Differential Gene Expression

• Differential gene expression and genomic equivalence are two linked concepts in biology
• Organism development and maintenance involve various stem cells, each described by its potential: pluripotent, multipotent, and unipotent, ending in differentiation/commitment

Differential Gene Expression

• Involves amplification steps, where expressing a protein occurs
• Reporter genes and deletion mapping are tools to examine how genes are regulated
• Nucleosomes' structure affects DNA binding, impacts gene regulation

Cis vs. Trans Regulatory Elements

• Impact transcription through different mechanisms
• Limited transcription factors can regulate all the genes in a cell
• Cell can maintain stable gene expression patterns through generations

Gene Regulation Coordination

• Can coordinate groups of genes during development or in response to external stimuli
• Prokaryotic genes for the same pathway coordinate through operons
• Distinguish between transcriptional and post-transcriptional regulation
• Cis-regulatory elements are located near their target eukaryotic gene
• All cells originate from other cells
• Gradual and specific steps are needed to produce functional, differentiated cells from stem cells
• Cellular commitment restricts cell fate

Stem Cells

• Retain ability to divide and self-renew, resulting in specialized cells through differentiation
• Level of commitment varies

Pluripotent Stem Cells

• Produce all cell types in the body(ex. embryonic stem cells)

Multipotent Stem Cells

• Produce related group of cells (ex. hematopoietic stem cells)

Unipotent stem cells

• Only produce their own type but self-renew (ex. muscle stem cells)
• Have the property of self-renewal required to be labeled a stem cell (ex. muscle stem cells)
• The epigenetic level of commitment can be changed
• Cells with different RNA and proteins have different structural/functional properties

Genomic Equivalence

• Every somatic cell contains the same DNA content
• To assess this, researchers are wondering if all cells have the same DNA or keep only necessary segments
• There are more highly expressed genes
• Gene activity depends on cell type and external stimuli

Proving Differential Gene Expression

• Demonstrated via detection of specific mRNA transcripts restricted to certain sites or times

Studying Gene Expression:

• Use two-dimensional gel electrophoresis
• Separate proteins by charge and mass
• RNA-Seq sequences all RNA, reverse transcribes to DNA, adds adaptors for amplification
• RNA-Seq helps study transcriptome changes, detect mutations, splicing variants
• “Sequence reads” from RNA-Seq data align to the genome to show gene expression levels
• Heatmaps often display transcriptome or proteome
• Heatmaps group similar genes or subtypes

Gene Regulation

• Occurs at every level
• Regulation levels are transcriptional control, RNA processing, RNA transport and localization, translational control, mRNA degradation, protein activity, and degradation

Transcriptional Regulators

• Binding through specific proteins recognizes DNA consensus sequences
• They are often non-covalent
• Cis-regulatory elements: DNA sequences on the same chromosome as the gene they affect
• Trans-regulatory elements: Supplied from any chromosome, influence molecules

Transcriptional Regulation

• Cis-regulatory elements and groups of many different proteins
• Mediator connects sites far from the promoter, influences gene expression
• Transcription factors or RNA polymerase interact
• Transcription factor can increase/decrease transcription by forming complexes
• A single gene is regulated by many transcription factors
• One transcription factor might regulate multiple genes
• Action of a TF depends on the context
• Dimerization makes binding very specific

TF Binding Sites

• Use ChIP-Seq
• ChIP-Seq involves cross-linking proteins to DNA, sequencing to find binding locations

Cis-Regulatory Elements

• Promoters are where RNA polymerase binds to start transcription
• Enhancers influence gene products made from a promoter
• DNA can loop around with many cis-regulatory elements

Enhancers

• One gene uses different enhancers based on cell type, time, signal
• Cis-regulatory sequences are identical in nearly all cell types
• Transcription factors combined with co-regulators differ each cell type

Studying Enhancers

• Use reporter gene expression to visualize and study them

Silencers

• DNA regulatory elements that prevent promoter use
• Stop transcription and restricts gene expression to the proper cells/times

Transcription Factors

• Act as activators or repressors, binding to cis-regulatory elements
• Co-activators and co-repressors are recruited to help
• DNA bending proteins can affect
• Nucleosomes affect TF binding depending on surface exposure
• Variant nucleosomes assist w/ more open DNA

Insulators

• Help organize loops such that each site has independent expression
• Combinations of regulatory factors act together

Regulators:

• Transcriptional activators
• Cooperate
• Combinations can regulate genes and cells because small amount of genes combine
• Eukaryotic regulators coordinate in networks
• Networks have both positive and negative feedback
• Not all regulators are proteins, use IncRNAs
• Maintains stable patterns of expression even across cell division

Stable Patterns

• Histone reader-writer propagate and maintain stability
• Histone modifications write code to pass on

Insulators

• Set boundaries to prevent effects from afar

Activators+

• Cause changes to chromatin structure

Prokaryotic Regulation

• Repressors turn genes off
• Activators turn them on
• Operons are for specific resources
• Share same element on DNA (operator)

Tryptophan Repressor:

• Binds operator in presence of Tryptophan
• When tryptophan is high, no need to make it

Lac Operon:

• Has 2 regulators which allows for preferred fuel choice
• Makes glucose
• Cap activates with glucose
• Lactose comes in and induces