary (for example, using CRISPR to delete the relevant region of the genome) can allow new promoter-enhancer contacts to form. This can affect gene expression nearby - such misregulation has been shown to cause limb malformations (e.g. polydactyly) in humans and mice. Computer simulations have shown that transcription-induced supercoiling of chromatin fibres can explain how TADs are formed and how they can assure very efficient interactions between enhancers and their cognate promoters located in the same TAD.

Positional enhancer blocking by an insulator

The position-dependence of enhancer blockers dictates that an enhancer targets a promoter via the intervening chromatin fibre prevents enhancer from acting on promoter CTCF is the only known enhancer blocker it has to be between the enhancer and promoter

PIC

The remodelling complex slides a nucleosome along, allowing TFIID to bind. Other basal transcription factors are recruited along with TFIID, allowing transcription initiation.

co-activators and co-repressors

They do not directly bind DNA Work through protein-protein (or RNA) interactions

general

Transcription factors are a group of proteins that can bind to specific sequences upstream of the 5′ terminus of target genes, typically considered the promoter region1,2. In this way these transcription factors can inhibit or enhance gene expression and ensure specific temporal target gene expression3. Under normal circumstances, promoter-specific transcription factors contribute in basic biological activities including differentiation4, development5, and metabolism6. Importantly, dysregulation of these transcriptional programs can lead to malignant growth and cancer formation7,8. Transcription factors can be subject to a variety of enzyme-catalyzed post-translational modifications (PTMs) in response to environmental changes, especially in disease occurrence and tumorigenesis9,10. These transcription factor PTMs are added and removed by the same enzyme families that are involved in histone modifications like acetylation, phosphorylation, and methylation11,12,13. Specific modifications have selective effects on transcription factor functions, resulting in specific gene expression alterations. It has been demonstrated in the literature that transcription factor phosphorylation and acetylation can promote carcinogenesis by regulating transcriptional activity14,15. We have greatly improved our understanding of transcription factor methylation with the development of mass-spectrometric techniques in the last few decades16. Protein methylation occurs at specific sites on substrates, with lysine methylation being one of the important forms17,18,19. The lysine (K) ε-amino group of protein substrates can accept up to three methyl groups, resulting in either mono-, di-, or trimethyl lysine, in a process termed lysine methylation20,21,22. Recent studies have revealed that a number of transcription factors have been found to be modified by lysine methyltransferases (KMTs)23,24,25, resulting in specific gene expression alterations26,27. The abnormal expression of methyltransferases in many tumor types, which has been proven to be associated with tumorigenesis and cancer development, has become the focus of anticancer research28,29,30. In addition to histone methylation31, transcription factor methylation modification is also an important aspect for the development of cancer27,32. To date, multiple studies have demonstrated that lysine methylation of transcription factors can directly regulate target gene expression by altering transcription factor stability and function. In this review, we summarize recent studies on lysine methylation of transcription factors, aiming to underline the biological significance and highlight the potential clinical value of lysine methylation of transcription factors in cancer.

