GENE REGULATION

Key control mechanism for dictating cell phenotypeand function

Two kinds of genes

• Housekeeping genes Needed for cell metabolism

• Required for nearly all cell types

• Regulated Genes Controls cell phenotype and function

Housekeeping Genes

• Pol I transcripts Ribosomal RNA genes (18S, 5.8S, and 28s rRNAs)

• Some Pol II transcripts e.g. Structural Proteins such as Actin

• Pol III transcripts 5S rRNA (ribosomal RNA) tRNAs (translation) snRNA (splicing)

Regulated Genes

• Pol II transcripts For e.g.

• Transcription factors• Cytokines• Kinases• Phosphatases• Ubiquitinases• etc., etc., etc.

• Pol III transcripts• miRNAs (siRNA like molecules that inhibit specific gene

expression)

Multiple Mechanisms of Gene Regulation

• Transcription• Initiation• Elongation

• Post-transcription RNA level

• RNA processing (alternative splicing, polyadenylation)• RNA transport• mRNA stability

Protein level• Translation initiation• Protein transport• Protein stability• Protein processing

• Epigenetic

Transcription

• Housekeeping Pol I transcription

• Ribosomal RNA genes (18S, 5.8S, and 28s rRNAs) Pol III transcription

• 5S rRNA (ribosomal RNA)• tRNAs (translation)• snRNA (splicing)• miRNAs (control of gene regulation)

• Regulated Pol II transcription

• Protein encoding transcripts

Pol I Transcription

• 18S, 5.8S, and 28S RNA genes transcribed as one 13 kb transcript.

• This transcript is subsequently processed (cleaved) to give individual 18S, 5.8S and 28S rRNAs.

• On chromosome, 13 kb transcription unit (along with a 27kb intergenic region) is repeated 30-40 times Helps provide higher expression of these

housekeeping gene products.

Pol I Transcription

Pol I Promoter Function

Pol III Transcription

Pol II Transcription

• Three types of cis elements: Core promoter elements (-45 to +40)

• Binds basal factors required for initiation and elongation.

Proximal promoter elements (-1kb to +200)• Binds regulatory transcription factors involved in activating or

suppressing basal transcription

Enhancers/Silencers (far up or downstream)• Binds regulatory transcription factors involved in activating or

suppressing basal transcription

Pol II Transcription

Core promoter function - facilitates transcription initiation and elongation- Pol II - Catalyzes RNA synthesis- TFIID - Provides scaffold for general transcription factors (TBP is at core of this complex and is associated with TAFs (TBP Associated Factors)- TFIIB - Binds TBP, selects start site and recruits Pol II- TFIIA - Stabilizes binding of TFIIB and TBP to promoter- TFIIF - Binds TFIIB and Pol II- TFIIE - Recruits TFIIH- TFIIH - Helicase and kinase (Unwinds DNA phosphorylates C-terminus of Pol II)

Pol II Transcription

Proximal promoter function - regulates core promoter functionProximal promoter binding factors activate transcription through multiple mechanisms:

- Recruit basal factors- Recruit coactivators and mediators (for example, CBP, p300, p/caf, etc)

- helps recruit basal factors- contain or recruit histone acetyl-transferases- recruit histone methyl-transferases- contain or recruit ATP dependent chromatin remodeling enzymes- helps form stable complexes between multiple proximal transcription factors

Regulation of promoter function by proximal promoter factors and enhancer/silencers

• Recruitment of basal factors*

• Recruitment of histone acetylases

• Recruitment of histone methylases

• Recruitment of ATP dependent chromatin remodeling complexes*

Histone modification

- Histone acetylation - generally associated with promoter activation (histone deacetyleses (HDACs) inhibit transcription

- Neutralizes basic charges on lysines and arginine residues - relaxes nucleosome- Allows direct binding of activating proteins to promoter bound histones

- Histone methylation- Arginine methylation associated with promoter activation- Lysine methylation associated with promoter inactivation

Promoter Elements Function in Either Orientation

Pol II Promoters Contain Multiple Transcription Factor Binding Sites

Human Insulin Promoter - red, specific for pancreatic beta cells

Reasons?- Coactivator interactions stabilized by binding to multiple transcription factors at the same time- Some factors cannot carry out all steps required for promoter activation (SP1)- Some elements play a role in signaling activation by different effectors

Solved DNA Interaction Motifs

DNA Recognition

Regulation of Transcription factor function

• Tissue specific expression of transcription factors

• Translocation of transcription factor to nucleus

• Post-transcriptional modification of transcription factors

Steroid Hormone Receptors

- Zinc fingers- Bind as dimers

Regulation of Steroid Hormone Receptors

Regulation of CREB

NF-kB Regulation

Other Mechanisms for Regulating Gene Expression

• Regulation of translation

• Regulation of mRNA stability

• Alternative promoter usage

• Alternative Splicing

• Alternative polyadenylation

Translational Control and RNA Stability Control

Tissue Specific Promoter Usage

Dystrophin GeneC - CoricalM - MuscleP - PurkinjeR - RetinalCNS - Central Nervous SystemS - Schwann cellG - General

Alternative Splicing

Alternative Splicing - Examples