Chapter 19 Regulation of Gene Expression in Eukaryotes © John Wiley & Sons, Inc.

Chapter Outline Ways of Regulating Eukaryotic Gene Expression: An Overview Induction of Transcriptional Activity by Environmental and Biological Factors Molecular Control of Transcription in Eukaryotes Posttranscriptional Regulation of Gene Expression by RNA Interference Gene Expression and Chromatin Organization Activation and Inactivation of Whole Chromosomes © John Wiley & Sons, Inc.

Ways of Regulating Eukaryotic Gene Expression: An Overview Eukaryotic gene expression can be regulated at the transcriptional, processing, or translational levels. © John Wiley & Sons, Inc.

Eukaryotic Gene Expression -Capping 5’ polyAed at 3” -RNA Splicing -Compartmentalization -Regulation ? © John Wiley & Sons, Inc.

Controlled Transcription of DNA Eukaryote: Both intracellular signaling and intercellular communication are important for transcriptional regulation in eukaryotes (cell surface to nucleus). Positive and negative regulator proteins called transcription factors bind to specific regions of DNA and stimulate or inhibit transcription. Prokaryote: Protein/DNA interaction: negative (lac repressor) and positive ( CAP/cAMP) RNA polymerase © John Wiley & Sons, Inc.

Alternate Splicing of RNA Splicing:-removing introns-spliceosomes Alternate splicing of transcripts makes it possible for a single gene to encode several polypeptides. is a prominent mechanism to generate protein diversity. © John Wiley & Sons, Inc.

Alternate Splicing of the Rat Troponin T Gene © John Wiley & Sons, Inc.

Cytoplasmic Control of mRNA Stability mRNA degradation mRNA stability is influenced by several factors The poly(A) tail The sequence of the 3’UTR Chemical factors (e.g., hormones) Small interfering RNAs (siRNAs) or microRNAs (miRNAs) © John Wiley & Sons, Inc.

Induction of Transcriptional Activity by Environmental and Biological Factors Eukaryotic gene expression can be induced by environmental factors such as heat and by signaling molecules such as hormones and growth factors. Lactose- inducer Tryptophan- repressor bacteria © John Wiley & Sons, Inc.

The Heat-Shock Genes (Proteins) When organisms are subjected to the stress of high temperature, they synthesize a group of proteins (the heat-shock proteins) that help to stabilize the internal cellular environment. The expression of the heat-shock proteins is regulated at the transcriptional level; transcription of the heat-shock genes is induced by heat. © John Wiley & Sons, Inc.

Induction of the Drosophila hsp70 Gene by Heat Shock 36.7 to 37ºC 41 to 42ºC © John Wiley & Sons, Inc.

Regulation of Gene Expression by Steroid Hormones Transcription factors © John Wiley & Sons, Inc.

Regulation of Gene Expression by Peptide Hormones Signal transduction (several) © John Wiley & Sons, Inc.

Hormone Response Elements Hormone response elements (HREs) are analogous to the heat-shock response elements. HREs are DNA specific sequences located near the genes they regulate that bind specific proteins that act as transcription factors. © John Wiley & Sons, Inc.

Activation of Transcription by Hormones A steroid hormone/cytosolic receptor complex binds to the HRE sequence to stimulate transcription. For peptide hormones, the receptor stays at the cell membrane; the signal is conveyed through the cytoplasm by other proteins, causing a transcription factor to bind to a regulatory sequence near a gene. © John Wiley & Sons, Inc.

Molecular Control of Transcription in Eukaryotes The transcription of eukaryotic genes is regulated by interactions between proteins and DNA sequences within or near the genes. © John Wiley & Sons, Inc.

DNA Sequences that Control Transcription Basal transcription factors are proteins that bind to specific DNA sequences within the promoter to facilitate RNA polymerase alignment. Special transcription factors are proteins that bind to response elements or to sequences called enhancers that are located near a gene and facilitate the action of basal transcription factors and RNA polymerase. © John Wiley & Sons, Inc.

Properties of Enhancers Enhancers act over relatively large distances. The influence of an enhancer of gene expression is independent of orientation. The effects of enhancers are independent of position. They may be upstream, downstream, or within an intron. © John Wiley & Sons, Inc.

Tissue-Specific Enhancers of the Drosophila yellow Gene © John Wiley & Sons, Inc.

There Are Several Types of Transcription Factors Basal factors (TFs) and RNA polymerase bind to promoter and TATAA box. Activators are proteins that recognize specific short DNA sequences inducing the efficiency of the promoters. Co-activators are proteins required for a more efficient transcription. They do not bind DNA. Regulators of chromatin structure Figure 25.2

Regulation of Transcription by Enhancers Proteins that bind to enhancers influence the activity of proteins that bind to promoters, including the basal transcription factors and RNA polymerase. These proteins are brought into contact with one another by the mediator complex. © John Wiley & Sons, Inc.

Proteins That Control Transcription Transcription factors usually have two domains (fragment) that may be in separate parts of the molecule or overlapping A DNA binding domain that binds the enhancer A transcriptional activation domain A transcription factor bound to an enhancer element may interact with other proteins bound at enhancers or with proteins bound at the promoter to facilitate RNA polymerase alignment. © John Wiley & Sons, Inc.