zig-zag model of nucleosome fibre

Two models for chromatin secondary structure.The solenoid model is characterized by interactions between consecutive nucleosomes (n, n + 1; a,b), whereas the zigzag model implies interactions between alternate nucleosomes (n, n + 2; c,d). The alternative nucleosomes are numbered from N1 to N8. In the solenoid model proposed by Rhodes and colleagues111, the 30 nm chromatin fibre is an interdigitated one-start helix in which a nucleosome in the fibre interacts with its fifth and sixth neighbour nucleosomes. Alternative helical gyres are coloured blue and magenta (b). In the zigzag model, the chromatin fibre is a two-start helix in which nucleosomes are arranged in a zigzag manner such that a nucleosome in the fibre binds to the second neighbour nucleosome108. Alternative nucleosome pairs are coloured blue and orange (d). The two models also differ in the trajectory and degree of bending of the DNA that connects two nucleosomes (linker DNA). Despite years of effort, the structure of the 30 nm fibre has not been resolved. Two competing models, the solenoid and zigzag arrangement of nucleosomes, have been proposed on the basis of in vitro data2 (FIG. 4). In the 'one-start' solenoid model, consecutive nucleosomes interact with each other and follow a helical trajectory with bending of linker DNA107. In the 'two-start' zigzag structure, two rows of nucleosomes form a two-start helix so that alternate nucleosomes (for example, N1 and N3) become interacting partners, with relatively straight linker DNA108 (FIG. 4). Twisting or coiling of the two stacks can produce different forms of the zigzag model. The main reason the structure of the 30 nm structure has not been resolved is that its highly compacted nature prevents the path of the DNA from being visualized by electron microscopy109. Crosslinking studies and the ultimate crystallization of a tetranucleosome array (albeit with a very short 167 bp nucleosome repeat length (NRL)) have provided the strongest evidence to date that the 30 nm fibre adopts a zigzag structure108,110. However, the biological relevance of this structure is questioned given that the short repeat length and the absence of linker histone H1 is not typical for higher eukaryotes111,112. Linker histone H1 (for which several variants and post-translationally modified versions have been identified) binds tightly to nucleosomes with linker DNA113,114 and stabilizes the higher-order structure of chromatin111. Although its biological role is still controversial115, it is clear that most nucleosomes in metazoans are bound by H1. Cryo-electron microscopy on long regular nucleosomal arrays with defined and constant repeat length, and in the presence of linker histone, resulted in a very tight compaction of reconstituted chromatin fibres, especially for repeats above 207 bp109,116. At that time, this was interpreted as evidence for a multiple- start interdigitated solenoid model111 because the observed high nucleosome packing ratio was not compatible with two-start zigzag or classic one-start solenoidal models. A more recent molecular modelling study showed that these compact structures were also consistent with a set of multi-start chromatin fibre models with extended nucleosome linker DNA higher salt conc - more squished up more compact (A and B) Electron microscopic evidence for the top and bottom-left model structures depicted in (C). (C) Zigzag variations. An interconversion between these three variations is proposed to occur by an accordion- like expansion and contraction of the fiber length. Differences in the length of the linker between adjacent nucleosome beads can be accommodated by snaking or coiling of the linker DNA, or by small local changes in the width of the fiber. Formation of the 30-nm fiber requires both histone H1 and the core histone tails; for simplicity, neither is shown here

A working model for HS4 barrier activity

USF binds at HS4 insulator recruits HATs and active chromatin modifiers which will lead to the modification of nearby nucleosomes so when heterochromatin is spreading along the chromosome it comes along the active modification sites one model is that the active modifications are preventing the spread of repressive modifications by recruiting many diff factors leading to histone methylation and acetylation and changing modifications to act as a high powered high active chromatin locus that repress the spreading of heterochromatin Ubiquitous DNA binding proteins, including USF, interact with the constitutive 5HS4 insulator element. USF mediates the recruitment of histone modifying enzymes, including those identified in this study. Nucleosomes in the immediate vicinity of the 5HS4 element become constitutively enriched in H3K4-Me and histone acetylation at multiple sites (Ac) as a result. Immediately upstream of the 5HS4 insulator is a 16 kb region of uninterrupted condensed chromatin that is deacetylated and enriched in H3K9-Me. Histone hyperacetylation and H3K4-Me at the 5HS4 element act as a specific barrier to the propagation of H3K9-Me by HMTs. We do not exclude the possibility that the 5HS4 element recruits as yet uncharacterized histone modifications that also oppose chromatin condensation pathways

Position-effect variegation in nature

Variable heterochromatin spreading in tissue stem cells is fixed during differentiation Variegated chromatin states are inherited in subsequent cell clones diff levels of spreading ( some on some off) this is inherited during mitosis

Natural use of PEV to generate variable phenotypes

Variegated expression of Agouti coat pigment gene in mice (silencing from transposon next to gene) Variegated expression of Dfr-B pigment gene in morning glory (silencing from transposon next to gene) Variegated expression of orange pigment gene in calico cats (silencing from random X-inactivation, so females only) position effect genes lead to human genetic disease ( env of gene causing phenotype not mutation)

"

We have studied developmentally regulated patterns of histone acetylation at high resolution across ∼54 kb of DNA containing three independently regulated but neighboring genetic loci. These include a folate receptor gene, a 16 kb condensed chromatin region, the chicken β-globin domain and an adjacent olfactory receptor gene. Within these regions the relative levels of acetylation appear to fall into three classes. The condensed chromatin region maintains the lowest acetylation at every developmental stage. Genes that are inactive show similarly low levels, but activation results in a dramatic increase in acetylation. The highest levels of acetylation are seen at regulatory sites upstream of the genes. These patterns imply the action of more than one class of acetylation. Notably, there is a very strong constitutive focus of hyperacetylation at the 5′ insulator element separating the globin locus from the folate receptor region, which suggests that this insulator element may harbor a high concentration of histone acetylases.

