How Genes Are Regulated Gene Regulation in Eukaryotes Overview of Eukaryotic Gene Regulation Control of Transcription Initiation Regulation After Transcription A Comprehensive Example: Sex Determination in Drosophila Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16

Overview of eukaryotic gene regulation Eukaryotes use complex sets of interactions Regulated interactions of large networks of genes Each gene has multiple points of regulation Themes of gene regulation in eukaryotes: Environmental adaptation in unicellular eukaryotes Maintenance of homeostasis in multicellular eukaryotes Genes are turned on or off in the right place and time Differentiation and precise positioning of tissues and organs during embryonic development

Compared to prokaryotes, eukaryotes have additional levels of complexity for controlling gene expression Eukaryotic genomes are larger than prokaryotic genomes Chromatin structure in eukaryotes makes DNA unavailable to transcription machinery Additional RNA processing events occur in eukaryotes In eukaryotes, transcription takes place in the nucleus and translation takes place in the cytoplasm

Key regulatory differences between eukaryotes and prokaryotes Attenuation is a regulatory feature found throughout Archaea and Bacteria causing premature termination of transcription.[1] Attenuators are 5'-cis acting regulatory regions which fold into one of two alternative RNA structures which determine the success of transcription.[2] The folding is modulated by a sensing mechanism producing either a Rho-independent terminator, resulting in interrupted transcription and a non-functional RNA product; or an anti-terminator structure, resulting in a functional RNA transcript.  Table 16.1

Multiple steps where production of the final gene product can be regulated in eukaryotes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16 there are many more steps in the process leading to an active product beyond transcription initiation. Fig. 16.1

Control of transcription initiation Three types of RNA polymerases in eukaryotes RNA pol I – transcribes rRNA genes RNA pol II – transcribes all protein-coding genes (mRNAs) and micro- RNAs RNA pol III – transcribes tRNA genes and some small regulatory RNAs

RNA polymerase II transcription RNA pol II catalyzes synthesis of the primary transcript, which is complementary to the template strand of the gene Most RNA pol II transcripts undergo further processing to generate mature mRNA RNA splicing – removes introns Addition of 5' GTP cap – protects RNA from degradation Cleavage of 3' end and addition of 3' polyA tail

cis-acting elements: promoters and enhancers Promoters – usually directly adjacent to the gene Include transcription initiation site Often have TATA box: Allow basal level of transcription Enhancers – can be far away from gene Augment or repress the basal level of transcription Fig. 16.2

Use of reporter genes to identify promoters and enhancers in eukaryotes Examples of reporter genes used in eukaryotes lacZ gene (Ch 15), blue color when X-gal used Gene for "green fluorescent protein" (GFP) Mutations in regulatory regions can be engineered and tested for effects on expression Fig. 16.3

trans-acting factors interact with cis-acting elements to control transcription initiation Activators and repressors bind to enhancers Direct effects of transcription factors: Through binding to DNA Indirect effect of transcription factors: Through protein-protein interactions Basal Factors bind to promoter Fig. 16.4

Use of reporter genes to identify trans-acting factors in transcriptional regulation Identifying trans-acting factors. (a) Trans-acting factors bind to regulatory regions (enhancers) to increase transcription. (b) A trans-acting mutation that reduces transcription identifies a regulatory protein. Fig. 16.5

Basal transcription factors Basal transcription factors assist the binding of RNA pol II to promoters (see Fig 16.6) Key components of basal factor complex: TATA box-binding protein (TBP) Bind to TATA box First of several proteins to assemble at promoter TBP-associated factors (TAFs) Bind to TBP assembled at TATA box RNA pol II associates with basal complex and initiates basal level of transcription

Basal factors bind to promoters of all protein-encoding genes Ordered pathway of assembly at promoter: 1. TBP binds to TATA box 2. TAFs bind to TBP 3. RNA pol II binds to TAFs Fig. 16.6

Activators are transcription factors that bind to enhancers Activators are responsible for much of the variation in levels of transcription of different genes Increase levels of transcription by interacting directly or indirectly with basal factors at the promoter 3-dimensional complex of proteins and DNA (Fig. 16.7) Mechanisms of activator effects on transcription Stimulate recruitment of basal factors and RNA pol II to promoters Stimulate activity of basal factors already assembled on promoters Facilitate changes in chromatin structure Although similar sets of basal factors bind to all the promoters of the tens of thousands of genes in the eukaryotic genome, a cell can transcribe different genes into widely varying amounts of mRNA. This enormous range of transcriptional regulation occurs through the binding of different transcription factors to enhancer elements associated with different genes.

