[IV] The Role of Chromatin Structure in Control of Gene Expression
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[IV] The Role of Chromatin Structure in Control of Gene Expression. Overview of levels of control of gene expression Chromatin structure in active or potentially active genes Alterations in DNA methylation in active or potentially active genes

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Iv the role of chromatin structure in control of gene expression

[IV] The Role of Chromatin Structure in Control of Gene Expression

  • Overview of levels of control of gene expression

  • Chromatin structure in active or potentially active genes

  • Alterations in DNA methylation in active or potentially active genes

  • Modification of histones in the chromatin of active or potentially active genes

  • Changes of chromatin sturcture in the regulatory region of active or potentially active genes

  • Other situations in which chromatin structure is regulated


Central dogma of molecular biology

Central Dogma of Molecular Biology

DNA

mRNA

Protein

RNA

Reverse Transcription

4

Translation

Transcription

3

2

  • Replication

  • Transcription

  • Translation

  • Reverse Transcription

1

Replication

This dogma was proposed by Francis Crick in 1957 to explain the process of information transfer within cells


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Growth of E. coli Cells in a Medium Containing Glucose and Lactose

  • Cells use up glucose first and then use up lactose after a delay of one hour

  • This is called “Diauxic” = auxilium in Latin

  • Jacob and Monod studied the metabolism of lactose in details and proposed that genes involved in metabolism of lactose cluster together


Genes involved in the metabolism of lactose

Genes Involved in the Metabolism of Lactose

Jacob and Monod discovered that the following three genes are involved in the metabolism of lactose in E. coli cells

lac Z: encodes b-galactosidase

lacY: encodes lactose permease

Lac A: encodes galactoside transacetylase

These genes are clustered together

A lac I gene encodes a protein which was found to play a regulatory role in the appearance of these enzymes

Lac operon

promoter

lac Z

lac I

lac Y

lac A

I gene product is a negative regulator of the lac operon


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Regulation of Lac Operon

  • I gene product, the repressor, binds to the operator site to block the transcription of operon by RNA polymerase

  • Binding of galactoside to the I gene product release it from the operator and thus induce the transcription of the operon. This is called “Negative “ regulation leading to induction

  • Binding of CAP to the promoter enhance the transcription of the operon—”Positive” regulation

  • Regulation of Lac operon has a positive regulation component and a negative regulation component


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Lac Repressor-Operator Interactions

  • The tetrameric Lac repressor binds to lacO1, near the site where RNA polymerase binds. It also binds to lacO3 and lacO2 sites simultaneously at equilibrium. Mutation of O2 and/or O3 will reduce repression of the operon

  • Strong promoter vs. weak promoter

    -35 -10

    TTGACAT--------15 – 17 bp-------TATAAT

    Strong Promoter


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Regulation of Tryptophan Operon

  • Synthesis of tryptophan is catalyzed by five enzymes encoded by five genes: EDCBA cistrons. This system was discivered by Charles Yanofsky at Standard University

  • trp R gene encodes a protein which does not bind to operator even after dimerization. Thus the trp operon is on

  • When the intracellular levels of tryptophan is high, it binds to the trp aporepressor and the complex, in turn, binds to the trp operator to turn off the operon

  • This type of regulation is concerned as “negative regulation” leading to repression


Active rna polymerase in bacterial cells

Active RNA Polymerase in Bacterial Cells

  • For active transcription in eubacteria, the RNA polymerase needs to bind to a protein, s factor (s70), to form a complete complex

  • Sigma factor (s70)binds to the promoter DNA at -10 (six bases) and -35 (seven bases) to bring the core enzyme of RNA polymerase to initiate transcription at +1 position

    -35 -10

    TTGACAT--------15 – 17 bp-------TATAAT

  • Sigma factor (s70) acts as an initiation factor for transcription since it falls off from the RNA polymerase I once the first few bases are transcribed. It is not required for elongation of the transcription

  • Sigma factor is considered as a positive transcription factor


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Interaction of Bound NtrC and s54-RNA Polymerase

  • Most E.coli promoters interact with s70-RNA polymrase in transcription of genes

  • The transcription of some genes in prokaryotes are accomplished by s54- RNA polymerase.

