The Challenges of Genes, Gene Expression and Genomics

The Challenges of Genes, Gene Expression and Genomics Scott A. Ness Molecular Genetics & Microbiology University of New Mexico HSC Albuquerque, NM

Genes and Gene Products Chromosome: Gene: Spliced mrna: Encoded Protein: Functional Activity: Enzymes, hormones, structural proteins, oncogenes, etc.

Chromosome: Genes and Gene Products Mutation Gene: Spliced mrna: Encoded Protein: Activated Oncogene Functional Activity: Enzymes, hormones, structural proteins, oncogenes, etc. e.g. p53, ras, myb

Results of the Human Genome Project Humans have at least 50,000 genes Genes encode functional RNAs, proteins Many genes encode multiple products At least 200,000 different gene products Genetic Differences = Diversity At least 1,000 allelic differences per person

Genomics I: Measuring Differences in Gene Expression Profiles Different cell types (e.g. liver vs. kidney, normal vs. tumor) express different genes Gene expression patterns can distinguish between different cell or tumor types (classification) Differences in gene expression can identify novel targets for drug development

Comparison of Gene Expression Profiles Cells or Tissue e.g. Normal vs. Tumor Purify RNAs, label with fluorescent tags Hybridize to microarray, detect fluorescence

Affymetrix GeneChip GeneChip Probe Array 1.28cm Human Genome Array > 50,000 genes > 1,200,000 features

Genomics Research at UNM SOM Human Genomics Cancer Biology Novel Targets, Diagnostic Tools Asthma, Diabetes, Drug Screening, Schizophrenia Epidemiology Risk Detection and Prediction Genomics in Animal Models Stroke, Neurological Studies, Toxicology Microbial Genomics DNA Repair and Mutagenesis Cell Cycle Control, Gene Regulation Biosensor, Antibiotic, Diagnostics Development Hantavirus Biology Host-Pathogen Interactions (Cystic Fibrosis)

Example: Lymphoma Gene Profiling - Willman Lab 392 Genes 142 Patients

Gene Expression in PKD Samples Under-expressed in disease samples

Gene Expression in PKD Samples Over-expressed in disease samples

Vertebrates Express Three Myb Transcription Factors c-myb A-Myb B-Myb Highly Conserved Central Trans- Negative DNA Binding Activation Domain Domain Regulatory Domain

Myb Proteins Affect Development B-Myb c-myb A-Myb blastocyst embryo adult offspring B-Myb knockout blocks cell proliferation c-myb knockout blocks definitive hematopoiesis A-Myb knockout affects testis, mammary gland development

Myb Proteins are Transcription Factors Target Genes Myb Proteins Regulate Differentiation Regulate Apoptosis Regulate Proliferation Myb Proteins Control Cell Fate by Regulating the Expression of Other Genes

c-myb Expression in Hematopoiesis Hematopoietic Stem Cell Myeloid/Erythroid Stem Cell Lymphoid Stem Cell c-myb CFU-E CFU-Meg CFU-Bas CFU-Eosin CFU-GM Pre-B Pre-T Erythrocyte Megakaryocyte Basophil Eosinophil Neutrophil Monocyte B-Cell T-Cell

c-myb Expression in Hematopoiesis Hematopoietic Stem Cell Myeloid/Erythroid Stem Cell Lymphoid Stem Cell v-myb CFU-E CFU-Meg CFU-Bas CFU-Eosin CFU-GM Pre-B Pre-T Erythrocyte Megakaryocyte Basophil Eosinophil Neutrophil Monocyte B-Cell T-Cell

v-myb Induces Myeloid Leukemias Normal Chicken Blood v-myb-induced Leukemia

Myb Protein Comparisons A-, B- and c-myb proteins have distinct expression patterns A-Myb: specialized epithelial, hematopoietic cells B-Myb: all dividing cells c-myb: immature hematopoietic, epithelial v-myb: oncogenic, induces leukemia Each protein has a unique biological activity All the Myb proteins have similar structures

Test the Activities of Myb Proteins on Endogenous Human Genes Myb Expression Vector Recombinant Adenoviruses Use Affymetrix GeneChips to measure changes in endogenous gene expression Human Cells

MCF-7: Mammary Epithelial Cells Estrogen-responsive mammary carcinoma cells Common model for estrogen-responsive breast cancer Express A-Myb, B-Myb and c-myb during cell cycle, in response to estrogen

Adenovirus Expression Vectors c-myb A-Myb B-Myb MybEng c-myb DNA Binding Domain Engrailed Repressor Domain

Filtering Microarray Data to Identify Myb Regulated Genes Affymetrix U95A, U95B, U95C, U95D, U95E arrays 60,000 genes 100 % Filter for genes expressed above background 18,063 genes 30 % Filter for genes induced or repressed >2.5X 215 genes 3.5 %