Binding of activators to enhancers increases transcriptional levels Low level transcription occurs when only basal factors are bound to promoter When basal factors and activators are bound to DNA, rate of transcription increases Fig. 16.7

Domains within activators Activator proteins have two functional domains Sequence-specific DNA binding domain (Fig 16.8) Binds to enhancer Transcription-activator domain Interacts with other transcriptional regulatory proteins Some activators have a third domain (Fig 16.9) Responds to environmental signals Example - steroid hormone receptors

DNA-binding domains of activator proteins Interacts with major groove of DNA Specific amino acids have high-affinity binding to specific nucleotide sequence The three best-characterized motifs: Helix-loop-helix (HLH) Helix-turn-helix (HTH) Zinc finger Fig. 16.8

Steroid hormone receptors are activators only in the presence of specific hormones Steroid hormones don't bind to DNA but are coactivators of steroid hormone receptors In the absence of hormone, these receptors cannot bind to DNA and so cannot activate transcription In the presence of hormone, these receptors bind to enhancers for specific genes and activate their expression Fig. 16.9

Coactivators Proteins and other molecules that play a role in transcriptional activation without binding directly to DNA are called coactivators. The hormone component of a DNA bound hormone- receptor activation complex is one example of a coactivator.

Many activators must form dimers to function Homodimers: multimeric proteins made of identical subunits Heterodimers: multimeric proteins made of nonidentical subunits Examples - Fos and Jun, both have leucine zippers Fos forms heterodimers with Jun, but cannot form homodimers Jun can form homodimers Jun-Jun and Jun-Fos dimers recognizes different sequences Jun-Jun and Jun-Fos dimers bind to the same enhancer sequences, but have different affinities Fig. 16.10

Dimerization domains make up another class of transcription factor domains Dimerization domains are specialized for polypeptide-polypeptide interactions Leucine zippers are a common dimerization motif in eukaryotes Amino acid sequence twirls into an a helix with leucines protruding at regular intervals Heterodimers greatly increases the number of potential regulatory transcription complexes Fig. 16.11

Repressor proteins suppress transcription initiation through different mechanisms Some repressors have no effect on basal transcription but suppress the action of activators Compete with activator for the same enhancer (Fig 16.12a) OR Block access of activator to an enhancer, quenching, (Fig 16.12b) Some repressors eliminate virtually all basal transcription from a promoter Block RNA pol II access to promoter Bind to sequences close to promoter or distant from promoter

Repressor proteins that act through competition with an activator protein Repressor binds to the same enhancer sequence as the activator Has no effect on the basal transcription level Fig. 16.12a

Repressor proteins that act through quenching an activator protein Quenchers bind to the activator but do not bind to DNA Type I: Repressor blocks the DNA-binding domain Type II: Repressor blocks the activation domain Fig. 16.12b

Corepressors These blocked activators still bind to their enhancers, but once bound, they are unable to carry out activation. Quenching polypeptides that operate in this manner are termed corepressors. Just like coactivators, corepressors associate indirectly with enhancers through their interaction with DNA- binding proteins.

Some repressors eliminate virtually all basal transcription by binding to DNA sequences very close to the promoter and thereby blocking RNA polymerase’s access to the promoter. OR bind to DNA sequences farther from the promoter and then reach over and contact the basal factor complex at the promoter, allow contact between the repressor and the basal factor complex.

The same transcription factor can play different roles in different cells Example: a2 repressor in yeast Determines mating type by binding to enhancers of certain genes In a haploids and a/a diploids, a2 binds to enhancers of a- specific genes but not to enhancers of haploid-specific genes In a/a diploids, binding specificity of a2 is altered so that it can bind to enhancers of haploid-specific genes Fig. 16.13

The Myc-Max mechanism can activate or repress transcription Myc − identified as an oncogene Regulates transcription of genes involved in cell proliferation Doesn't have DNA binding activity on its own Max − identified through its binding to Myc Max/Max homodimers and Myc/Max heterodimers bind to the same enhancers Max/Max represses transcription Myc/Max activates transcription

Comparative structures of Myc and Max Myc/Myc homodimers cannot form Max/Max homodimers and Myc/Max heterodimers can form Max/Max homodimers bind DNA but cannot act as activators Fig. 16.14

Dimer structure and subunit concentrations of Myc and Max affect activation or repression Max is constitutively expressed but Myc expression is tightly regulated Max/Max acts as a repressor when Myc is not expressed Fig. 16.15a

Dimer structure and subunit concentrations of Myc and Max affect activation or repression When Myc is present, Myc/Max heterodimers form and activate transcription Myc - Max affinity is higher than Max - Max affinity Fig. 16.15b

Complex regulatory regions enable fine-tuning of gene expression Each gene can have many regulatory proteins In humans, ~2000 genes encode transcriptional regulatory proteins Each regulatory protein can act on many genes Each regulatory region can have dozens of enhancers

Not just a matter of turning genes on and off It also entails fine-tuning the precise level of transcription higher or lower in different cells, higher or lower in cells of the same tissue but at different stages of development. It also includes mechanisms that allow each cell to modify its program of gene activity in response to constantly changing signals from its neighbors.

