GENE REGULATION Key control mechanism for dictating cell phenotype and function

Two kinds of genes Housekeeping genes Regulated Genes Needed for cell metabolism Required for nearly all cell types Regulated Genes Controls cell phenotype and function

Housekeeping Genes Pol I transcripts Some Pol II 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 Pol III 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 Regulated 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 10_02.jpg

Pol I Promoter Function 10_03.jpg

Pol III Transcription 10_04.jpg

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)

Pol II Transcription 10_05.jpg 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 10_05.jpg Proximal promoter function - regulates core promoter function Proximal 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 10_01.jpg 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 10_07.jpg

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 10_06.jpg

Solved DNA Interaction Motifs 10_08.jpg

DNA Recognition 10_09.jpg

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 10_10.jpg - Zinc fingers - Bind as dimers

Regulation of Steroid Hormone Receptors 10_11.jpg

Regulation of CREB 10_12.jpg

NF-kB Regulation 10_12_2.jpg

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 10_13.jpg

Tissue Specific Promoter Usage 10_14.jpg Dystrophin Gene C - Corical M - Muscle P - Purkinje R - Retinal CNS - Central Nervous System S - Schwann cell G - General

Alternative Splicing 10_15.jpg

Alternative Splicing - Examples 10_16.jpg