601 Exam 2

regulatory region of a gene

umbrella term for sequences like the promoter, enhancer, silencers, TF binding sites, etc. other names in prokaryotes.

Anatomy of a gene: 5’ to 3’

5’ regulatory region (can differ), transcription start site (DNA), 5’ UTR (mRNA), AUG (mRNA), exons and introns (both), UAG (mRNA), 3’ UTR (mRNA), termination region (DNA)

mRNA processing steps

addition of the 5’ cap and poly A tail; intron splicing

purpose of the 5’ cap

exonuclease protection, ribosomal recognition

purpose of the poly A tail

enzymatic protection, nuclear transport, translation efficiency

coding region of a gene

from the start codon to the stop codon, exons only, doesn’t include the regulatory region

what’s more similar in terms of gene expression: a dog and a human brain or a human brain and a human liver

dog brain and human brain

two main categories of epigenetic modifications

histone modifications and cytosine methylation

molecular memory

histone and cytosine modifications can keep DNA in its current expressive state

the main driver of cell type variability

gene expression! they all have the same genome duh

different reasons gene expression should differ (4)

cell type, brain region for the same cell type (ie temporal vs frontal lobe neurons), species for the same cell type, developmental lineage (progenitor vs committed cell)

diseases with multiple seemingly unrelated affected systems could be explained by

a mutation in a progenitor cell common to all of those lineages

TATA box

a DNA sequence at the start of genes where the RNA polymerase assembly occurs. Easy to unwind (AT rich), and conserved in about 15% of human genes

TBP transcription machinery

TATA box binding protein: recognizes TATA box and recruits other transcriptional machinery (TFIIA thru TFIIH essentially)

TFIIB transcription machinery

recognizes BRE: TFIIB recognition element

TFIID transcription machinery

recognizes INR (initiator, contains the TSS) and DPE (downstream promoter element)

enhancer elements

can be upstream or downstream of a gene, and very far away. allow for additional transcription factors to bind and influence gene expression

components of the RNA polymerase assembled complex

pol II, TBP, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

names for the complexes that bridge enhancers and promoters, especially when they’re very far away

activator (sequence-specific DNA binding protein) and mediator (bridge between activator and RNA polymerase)

transcription inititation

once the transcription initiation complex is assembled (RNAp in the right spot, enhancer binding proteins bridged, etc), RNAp begins to move, hydrolyzing ATP, bringing in bases and polymerizing them

components of the regulatory environment in eukaryotic cells

cis regulatory elements (enhancer, promoter); TF availability; histone modifications; nucleosome occupancy (histone presence/density); chromatin environment (remodeling, DNA accessibility)

molecular mechanism of DNA being locked into loops (transcription)

Condensin is a ring-shaped protein that slides along chromatin, pulling DNA out into a loop until it hits a bound CTCF protein. CTCF proteins are directionally specific and recognize specific DNA sequences. The loop is finalized based on when the directional CTCF protein locks it into place. The locked DNA loop can help the enhancer and promoter interact from far away and create local domains of gene expression or repression.

why might a cell want to lock DNA into loops (transcription)?

genes on loops often have common functionality, so they can be expressed or repressed together

smallest scale of DNA regulatory environment

compaction of DNA into chromatin, nucleosome wrapping

intermediate scale of DNA regulatory environment

chromatin loops and topologically associating domains (TADs) (can be made by CTCF); epigenomic compartments (active vs inactive)

large scale of DNA regulatory environment

chromosome territories, regulation of contact between them

in the location of CTCF binding, what would happen to DNA-DNA interactions

an absence! proteins are bound to the DNA and DNA gets looped out, both of which decrease DNA-DNA interactions in the area. It also helps with enhancer and promoter connection via activators and mediators.

How do DNA-DNA interactions look on a Hi-C map when plotted against CTCF and cohesin binding sites

In the areas with CTCF and cohesin binding, there are low DNA-DNA interactions. This looks like less colored areas that make up the bases of triangles. The peak of the colored triangles comes in the middle of large gaps without CTCF and cohesin binding sites.

How might the graph of CTCF and cohesin binding sites along with DNA-DNA interactions look different in different cell types?

Different cell types have different expression profiles and local expression domains, so their graphs of where CTCF and cohesin bind (decreasing DNA-DNA interactions) will differ! These maps can give you an idea of the different expression profiles of different cells.

most common modifications on histone tails

acetylation, methylation, phosphorylation

4 major classes of proteins that do work on histones

writers, erasers, readers, remodelers (remodel chromatin by moving histones)

examples of epigenetic writers

histone methyltransferase, histone acetyltransferase

examples of epigenetic erasers

histone demethylase, histone deacetylase

examples of epigenetic readers

bromodomains, chromodomains, tudor domains, PHD fingers, PWWP domains, malignant brain tumor domains (MBT). These are very often affected in cancer.

Major classes of transcription factors

zinc finger, leucine zipper, helix-turn-helix (HTH/homeodomain), helix-loop-helix (HLH)

domains possible for TFs to have

DNA bindgin domains, ligand binding domains (other proteins, histones, signals, etc), enzymatic activity domains

TF role in cell type identity

TFs help genes be expressed in a tissue specific manner. This establishes cell type identity and directs organism development

example of TF acting as intermediate between extracellular signaling and gene expression change

external ligand causes cAMP to activate a kinase that phosphorylates CREB (CRE = cAMP response element). Phosphorylated CREB is active and can bind DNA, activating transcription of target genes

sequence logo

visualization of the consensus sequence at a TF’s DNA binding site. height of the letters indicates how often that letter is seen at that position. Shows you how tightly the TF needs to match its target sequence in order to bind.

Body patterning is dictated by

spatial expression of specific transcription factors (classic ex is fruit fly, where the TFs are clustered on the chromosome in the order of the body segments)

Main difference between the body patterning TFs in simple and complex organisms

Expansion of families, like duplications that gained new related functions. Many of the ancestral genes are paralogs of the current human genes.

what drives the differentiation of pluripotent stem cells into their eventual cell types (and example)

transcription factors duhhhh (the Ets family of TFs play a large role in hematopoiesis)

yamanaka factors

TFs expressed very early in dvelopment can also reprogram mature cells back into stem cells (iPSCs, which can be redifferentiated for a variety of purposes)