Unit II- Gene Regulation

Question 1

Gene regulation

Answer 1

• the process of turning on and off the expression of genes in response to developmental, environmental and hormonal signals
• for a multicellular organism, this is important for the organism to develop specialized cell types,such as muscle, nerve and blood
• this differentiation arises from the accumulation of different sets of proteins due to altered states of gene regulation

Question 2

Gene regulation at every stage

Answer 2

1) transcriptional control *key step
2) RNA processing
3) RNA transport and localization control
4) mRNA degradation control
5) translation control
6) protein activity control

Question 3

How is transcription switched on and off

Answer 3

1) Transcription Regulatory Proteins- protein-DNA interactions
2) Control of Transcription in Bacteria (logic of regulatory circuits)
3) Control of Transcription in Eukaryotes (transcription factors, enhancers, chromatin structure, cell-type specificity)

Question 4

Transciptional Regulatory Proteins

Answer 4

• gene regulatory proteins, “transcription factors” bind the regulatory DNA sequences and control the rate of initiation by RNA polymerase
• hundred of regulatory DNA sequences
• gene regulatory proteins (TFs) are repressors or activators
• CAP is an E.coli “activator”

Question 5

Transcription Regulatory Proteins and different motifs

Answer 5

• common eukaryotic DNA binding motifs are found within transcription regulatory protein (the motifs are the part of the protein that contacts DNA)
• the motifs usually contain a recognition alpha helix that inserts in the major groove of DNA and makes multiple contacts

Question 6

DNA recognition

Answer 6

• amino acid bone with specific base pairs in DNA, recognized at their edges, without the need to open the double helix
• the protein-DNA interface consists of 10 to 20 protein and base pair contacts, each involving a different amino acid and each contributing to the strength of the protein-DNA interaction

Question 7

Control of Transcription in Bacteria

Answer 7

• genes are often clustered in operons and coordinately regulated
• mRNAs are polycistronic (multiple proteins from single mRNA)
• operators are binding sites for repressor proteins

Question 8

Bacterial repression

Answer 8

• the level of trytophan inside the cell is low, the trp repressor protein does not bind tryptophan and thus cannot bind to its control region, the operator, within the promoter
• RNA polymerase can thus bind to the promoter and transcribe the five genes of the tryptophan operon (left)
• if the level of trytophan is high, like in the gut after a large mean, the repressor protein binds trytophan, causing an allosteric change so now it binds the operator, which it blocks the binding of RNA polymerase
• when the level of intracellular trytophan drops, the repressor releases its trytophan and is released from the DNA, allowing the polymerase to again transcribe the operon
• trp repressor expression is constitutive

Question 9

Activation of the lac operon by the CAP activator

Answer 9

• activator proteins function in the opposite way from repressor proteins
• they act on weak promoters that are only marginally functional in binding RNA polymerase
• upon binding to their recognition site near the promoter, activators help recruit and/or stimulate RNA polymerase to begin transcription
• the ability of the E. coli activator protein is CAP is regulated by a small molecule (cAMP)
• cAMP is abundant when glucose is unavailible, allowing CAP to turn on transcription of operons ordinarily repressed by glucose

Question 10

Control of Transcription in Eukaryotes

Answer 10

• eukayotic TFs are modular
• the two main functions- DNA binding and transcription activation (or repression) are separable (encoded in separate protein domains)
• most transcription factors function as dimers or interact with other TF to form heterodimers
• some like steroid receptors require ligand (hormone) binding for nuclear entry and DNA binding to occur

Question 11

Enhancers

Answer 11

• activators stimulate transcription by helping assemble RNA pol II at the promoter
• typical activators work via a large “mediator complex”
• DNA looping explains why a sequence so far away can impact a given promoter
• cell type specific because the proteins that bind them are differently expressed
• cis acting sequences (that bind activators)

Question 12

Why do I care about enhancers?

