Genetics Test 3

Why regulate gene expression?

1. Respond to environmental conditions

2. Express genes appropriate to developmental stages of life cycle

3. Express genes appropriate to cell type

Transcriptional Regulation in Eukaryotes

1. Regulatory Transcription factors

2. Chromatin / Histone Modifications

3. DNA methylation

General transcription factors

• required for binding of RNA pol to core promoter and progression to elongation stage

• necessary for basal transcription

• Recall TFIID, TFIIH, etc.

Regulatory transcription factors

• regulate the rate of transcription of target genes

• influence ability of RNA pol to begin transcription of a particular gene

• **2-3% of human genes encode transcription factors

regulatory transcription factors recognize

cis regulatory elements near the core promoter

regulatory TF’s: activators

proteins with DNA-binding motifs

regulatory elements (control elements / regulatory sequences): enhancers

• most upstream of promoter, within few hundred bps

• many orientation-independent

______ bind ________ and up-regulate gene expression

activators, enhancers

regulatory TF’s: repressor

proteins with DNA-binding motifs

(repressor binds silencer)

regulatory elements (control elements, regulatory sequences): silencers

• most upstream of promoter within a few hundred bps

• many-orientation dependent

• repressor - silencer

_____ binds ______ and down regulates expression

repressors, silencers

Structural features of regulatory TFs

-Regulatory TF’s contain motifs that contribute to their activity

• DNA binding domain

• binding site for effector molecules

• dimerization domains

mechanism of action

most regulatory TFs don’t bind directly to RNA poymerase

1) Interact with TFIID

• Transcription activation occurs when activator complex recruits TFIID

• Transcription repression occurs when repressor inhibits binding of TFIID to core promoter

2) Interact with mediator

• Activation: activator protein + mediator, phosphorylation of C-terminal domain of RNA pol enhanced, GTFs released and RNA poly proceeds to elongation

• Repression: repressor protein + mediator, prevents phosphorylation of C-terminal domain of RNA pol, inhibits switch to elongation stage

steroid hormones

produced by endocrine glands, secreted into the bloodstream, diffuse into cells

steroid receptors

regulatory TFs that respond to steroid hormones

GRE (Glucocorticoid Response Elements)

Enhancers where steroid receptors bind (near dozens of different genes)

responding to extracellular signals

• most signaling molecules can’t enter cells

• detected at surface, message relayed to TF’s inside the cell]

• ex. G-coupled protein receptors (cAMP levels)

Cyclic AMP Response Element Binding protein (CREB)

activator - activated by increased cAMP levels and phosphorylation

Cyclic AMP Response Element (CRE)

enhancer

gene body

A region DNA whose nucleotide sequence encodes for a transcribed RNA and for those that go on to be translated to protein it typically contains exons and introns that will
be used to create primary RNA transcript

5’ UTR

A region of DNA/RNA from the 5’ end to the position of the first codon used in translation initiation (post-transcriptional control)

3’ UTR

A region of DNA/RNA from the 3’ end to the position of the last codon near the termination of transcription and used in translation for stability (post-transcriptional control)

Enhancer

A region or site of DNA to which transcription factor and co-activator binding occurs and regulates promoter availability

silencer

A region of DNA that binds negative regulatory elements for transcription suppression

promoter

A region of DNA on which transcription machinery assembles including RNA polymerase

insulators

A region of DNA that binds proteins to help define chromatin domains and protect genes from inappropriate signals, in human this is a CTCF binding site

Chromatine Structure

• DNA is condensed/compact

• several levels of compaction

• space constraints

• organization of chromosomes into domains

• affects access to transcription machinery

chromatin

DNA plus associated proteins

chromatin structure is

dynamic

DNA wraps around

histone proteins (nucleosome)

• 146 bps DNA in wrap

• linker DNA more accessible

core histones (octamer)

2* H2A, H2B, H3, H4

linker histone

H1

histones

• basic(positively charged) proteins

• contain many Lys and Arg

• bind to phosphate in DNA backbone

Histone structure

• globular domain

• flexible, charged ‘tail’

nucleosomes associate to form a

30 nm fiber - histone H1 involved

• structure still not fully elucidated

chromatin remodeling

chromatin structure is dynamic, and influences levels of gene expression

ATP-dependent chromatin remodeling can alter

chromatin structure

• small local changes or large-scale

• multiprotein complexes move or modify nucleosomes

• this changes compaction level and accessibility to transcription

histone modifications can alter

transcription levels

acetylation

looser wrap

methylation

tighter wrap

histone code

histone modifications occur in patterns that are recognized by proteins

• pattern of modifications provide binding sites for proteins that specify alterations to be made to chromatin structure

histone variants

• human genome contains over 70 histone genes

• most encode standard histones

• few have accumulated mutations

nucleosome-free regions (NFR)