  • In this case, it is regulated by an activator binding to a cis-acting element named enhancer located at –80 — -160 bp upstream from the start site

  • The promoter of gln gene is bound by NtrC (nitrogen regulatory protein C, an activator protein) which , after activation by NtrB (a protein kinase), can bind to s54-polymerase at the promoter region and initiate transcription

  • NtrC has ATPase activity, and hydrolysis of ATP is required to activate s54-polymerase


Summary

Summary

In prokaryotes, regulation of gene expression are:

Regulation of operons is achieved by positive and/or negative regulation and the RNA polymerase involved is s70-polymerase

Regulation of transcription involving s54-polymerase is achieved by two-component system (activator and another component). This format of regulation is very similar to the regulation of transcription in eukaryotic cells

Reading List:

Nobel Prize lecture by Monod (1965)

A second paradigm for gene activation in bacteria (2006)

In eukaryotes, regulation of gene expression is far more complex. Why?? How complex??


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Example I: Estrogen Control of Gene Expression

Estrogen induces ovalbumin synthesis only in chicken oviduct, but vitellogen synthesis only in the liver of both male and female chicken. These results suggest tissue specific gene expression induced by a hormonal factor, estrogen


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Example II: Developmental fate of cells can be influenced by culture medium

Results of the experiment showed that cartilage cells are capable not only of maintaining their differentiated phenotype in a particular medium supplying appropriate signals, but also remembering that phenotype in the absence of such signals. When they are placed in a medium containing the particular signal, correct differentiation is expected

This shows the stability of the commitment of the cells


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Stability of Commitment in Drosophila Imaginal Discs

  • This experiment was conducted by Professor E. Hadorn in 1963

  • This experiment demonstrated that disc cells maintained their commitment characteristics to develop into specific adult structures even after many generation of culturing in adult hemoceol

  • It clearly suggested the presence of a mechanism to maintain the long term commitment of these cells.

  • However occasionally, long term culturing of disc cells in adult flies may result in changing the commitment, i.e., homeotic transformation


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Homeotic Transformation of Cultured Drosophila Imaginal Disc Cells

  • Genital disc cells can develop into leg and/or antenna structures, Leg disc cells can develop into labial, antenna disc cells can developed into wing etc.

  • Homeotic mutation

(a). antenna

(b). Antenna-pedia mutant


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In higher eukaryotes, the expression of genes follows a cell type specific and developmental stage specific manner. How is this achieved?


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Overview of Four Basic Molecular Genetic Processes


Overview of control of gene expression

Overview of Control of Gene Expression

Regulation at transcriptional level:

Regulation of initiation of transcription

Chromatin-mediated transcriptional control

Activators and repressors interaction with transcription complex

Regulation at post-transcriptional level:

Regulation of alternative splicing leading to production of multiple isoforms of proteins

Regulation of transport of mRNA into cytoplasm

Regulation at the translational or post-translational level

Modification of the translational apparatus or specific protein factors

Micro RNAs

RNA intereference (RNAi or siRNA)

Cytoplasmic polyadenylation

mRNA degradation

Localization of mRNA in the cytoplasm


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TATA Box -25 - -35 bp

+1

Transcription

Distal promoter

Proximal promoter

Transcription start site

Regulatory region (regulatory cis element)

Structural gene

Structure of Protein Coding Gene

Two key features of transcription control:

  • Chromatin-mediated transcriptional control

  • Activators and repressors interaction with transcription complex


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Differentiation of Transcription-Active from Transcription-Inactive Chromatin

  • Transcription active chromatin can be differentiated from inactive chromatin by digestion with DNase I. This is due to the fact that inactive chromatin has a compact structure that is resistant to digestion by DNase I

  • Figure on the left depicts the protocol used to differentiate active chromatin from the inactive chromatin

  • The same method can also be used to demonstrate the presence of DNase-I hypersensitive site on the chromatin


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In Adult Erythroid Cells, the Adult b-Globin Gene is Highly Sensitive to DNase I Digestion

Chromatin isolated from erythroid cells, digested with various doses of DNase I, DNA recovered and resolved on agarose gels. Following blotting to a nylon membrane, the blot is hybridized to embryonic b-globin and adult b-globin gene