The chicken b-globin neighbourhood

What prevents the β-globin LCR activating FOLR1? What prevents FOLR1 HSA/B from activating the β-globin genes?

Bromodomains ( proteins ) bind

acetylated lysine Acetylated lysine residues are bound by bromodomain-containing proteins e.g. HATs ( + feedback ) , SWI/SNF remodelling complexes. The acetylated lysine recruits protein complexes that have bromodomains, such as: • nucleosome remodelling complexes eg SWI/SNF • co-activators eg CBP/P300 These promote the binding of other transcription factors and the mediator complex, leading to RNA pol II recruitment and formation of the pre-initiation complex (PIC) .. promote initiation of transcription by stabilising the pre initiation complex some bromodomains are inhibited by drugs called ( BET inhibitors currently in clinical trials for several cancers)

enhancers

an enhancer is a short (50-1500 bp) region of DNAthat can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur.[1][2]These proteins are usually referred to as transcription factors. Enhancers are cis-acting. They can be located up to 1 Mbp (1,000,000 bp) away from the gene, upstream or downstream from the start site.[2][3] There are hundreds of thousands of enhancers in the human genome.[2] They are found in both prokaryotes and eukaryotes.[4]

The chicken b-globin locus

beta globin comes in 4 diff genes that are expressed at diff times in development beta globin works with alpha globin to bind oxygen to red blood cells and the affinity for o2 is imp in the embryos of human and chickens the affinity for oxygen is higher because the blood of the embryo needs to bind oxygen more tightly than the surrounding env whereas when the baby Is born it docent need to bind oxygen so tightly / weaker o2 affinity Betahatchiing expressed when hatched these genes are expressed in RBC this locus is near other genes ( olfactory epithelium) and FOLR1 a diff cell type to RBC

HMGN proteins

bind to the nucleosome core particle .. 2 hmgn proteins is bound to the nucleosome and we were wondering how do they work to regulate gene expression

histone modifications mark transcription events

by screening where those modifications occur across the genome and lining them up with genes we can get average profiles of modifications active gene - low methylation activity at start site compared to other parts which helps gene expression but its not entirely clear repressed - methylation across the body of genome di/tri on an average you'll find one or other poised - many tf's but not activey transcribed they are just ready to get transcribed

histone acetyltransferase (HAT)

complexes recruited by TF's they don't have sequence specific DNA binding activity and they don't know where to go within the genome which is why they are recruited by TF's. Gnc4 - has DNA binding domain and activation domain which recruits Gcn5 is HAT.. acetylates the nucleosomes in the vicinities of tf binding site UAS- upstream activation sequence Examples of HATs are Gcn5, PCAF, P300/CBP ( 2 very similar proteins)

aims

define epistasis / order gene product action by epistasis/understand the molecular basis of epistasis eg : worm lab

Studying enhancer interactions at the human b-globin locus

erythroid-specific transcription factors bind to LCR enhancers Stage-specific transcription factors bind to globin promoters chicken-globing locus is small so we can't really get quality/diffrential data since everything is close to each other with the human beta globin locus is larger and easier to get differential data from as everything is distant - locus control region : cluster of strong enhancers to drive transcription - Ggamma/Agamma genes = development - alpha/beta expressed later on

The HS4 insulator reduces variability in transgenic cells

experiment: Viral vector delivery of the human b-globin gene into mouse erythroid cells no flanked insulator - expression variable if transgene flanked - expression similar across diff lanes 1) contain barrier elements 2) ctcf so enhancers can't over activate a gene high expression at some points its cut they're next to an enhancer

HDACs

histone deacetylases remove acetyl groups from histones HDAC (histone deacetylase) complexes are usually recruited by repressive transcription factors. URS1 ( upstream repressor sequence is bound with dna binding domain together with a repressor domain which recruits the SIN3 complex that contains HDACs ( Rpd3) that therefore deacetylases histone N terminal tails

CTCF,loops and TADs

how does the cell guarantee enhancer promoter specificity Enhancers are often within range of neighboring genes In the absence of enhancer-promoter specificity, what prevents cross-talk between neighboring gene loci of different expression programs?