• found at the beginning and end of many genes

• less regularly distributed elsewhere

tightly packed nucleosome

inaccessible protein

caveats to protein accessibility

epigenetic modifications and methylation/acetylation

DNA methylation

silences gene expression

DNA methylation enzyme

DNA methyltransferase

DNA can be

methylated on cytosine

• CpG islands near promoters of many genes

methylation of CpG islands

silences gene expression

• methylation influences TF binding

• methyl-CpG-binding proteins recruit factors that increase chromatin compaction

housekeeping genes

• expressed in most cell types

• CpG islands unmethylated

tissue-specific genes

• CpG islands unmethylated in tissues where expression is needed

• CpG islands methylated in non-target tissues

Transcriptional silencing by methylation

a. methylation inhibits the binding of an activator protein

b. Methyl-CpG-binding protein recruits proteins that close chromatin structure

DNA methylation is

heritable

• Methylated DNA sequences are inherited during cell division

• de novo methylation is infrequent, highly regulated

• genomic imprinting, epigenetics

combinatorial control

multiple factors can contribute to regulation of one single gene

activator and repressor activity can be modulated

effectors, protein-protein interactions, covalent modification

regulatory proteins can alter

nucleosomes near the promoter

DNA methylation inhibits transcription by

preventing binding of the activator and recruiting proteins that compact chromatin

Transcriptional regulation of gene expression

• regulatory TFs

• DNA methylation

• nucleosome location/composition

Translational regulation of gene expression

• small non-coding RNA

• RNA stability

• feedback inhibition

• covalent modifications

Histone acetyltransferases are directly involved in which of the following?

chemical modification of histones

Transcription factors are proteins that influence the ability of the RNA polymerase to transcribe a gene.

true

Activator proteins bind to silencer sequences and repressor proteins bind to enhancer sequences.

false

A heterodimer occurs when two identical transcription factors interact on a sequence of DNA.

false

A repressor protein would enhance the ability of TFIID to bind to the TATA box of the promoter.

False

Steroid hormones are an example of an effector which regulates regulatory transcription factor activity.

True

DNA that contains actively transcribed genes would most likely contain chromatin in the closed configuration.

False

Nucleosome location may be changed by a process called ATP-dependent chromatin remodeling.

True

DNA methylation usually activates gene expression.

False - it inhibits it

Which of the following is the most likely location of an insulator sequence?

Between two genes

What general transcription factor is most often affected by regulatory transcription factors?

TFIID

CpG islands are associated with which of the following?

DNA methylation

Housekeeping genes are unmethylated and active in most cells

true

A particular cell contains all of the standard histones but lacks several histone variants. Which of the following MAY be true of this cell?

The cell will express different sets of genes than other cells in the same organism

The activity of some transcription factors can be regulated by covalent modifications.

True

nucleosome positioning

structure is fluid and can be changed by modifications, evictions, variants

schizophrenia and chromatin accessibility

• Very few coding mutations discovered

• must mutations are SNPs lying in non-coding regions

transposable elements

repetitive DNA

DNA transposases

enzymes that move discrete segments of DNA from one location in the genome to a new site

retrotransposons

use RNA instead of DNA

Tn5

bacterial cut and paste, inserts a fragment of DNA in a new location

• altered to carry and insert two double stranded adaptors

what is epigenetics

• changes in gene expression without change in DNA sequence

• passed from cell to cell

• can last lifetime of individual

• not permanent over multiple generations

• reversible

epigenetic inheritance

from parent to offspring

epigenetics deals with

the markings that are placed ‘on’ the genome

• marks can be ON DNA itself (CpG islands)

• marks can be on the histones

mechanisms of Epigenetic gene regulation

• DNA methylation

• Chromatin remodeling

• Covalent histone modification

• Localization of histone variant

HP1 and PTMs

specific proteins bind to them

PTM

post-translational modifications

HP1

• binds methylated histones

• pulls nucleosomes closer together

• has a reader domain

role of heterochromatin

• gene silencing

• prevent transposable element movement

• prevent viral proliferation

heterochromatin gene silencing

limit access to DNA-binding proteins

Heterochromatin prevents transposable element movement

• random insertion in genes, can disrupt function

• block movement of these DNA regions

Heterochromatin prevents viral proliferation

• viral DNA integrated on genome

• prevent viral activation/transcription

DNA methylation maintenance of heterochromatin after cell division

hemi-methylated DNA fully methylated via maintenance methylation

Histone modifications - maintenance of heterochromatin

histones recruit chromatin-modifying/remodeling enzymes to new histones/nucleosomes

DNA polymerase - maintenance of heterochromatin

recruits chromatin-modifying complexes

epigenetics and development

• genetically programmed stages of development

• many changes maintained by epigenetic regulation

genomic imprinting

• occurs during gametogenesis

• offspring expresses allele from one parent only

• in germ cells, base imprint erased and reestablished based on sex of individual

X chromosome inactivation

• occurs during embryonic development of female mammals

• ‘count’ number of X chromosomes, silence all but 1

• inactivation occurs at X inactivation center

• dosage compensation

Before inactivation

• Tsix expressed from both X chromosomes

• expression stimulated by pluripotency factors

• Tsix expression recruits DNA methyltransferase, silence Xist promoter