Chromatin isolated from erythroid cells, digested with various doses of DNase I. DNA is recovered and resolved on agarose gel. The DNA is hybridized to ovalbumin gene

The results showed that embryonic b-globin is less active than adult b-globin gene and ovalbumin gene is totally inactive


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Structures of Active and Inactive Genes


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Conversion Chromatin from Inactive to Active State

  • Inactive genes are assembled into compact chromatin, unavailable

    for transcription

  • Activator proteins bind to specific DNA (cis-acting control elements) and interact with mediators to decondense chromatin

  • This process will lead to conformational change of chromatin and result in genes available for transcription

Question: How is this achieved??


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Epigenetics Change


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Irreversible and Reversible Genetic Changes

  • Irreversible genetic change: genetic change as the consequence of mutation or loss of genetic materials

  • Reversible genetic change: Genetic change as the consequence of modifying the DNA such as epigenetic changes or other change resulting in heterochromatin formation


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Epigenetic Effects

  • Several different types of structures have epigenetic effects:

    • Covalent modification of DNA (methylation of a base)

    • A proteinaceous structure that assembles on DNA

    • A protein aggregate that controls the conformation of new subunits as they are synthesized

Assigned Reading: Perception of epigenetics (Nature 447: 396-398, 2007


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Replication of a Methylated Base

Epigenetic Effect on Heterochromatin


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Methylation of Cytosine

  • Between 2% to 7% of the cytosine in eukaryotic DNA can be methylated at C5 position

  • About 90% of the methylated C is followed by 3’G residue, this sequence forms part of the recognition sequence (CCGG) for two restriction enzymes, MspI and Hapa II

  • MspI will cut DNA whether or not the second C is methylated, but HapaII will only cut the DNA when the second C is not methylated. Therefore this pair of restriction will be used to determine whether the second C at a sequence CCGG is methylated or not

  • Experiment outline below help to detect DNA methylation


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Example of Tissue-Specific Methylation of Msp/HpaII Sites of Chicken Globin Gene

  • The results show that the CCGG sequence of the globin gene in the red blood cells is unmethylated but in brain cells is methylated

  • Similarly, the tyrosine amino-transferase gene which is expressed in the liver cell is under methylted

  • From this type of study, a good correlation can be drawn: the CCGG sequence of an active gene is under methylated


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DNA Methylation Regulates Chromatin Structure

  • Introduction of globin gene containing 5’methyl-C into cells resulted in non-expression of globin gene, whereas introduction of un-methylated globin gene results in expression of globin gene

  • The methylated globin gene is insensitive to DNase I digestion

  • Treating undifferentiated fibroblast cells with 5-azacytidine, an analog of cytidine, results in activation of some key regulatory genes and leading to differentiation of these cells into multinucleated, twitching striated muscle cells

  • Treating of undifferentiated HeLa cells with 5-azacytidine and fused with mouse muscle cells will result in expression of mouse muscle-specific genes, suggesting un-methylation in C will allow gene expression


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DNA Methylation Recruit Proteins to Compact Chromatin Structure

  • Evidence available indicating the importance of DNA methylation in modulating the structure of chromatin from active to inactive state. How is this achieved??

  • There are two possible mechanisms: (i) A protein binds to the unmethylated site that insures chromatin to maintain in active state; (ii) An inhibitory protein that binds to the methylated site and thus recruit other proteins to result in compaction of chromatin

  • The discovery of MeCP2 and HDAC support the mechanism described in (ii)

  • MeCP2 (methyl CpG binding protein 2) is involved in turning off genes by binding to methylated CpG. In human this protein comprises a family of proteins, MBD1, MBD2, MBD3 and MBD4. It also binds to HDAC


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CpG Island and Methylation

  • Methylation of DNA occurs at CpG island

  • Fully methylated vs. hemimethylated

  • DNA methylase (Dnmt): De novo methylase (Dnmt3A and Dnmt3B) and perpetuation methylase

  • Demethylase: removal of methyl group from the CpG island


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DNA Methylation and Heterochromatin Formation

