[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

DNA mRNA Protein

Transcription Translation

Replication

1. Replication

2. Transcription

3. Translation

4. Reverse Transcription

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

RNA

Reverse Transcription

12

3

4

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

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

lac Z: encodes -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 I promoter lac Z lac Y lac A

Lac operon

I gene product is a negative regulator of the lac operon

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

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

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

• For active transcription in eubacteria, the RNA polymerase needs to bind to a protein, factor (70), to form a complete complex

• Sigma factor (70) 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 (70) 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

Interaction of Bound NtrC and 54-RNA Polymerase

• Most E.coli promoters interact with 70-RNA polymrase in transcription of genes

• The transcription of some genes in prokaryotes are accomplished by 54- RNA polymerase

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

• The promoter of gln gene (glutamyl-tRNMA gene) is bound by NtrC (nitrogen regulatory protein C, an activator protein) which , after activation by NtrB (a protein kinase), can bind to 54-polymerase at the promoter region and initiate transcription

• NtrC has ATPase activity, and hydrolysis of ATP is required to activate 54-polymerase

• Many other bacterial responses are controlled by “two-component” regulatory systems similar to NtrB and NtrC

Two-Component Regulatory System• Control of the transcription of E. coli glnA

gene depends on a two-component system NtrC and NtrB proteins

• At high concentration of glutamine, glutamine binds to sensor domain of NtrB and causing a conformational change in the protein that inhibits its histidine domain of NtrC blocks the DNA binding domain from binding to glnA gene enhancer

• Under the condition of low glutamine, glutamine dissociates from the sensor domain of NtrB, leading to activation of a histidine kinase transmitter domain in NtrB that transfer -phosphate of ATP to the histidine residue in the NtrC and resulting in activation of the transmitter domain. This phosphohistidine then transfer the phosphate to aspartic acid residue in NtrC to unmask the DNA binding domain so that it can binding to enhancer of glnA gene

Summary• In prokaryotes, regulation of gene expression are:

Gene expression in prokaryotes is regulated primarily by the mechanisms that control the initiation of transcription

Regulation in operons is achieved by positive and/or negative regulation and the RNA polymerase involved is 70-polymerase

Regulation of transcription involving 54-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??

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

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

Example III: 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

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

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

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

Overview of Four Basic Molecular Genetic Processes

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

+1

Transcription start site

TATA Box -25 - -35 bp

TranscriptionProximal promoter

Distal promoter

Regulatory region (regulatory cis element) Structural gene

Structure of Protein Coding Gene

• Chromatin-mediated transcriptional control• Activators and repressors interaction with transcription

complex

Two key features of transcription control:

Transcription-Active and Transcription-Inactive Chromatin

• Transcription active chromatin can be differentiated from transcription 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

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

• Chromatin isolated from adult erythroid cells, digested with various doses of DNase I. Following digestion, DNA is recovered, digested with restriction enzyme, DNA resolved on agarose gels and transferred to a nylon membrane. The blot is hybridized to radiolabelled embryonic-globin, adult -globin and ovalbumin genes

• The results showed that while adult globin gene is sensitive to digestion by DNase I, embryonic globin gene and ovalbumin gene are insensitive to digestion by DNase I

Structures of Active and Inactive Genes

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??

Epigenetics Change

Epigenetic change refers to the inherited changes in the phenotype of cell that do not result from changes in DNA sequence:

DNA methylation and chromatin remodelingRNA transcription and their encoded proteinsmiRNA and siRNA mediated epigenetics

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

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 --Epigenetics

Methylation of Cytosine on DNA 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

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

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

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

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 (b)

• 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 (histone deacetylase)

DNA Methylation Leading to Heterochromatin Formation

• When chromosomal DNA is replicated, the parental histones randomly associated with two daughter molecules while unmethylated histones synthesized during S phase comprised other nucleosomes on the sister chromosome.

• The histone H3 lysine 9 methyl transferase (H3K9 HMT) associated with parental nucleosomes bearing with H3K9 will methylate the unmethylated H3K9 residue

• Trimethylation of lysine 9 in histone H3 in nucleosomes can bind with heterochromatin binding protein 1 (HP1) and lead to condensation of chromatin to form heterochromatin

• KAP1 (KRAB-associated protein 1 (also called as Tripartite motif-containing 28) co-repressor is the example

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 of 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.

