Chapter 15: Eukaryotic Gene Regulation

SWI and SNF Mutants

- SWI: yeast have defective mating type switching - SNF: yeast are sucrose non fermenting

Chromatin Immunoprecipitation Sequencing (ChIP-Seq) (3)

- allows determination of nucleosome location, histone variants location, and where covalent modifications of histones occur - able to determine where nucleosomes are not by comparing DNA acquired to the known sequence - found that the transcriptional start site and the transcriptional termination site are most often free of nucleosomes

CREB protein (3)

- cAMP response element-binding protein - activated in response to cell-signaling molecules that cause and increase in the cytoplasmic concentration of cAMP (G proteins) - recognizes cAMP response element (CRE) located on target gene

Chromatin Remodeling (3)

- carried out by ATP dependent chromatin remodeling complexes with DNA translocase activity - move, evict, or change nucleosome composition (replace with variants) - multiple families including: SWI/SNF, ISWI, INO80, MI-2

DNA Methylation (4)

- change in chromatin structure by DNA methyltransferase that silences gene expression in eukaryotes - CpG islands: found near promoters and are targets for methylation (1000 to 2000 bp long) - housekeeping genes: CpG islands are unmethylated and genes tend to be expressed in most cell types - tissue specific genes: expression of some genes is silenced by CpG island methylation

Combinational Control (4)

- combination of TFs from same family stimulate or inhibit transcription - activators/repressors may be modulated by small effector molecules, protein-protein interactions, and covalent modifications - regulatory proteins may alter nucleosomes near the promoter - DNA methylation may inhibit transcription by preventing binding of activator protein/recruitment of proteins that alter chromatic compaction

Structural Features of Regulatory Transcription Factors (2)

- contain domains with specific functions, such as binding DNA - motif is a domain that has very similar structure in many different proteins, such as dimerization motifs

Enhancers and Silencers (4)

- enhancers bind TFs and increase the rate of transcription (up regulate 10-1000 fold; ~300 fold) - silencers bind TFs and decrease the rate of transcription (down regulate) - orientation independent/bidirectional - found up to several thousand bp away, downstream from promoter, and in introns

Heritability of DNA Methylation (2)

- methylated DNA sequences are inherited during cell division - explains genomic imprinting where specific genes are methylated in gametes from mother or father and remain methylated in offspring (such as gene for placenta formation)

Steroid Hormones (3)

- non polar ring structures capable of crossing plasma membrane and binding transcription factors in the cytoplasm - glucocorticoids: influence nutrient metabolism (glucose utilization, fat mobilization, protein breakdown) - gonadocorticoids: estrogen and testosterone influence growth and function of gonads

Histone Variants (4)

- over 70 moderately repetitive histone genes, a few of which have mutations - H3.3: promotes open conformation - H2ABBD: promotes open conformation - macroH2A: drives compaction (Barr bodies)

Insulators (2)

- segments of DNA that insulates gene from regulatory effect of other genes - recruit proteins that act as barriers to remodeling or block the effect of enhancers by chromosome looping

Histone Code (3)

- shows patterns of modifications to the NTD of histones and how they affect the level of transcription by changing nucleosome interactions - acetylation: loosens nucleosome by acetylating lysine using histone acetyl transferase (HATs); chromatin retightened by histone deacetylase (HDAC) - phosphorylation and methylation may also occur

Mechanism of Promoter Bashing 4 Steps

- used to locate a promoter when do not have protein that binds it 1. Amplify region upstream from a known exon and insert in to a promoter vector containing antibiotic resistance and reporter gene (beta-gal or firefly luciferase) 2. Transinfection in to mammalian cancer cells of specific tissue 3. Cell transcribes reporter gene once inside nucleus 4. Break cells and assay reporter gene activity - if high expression of reporter gene, upstream region contains promoter - shorten upstream region with primers and rerun promoter bashing to isolate the promoter location

Mediator Activation/Repression (3,2)

