• 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

• Your Presentations

• Happy New Year!

Molecular GeneticsThe Human Genome: Biology and Medicine

http://priede.bf.lu.lv/ Studiju materiāli / MolekularasBioloģijas / MolGen / EN

http://priede.bf.lu.lv/ 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

2Gene OFF Gene 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)

MeCP2protein

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, 439-446

o Promoters – recognition sequences for binding of RNA polymerase

o Enhancers – increase transcription of a related geneo 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 promoters2. barrier sequences – prevent spread of heterochromatin3. 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 moduleso in all promoterso BRE, TATA, Inr, DPEo recognised by general transcription factors

Basal promoter moduleso present in many RNA polymerase II promoterso set the basal level of transcription initiation, without responding to any tissue-specific signalso the CAAT box (recognised by the activators NF1 and NFY), the GC box (SP1)…

Response moduleso found upstream of various geneso enable response to general signals from outside of the cello CRE (the cyclic AMP response element) recognised by the CREB activator; SRE (serume response element), recognised by serume response factor…

Cell-specific moduleso are located in the promoters of genes that are expressed in just 1 type of tissueo the erythroid module, which is binding site for the GATA-1 activator; the myoblast module, recognised by MyoD…

Modules for developmental regulatorso 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 – enhancerEB - enhancer-blocker proteins

A model for establishment of silencing and barrier activity

2010, 11, 439-446

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, 703-713

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

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