Master Controlling Elements in Stem Cells

Master gene regulators Mapping the New Frontier: Human Regulatory Circuitry Gene regulation - control of cell state - master gene regulators Mapping gene regulatory circuitry Importance of maps - human biology - future medicine Gene Regulation is Key to Human Development Master gene regulators establish gene expression program Cell state is a product of that gene expression program Master Gene Regulators are Generally Transcription Factors Transcription Factor Binds specific DNA sequence in control region of gene DNA TFIIH Transcription RNA Translation Protein Master Gene Regulators are Known for Many Cell Types Embryonic stem cells OCT4, NANOG, SOX2 PcG Brain and Spinal Cord SOX1-18, PAX6, OCT6, NGN, NEUROD Cerebrum Cerebellum Ganglia & nerves Circulatory System Myocardin, GATA4, TBX5, NKX2.5, MEF2, HAND Heart Vascular system Digestive System HNF1, HNF4, HNF6, CBP, PGC1, FOXA, PDX1 Esophagus Stomach Intestines Liver Pancreas Urinary System HNF1B, HNF4, CDX, FTF C/EBP, FOXA, GATA3 Kidney Urinary tract Respiratory System HNF-3, NKX2.1 and GATA6 Airways Lungs Reproductive Organs ERα, SF1, DAX1, C/EBPβ Ovary Uterus Breast Testis Skeletal and Muscular MYOD, MEF2, MRF4, MYF5 Myogenin Bone Muscle Cartilage Hematopoietic System TAL1, LMO1, LMO2, E2A, XBP1, AML1, MLL1, PU.1, C/EBPα Bone marrow Blood Embryonic Liver Immune System STAT1, STAT3 STAT5, IRF1, IRF3, IRF5, NFκB family, FoxP3 Thymus Spleen Lymph nodes Sensory Organs SOX1-18, OCT6, PAX3, PAX6, NGN, SKIN1 Eye Ear Olfactory Skin Tongue Master Gene Regulators Can Reprogram Cell State Oct4, Sox2, KLF4, cMyc Takahashi Cell (2006) Maherali CellSC (2007) Okita Nature (2007) Wernig Nature (2007) MyoD Davis Cell (1987) Weintraub PNAS (1989) Weintraub Science (1991) iPS cells Fibroblasts Neurod1 Skeletal muscle Kojima Nature (2003) CEBPβ Shen NatureCB (2003) Hepatocytes Pancreatic-like cells Mutations in Master Regulators Cause Major Diseases Diabetes Hnf1α, Hnf1β, Hnf4α, Pdx1, NeuroD1 Coronary Heart Disease and Hypertension NKX2.5, MEF2, NR3C2, GCCR Cancer TAL1, LMO1, LMO2, E2A, AML1, p53, PLZF, PML, Rb, WT1 Developmental Defects GATA1, VDR, CRX, CBP, MeCP2 Autoimmunity and Immunological Disorders FoxP3, RFX5, WHN Central Nervous System Disorders PAX3, EGR-1, EGR-2, OCT6, SOX family Obesity PPARγ, SIM1 Gene Regulation I: Transcription Factors Control RNA Polymerase, Which Produces RNA Transcripts HATs H3K4 methyase H3K36 methylase Active Gene Transcription factors RNA Polymerase Initiation Elongation Inactive Gene Repressors We understand how only a few genes are regulated by transcription factors PcG H3K9 methylase HDACs Human Gene Regulation Human genome • 25,000 genes • 1500 genes encode transcription factors • 3 times more DNA devoted to gene control than to protein-coding • regulation is understood for only a few genes Transcriptional Regulatory Circuitry: How 1500 Transcription Factors Regulate 25,000 Genes Transcription Factors The mapping of transcriptional regulatory circuitry will: • reveal how cell state, differentiation and response to stimuli are controlled Target Genes • suggest new strategies to combat disease Mapping the New Frontier: Human Regulatory Circuitry Summary so far: Master regulators (transcription factors) establish gene expression program Master regulators are powerful - they can reprogram gene expression Defects in master regulators can cause disease Basic principles of gene regulation established How 1500 transcription factors regulate 25,000 genes is not known Map of transcriptional regulatory circuitry should reveal how cell state is controlled and suggest new strategies to combat disease Mapping the New Frontier: Human Regulatory Circuitry Gene regulation - control of cell state - master gene regulators Mapping gene regulatory circuitry Importance of maps - human biology - future medicine Where Do We Start? ES Cells Can Differentiate into Most Any Cell Type Founding Circuitry of Human Development ES Cells Technology: Mapping Transcription Factor - DNA Interactions Genome-wide Crosslink protein to DNA binding sites in living cells Harvest cells and fragment DNA Enrich for protein-bound DNA fragments with antibodies Differentially label the two pools of DNA Hybridize to microarray Obtain binding ratio Identification of All Genes Regulated by a Transcription Factor Transcription Factor 1 Gene A TFIIH Gene B Gene C Gene D This can be repeated for each transcription factor to build a map showing all target genes for all transcription factors Map of Core Regulatory Circuitry of Human Embryonic Stem Cells Oct4, Sox2 and Nanog: • Master regulators of embryonic stem cells • Regulate their own genes • Regulate regulators of development Mapping is a Big Problem: Master Regulators Change with Each Cell State Challenge is to map all regulators in all cell states NIH is funding major new efforts to do this This is Great, But We Have Noticed a Problem… Transcription factors typically recognize 