Introduction Basic Genetic Mechanisms Eukaryotic Gene Regulation The Human Genome Projects Test 1 Genome I - Genes Genome II – Repetitive DNA Genome III - Variation Test 2 Monogenic and Complex Diseases Finding ‘Disease’ Genes Pharmacogenomics Test 3 Your Presentations Happy New Year! Molecular Genetics The Human Genome: Biology and Medicine Studiju materiāli / MolekularasBioloģijas / MolGen / EN

Two ways of genome regulation Genomes, 3rd Edition

Chromatin and gene activity

The types of interphase chromatin The Positional effect – variability in gene expression that occurs after a new gene has been inserted into eukaryotic chromosome 1. Euchromatin –the ‘usual ‘ form –contains (potentially) active genes 2. Heterochromatin –more condensed form –additional proteins (HP1) – constitutive no genes feature of all cells e.g., centromeric, telomeric DNA – facultative in some cells some of the time inactive genes Molecular Biology of the Cell, 5th Edition

Two ways in which chromatin structure can influence gene expression Genomes, 3rd Edition, modified heterochromatin – genes unaccessible euchromatin - genes accessible 1 2 Gene OFFGene ON

Types of alterations in euchromatin structure 1. Modifications of histones activating (here) repressing 2. Nucleosome remodeling remodeling (here) histone replacement histone removal

The major types of histone modifications Recombinant DNA, 3rd Edition

Modifications of histones convey a specific meaning to chromatin (‘Histone code’)

Gene activity is substantially affected by DNA methylation CG sequences; leads to silencing; CpG islands - usually in promoters

A model for the link between DNA methylation and gene silencing Genomes, 3rd Edition (modificēts) MeCP2 protein methyl-CpG-binding protein (MeCP) components of a histone deacetylase complex (HDAC)

Regulatory sequences and gene activity

Eukaryotes, like you, have many regulatory elements Molecular Biology of the Gene, 5th Edition Regulatory sequence – DNA sequence to which a gene regulatory protein binds

Major types of regulatory DNA elements in eukaryotes S – silencer P – promoter I – insulator E – enhancer TF – transkrition factor heterochromatin 2010, 11, o Promoters – recognition sequences for binding of RNA polymerase o Enhancers – increase transcription of a related gene o Silencers – decrease transcription of a related gene o Insulators or boundary elements – block undesirable influences on genes: 1. enhancer blockers – prevent ‘communication’ between enhancers and unrelated promoters 2. barrier sequences – prevent spread of heterochromatin 3. combined o LCR – locus control regions – activate some gene clusters

Composition of regulatory DNA elements is modular Genomes, 3rd Edition RNA polymerase II promoter modules (Genomes, 3rd Ed.) The core promoter modules o in all promoters o BRE, TATA, Inr, DPE o recognised by general transcription factors Basal promoter modules o present in many RNA polymerase II promoters o set the basal level of transcription initiation, without responding to any tissue-specific signals o the CAAT box (recognised by the activators NF1 and NFY), the GC box (SP1)… Response modules o found upstream of various genes o enable response to general signals from outside of the cell o CRE (the cyclic AMP response element) recognised by the CREB activator; SRE (serume response element), recognised by serume response factor… Cell-specific modules o are located in the promoters of genes that are expressed in just 1 type of tissue o the erythroid module, which is binding site for the GATA-1 activator; the myoblast module, recognised by MyoD… Modules for developmental regulators o mediate expression of genes that are active at specific developmental stages

Levine M & Tjian R (2003) Nature, 424, 147 This ensures efficient combinatorial control of gene expression

A model for the control region of the human  -globin gene

What type of promoter recognition is characteristic to you? Genomes, 3rd Edition

Transcription initiation in eukaryotes requires General Transcription Factors (TF)

Some genes have alternative promoters, eg, dystrophin gene Genomes, 3rd Edition Cilvēka distrofīna gēns Alternative promoters C, cortical tissues; M, muscle; Ce, cerebellum; R, retinal tissue (and also brain and cardiac tissue); CNS, central nervous system (and also kidney); S, Schwann cells; G, general (most tissues other than muscle).

Eukaryotic transcription initiation also needs activator proteins Genomes, 3rd Edition

Activators aid the assembly of the transcription initiation complex Activator Chromatin remodelling or modifying complexes Multi-subunit cofactors (mediators) Levine M & Tjian R (2003) Nature, 424, 147

Activators may act from a large distance Two models: (i) direct-contact model (here); (ii) tracking model

Writing and reading the histone code during transcription initiation

INSULATORS divide genome into functional domains gene A enhancer gene B insulator (enhancer blocker) insulator (barrier sequence)

Insulators prevent inappropriate activation from enhancers thus maintaining the independence of a functional domain Molecular Biology of the Gene, 5th Edition

Models for enhancer-blocking activity E – enhancer EB - enhancer-blocker proteins

A model for establishment of silencing and barrier activity 2010, 11, S – silencer, B – barrier elements, TF – transcription factor, CR – chromatin remodellers, HM – histone-modifying enzymes, R – repressor proteins

Activity of some gene clusters requires LCRs

Composition of chicken  -globin LCR Vol. 7,

RNA interference miRNA (micro RNA) Regulation of gene expression – at least 30% of human genes Precursor (pre-miRNA) – single-stranded RNA Origin – transcripts of cell siRNA (small interfering RNA) Defense against viruses and expansion of transposons Precursor – double-stranded RNA Origin - various Phenomenon when very short RNAs (21-22 nts) repress – or silence – expression of genes with homology to those RNAs

RISC = RNA-induced silencing complex dsRNA = double-stranded RNA Mechanism of RNA interference Molecular Biology of the Gene, 6th Edition (modified)

miRNA processing and mechanism of action

miRNAs are coded in both coding and noncoding sequences Molecular Biology of the Gene, 6th Edition (modified)

EPIGENETICS Waddington, 1942: “[T]he branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being” ( epi [genesis] + genetics ). Literally: epi (Greek: επί- on, over) genetics – something working above DNA.

EPIGENETIC INHERITANCE Any heritable difference in the phenotype of a cell that does not result from changes in the nucleotide sequence of DNA Specific epigenetic phenomena: 1. dosage compensation: a) X-chromosome inactivation; b) mono-allelic expression; 2. imprinting (parent-of-origin specific differences in gene activity)

Inheritance of DNA methylation DNMT1 (DNA methyltransferase 1)

Inheritance of histone modifications

Imprinting

Mechanism of imprinting in the mammalian Igf2 locus Molecular Biology of the Gene, 5th Edition

X chromosome inactivation

Mechanism of mammalian X chromosome inactivation XIC : X-inactivation center XIST: X-inactivation specific transcript

Molecular Biology of the Gene, 6th Edition

Identical twins are not identical

Phenotype = Genotype + Epigenotype + Environment