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

• Consequently nucleosome 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

Wild Type Eye Color Position Effect Variegation

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

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

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

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)”

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 spreads 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

• 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 (specialized chromatin 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

Reading List IV:

MeCP2 (CpG Binding Protein 2) (from Wikipedia)

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 imprinted gene

Oppositely Imprinted Genes is Controlled by a Single Center

• Methylation of the “Differentially methylated domains (DMDs)” or “Imprinting control regions (ICRs)” is responsible for controlling imprinted genes. Deletion of these sites removes imprinting of target genes

• Taking igf-2 and H19 as example, methylation of ICR in paternal allele results in inactivation of H19 and activation of igf-2 gene, whereas unmethylation of ICR in maternal allele results in inactivation of igf-2 gene and activation of H19 in maternal allele

• The ICR contains an insulator sequence that prevents an enhancer from activating igf-2. The insulator functions only when CTCF (CCCTC binding protein) binds to unmethylated DNA

The ICR is methylated on the paternal allele, where igf-2 is active and H19 is inactive. When ICR is unmethylated on the maternal allele, igf-2 is inactive and H19 is active

Epigenetic Controlled by Polycomb and Trithorax Complexes (I)

• Polycomb proteins and trithorax proteins are involved in another form of epigenetic change which is responsible for cell type specific repression of gene expression during embryonic development

• Polycomb Recessive Complex 2 (PRC 2): One of the polycomb proteins which has histone methyltransferase activity for trimethylating histone H3 on lysine 27

• Protein Regulator of Cytokinesis 1 (PRC 1): Working together with PRC 2 to repress the expression of Hox genes

• Hox genes: are a group of related genes that control the body plan of an embryo along the anterior-posterior (head-tail) axis

Epigenetic Controlled by Polycomb and Trithorax Complexes (II)

• Besides trimethylation activity in the SET domain, PRC 2 complexes also contain histone deacetylase activity that inhibit transcription

• The PRC 1 complex binds to methylated nucleosomes through dimeric Pc subunit (CBXs in mammals). Binding of the dimeric Pc submits to neighboring nucleosomes will condense the chromatin into structure that inhibits transcription

• PRC 1 also contains a ubiquitin ligase that monoubiquitinate histone H2A at lysine 119, leading to inhibition of Pol II elongation through chromatin inhibiting the association of a histone chaperone required to remove histone octamers from DNA as Pol II transcribes through a nucleosome

Epigenetic Controlled by Polycomb and Trithorax Complexes (III)

• Trithorax proteins counteract the repressive mechanism of Polycomb proteins

• Figure in the left shows the expression of Hox transcription factor Abd-B in the Drosophila embryo with influence of Polycom wildtype and Trithorax genes: Wild type: Expression of Abd-B

restricted in the posteror Homozygous Polycomb mutant

(Scm-): Expression of Abd-B from posteror to anterior

Homozygous Trithorax mutation: Expression of Abd-B is only observed in posteror

X-Chromosome Inactivation in Mammals

• X-chromosome inactivation in female mammals is one of the examples of epigenetic repression mediated by a long, non-protein-coding RNA of about 100-kb on the X-chromosome (X-chromosome inactivation center)

• X-chromosome inactivation center encodes several non-coding RNAs (ncRNAs). These ncRNAs are Xist (~17-Kb), Txit (~40-Kb) and RepA RNA (~1.6-Kb)

• The expression of these ncRNAs from the X-chromosome inactivation center of both X-chromosomes inactivate one of the X-chromosomes

Probed with Xist

Probed for X-chromosome

Overlay photo

The region of the human X-inactivation center encoding the noncoding RNAs Xist, RepA and Tsix

Epigenetic Control Non-Coding RNAs and siRNA

• Besides X-chromosome inactivation, long non-coding RNA and siRNA can also exert epigenetic control of gene expression

• RNAs transcribed from a region encoding a cluster of HOX genes (the HOXC locus) can repress genes in the HOXD locus (~40 – Kb region on another chromosome) encoding several HOX proteins and multiple other encoding RNAs

• Assays similar to chromatin immunoprecipitation revealed that this noncoding RNA (HOX antisense intergenic RNA, HOTAIR) interacts with Polycomb PR C2 complexes leading to di- and trimethylation of histone H3K27, and with PRC 1 leading to demethylation at histone H3K4. All of these will lead to repression of transcription in trans