1. Activation - transcriptional activator stimulates the function of mediator - enables RNA pol to form pre initiation complex - mediator controls the phosphorylation activity of TFIIH and/or may phosphorylate RNA pol on its own 2. Repression - transcriptional repressor inhibits the function of mediator - transcription is repressed b/c RNA pol cannot be hyperphosphorylated

Transcriptional Activation Via Chromatin Remodeling 4 Steps

1. Binding of activator to enhancer in a nucleosome free region (NFR) 2. Activator recruits a chromatin remodeling complex and histone modifying enzymes. Nucleosomes may be moved, evicted, or replaced by histone variants depending on recruited proteins 3. General TFs and RNA pol II are able to bind core promoter to form pre-initation complex 4. During elongation, nucleosomes ahead of open complex are evicted by acetylation and behind are rebound by deacetylation

3 Modulations of Regulatory Transcription Factors

1. Binding of small effector molecule - inducer/repressors/inhibitors 2. Protein-protein interactions - concentration driven formation of dimers 3. Covalent modulation - phosphorylation, acetylation, methylation

2 Things Gene Regulation is Necessary to Ensure

1. Control of expression patterns throughout stages of development 2. Differences among distinct cells types

Mechanism of Methylation of CpG Islands 3 Step

1. DNA methyltransferase methylates CpG island 2. CpG recruits methyl-CpG binding proteins that recruit histone deacetylase 3. Histone deacetylase compacts chromatin

Mechanism of CREB Protein 3 Step

1. Extracellular signaling molecule activates G protein resulting in high cytoplasmic cAMP 2. cAMP activates protein kinase A, which enters nucleus to phosphorylate CREB dimer bound to CRE 3. Phosphorylation of CREB activates gene expression

2 Types of Transcription Factors (2,2)

1. General/basal TFs - required for binding of RNA pol to the core promoter and progression to the elongation state - necessary for basal transcription 2. Regulatory TFs - serve to regulate the rate of transcription of target genes by influencing initiation of RNA pol - recognize cis regulatory elements like enhancers/silencers and promoter

Mechanism of Glucocorticoid Gene Regulation 4 Steps

1. Glucocorticoid enters the cell and binds to glucocorticoid receptor 2. Binding results in the dissociation of heat shock protein 90 exposing the nuclear localization signal (NLS) 3. Glucocorticoid receptors dimerize and enter the nucleus 4. Glucocorticoid receptor dimer binds to glucocorticoid response element (GRE) (enhancer). Activates genes expression

3 Common Structural Motifs of Regulatory Proteins (3,2,2)

1. HTH/HLH - 60 AA dimerization domain including 2 alpha helices - DNA binding domain (bHLH) - master regulators 2. Leucine Zipper - alternating leucine residues in both proteins interact (hydrophobic) resulting in protein dimerization - Leu 7 AA apart, 3.4 AA per turn 3. Zn Finger - composed of one alpha helix and two beta sheets held together by zinc metal ion - often cysteine interacting with zinc

3 Common Regulatory TFs

1. TFIID directly or through coactivator 2. Mediator 3. Proteins that affect nucleosome composition

ChIP-Seq Mechanism 6 Steps

1. Treat cells with formaldehyde cross linker to "glue" proteins to DNA 2. Lyse cells and treat with sonicator to break dsDNA in to pieces (500-1000bp) 3. Add antibodies against histone of interest plus sugar beads with protein A/G - antibodies bind sugar beads with Fc region and the dsDNA with variable region 4. Centrifuge to acquire histone+bead+antibody pellet 5. Dissociate the beads/antibodies to isolate protein bound DNA fragments 6. Treat with a reverse cross linker and add linkers the ends of the remaining DNA strands to allow for PCR amplification

TFIID Enhancement/Repression

Enhance: - activator/coactivator complex recruits TFIID at trans activation domain to the core promoter and/or activates its function enhancing transcription Repress: - repressor protein inhibits binding of TFIID to the core promoter or inhibits it function silencing transcription

Homo vs. Heterodimers

Homo: - formed by two identical transcription factors Hetero: - formed by two different transcription factors