Complex regulatory regions enable fine-tuning of gene expression Enhanceosome – multimeric complex of proteins and other small molecules that associate with an enhancer Enhancers can be bound by activators and repressors with varying affinities Different sets of cofactors and corepressors compete for binding to activators and repressors These changes lead to the assembly of an altered enhanceosome, which recalibrates gene activity. In short, a large, exquisitely controlled machinery determines the level of primary transcript produced.

Control of the string gene in drosophila The enhancer regions of some genes are very large, containing multiple elements that make possible the fine-tuned regulation of a gene. Those genes in multicellular eukaryotes that must be expressed in many different tissues.

Different enhancers for the string gene in Drosophila are used in different cell types Cells in different parts of Drosophila embryos go through the 14th mitosis at different times The string gene product activates the 14th mitosis Different activators for string are expressed at different times in different tissues Fig. 16.16

Chromatin structure and epigenetic effects Chromatin structure can affect transcription Nucleosomes can sequester promoters and make them inaccessible to RNA polymerase and transcription factors Histone modification and DNA methylation Chromatin remodeling and hypercondensation Epigenetic changes – changes in chromatin structure that are inherited from one generation to the next Altered chromatin structure can cause changes in gene expression and therefore phenotypic changes in a cell or an organism. DNA sequence is not altered

Chromatin reduces transcription (a) DNA molecules containing a promoter and an associated gene can be purified away from chromatin proteins in vitro. The addition of basal factors and RNA polymerase to this purifi ed DNA induces high levels of transcription. (b) Within the eukaryotic nucleus, DNA is present within chromatin. Promoter regions are generally sequestered within the nucleosome and only rarely bind to basal factors and RNA polymerase. Thus, the chromatin structure maintains basal transcription at very low levels. Fig. 16.17

Effects of chromatin structure on transcription: Histone modification and DNA methylation N-terminal tails of histones H3 and H4 can be modified Methylation, acetylation, phosphorylation, and ubiquitination Can affect nucleosome interaction with other nucleosomes and with regulatory proteins Can affect higher-order chromatin structure the differentiation of pluripotent stem cells into specific tissue lineages, development DNA methylation occurs at C5 of cytosine in a CpG ? dinucleotide Associated with gene silencing CpG is shorthand for 5'—C—phosphate—G—3' , that is, cytosine and guanine separated by only one phosphate; phosphate links any two nucleosides together in DNA.

Determining methylation state of DNA using two restriction enzymes and Southern blotting HpaII and MspI have the same recognition sequence (CCGG), but different sensitivity to DNA methylation HpaII doesn't cut if 2nd C is methylated Methylation doesn't affect MspI cutting A determination of the methylation status of a DNA region can be made using a pair of restriction enzymes that both recognize the same base sequence, with one being able to digest methylated DNA, while the other can’t. In this example, the restriction site is CCGG, and the enzymes are MspI, which can digest both methylated and unmethylated sites, and HpaII, which cannot digest methylated sites. If a methylated site is present between two unmethylated sites, HpaII digestion will leave a larger fragment than MspI. After electrophoresis and Southern blot analysis with a probe that hybridizes to a sequence on one side of the methylated site, there will be a clearly observable difference in band size. Fig. 16.18

Chromatin remodeling can expose the promoter Nucleosomes can be repositioned of removed by chromatin remodeling complexes After remodeling, DNA at promoters and enhancers becomes more accessible to transcription factors Can be assayed using DNase digestion (DNase hypersensitive sites, DH sites) DH sites show up at the 5’ ends of genes that are either undergoing transcription or are being prepared for transcription in a later step of cellular differentiation Fig. 16.19

The SWl-SNF remodeling complex One of best-studied remodeling complexes in yeast Uses energy from ATP hydrolysis to alter nucleosome positioning chromatin decompaction gives basal factors much greater access to promoter regions, and consequently, transcription rapidly accelerates In Drosophila Human cells Fig. 16.20

Hypercondensation of chromatin results in silencing of transcription Examples of heterochromatin – inactive X chromosome, centromeres, telomeres Heterochromatin has methylation of CpG dinucleotides and methylation of lysine in histone H3 tails Fig. 16.21