Does RNAi play a role in heterochromatin formation or maintenance?

imp to keep centromeres and telomeres silenced

Repressive chromatin

in mammalian cells the vast of the genome is not coding for protein so generally the chromatin env is repressive so transposons need to be silenced and shut up as well as random recombination .. maintaining genome stability .. It can spread along the chromosomes from one region to another eg: a nucleosome is methylated by lysine 9 and the methyl marker is recruiting HP1 which is in a complex with suv39h and that can methylate the next nucleosome and so it spreads the dominant spreading mechanisms leading to silencing and repressing modifications are spread throughout the genome

30 nm fiber 2nd level of folding

interactions between nucleosomes cause the thin fiber to coil or fold into this thicker fiber thought it was solenoid of nucleosomes

Transgenes are protected from chromosomal position effect silencing when flanked by HS4

is a transgene is taken and insert it into sells and don't select for that transgene at first it will be highly expressed however over time it will be silenced and that's because most of the time that gene would've landed in a heterochromatin region cut most of our genome is heterochromatin and that will have spread over and silenced gene ( 3 week tine ) however if you flank it with HS4 from both sides, the expression from the transgene will be constant this means the spreading of heterochromatin is stopped and silencing that transgene

H3 N-terminal tail

lysine - K several lysine and arginine and sirines ( + charged) in the tail .. they both can be modified

epigenetic mechanisms for gene silencing

lysine methylation can be associated with activation or repression however this depends on what lysine is methylated and its outcome lysin-4 = associated with activated transcription found at promoters

K-9 & K-27

lysing 9 can be methylated by HMT ( suv39h ) and that is the writer that's depositing the modification .. HP1 then binds to K9 which is the reader at lysine 27 = a different complex is recruited which is the PRC2 complex and has HMT activity .. EZH2 is the one that's doing the methylation this is read by a complex of proteins called PRC1 HP1 and PRC1/2 cause transcriptional silencing and heterochromatin formation. They bind methyl-lysine using their chromodomains.

DNA methylation is an important epigenetic mark

mainly occurs in cytosine residues Approximately 80% of CpG dinucleotides in human somatic cells are subject to cytosine methylation. palindromic so if a cytosine is methylated on one strand, the cytosine on the other strand ( n+1) is also methylated most of cpg dinucleotides are methylated in the non coding regions and the coding exon as well

Transcriptional repression mediated by DNA methylation

methylation of a specific DNA sequence can prevent the tf binding

post translational modification of core histone tails

microRNA, methylation , acetylation

Enhancers as cis-regulatory DNA elements in gene activation

nhancers are distal sequences that lie upstream or downstream of the core promoter, and can activate or regulate the level of transcriptional initiation by recruiting transcription factors necessary for PIC assembly at the core promoter (Fig. 1) (26-28). In yeast, upstream-activating sequences, also known as enhancer-like sequences, are required for transcription, and are typically positioned much closer to the core promoter (2, 29). Enhancers act independently of their orientation, and their genomic location is believed to be responsible for the accurate surveillance of spatiotemporal transcription patterns during development and/or in different cell types. For example, the first mammalian enhancer was discovered downstream of the immunoglobulin (Ig) heavy-chain gene, which is necessary for the proper expression of Ig, and only exhibits enhancer activity in lymphocyte-derived cell lines and during B lymphocyte differentiation (30, 31). Enhancers help recruit RNAPII to promoters and can attract various chromatin-modifying enzymes to DNA to establish and/or maintain an active chromatin conformation via PICs (32). Enhancers can also recruit pioneer factors and lineage-specific transcription factors (TFs) as early as the ESC stage (17-22, 33). Promoters, on the other hand, are less likely to be occupied by developmentally important and lineage-specific TFs (34). Notably, enhancers are frequently marked with H3K4me1 and H3K27ac, but not H3K4me3, unless the enhancer is highly transcribed (35-38). Accordingly, putative enhancers are commonly annotated by comparing the ratio of H3K4me1 to H3K4me3, the presence of H3K27ac, the replacement of canonical histones with histone variants like H2A.Z, the binding of co-factors such as CBP/p300, and the clustered binding of multiple master TFs (20, 39-41). Enhancers can work with both homologous and heterologous promoters to increase the transcription of target genes, and can function independently of their position and orientation. Displaying DNase I hypersensitivity remains a primary criterion for identifying enhancers in mammalian genomes.