  • UHRF1 (Ubiquitin-like, containing PHD and RING finger domains 1), is a protein that can recognize hemimethylated DNA. It can recruit a maintenance methylase to the hemimethylated DNA and methylate the unmethylated group

  • UHRF1 can also bind to HP1 which in tern bind to methylated histone 3 (H3K9). By this way, Dnmt assists to stablize inactive chromatin

  • Methylation has several functional targets. Gene promoters are the most common target. There are several diseases associated with Dnmt mutation


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Drosophila Eye Colors

Wild Type Eye Color

Position Effect Variegation

Position-effect variegation in eye color of Drosophila results when the white gene is integrated near heterochromatin. Cells in which white is inactive give patches of white eye, whereas cells in which white is active give rise to red patches. The severity of the effect is determined by the closeness to the integrated gene heterochromatin


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Extension of Heterochromatin Inactivates Genes

  • The figure in the left explain the phenomenon of eye variegation in Drosophila

  • The inactivation of the white gene spreads from heterochromatin into the adjacent region for a variable distance. In some cells, it goes far enough to inactive a near by gene

  • The closer a gene lies to heterochromatin, the higher the probability that it will be inactivated

  • Telomeric silencing in yeast is analogous to position effect variegation in Drosophila


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De Novo Methylation and Maintenance of Methylation of Cytidine Residue

  • Several DNA methyltransferase enzymes that methylate DNA have been found to be essential for development in mammals

  • Dnmt 3a, Dnmt3b and Dnmt1 are three methyltransferases that are essential for development in mammals

  • When one the CG dinucleotide of a DNA strand is methylated, the other C in the other strand of the DNA will also be methylated. Enzyme involves in the de novo methlation is Dnmt 3a or Dnmt 3b

  • Dnmt 1 only recognizes the hemimethylated C and rapidly methylates the second unmethylted C. This is how a methylated pattern is preserved


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De Novo Formation of Unmethylated Genomic DNA

Unmethylated C residue in the methylated DNA can be achieved during DNA replication by mechanisms indicated in (a) or (b)

By these mechanisms, cells with full methylated and unmethylated C will be maintained

This is exactly observed in stem cells during cell division


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Differentiation of Stem Cells during Embryonic Development

  • During embryonic development, a stem cell divides to yield two daughter cells, one remainds stem cell lineage and the other differentiates into adult cell types

  • The daughter cell remains stem cell lineage has the same methylation pattern as its mother cell while the one that goes into differentiation is unmethylated

  • DNA methylation processes provide a means of explaining the stability of the committed state, while allowing for its modification in stable circumstances

  • Therefore, change of DNA methylation pattern in any cell leading to transdetermination (or transdifferentiation) require DNA synthesis and inhibition of methylation at particular sites


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Reading List IV:

  • MeCP2 (CpG Binding Protein 2) (from Wikipedia)


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DNA Methylation and Imprinting

  • Patttern of methylation of germ cells is established in each sex during gametogenesis by a two-stage process:

    • Removal of existing pattern by a genome wide demethylation in primordial germ cells

    • Pattern specific for each sex is imposed during meiosis

  • Figure in the left showed the pattern of imprinting paternal and maternal genes. In the embryo, if the maternal gene is methylated and paternal gene is not methylated, In the subsequent generation, the paternal gene in the male gametes will still be unmethylated and the maternal gene will still be methylated. The imprinting of IGF-II gene follows this pattern

  • The imprinting pattern of some genes follows the opposite pattern. IGF-IIR gene is methylated in paternal source and unthelylated in maternal source

  • Therefore the consequence imprinting is that an embryo is hemizygous for any implanted gene


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Epigenetic Inheritance

  • In the case of a heterozygous cross where the allele of one parent has an inactivating mutation:

    • the embryo will survive if the wild type allele comes from the parent in which this allele is active

    • The embryo will die if the wild type allele comes from the parent in which this allele is inactive (imprinted)

  • This type of dependence on the directionality of the cross which is in contrast with Mendelian genetics is called “epigenetic inheritance”

  • The imprinted genes are estimated to comprise 1% - 2% of the mammalian transcriptome. These genes are sometimes clustered