Gene silencing by the SIR complex in yeast Gene for a2 repressor is at the MAT (mating type) locus HML and HMR are copies of MAT locus but are silenced by SIR complex SIR mutations cause sterility because HML and HMR genes not silenced and both a- and a- specific genes are expressed They allow the simultaneous expression of a and α information. The resulting cells, which behave as diploids, do not mate. Fig. 16.22

Genomic imprinting results from transcriptional silencing Mendal states that the parental origin of an allele not affect its function in the F 1 generation ???? NOT CASE ALWAYS!! Genomic imprinting – expression pattern of a gene depends on whether it was inherited from the mother or father Occurs with some genes of mammals Epigenetic effect (no change in DNA sequence) Paternally imprinted gene is transcriptionally silenced if it was transmitted from the father Maternal allele is expressed Maternally imprinted gene is transcriptionally silenced if it was transmitted from the mother Paternal allele is expressed

For most genes, we inherit two working copies -- one from mom and one from dad. But with imprinted genes, we inherit only one working copy. Depending on the gene, either the copy from mom or the copy from dad is epigenetically silenced. they are reset during egg and sperm formation. Regardless of whether they came from mom or dad, certain genes are always silenced in the egg, and others are always silenced in the sperm.

In mice, deletion of Igf2 causes a mutant phenotype only when transmitted by father Maternal imprinting of Igf2 gene Maternally-inherited Igf2 allele is silenced Igf2 deletion heterozygotes have normal phenotype if the mutant allele was inherited from the mother The phenotypic effect of an Igf2 deletion is determined by the parent transmitting the mutant locus. This parent-of-origin effect can be demonstrated in the two-generation cross illustrated here. Fig. 16.23a

Methylation of complementary strands of DNA in genomic imprinting Epigenetic state can be maintained across cell generations through the action of DNA methylases One important component is the methylation of cytosines in CG dinucleotides within the imprinted region Fig. 16.23b

H19, found just 70 kb downstream of Igf 2, Imprinted but in opposite way With H19, the copy inherited from the father is silenced and the copy inherited from the mother is active in normal mice. identified an enhancer region downstream of the H19 gene that can interact with promoters for both genes ( Fig. 16.23c ). In the region between the two genes lies another type of transcriptional regulation element called an insulator an insulator becomes functional, it stops communication between enhancers on one side of it and promoters on the other side.

Insulators limit the chromatin region over which an enhancer can operate An insulator that binds CTCF is involved in reciprocal imprinting of the Igf2 and H19 genes Methylation of the H19 promoter and the insulator occurs in spermatogenesis In the Igf2-H19 region, the insulator DNA becomes functional by binding a protein called CTCF. The binding normally occurs on the maternal chromosome. As a result, the enhancer element on the maternal chromosome can interact only with the promoter of H19; this interaction, of course, turns on the H19 gene. In such a situation, the Ig f2 gene remains unexpressed. On the paternal chromosome, by contrast, both the insulator and the H19 promoter are methylated. Because methylation of the insulator prevents the binding of CTCF, the insulator is not functional; and without a functional insulator, the enhancer downstream of H19 can reach over a great distance to activate transcription from the Igf2 promoter. In addition, methylation of the H19 promoter suppresses transcription of the paternal H19 gene. Imprinting of the paternal chromosome by methylation thus turns on transcription of Ig f2 and prevents transcription of H19. A methylase is essential for imprinting of the H19 gene Fig.16.23c

The resetting of genomic imprints during meiosis Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16 Epigenetic imprints remain throughout the lifespan of the mammal In germ cells, epigenetic imprints are reset at each generation During meiosis, imprints are erased and new ones are set Fig. 16.23d

With imprinting, it is the sex of the parent carrying a mutant allele that counts, and not the sex of the individual inheriting the mutation An inactivating mutation in a maternally imprinted gene could pass unnoticed from mother to daughter for many generations (because the maternally derived gene copy is inactive due to imprinting). If, however, the mutation passed from mother to son, the son would have a normal phenotype (having received an active wild type allele from his father), but the son’s children, both boys and girls, would each have a 50% chance of receiving a mutant paternal allele.

