Genetic Regulation of Gene Expression

Classic view of gene regulation

Organism' complexity generally correlates with the number of non-coding genome

2% of human genome encodes proteins
98% of human genome is non-coding

Genome Structure

Chromosomal territories > Chromatin > TADs > Chromosome loops > DNA sequence

TADs: Topologically Associating Domains, a self-interacting genomic region

Recall:

Euchromatin: loosely packed, transcriptionally active

Heterochromatin: tightly packed, transcriptionally inactive

Genome give rise to different cell types

All cells in an organism have the same genome (Normally)
Specialized tissues need different proteins which need different genes

House-keeping genes vs. Tissue-specific genes

	House-keeping genes	Tissue-specific genes
Distribution	All cells	Specific cells
Percentage of genes	40%	50%
Specialized gene regulation	No	Yes
Examples	ACTB, GAPDH	Hemoglobin

Comptemporary view of gene regulation

Gene locus: a regulator unit, contains regulatory elements

How to map regulatory elements to target genes?

Conservation
- Conservation studies: compare across species
Biochemical
- Chromatin accessibility (DNase-seq)
- Histone modification (ChIP-seq)
- Transcription factor binding (ChIP-seq)
- Chromosome interaction (ChIP-seq)
Genetic dissection and functional probing

Cis-regulatory elements and Trans-acting regulators

Trans-acting regulators:
- DNA-binding proteins (Direct binding)
- Co-factors (Indirect, bind via other proteins)
Cis-regulatory elements:
- Promoters - Regions upstream of genes where transcription is initiated. Binding site for RNA polymerase and general transcription factors.
- Enhancers - Distal elements that activate gene transcription by interacting with promoters. Binding sites for tissue-specific transcription factors.
- Insulators - Boundary elements that constrain enhancer-promoter interactions. Prevent spread of heterochromatin.

Promoters:

Located upstream of genes, initiation site of transcription
Binding site for RNA polymerase II and general transcription factors
Have open chromatin structure revealed by ATAC-seq
Histone modification H3K4me3 enriched at promoters
Spiky peaks in chromatin accessibility profiles

Enhancers:

Relay information to the promoter
Distal regulatory elements that activate gene transcription
Have open chromatin structure but not unique to enhancers
Histone modifications H3K27ac and H3K4me1 enriched at active enhancers
Binding sites for multiple tissue-specific transcription factors
Can be located far from promoters but interact through 3D proximity
Often form clusters and work cooperatively

Insulators:

Boundary elements that constrain enhancer-promoter interactions
Binding sites for CTCF and cohesin proteins
Help form chromatin loops to delimit domains
Prevent spread of heterochromatin
Strength of insulation varies by element and context
Can block enhancer-promoter contacts or prevent spread of enhancer activity

Central Dogma of Molecular Biology

DNA -> Transcription -> RNA -> Translation -> Protein

Transcription control:
- Trans-acting regulators (TFs)
- Cis-regulatory elements (promoters, enhancers, insulators)
Post-transcriptional control:
- RNA processing
- RNA transport
- mRNA stability

Alternative splicing

Alternative RNA splicing is a process that removes the introns from pre-mRNA and joins the exons to enable translation of mRNA into a protein. Over 90% of the human protein-coding genes undergo some kind of alternative splicing, which can produce different forms of protein from the same gene. Splicing is mediated by large ribonucleoprotein complexes known as spliceosomes .

mRNA splicing:

Splice donor site: the 5' end of an intron (GU)

Splice acceptor site: the 3' end of an intron (AG)

Branch site: located close to the splice acceptor site, initiates the splicing reaction (A)

Splice enhancer sequeces: located in exons, promote splicing

Splice suppressor sequences: located in exons, inhibit splicing

miRNA (non-coding RNA)

translational repression, mRNA degradation
regulate about 30% of human genes

Cis-regulatory elements (on alpha-globin gene regulation)

Enhancer Experiments:

Deletion of enhancers R1/R2 nearly abolishes alpha globin expression, showing they are critical
Deletion of R3/R4/R5 reduces expression by 40%, suggesting facilitator role R3, R4, R5 work as a unit with redundancy between elements
Inversion of the enhancer cluster substantially reduces expression, indicating orientation is important

Facilitator concept:

R3/R4/R5 proposed as facilitator elements that support enhancer activity
They lack intrinsic enhancer activity but are important for R1/R2 function
Highlights enhancer clusters can work cooperatively, not just additively

Insulator Experiments:

Deletion of 5' insulator does not affect alpha globin expression But allows enhancer activity to spread to neighboring genes
Deletion of 3' insulator also does not affect alpha globin expression Active transcription of genes may block/insulate enhancer activity
Varying strength of different insulators based on position and context Insulators constrain but do not confer specificity to enhancer-promoter interactions

DNA variants and genetic diseases

DNA variants

Single nucleotide variant (SNV): a single nucleotide change
Structural variant (SV): a large DNA sequence change
- Deletion: a segment of DNA is missing
- Insertion: a segment of DNA is inserted
- Tandem duplication: a segment of DNA is duplicated and inserted next to the original segment
- Interspersed duplication: a segment of DNA is duplicated and inserted at a different location
- Inversion: a segment of DNA is reversed
- Translocation: a segment of DNA is moved to a different chromosome
- Copy number variant (CNV): a segment of DNA is duplicated or deleted

Mutations in the genome

Exome is easier to identify mutations
But highest % of mutations are in non-coding regions
Significance will depend on their location and function

Disruption on enhancer, promoter, insulator, splicing site, etc. can cause diseases

Degradation of miRNA can cause diseases