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Oppositely Imprinted Genes is Controlled by a Single Center

  • Differentially methylated domains (DMDs) or Imprinting control regions (ICRs) are responsible for controlling imprinting genes

  • Taking IGF-II and H19 as example, methylation of ICR in paternal allele results in inactivation of H19 and activation of IGF-II gene, whereas unmethylation of ICR in maternal allele results in inactivation of IGF-II gene and activation of H19 in maternal allele

  • The ICR contains an insulator that prevents an enhancer from activating IGF-II. The insulator functions only when CTCF (CCCTC binding protein) binds to unmethylated DNA


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Chemical Modification of Histone Tails


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Modification of Histones and Their Effects on Transcription

  • Histones H2A, H2B, H3 and H4 are subjected to different modifications such as acetylation, methylation, phosphorylation and ubiquitation. All of these modifications have been implicated in the regulation of chromatin structure and therefore of gene expression

  • Acetylation: lysine residue; Methylation: lysine and arginine; ubiquitilation: lysine; Phosphorylation: serine and threonine; Sumoylation: lysine


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Factors Affecting Acetylation of Histones Leading to Activation or Inactivation of Genes in Chromatin

(a). An activator (A) that directly acetylate histone resulting in opening the chromatin structure

(b). An inhibitory molecule (R) that can deacetylate histone leading to opposite effect on chromatin structure

  • Acetylation of lysine residues on histone happens on the amino group of specific lysine residue. Acetylation will result in reducing net positive charge histones and causing the dissociation of histone from the DNA

  • Addition of sodium butyrate to cells will lead to inhibition of cellular deacetylation activity and hence increasing histone acetylation

  • Increase of acetylation on histones is related to allowing chromatin to be more sensitive to DNase I digestion


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Effect of MEF2 and HDAC on Regulation of Expression of Myotube-Specific Gene

  • The example of MeCP2 protein which binds to CpG dinucleotides that can recruit a HDAC activity, thereby linking histone deacetylation to the repressive effect of DNA methylation

  • Therefore, activators recruit acetylases and repressors recruit deacetylases to regulate the structures of chromatin for transcription

  • Acetylase and deacetylase themselves can also be regulated. This is seen in muscle differentiation

  • In myoblasts, the transcription activator (MEF2) is associated with HDACs. When differentiation from myoblasts to mature myotubes, the HDAC is phosphorylated which induces to move to the cytoplasm, thereby freeing MEF2 to activate transcription

  • MEF2: myocyte enhancer factor-2 (MEF2) proteins are a family of transcription factors through which control gene expression


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Acetylation of Histone Proteins May Affect Nucleosome Structures

  • The lysine moieties in N-terminal region of histones projects out of nucleosomes after acetylation, and the acetylated group interactes with the N-terminal ends of adjacent nucleosomes or with non-histone proteins

  • This interaction may result in:

    • Improved access of to the DNA for factors that my stimulate transcription (as in a), or

    • A looser association could facilitate displacement of nucleosomes by forming chromatin-remolding complex, thus leading to easy access by transcription activators


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Alternative Consequences of Acetylation of Histones

  • Alternative consequences of histone acetylation could be:

    • Acetylation of histones may result in binding of positively or negatively acting factor to DNA leading to destabilization of the 30 nm chromatin fiber and transcriptional activation

    • Alternatively, acetylation may disrupt the association of nucleosones with inhibitory proteins involved in maintaining the close structure of chromatin


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Important Terms

  • Histone code: The situation of acetylation, methylation, phosphorylation, ubiqutination and sumolation of the histone tails. The pattern of modification affects the activity of the chromatin

  • Chromododomain (chromatin organization modifier) ): A protein structural domain of about 40-50 amino acid residues found in association with remodeling and manipulation of chromatin

  • Chromo shadow domain: A protein domain which is distantly related to the chromodomain. Proteins containg a chromoshadow domain include Su(var)205 (HP1) and mammalian modifier1 and modifier 2

  • Bromodomain:A protein domain that recognizes lysine residues in the histone tail