6 base pair DNA sequence A 6 base pair DNA sequence should occur every 4n base pairs (46) So we should observe one binding event every ~4000bp Actually see much lower frequency: perhaps one binding event every ~1,000,000bp Why? The Genome Packaging Problem Each base pair in DNA double helix is 3.4 x 10-10 m Human genome is 6 billion base pairs (3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m Most human cells are roughly 20 um across, with nuclei about 10 um (10-5 m) Human Genome Packaging Nucleosome Structure Nat Rev Cancer 1:194 (2001) Chromatin Hamkalo and Foe in Essential Cell Biology (1998) Human Chromosomal Karyotype (NIH) Quiz: What is the total length of DNA fiber in your body? One cell: Each base pair in DNA double helix is 3.4 x 10-10 m Human genome is 6 billion base pairs (3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m DNA fiber length in Human: We have about ~10 trillion cells 1013 cells x 2.4m = 2.4 x 1013 m Is this: 1) Distance around earth? 2) Distance to moon? 3) Distance to sun? 4) Diameter of solar system? 4 x 107 m 4 x 108 m 1.5 x 1011 m 1.6 - 2.4 x 1013 m Quiz: What is the total length of DNA fiber in your body? One cell: Each base pair in DNA double helix is 3.4 x 10-10 m Human genome is 6 billion base pairs (3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m DNA fiber length in Human: We have about ~10 trillion cells 1013 cells x 2.4m = 2.4 x 1013 m Is this: 1) Distance around earth? 2) Distance to moon? 3) Distance to sun? 4) Diameter of solar system? 4 x 107 m 4 x 108 m 1.5 x 1011 m 1.6 - 2.4 x 1013 m Quiz: What is the total length of DNA fiber in your body? One cell: Each base pair in DNA double helix is 3.4 x 10-10 m Human genome is 6 billion base pairs (3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m DNA fiber length in Human: We have about ~10 trillion cells 1013 cells x 2.4m = 2.4 x 1013 m Is this: 1) Distance around earth? 2) Distance to moon? 3) Distance to sun? 4) Diameter of solar system? 4 x 107 m 4 x 108 m 1.5 x 1011 m 1.6 - 2.4 x 1013 m Quiz: What is the total length of DNA fiber in your body? One cell: Each base pair in DNA double helix is 3.4 x 10-10 m Human genome is 6 billion base pairs (3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m DNA fiber length in Human: We have about ~10 trillion cells 1013 cells x 2.4m = 2.4 x 1013 m Is this: 1) Distance around earth? 2) Distance to moon? 3) Distance to sun? 4) Diameter of solar system? 4 x 107 m 4 x 108 m 1.5 x 1011 m 1.6 - 2.4 x 1013 m Quiz: What is the total length of DNA fiber in your body? One cell: Each base pair in DNA double helix is 3.4 x 10-10 m Human genome is 6 billion base pairs (3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m DNA fiber length in Human: We have about ~10 trillion cells 1013 cells x 2.4m = 2.4 x 1013 m Is this: 1) Distance around earth? 2) Distance to moon? 3) Distance to sun? 4) Diameter of solar system? 4 x 107 m 4 x 108 m 1.5 x 1011 m 1.6 - 2.4 x 1013 m Quiz: What is the total length of DNA fiber in your body? One cell: Each base pair in DNA double helix is 3.4 x 10-10 m Human genome is 6 billion base pairs (3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m DNA fiber length in Human: We have about ~10 trillion cells 1013 cells x 2.4m = 2.4 x 1013 m Is this: 1) Distance around earth? 2) Distance to moon? 3) Distance to sun? 4) Diameter of solar system? 4 x 107 m 4 x 108 m 1.5 x 1011 m 1.6 - 2.4 x 1013 m Quiz: What is the total length of DNA fiber in your body? One cell: Each base pair in DNA double helix is 3.4 x 10-10 m Human genome is 6 billion base pairs (3.4 x 10-10 m/bp) (6 x 109 bp) = 2.4m DNA fiber length in Human: We have about ~10 trillion cells 1013 cells x 2.4m = 2.4 x 1013 m Is this: 1) Distance around earth? 2) Distance to moon? 3) Distance to sun? 4) Diameter of solar system? 4 x 107 m 4 x 108 m 1.5 x 1011 m 1.6 - 2.4 x 1013 m So… Transcription Factors Have to Overcome Constraints Due to Human Genome Packaging Nucleosome Structure Nat Rev Cancer 1:194 (2001) Chromatin Hamkalo and Foe in Essential Cell Biology (1998) Human Chromosomal Karyotype (NIH) Gene Regulation II: Transcription Factors and Chromatin Regulators Collaborate HATs H3K4 methyase H3K36 methylase Active Gene Transcription factors Initiation Elongation Inactive Gene Repressor PcG HDACs H3K9 methylase Regulatory Circuitry of Human Cells Environmental Stimulus Signal Transduction Signal transduction Transcription factors Chromatin regulators miRNA regulation Chromatin Regulators miRNA Transcription Factors Target Genes Mapping the New Frontier: Human Regulatory Circuitry Gene regulation - control of cell state - master gene regulators Mapping gene regulatory circuitry Importance of maps - human biology - future medicine Maps Are Key to Development of Therapeutics But Only Protein Pathways Have Been Mapped Signaling Pathways Regenerative Medicine: Reprogramming With Maps and Without Viral Vectors • Small molecule drugs • Artificial transcription factors • RNAi