PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker CHAPTER 15 GENE REGULATION IN EUKARYOTES Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

INTRODUCTION Eukaryotic organisms have many benefits from regulating their genes For example They can respond to changes in nutrient availability They can respond to environmental stresses In plants and animals, multicellularity and a more complex cell structure, also demand a much greater level of gene expression 15-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

INTRODUCTION Gene regulation is necessary to ensure 1. Expression of genes in an accurate pattern during the various developmental stages of the life cycle Some genes are only expressed during embryonic stages, whereas others are only expressed in the adult 2. Differences among distinct cell types Nerve and muscle cells look so different because of gene regulation rather than differences in DNA content 15-3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 15.1 15-4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

15.1 REGULATORY TRANSCRIPTION FACTORS Transcription factors are proteins that influence the ability of RNA polymerase to transcribe a given gene There are two main types General transcription factors Required for the binding of the RNA pol to the core promoter and its progression to the elongation stage Are necessary for basal transcription Regulatory transcription factors Serve to regulate the rate of transcription of nearby genes They influence the ability of RNA pol to begin transcription of a particular gene 15-5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Regulatory transcription factors recognize cis regulatory elements located near the core promoter These sequences are known as response elements, control elements or regulatory elements The binding of these proteins to these elements, affects the transcription of an associated gene A regulatory protein that increases the rate of transcription is termed an activator The sequence it binds is called an enhancer A regulatory protein that decreases the rate of transcription is termed a repressor The sequence it binds is called a silencer Refer to Figure 15.2 15-6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Functional Domains of Eukaryotic Transcription Factors DNA binding domain – DBD Binds specific sequence of base pairs Transcriptional activation domain – TAD Interacts with basal txn factors or directly with RNA polymerase II Protein-protein interaction domain – PPID Homo- & Hetero dimerization domains Interaction with other txn factors (besides the basal apparatus)

Transcription Factor Families Family Representative Pit-1,Unc-86, Oct-2, Brn-5 POU Hoxa-1, Antp, eve, en Hox Paired Box Pax-1,2,6, Prd Lim-1, Isl-1 LIM Homeodomain Forkhead/Winged Helix Fkh, HNF-3 AP1, Myc, C/EBP leucine-zipper Basic MyoD, Ac, da, emc helix-loop-helix WT-1, Krox-20, Kr, hb monomeric GR, ER, RAR, kni steroid receptors Zinc-finger Sry, SoxD, Sox2 Sox MEF-1, MCM-1, AG, DEF, SRF MADS box

Structural Features of Regulatory Transcription Factors Transcription factor proteins contain regions, called domains, that have specific functions One domain could be for DNA-binding Another could provide a binding site for effector molecules A motif is a domain or portion of it that has a very similar structure in many different proteins Figure 15.3 depicts several different domain structures found in transcription factor proteins 15-8 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Composed of one a-helix and two b-sheets held together by a zinc (Zn++) metal ion Figure 15.3 15-10 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Zinc Finger DNA Binding Domain

Zinc Finger DNA Binding Domains Interacting with DNA

Protein-DNA Contacts of Zif268

Specificity of Amino Acids for DNA Site Recognition

Steroid Hormone Receptor Dimers Binding DNA

Helix-Turn-Helix DNA Binding Motif The Homeodomain

Hydrogen bonding between an a-helix and nucleotide bases is one way a transcription factor can bind to DNA Figure 15.3 15-9 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Helix-turn-Helix Motif Found in l Repressor

Homeodomain First Identified in Genes Causing Homeotic Transformations Antennapedia Ultrabithorax

Note: Helix-loop-helix motifs can also mediate protein dimerization Figure 15.3

Basic Domain Coupled with Helix-loop-Helix (bHLH)

Leucine Zipper and bZip domains Alternating leucine residues in both proteins interact (“zip up”), resulting in protein dimerization Two a-helices intertwined due to leucine motifs Homodimers are formed by two identical transcription factors; Heterodimers are formed by two different transcription factors Figure 15.3