Genomic imprinting and human disease Examples: two syndromes associated with small deletions in chromosome 15 At least two genes within this region are differently imprinted Praeder-Willi syndrome occurs when the deletion is inherited from the father Angelman syndrome occurs when the deletion is inherited from the mother Affected individuals have mental retardation and development disorders

Inheritance patterns of disorders resulting from mutations in imprinted genes These pedigrees may appear to be instances of incomplete penetrance, but are distinctly different In each pedigree, affected individuals (represented by filled in, orange circles and squares) are heterozygotes for a deletion removing a gene that has either a paternal or a maternal imprint. In these pedigrees, a dotted symbol indicates individuals carrying a deleted chromosome but not displaying the mutant phenotype. Fig. 16.23e

Regulation after transcription Posttranscriptional regulation can occur at any step At the level of RNA Splicing, stability, and localization Example – alternative splicing of mRNA Generates more diversity of proteins Common feature in eukaryotes At the level of protein Synthesis, stability, and localization

Expression of the Sxl mRNA in early Drosophila development Sex lethal (Sxl) gene encodes a protein required for female-specific development (see Comprehensive example) In early embryos, Sxl is transcribed only in females Fig. 16.24a

Differential RNA splicing of Sxl mRNA during Drosophila development Later in development, Sxl gene is transcribed in both sexes from different promoter Sxl protein regulates alternative splicing of its own mRNA Later in development, the transcription factors activating the Sxl early promoter in females disappear; but to develop as females, these animals still need the Sxl protein. How can they still make the Sxl protein they need? Fig. 16.24b

Some small RNAs are responsible for RNA interference (RNAi) Specialized RNAs that prevent expression of specific genes through complementary base pairing Small (21 – 30 nt) RNAs Micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) First miRNAs (lin-4 and let-7) identified in C. elegans Nobel prize to A. Fire and C. Mello in 2006 Posttranscriptional mechanisms for gene regulation mRNA stability and translation May also affect chromatin structure

Primary transcripts containing miRNA Most miRNAs are transcribed by RNA polymerase II from noncoding DNA regions that generate short dsRNA hairpins Fig. 16.25a

miRNA processing Drosha excises stem-loop from primary miRNA (pri-miRNA) to generate pre-miRNA of ~ 70 nt Dicer processes pre-miRNA to a mature duplex miRNA One strand is incorporated into miRNA-induced silencing complex (RISC) Fig. 16.26

Two ways that miRNAs can down-regulate expression of target genes When complementarity is perfect: Target mRNA is degraded When complementarity is imperfect: Translation of mRNA target is repressed Fig. 16.27

siRNAs detect and destroy foreign dsRNAs Two biological sources of dsRNAs that are precursors of siRNAs (pri- RNAs) Transcription of both strands of an endogenous genomic sequence Arise from exogenous virus pri-RNAs are processed by Dicer siRNA pathway targets dsRNAs for degradation siRNAs are very useful experimental tools to selectively knock down expression of target genes To study function of a gene, dsRNAs for that gene can be introduced into cells

Posttranslational modifications of proteins Ubiquitination – covalent attachment of ubiquitin to other proteins targets those proteins for degradation by the proteosome Cascades of phosphorylation and dephosphorylation Transmission of signals across the cell membrane to the nucleus (discussed more in Chapter 17) Sensitization – tissues exposed to hormones for long periods of time lose ability to respond to the hormone Example: binding of epinephrine to β-adrenergic receptors on surface of heart muscle cells

Phosphorylation and desensitization of β-adrenergic receptor Phosphorylation of receptor has no effect on its binding to epinephrine, but blocks its downstream functions Fig. 16.28

A comprehensive example of gene regulation: Sex determination in Drosophila In Drosophila, ratio of X chromosomes to autosomal chromosomes (X:A ratio) determines sex, fertility, and viability X:A ratio influences sex through three independent pathways: Male vs female appearance and behavior Development of germ cells as eggs or sperm Dosage compensation – males have increased transcription of X- linked genes

Sex-specific traits in Drosophila Fig. 16.29

How chromosomal constitution affects phenotype in Drosophila Table 16.2

Drosophila mutations that affect the two sexes differently Table 16.3 *Sxfl is a recessive mutation of Sex lethal **SxlML is a dominant mutation of Sex lethal

The X:A ratio regulates transcription of the Sxl gene Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16 Several HLH transcription factors are key regulators of sex determination X-linked genes are "numerator elements" Autosomal genes are "denominator elements" Numerator/denominator determines ratio of HLH homodimers and heterodimers Only the homodimer activates transcription of Sxl Fig. 16.30

Sxl protein regulates splicing of tra mRNA The Sxl protein triggers a cascade of splicing of transformer (tra) and double-sex (dsx) mRNAs Sxl protein regulates splicing of tra mRNA Tra protein regulates splicing of dsx mRNA Fig. 16.31

Dsx-F is a transcriptional activator and Dsx-M is a transcriptional repressor Fig. 16.32

The Tra protein regulates splicing of fruitless (fru) mRNA Fig. 16.33