Why regulate gene expression?
Respond to environmental conditions
Express genes appropriate to developmental stages of life cycle
Express genes appropriate to cell type
Transcriptional Regulation in Eukaryotes
Regulatory Transcription factors
Chromatin / Histone Modifications
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