Answer 12

• because 5-10% of T-cell acute lympoblastic leukemias caused by a t(10:14) translocation that juxtaposes the strong T-cell enhancer with the HOX11 gene
• result HOX11 homeodomain protein is overexpressed in hematopoietic cells causing uncontrolled growth
• Rubinstein-Taybi syndrome

Question 13

Eukaryotic Transcription Regulators and chromatin modifiers

Answer 13

• chromatin needs to be loosened up for RNA pol II to transcibe gene
• some transcription regulators recruit chromatin modifying enzymes
• two major types of chromatin modifications are: (covalent histone modifications, ATP-dependent nucleosome remodeling (move, slide, exchange)

Question 14

Major histone modification

Answer 14

Acetylation of Lys (H3)- activation
De- acetylation of Lys (H3)- repression

Methylation of Lys, Arg (H3)- activation or repression
Phosphorylation of Ser/Thr (H2A, H3) (coupled to acetlyation)

HATs-activate
HDACs-repress transcription

Question 15

TFs work together

Answer 15

• regulatory proteins work together as a committee to control the expression of a eukaryotic gene
• once bound to DNA, eukaryotic gene activator proteins increase the rate of transcription initiation
• they do this by acting directly on the transcriptional machinery (different activators target different members of the transcriptional machinery) and by changing the local chromatin structure

Question 16

Single TF may control multiple genes

Answer 16

• their effect on levels of activation (or repression) of a given gene may depend upon what other regulators are bound to that gene
• 1,800 factors in human genome for controlling more than 30,000 genes
• may be root cause of side effects of a drug

Question 17

Synthesis and activity of transcription factors

Answer 17

• have their production and activity regulated by a number of mechanisms, including covalent modification (like phosphorylation), intracellular trafficking or selective degradation
• each of these mechanisms is typically controlled by extracellular signals which are communicated across the plasma membrane to the gene regulatory proteins in the cell

Question 18

TFs development of different cell-types

Answer 18

• specialized cell types in a multicellular organisms differ by virtue of the different sets of RNA and protein molecules made
• what governs differential synthesis of such RNAs and proteins over space (tissue specificity) and time (developmental control)
• it involves a combination of the cell’s internal programming and developmental history, and the presence at any given moment of particular spatial cues and environmental signals, all of which impinge on function of regulatory proteins and RNA polymerase II at gene control ergions unique to each gene

Question 19

Transcriptional profiling

Answer 19

• gene expresiion profiling the measurement of the mRNA abundance as measured by DNA microarray of thousands of genes at once, to create a global picture of gene expression and hence cellular function
• cluster analysis of the total pattern can reveal a characteristic gene expression pattern for a given stage of tumor

Question 20

Transcriptional Regulation in the Production of Human Hemoglobin

Answer 20

• B-globin chains from the B globin gene
• B globin gene has two enhancers, one upstream and one downstream of the gene
• a complex array of gene regulatory proteins controls expression of the beta globin gene , some acting as activators and some repressors
• once bound to DNA gene regulatory proteins (like GATA-1, CP1) recruit chromatin remodeling complexes, histone modifying enzymes and the general transcription factors and RNA polymerase to the promoter

Question 21

LCR

Answer 21

• in addition to the individual regulation of each of the globin genes, the entire cluster is subject to a shared control region called the locus of control region (LCR)
• LCR lies far upstream from the gene cluster
• in cells where the globin genes are not expressed (such as brain or skin cells), the whole gene cluster appears tightly packed into a heterochromatin-like structure
• in erythroid cells, by contrast the higher order packaging of the chromatin has become decondensed

Regulation: first the chromatin of the entire globin locus becomes decondensed, second the remaining gene regulatory proteins assembly on the DNA and direct the expression of individual genes

-deletion of LCR silences entire B-like globin gene cluster

Question 22

Cell memory transmitted through transcriptional mechanisms

Answer 22

• autoregulation

- epigenetic inheritance via modification of chromatin or DNA

Question 23

Autoregulation

Answer 23

-Txn factor activates other genes, but also its own gene

Question 24

Epigenetic inheritance- modification of histones

Answer 24

• acetlyation, methylation
• the inheritance of an active or repressed chromatin state keeps the genes expressed at similar levels in daughter cells

Question 25

Epigenetic inheritance- modification of DNA

Answer 25

• associated with transcription repression
• modification of DNA itself called imprinting is prevalent in mammels and results in gene silencing
• newer data indicates that methylated CpG islands attract histone modifiers (HDACs) which leads to condensed chromatin and transcription repression
• the propagation of the epigenetic mark following DNA replication and cell division requires that the hemi-methylated DNA is targeted by DNA methyltransferases to restore methylation on both strands