Structural Motifs (smaller fragment) of Transcription Factors © John Wiley & Sons, Inc.

-----control elements Anatomy of a Typical Eukaryotic Gene, with Its Core Promoter and Proximal Control Region -----control elements Enhancer Silencer monocistronic

Model for Enhancer Action Suppressor or silencer DHAC: De-acetylase complex MTC: Methyl transferase complex Co-repressor HAT: histone acetyl transferase Co-activator Remodeling complex

Combinatorial Model for Gene Expression

Post-transcriptional Regulation of Gene Expression by RNA Interference Short noncoding RNAs may regulate the expression of eukaryotic genes by interacting with the messenger RNAs produced by these genes. © John Wiley & Sons, Inc.

RNA Interference Small, noncoding RNAs base pair with target sequences in mRNA. The small RNAs interfere with expression of the target mRNAs. RNAi has been documented in C. elegans, Drosophila, Arabidopsis, and in mammals, including humans. © John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

MicroRNAs Some molecules that induce RNAi are derived from microRNA (mir) genes. The mir transcript forms a stem-loop structure that is processed by the enzymes Drosha and Dicer to form an miRNA © John Wiley & Sons, Inc.

Sources of siRNA and miRNA miRNAs are derived from endogenous transcripts of the mir genes. Long double-stranded RNA that is transfected or injected into a cell can be processed to form an siRNA. This can be used experimentally to knock down expression of a gene. © John Wiley & Sons, Inc.

Gene Expression and Chromatin Organization Various aspects of chromatin organization influence the transcription of genes. © John Wiley & Sons, Inc.

Chromatin packaging: Eu- or hetero-chromatin Position-effect variegation: change in the position of the interaction of DNA/histones may move the conversion of eu- to hetero-chromatin Eye color phenotype © John Wiley & Sons, Inc.

Molecular Organization of Transcriptionally Active DNA Transcriptionally active DNA is more sensitive to DNase I than non-transcribed DNA. The nuclease sensitivity of transcriptionally active genes depends on the presence of two small nonhistone proteins, HMG14 and HMG17. The promoter and enhancer regions of active genes contain DNase I hypersensitive sites. © John Wiley & Sons, Inc.

The -Globin Gene Cluster The locus control region (LCR) contains several DNase I hypersensitive sites. The -globin genes are spatially and temporally regulated. The LCR is dependent on orientation, unlike enhancer elements. The LCR insulates the -globin genes from nearby chromatin. © John Wiley & Sons, Inc.

Chromatin Remodeling In preparation for transcription, nucleosomes are altered by multiprotein complexes in a process called chromatin remodeling. © John Wiley & Sons, Inc.

Types of Chromatin Remodeling Complexes Histone acetyl transferases (HATs) transfer acetyl groups to lysine side chains on histone proteins. Other remodeling complexes disrupt nucleosome structure near a gene’s promoter by sliding or transferring histone octamers to new locations (e.g., the yeast SNI/SNF complex) © John Wiley & Sons, Inc.

Reverse Remodeling Active chromatin can be made inactive by biochemical modifications to histones. Histone deacetylases (HDACs) remove acetyl groups from histone proteins. Histone methyl transferases (HMTs) add methyl groups to histone proteins. DNA methyl transferases (DNMTs) add methyl groups to nucleotides to inactivate transcription. © John Wiley & Sons, Inc.

Euchromatin and Heterochromatin Heterochromatin stains deeply. Euchromatin stains more lightly. Euchromatin contains the majority of eukaryotic genes. © John Wiley & Sons, Inc.

Chromatin Is Divided into Euchromatin and Heterochromatin Heterochromatin is more densely packed than euchromatin Heterochromatin : -permanently condensed -consists in DNA sequence repeats (not transcribed) -reduced density of genes (inactivated) -replicates at late states of the S phase. -interacts with Histones Regions of heterochromatin remain densely packed throughout interphase.

Types of Heterochromatin Centric heterochromatin is located around the centromeres. Intercalary heterochromatin is dispersed throughout eukaryotic chromosomes. Telomeric heterochromatin is located at the ends of the chromosomes. © John Wiley & Sons, Inc.

DNA Methylation Most methylated cytosines are found in the dinucleotide sequence CG, denoted mCpG. The restriction enzyme HpaII recognizes and cleaves the sequence CCGG, but cannot cleave the sequence when the second cytosine is methylated. © John Wiley & Sons, Inc.

CpG Islands CpG dinucleotides occur less often that expected in mammalian genomes, probably due to mutation into TpG dinucleotides over the course of evolution. The distribution of CpG dinucleotides is uneven. Most CpG islands are located near transcription start sites; cytosines in these regions are rarely methylated. © John Wiley & Sons, Inc.

Gene Expression Is Associated with DNA De-methylation Gene expression is associated with de-methylation Me Me

Methylated DNA is Associated with Transcriptional Repression The inactive X chromosome in female mammals is extensively methylated. Regions of mammalian genomes containing repetitive sequences are methylated. Proteins that repress transcription have been shown to bind to methylated DNA. DNA methylation in mammals is responsible for imprinting, in which the expression of a gene is controlled by its parental origin. © John Wiley & Sons, Inc.