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

1. [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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

11. 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??

12. 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

13. 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

14. 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

15. 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

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

17. Overview of Four Basic Molecular Genetic Processes

18. 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

19. 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

20. 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

21. 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

22. Structures of Active and Inactive Genes

23. 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??

24. Epigenetics Change

25. 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

26. 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

27. Replication of a Methylated Base Epigenetic Effect on Heterochromatin

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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

37. 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

38. 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

39. Reading List IV: • MeCP2 (CpG Binding Protein 2) (from Wikipedia)

40. 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

41. 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

42. 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

43. Chemical Modification of Histone Tails

44. 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

45. 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

46. 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

47. 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

48. 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

49. 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

50. 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