  • PHD finger: Cys4-His-Cys3 motifHAT3 . It relates to epigenetis

  • TUDOR domain: A protein that recognizes methylated histones


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Binding of Acetylated Histone to Bromodomain Containing Activator Protein (BD)

Bromodomain Protein

A bromodomain is a protein domain that recognizes acetylated lysine residues such as those on the N-terminal tails of histones. This recognition is often a prerequisite for protein-histone association and chromatin remodeling. The domain itself adopts an all a-protein folds, a bundle of four a-helices. This binding of BD to acetylated histones will result in opening of the chromatin structure available for transcription


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Methylation of Lysine and Arginine on Histones

  • Methylation of lysine or argine will result in a more open chromatin structure or more campact chromatin structure. For instance, methylation of arginine at position 2,9,17 and 26 and lysine at position 4, and 36 on histone H3 will result in a more open chromatin structure, but methylation of lysine at position 9 and 26 will result in a more compact chromatin structure

  • Methylation of arginine at position 3 of histone H4 promotes a more open chromatin structure and facilitate transcription activated by nuclear hormone receptor

  • In Drosophila, methylation of lysine 4 of histone H3 results in activation of transcription by ecdysone-receptor complex

  • In yeast, methylation of lysine 4 of histone H3 results in transcriptional activation mating-type locus

  • Methylation of lysine 4 of histone H3 will activate gene expression in human


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Polycomb (PC) Proteins

  • The balance of methylation at different histone sites plays a critical role in determining chromatin structures

  • In Drosophila, a particular homeotic mutant that showed severe transformation of multiple tissues. This type of mutant is called polycomb which is a recessive mutation

  • For instance: polycomb complex which is involved in transcriptional repression contains a histone methyl transferase enzyme that methylates H3 on lysine 9 and 27. Conversly the trithorax proteins act to open the chromatin by methylating H3 at lysine 4 and promotes demethylation of lysine at 9 and 27.

Trithorax: a homeotic mutant first identified in Drosophila


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HP1 and Histone Methyltransferase (HMT)

  • HP1: Chromodomain protein that binds to methylated lysine in histone tails, and it can also bind to histone methyltransferase (HMT)

  • Nucleosome with H3 methylated lysine 9 can recruit HMT, and the resulting complex will methylate the adjacent nucleosome to methyylate the unmethylated H3 lysine 9

  • By this progress methylation by HP1 and HMT, the adjacent unmethylated nucleosomes will be methylated and the open chromatin will be close structure


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Formation of Hetero-chromatin by Binding of HP1 to H3 Tri-methylated at Lysine 9

  • Histone code is read by proteins that bind to the modified histone tails and in turn promotes condensation or decondensation of chromatin, forming “closed” or “open” chromatin structure

  • Chromodomain (HP1): Some proteins contain chromodomain that can bind to histone tails when they are methylated at the specific lysine (lysine 9)

  • Chromoshadow domain: A second domain on HP1 which can bind another chromoshadow domain, histone methyl transferase (HMT)

  • HMT can methylate L9 of another nuclosome and binding to another HP1, thus extending the hetrochromatic region.


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  • Chromoshadow domain can also bind to the enzyme , histone methyl transferase (HMT), that can methylate H3 lysine 9

  • Consequently nucleosone adjacent to a region of HP1 containing heterochromatin becomes methylated at lysine 9 and creating a binding site for another HP1 that can bind to the H3K9 histone methyl transferase resulting in the spreading of the heterochromatin structure until it meet the boundary element

  • Boundary element: A region in the chromatin where several non-histone proteins bind to the DNA

  • In summary, multiple types of covalent modifications of histone tails can influence chromatin structure by altering nucleosome-nucleosome interactions and interactions with additional proteins that participate in or regulate processes such as transcription and DNA replication

  • One of the X chromosoms in human females is randomly inactivated during embryonic development. This will lead to dosage compensation in female.

