Regulation Of Gene Expression Eukaryotes

Regulation Of Gene Expression EukaryotesPresenter : Saranya.SModerator : Dr.V.Balasubramaniyan1

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CONTENT Regulation at Chromosomal level

Regulation at Transcriptional level

Summary 2

Evolution Of Gene Regulation Eukaryotes

MulticellularEvolved to maintain constant internal conditions while facing changing external conditionsHomeostasisRegulate body as a wholeGrowth & developmentLong term processesSpecializationTurn on & off large number of genesMust coordinate the body as a whole rather than serve the needs of individual cells

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Points Of ControlPacking/unpacking DNANucleosome movementNucleosome positionHistones modificationsTranscriptionPromotors EnhancerInsulator

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DNA PackingHow do you fit all that DNA into nucleus?DNA coiling & foldingDouble helixNucleosomesChromatin fiberLooped domainsChromosome

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from DNA double helix to condensed chromosome

Nucleosomes Beads on a string1st level of DNA packingHistone proteins 8 protein moleculesPositively charged amino acids Bind tightly to negatively charged DNA

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DNA Packing As Gene ControlDegree of packing of DNA regulates transcriptionTightly wrapped around histones No transcriptionGenes turned off

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Heterochromatindarker DNA (H) = tightly packedEuchromatinlighter DNA (E) = loosely packed

Nucleosome Movement Catalyzed By Nucleosome-remodeling ActivitiesDNA sites closest to the entry and exit points are the most accessible,Sites closest to the midpoint of the bound DNA are least accessible8

Two Modes Of DNA - binding Protein-dependent Nucleosome PositioningAssociation of many DNA-binding proteins with DNA is incompatible with the association of the same DNA with the histone octamerRemains nucleosome freeA subset of DNA-binding proteins has the ability to bind to nucleosomes.

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Nucleosome requires more than 147 bp of DNA to form, if two such factors bind to the DNA less than this distance apart, the intervening DNA cannot assemble into a nucleosome

Once bound to DNA, such proteins will facilitate the assembly of nucleosomes immediately adjacent to the proteins DNA-binding site

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Histones H1, H2A, H2B,H3, and H4Histones H2A, H2B, H3, and H4 are the core histonesHistone H1 Linker histone

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Histone ModificationsAcetylationMethylationPhosphorylationUbiquitinylationOccur on the tail regionsThere are occasional modifications within the histone foldE.g. Methylation of lysine 79 of histone H311

Histone AcetylationAcetylation of histones unwinds DNA : Histone acetyltransferase (HAT)Loosely wrapped around histonesEnables transcriptionGenes turned onAttachment of acetyl groups (COCH3) to histones Conformational change in histone proteinsTranscription factors have easier access to genesDeacetylationHistone Deacetylase (HDATs) : repressing the expression12

Histone MethylationMethylation of histone H4 on R4 (arginine residue at the 4th position) opens the chromatin structure leading to transcriptional activation

Methylation of histone H3 on K4 and K79 (lysines residues at the 4th and 79th position) opens the chromatin structure leading to transcriptional activation

Methylation of histone H3 on K9 and K27 (lysines residues at the 9th and 27th position) condenses the chromatin structure leading to transcriptional inactivation

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Other ModificationsUbiquitinationUbiquitination of H2A Transcriptional inactivationUbiquitination of H2B - Transcriptional activation

Methylation of DNATarget sites of methylation are - The cytidine residues which exist as a dinucleotide, CG (written as CpG)methylated cytidine -- Transcriptional activity

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DNA Methylation15

Unmethylated CpG islandTFRNA pol

Active transcription

Methylated CpG islandTFRNA pol

CH3

CH3

CH3

Repressed transcription

How Does Histone Modification Alter Nucleosome Function?Acetylation and phosphorylation each acts to reduce the overall positive charge of the histone tailsAcetylation of lysine neutralizes its positive charge - this loss of positive charge reduces the affinity of the tails for the negatively charged backbone of the DNAModification of the histone tails affects the ability of nucleosome arrays to form more repressive higher-order chromatin structureHistone amino-terminal tails are required to form the 30-nm fiber, and modification of the tails modulates this function16

Effects Of Histone Tail Modifications

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Transcriptional RegulationTranscription : The synthesis of RNA molecules using DNA strands as the templates so that the genetic information can be transferred from DNA to RNADNA regions that can be transcribed into RNA are called structural genesRNA Polymerase :RNA polymerase IRNA polymerase IIRNA polymerase III

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At Transcriptional LevelFor the transcription ,interactions of transcription machinery is essential Cis-acting elements - DNA sequences close to a gene that are required for gene expressionPromotors EnhancersInsulatorsTrans acting elementsRNA polymeraseTranscription factorsDomains of trans-acting factors

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TF For Eukaryotic Transcription

20Transcription factorsFunctionTBPRecognizes TATA boxTFII AStabilizes binding of TFIIB / TBP to promoterTFII BRecruits RNAPTFII DRegulation of transcriptionTFII EATPase and Helicase activityTFII FPrevents binding of RNAP to nonspecific DNA sequencesTFII HUnwinds DNA at promoter

PromotersThe region necessary to initiate transcriptionConsists of short nucleotide sequence that serve as the recognition point for binding of RNA polymeraseLocated immediately adjacent to the genes they regulate, upstream from the transcription start pointPromoters for RNA polymerase II include:

Promoters for RNA polymerase I & III have a different sequence and bind different transcription factors

21TATA box,CAAT box,GC box, Octamer box

Promoters

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Enhancers DNA sequences interact with regulatory proteins

Increase the efficiency of initiation of transcription Increase its rate23

How Enhancers Can Control Transcription Although They Are Located Away From The Transcription Site???Enhancers bind to transcription factors by atleast 20 different proteins

Form a complex Change the configuration of the chromatin

Folding, bending or looping of DNA.24

DNA looping will bring the distal enhancers close to the promoter site to form activated transcription complexes, then the transcription is activated, increasing the overall rate of RNA synthesis.

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Activators Recruits Transcriptional Machinery

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Activators Recruits Nucleosome Modifiers

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Insulator Block TranscriptionWhen distant enhancers activates a specific gene, it does activate other genes present in that rangeThis happens by the specific elements called insulators This does not inhibit the activity of different enhancers , that are placed in downstream of the promoterThe protein that bind insulators block the communication between the two27

Signal Integration To integrate signals, multiple activators work together i.e. synergyThis is achieved by cooperativitySynergy is critical for the signal integration by activators

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Summary DNA in eukaryotic cell is wrapped in histones to form nucleosomesImportant mechanism of transcription activation is removal of nucleosome at core promotorEnzyme RNA polymerase is highly conserved. Binds to approx. 50 proteins to form large protein complexActivator can interact with one or more of many different component of transcription machinery Insulators help ensure the activators work only on correct gene

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Thank you30

THANK YOUREFERENCES Lehninger Principles of Biochemistry, 5th Edition - David L. Nelson, Michael M. CoxMolecular biology of the gene- 5th edition -James D.Watson et al