The chicken β-globin locus as a paradigm for understanding the relationship between chromatin environment and gene expression; enhancers and 3C (chromosome conformation capture)

nucleated The chicken β-globin locus is a paradigm chromatin domain Integrating studies of - gene regulation during development - chromatin structure - histone modifications - enhancers - CTCF and chromosomal looping - insulators and barriers

acetylation of histones

opens chromatin, allowing transcription Acetylation is the transfer of acetyl groups to the epsilon position of lysine residues in the tail of nucleosomal histones. It is important to distinguish this modification from N-alpha acetylation of eukaryotic structural proteins that occurs on alanine, serine, and methionine residues, and that is catalyzed by N-acetyl transferases. Histone acetylation is catalyzed by a family of enzymes called histone acetyltransferases (HAT), and it is functionally correlated with transcriptionally competent chromatin. The removal of acetyl groups is catalyzed by a family of enzymes called histone deacetylases (HDAC or HD) and is functionally correlated with transcriptionally inactive chromatin Hence, the ratio of HDAC to HAT activity determines the level of histone acetylation. forms new binding platform for proteins HDACs can be inhibited by compounds too ( TSA) shifts equilibrium and makes more acetylation happens // associated with active transcriptions

Inheritance of DNA methylation patterns following DNA replication

pattern of DNA methylation 2 parental strands are split up one encodes for new daughter chromosome and the other one for the other one when they're split up each will carry the original methyl markers and is now called hemi-methylated ( parental) strand and now synthesised strand is not methylated DNMT1 - recognized hemi-methyl state so if it sees a methyl marker on one strand it will add another one on the other strand ..DNMT1 helps to copy the pattern of methylated markers on the parental strands to the daughter strand during mitosis

epigenetic regulation

process affecting the expression of a particular gene or genes, without affecting the sequence of nucleotides making up the gene itself Epigenetic regulators (histone acetyltransferases, methyltransferases, chromatin-remodelling enzymes, etc) play a fundamental role in the control of gene expression by modifying the local state of chromatin. However, due to their recent discovery, little is yet known about their own regulation work to do two things as shown in the figure

Super enhancers

super-enhancer is a region of the mammalian genome comprising multiple enhancers that is collectively bound by an array of transcription factor proteins to drive transcription of genes involved in cell identity.[1][2][3] Because super-enhancers are frequently identified near genes important for controlling and defining cell identity, they may thus be used to quickly identify key nodes regulating cell identity.

Epigenetics

the study of environmental influences on gene expression that occur without a DNA change Meiotically and mitotically heritable changes in gene expression that are not coded in the sequence itself.( not always the case) The structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states (Adrian Bird, 2007) Allphenomenona/mechanisms that impact upon DNA function (Adele Murell, 2014)

enhancers are found to use chromosomal looping when activating their target genes

they loop over to interact with promoters

next

they wanted to see if the Hmgn3 actually binds to glyt1 is it a direct effect or is it indirect which is regulated by another factor that activates glyt1 .. crosslinking chip .. enrichment of parent cells can be shown in green .. but when hmgn3 is over expressed gly1 binds but across the gene not specifically in the promoter and that's been seen for other genes too .. then they wanted to see how does it affect histone modifications and what can be seen is that there's a peak of h-N3a cells at the start of transcription site ( promoter) this modification is not altered . HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys4 of histone H3) and H3K9ac (acetylated Lys9 of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys14 of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production.

how did it start

took out cells that didn't express hmgn3 and then made new cell lines that do express it to see what does it do to gene expression and microarrays were done and there's an increase in glycine transporter 1 this was confirmed by rt-pcr and an increase is shown

Mosaic eye colour in Drosophila

white is a red pigment gene normally located in euchromatin Transposon-mediated inversion places white flips and close to centromeric heterochromatin ( repressed and silenced) Variable heterochromatin spreading Fixed and inherited -> mosaic expression variable spreading inherited during mitoses you get a mosaic expression during development as cells multiply retain expression patterns ( daughter cells)