  • X-chromosome inactivation is an epigenetic process and it is inherited by daughter cells


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Spreading of the Heterochromatic Region

Heterochromatic condensation can continue to spread along a chromatin because HP1 binds a histone methyltransferase (HMT) that methylate lysine 9 of H3 until it encounters a “boundary element (insulator)”


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Boundary Sequence (Insulator Sequence)

  • Insulator: Chromosomes are partitioned into different regions that can be regulated independently. Insulators fulfill this function by serving as barrier or boundary elements preventing the passage of activating or inactivating effects

  • Properties of insulators:

    • When an insulator is located between an enhancer and a promoter, it prevents the enhancer from activating the promoter. This may explain why an enhancer is active only for specific promoter

    • When an insulator is located between an active gene and heterochromatin, it provides a barrier that protects the gene against the inactivating effect of the spreeds from the heterochromatin

  • Some insulators have both properties while others have only one property

Insulator blocks the activity of an enhancer. It can also block the sprading of heterochromatin


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  • Insulator sequence was first observed in Drosophila polytene chromosome

  • Upon heat shock, Two chromosome puffs are found in band 87A; these two puffs contain Hsp70 mRNA

  • Upon analysis of Drosophila genome, two scs sites are found

  • These two sites are found at the band flanking the Hsp70 gene

  • These two sites are flanked by Dnase I hypersensitive sites suggesting they are in adjacent with euchromatin region


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Drosophila Eye Colors

  • Position-effect variegation in eye color of Drosophila results when the white gene is integrated near heterochromatin

  • Cells in which white is inactive give patches of white eye, whereas cells in which white is active give rise to red patches.

  • The severity of the effect is determined by the closeness to the integrated gene heterochromatin. The closer the white to the heterochromatin, the white patch will be greater


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Locus Control Region

  • LCR (locus control region) is a region at the 5’ end of a chromosomal domain and are typically containing multiple DNase I hypersensitive sites.

  • The role of the LCR is complex; in some ways it behaves as a “super enhancer” that poises the entire locus for transcription

  • The 3’ hypersensitive site is known to physically interact with LCR, but it precise function is still not entire clear

  • In addition to the individual promoters, the LCR is required to regulate the expression of the globin genes in the entire locus

  • The figure below showers the LCR of b-globin gene locus


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  • LCR could regulate the expression of genes located on different chromosomes. The example of this is TH2 LCR that coordinately regulates the expression of interleukin genes in the T helper type 2 cytokine locus located on chromosome 11. It also interacts with the promoter of INFg gene located on chromosome 10

  • The method of chromosome conformation capture (3C) has been used to determine the interaction of LCR and the individual genes that it regulates their expression. This method involves isolation of chromatin, formaldehyde crosslinking the chromatin, restriction enzyme digestion of the cross-linked chromatin, intra-molecular ligation of the DNA under dilute concentration, recover of the cross-linked DNA and PCR analysis of the product


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Physical Interaction of LCR the Genes It Regulates

The physical interaction of LCR with the genes it regulates on chromosome in vivo determined by the 3C method. B, b-globin locus; TH2 T helper type 2 cytokines locus


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Conclusion

  • The process of commitment to a particular differentiation pathway is the consequence of change of chromatin structure of genes. These changes include methylation of DNA at the CG dinucleotides, histone modification including methylation, acetylation, phosphorylation and ubquitilation, and ATP-dependent remolding of the chromatin structure by complexes such as SWI-SNF and NURF

  • Regulatory RNAs such as siRNAs, antisense RNA, XIST and TSIX are also involved in regulating chromatin structures

  • There are three chromatin structures in a cell: (i) nucleosome free regulatory region; (ii) active gene in beads-on-string 10 nm-fiber structure; (iii) 30 nm-fiber structure of inactive genes


Iv the role of chromatin structure in control of gene expression

Assigned Reading [IV]

  • Central dogma of molecular biology

  • Nobel Prize Lecture by Monod

  • A second paradigm for gene activation in bacteria

  • Perception of epigenetics

  • MeCP2 (CpG binding protein 2)

  • DNA methylation and histone modifications: teaming up to silence genes

  • The complex language of chromatin regulation during transcription

  • Reading and function of a histone code involved in targeting co-repressor complex in repression

  • HP1: a functionally multifaceted protein

  • Bromodomain structure

  • Transcription and RNA interference in the formation of heterchromatin

  • The key to development: interpreting histone code?

  • The Swi/Snf family: nucleosome-remodeling complex and transcriptional control


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