• In plants and fission yeast (i.g., Schizosaccharomyces pombe) , short interfering RNA (siRNA) can also me3deiate methylation of histone H3K9 and leading heterochromatin formation. The detail is shown in next slide

1) Poly II transcribe the repeated non-protein coding sequences of the centromere region at low level

2) The nescent RNA binds to RIST complex by base-pairing of siRNA associated with Agho 2 subunit of the RIST complex and the interaction of the Chp 1 with histone H3 methylated on K9

3) RIST complexes with the RDRC complex with an RNA-dependent RNA polymerase that converts the nacent Pol II transcript into double stranded RNA

4) The double stranded RNA is converted to RNA of ~22 bp by Dicer5) One of the double stranded RNA is bound Ago 1 of the RITS complex6) The RITS complex also associates with histone H3K9 methyltransferase

which methylates histone H3 in the centromere region and lead to heteromatin condensation

Model for Generation of Heterochromatin at S. pombe

Centromere by Non-coding RNA

Epigenetic Regulation of Transcription (I)

• Epigenetic control of transcription refers to repression or activation of genes maintained after cells replicate as the result of DNA methylation and/or post-translation modification of histones, especially histone methylation

• Methylation of CpG sequences in the CpG island promoters in mammals generate binding sites for a family of methyl-binding proteins (MBTs) that associate with histone deacetylase, including hypoacetylation of the promoter regions and transcriptional repression

• Histone H3 lysine 9 di- trimethylation creates binding sites for heterochromatin-associated protein HP1, which results in the condensation of chromatin and transcriptional repression. These post-translational modifications are perpetuated following chromosome replication because the methylated histones are randomly associated with the daughter DNA molecules and associate with histone H3 lysine 9 methyltransferases that methylate histone 3 lysine 9 on newly synthesized histone H3 assembled on the daughter DNA

Epigenetic Regulation of Transcription (II)• Polycomb complexes maintain repression of genes

initiallyrepressed by sequence-specific binding transcription factorrepressors expressed e4arly during embryogenesis. One class of Polycomb repression complexes, PRC2 complexes, is thought to associate with these repressors in early embryonic cells, resulting in methylation of histone H2 lysine 27. This creates binding sites for submits in the PRC2 complex and PRC1-type complexes that inhibit transcription elongation. Since parental histone octomers with H3 methylated at lysine 27 are distributed to both daughter DNA molecules following DNA replication, PRC2 complexes that associate with these nucleosomes maintain histone H3 lysine 27 methylation through cell division

• Trithorex complexes oppose repression by Polycomb complexes by methylating H3 at lysine 4 and maintaining this activating mark through chromosome replication

Epigenetic Regulation of Transcription (III)• X-chromosome inactivation in female mammals requires a long

noncoding RNA (ncRNA) called Xist that is transcribed from the X-inactivation center and then spreads by a poorly understood mechanism along the length of the same chromosome. Xist is bound by PRC2 complexes at an early stage of embryogenesis, initiating X inactivation that is maintained throughout the remainder of embryogenesis and adult life

• Long ncRNAs also have been discovered that lead to repression of genes in trans, as opposed to cis inactivation imposed by Xist. Repression is initiated by their interaction with PRC2 complexes. Much remains to be learned about how they are targeted to specific chromosomal regions, but the discovery of about 1600 long ncRNAs conserved between mammals raises the possibility that this is a widely utilized mechanism of repression

Chemical Modification of Histone Tails

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

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

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

(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

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

The example of MeCP2 protein which binds to methylated 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

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

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

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

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

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 -protein folds, a bundle of four -helices. This binding of BD to acetylated histones will result in opening of the chromatin structure available for transcription

Bromodomain Protein

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

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

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

• 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

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

Assigned Reading [IV]

1. Central dogma of molecular biology2. Nobel Prize Lecture by Monod3. A second paradigm for gene activation in bacteria4. Perception of epigenetics5. MeCP2 (CpG binding protein 2)6. DNA methylation and histone modifications: teaming up to

silence genes7. The complex language of chromatin regulation during

transcription8. Reading and function of a histone code involved in targeting

co-repressor complex in repression9. HP1: a functionally multifaceted protein10. Bromodomain structure11. Transcription and RNA interference in the formation of

heterchromatin12. The key to development: interpreting histone code?13. The Swi/Snf family: nucleosome-remodeling complex and

transcriptional control14. Epigenetics