Probing for regulatory elements in chromatin using DNaseI

Ø Digest nuclei with increasing concentrations of DNaseI Ø Prepare the digested genomic DNA - Southern blot Ø Hybridise with a radiolabelled genomic DNA probe of interest Ø A band within the smear indicates a site of preferential cutting called a DNaseI hypersensitive site (DHS) - could be a TF binding site Dnase1 - cuts dsDNA wherever successful it cuts more if there's distortions in the double helix ( generated by protein/TF binding) DHS is always there and docent depend if beta globin is expressed int hose cell types ( not expressed in brain but can see DHS )

Summary

Ø Gene transcription is regulated within a three dimensional chromatin environment Ø Enhancers reach over long distances to open up chromatin Ø Enhancer blockers such as CTCF restrict the action of enhancers to prevent cross-talk between neighbouring gene loci Ø Insulators also act as barriers to the spread of heterochromatin Ø Insulators can therefore limit position effects and cross talk to ensure accurate gene regulation during development Ø Chromosomes are organised into TADs and LADs, and this organisation is crucial for correct gene regulation. Ø Transcribed RNA and the RNAi machinery is important for heterochromatin assembly in yeast and other organisms.

How does DNA methylation lead to transcriptional silencing?

Ø Methylation of a transcription factor binding site can prevent binding of the factor to DNA( changed structure of recognition site ) Ø DNA methylation combines with certain histone modifications and repressor proteins to form a repressive chromatin structure. This silences the majority of the genome (including centromeric regions and telomeres) and inactive genes.

Enhancer elements control the activation and chromatin remodelling of cell-type specific genes

Ø cis-regulatory ( same chromosomes as gene they're regulating ) DNA elements required for gene activation Ø mostly found at genes with cell-type specific roles Ø functionally defined by reporter or genomic deletion experiments Ø contain binding sites for transcriptional activator proteins Ø can function over very large distances (tens of kb to over a Mb!) Ø can be located either 5' or 3' of a gene Ø can be many enhancers for one gene Ø clusters of enhancers can drive chromatin opening of a gene locus Ø called Locus Control Regions

Common features in small RNA-mediated chromatin modifications

• • • A study in Caenorhabditis elegans shows that small regulatory RNAs inhibit RNAPII transcription and accumulate H3K9me at the target site . Also several reports have hinted at the occurrence of induced siRNA-directed chromatin modification events in mammalian tissue culture cells. However, these effects often appear to be relatively mild . It is possible that a weak or mixed response occurs in differentiated somatic cells because these processes are tailored to occur during gametogenesis or early development.In support of this view, a striking example is the reprogramming role played by companion cells in germline formation in the plant Arabidopsis thaliana . In the vegetative nucleus of mature pollen, the key chromatin remodeler DDM1 is specifically downregulated, which reactivates and thereby reveals transposable elements (TEs). These active TEs trigger an RNAi response, and homologous siRNAs migrate into the nucleus of adjacent sperm cells thereby silencing their TEs. This mechanism ensures that active threatening TEs are not transmitted to the next generation, since only the sperm cells contribute the paternal genetic material to the zygote and endosperm, whilst the vegetative nucleus does not. A similar process involving Ago9-associated small RNA expressed in companion cells is required to disable TEs in developing female gametes Examples from Tetrahymena, C. elegans, Arabidopsis, Drosophila.Increasing evidence that this can happen mammalian cells. How does DNA methylation fit in? Transcribed RNA can also act as a scaffold to recruit chromatin factors without the RNAi machinery Common features in small RNA-mediated chromatin modifications. A schematic representation of the conserved processes and machineries involved in epigenetic reprogramming is depicted. During cellular differentiation (e.g. gametogenesis or early development) the epigenetic landscape is altered at the chromatin level via histones or DNA modifications (e.g. methylation). Transcription at target loci produces RNA transcripts that are processed into small RNA molecules. The PIWI/Argonaute surveillance machinery binds to the small RNAs to mediate genome regulation or defense activities. Chromatin modifiers interact with the PIWI/Argonaute proteins that target genomic loci with sequences complementary to the small RNAs. Hence, local changes in chromatin are achieved in a sequence-specific manner through an RNA intermediate. Blue balls, histone octamers; Red Me, methylation on histones; Yellow Me, methylation on DNA.