Basic Domain Coupled with Leucine Zipper (bZip)

Types of Activation Domains Another activation domain is a S/T rich sequence

Chimeric Transcription Factors Demonstrate Independent Domain Functions

Figure 15.2 15-7 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Mediation of TFIID binding Transcriptional activator recruits TFIID to the core promoter and/or activates its function Thus, transcription will be activated Transcriptional repressor inhibits TFIID binding to the core promoter or inhibits its function Thus, transcription will be repressed ON Activator protein Coactivator TFIID Coding sequence Enhancer Core promoter The activator/coactivator complex recruits TFIID to the core promoter and/or activates its function. Transcription will be activated.

Regulation of Transcription Factors Binding of an effector molecule Protein-protein interactions Covalent modification

Steroid Hormone Receptors Heat shock protein Heat shock proteins leave when hormone binds to receptor Nuclear localization Sequence is exposed Formation of a homodimer Glucocorticoid Response Elements These function as enhancers Transcription of target gene is activated Figure 15.6

The CREB Protein The CREB protein is another regulatory transcriptional factor functioning within living cells CREB is an acronym for cAMP response element-binding CREB protein becomes activated in response to cell-signaling molecules that cause an increase in cAMP Cyclic adenosine monophosphate The CREB protein recognizes a response element with the consensus sequence 5’–TGACGTCA–3’ This has been termed a cAMP response element (CRE) 15-21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Regulation by phosphorylation CREB Could be a hormone, neurotransmitter, growth factor, etc. Acts as a second messenger Activates protein kinase A Phosphorylated CREB binds to DNA and stimulates transcription Unphosphorylated CREB can bind to DNA, but cannot activate RNA pol

Positioned at regular intervals from -3,000 to + 1,500 Disruption in nucleosome positioning from -500 to + 200 Changes in nucleosome position during the activation of the b-globin gene Figure 15.3 15-40 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Chromatin Remodeling As discussed in Chapter 12, there are two common ways in which chromatin structure is altered 1. Covalent modification of histones 2. ATP-dependent chromatin remodeling

Removes acetyl groups, thereby restoring a tighter interaction 1. Covalent modification of histones Amino terminals of histones are modified in various ways Acetylation; phosphorylation; methylation Adds acetyl groups, thereby loosening the interaction between histones and DNA Removes acetyl groups, thereby restoring a tighter interaction Figure 12.13

2. ATP-dependent chromatin remodeling The energy of ATP is used to alter the structure of nucleosomes and thus make the DNA more accessible Proteins are members of the SWI/SNF family Mutants in SWI are defective in mating type switching Mutants in SNF are sucrose non-fermenters Figure 12.13 These effects may significantly alter gene expression

DNA Methylation DNA methylation is a change in chromatin structure that silences gene expression It is common in some eukaryotic species, but not all Yeast and Drosophila have little DNA methylation Vertebrates and plants have abundant DNA methylation In mammals, ~ 2 to 7% of the DNA is methylated

Only one strand is methylated Both strands are methylated CH3 (or DNA methylase) Only one strand is methylated CH3 Both strands are methylated Figure 15.15 15-48

DNA methylation usually inhibits the transcription of eukaryotic genes Especially when it occurs in the vicinity of the promoter In vertebrates and plants, many genes contain CpG islands near their promoters These CpG islands are 1,000 to 2,000 nucleotides long In housekeeping genes The CpG islands are unmethylated Genes tend to be expressed in most cell types In tissue-specific genes The expression of these genes may be silenced by the methylation of CpG islands

Figure 15.16 Transcriptional silencing via methylation Transcriptional activator binds to unmethylated DNA This would inhibit the initiation of transcription Transcriptional silencing via methylation Figure 15.16

Transcriptional silencing via methylation Figure 15.16

DNA Methylation is Heritable Methylated DNA sequences are inherited during cell division 15-52 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display