... Selected Gene Tree: Combined 215 smooth Colored by: MCF7 expressed genes Default Interpretation Gene List: Combined 2.5X up or down (320) 215 Myb Regulated Genes in MCF-7 Each Myb Protein Regulates a Different Set of Genes Uninf Cont A-Myb B-Myb c-myb MybEng Genes Repressed by A-Myb Genes activated by c-myb Genes activated by A-Myb Genes activated by B-Myb 4.0 3.0 2.5 2.0 1.5 1.2 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 1 4

Comparison of Myb Gene Activation in MCF-7 A-Myb Activated (119) 61 46 49 B-Myb Activated (100) 4 8 1 30 c-myb Activated (43) Genes activated 2.5X in independent replicate assays. More than 12,000 genes tested on U95A GeneChips.

Activation of Hep27 and DSIPI 8 7 6 Legend Hep27 DSIPI 5 4 3 2 1 0 Uninf Control c-myb MybEng Amyb Bmyb Affymetrix GeneChip Data

Myb Binding Sites in the Hep27 and DSIPI Gene Promoters Hep27 Promoter 1-1500 NFIL6 Myb Ets Ets Myb DSIPI Promoter -1114 YY1 Myb YY1 SP1

Swap Domains Between c-myb and A-Myb Conserved Restriction Sites A-Myb CHA AHC c-myb DNA Binding Domain Transcriptional Activation Domain

Selected Gene Tree: AC 30 genes >2.5X >2500 A-Myb or c-myb PEARSON Colored by: AC Swap Comparison Default Interpretation Gene List: AC 2.5X up >2500 A-Myb or c-myb (30) 214079_at Homo sapiens cdna FLJ2... 206463_s_at HEP27 217906_at LCP 204805_s_at H1FX 209343_at PP3051 209800_at KRT16 210592_s_at SSAT 202376_at SERPINA3 204542_at STHM 218010_x_at MGC2479, FLJ21046, dj6... 204698_at HEM45 33304_at HEM45 207076_s_at ASS1, CTLN1 202017_at MEH, EPHX 205353_s_at PBP 201508_at IGFBP4 202468_s_at CLLP 211456_x_at MT1H 208581_x_at MT1 212185_x_at MT2 204326_x_at MT1 215438_x_at GSPT1 206461_x_at MT1 218035_s_at FLJ20273 201506_at CSD, CDB1, CSD1, CSD2,. 212071_s_at Homo sapiens cdna FLJ1... 202949_s_at DRAL, SLIM3 217764_s_at RAB31 208763_s_at DIP, GILZ, TSC-22R 200878_at EPAS1 A-Myb and c-myb Specificity in Human Cells Ø A CHA AHC C Activated by A- Myb and c-myb Activated by the c-myb transactivation domain Specific for the A-Myb DNA binding domain 4.0 3.0 2.5 2.0 1.5 1.2 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 1 4

A-Myb and c-myb Deletions A-Myb AP AB AN c-myb CCA CB CN

Domains that Affect Gene Activation Required for Required for activation of activation of Hep27 TGFBI A AP AB AN C CA CB CN Must Must be be removed removed to activate to activate DSIPI TGFBI Northern Blots The ability to activate many genes is determined by the C-terminal domains of c-myb and A-Myb Hep27 TGFBI α-catenin DSIPI

Structure of the Human c-myb Gene 1A 8A 9A 9B 10A 13A 1 2 3 4 5 6 7 8 9 10 1112 13 14 DBD TAD NRD Alternative splicing can yield > 64 different c-myb mrnas Myb proteins with alternative C-termini are likely to have different specificities and to activate different genes

Alternative c-myb Gene Exons Expressed in Normal Cells Exons 8A, 9A, 9B, 10A and 13A are all detected

Alternative c-myb Gene Exons in Leukemia Samples Leukemias Express Additional c-myb Transcripts

c-myb Alternative Splice Products c-myb +Exon 8A +Exon 9A +Exon 9B +Exon 10A +Exon 13A Alternative C-termini = Alternative Activities

Compare the Effects of Myb Proteins in Different Cell Types Myb-Expressing Adenoviruses Do Myb proteins activate the same genes in different cell types?

75 Myb Regulated Genes in Lung Epithelial Cells A-Myb 31 genes 7 5 8 B-Myb 28 genes 10 9 5 31 c-myb 55 genes Genes activated 2.5X in independent replicate assays. More than 12,000 genes tested on U95A GeneChips.

339 Myb Regulated Genes in Lung Fibroblast Cells A-Myb 225 genes 95 25 49 B-Myb 142 genes 56 49 12 53 c-myb 170 genes Genes activated 2.5X in independent replicate assays. More than 12,000 genes tested on U95A GeneChips.

Myb Activities are Context-Specific MCF-7 A-Myb LE MCF-7 B-Myb LE MCF-7 c-myb LE 107 2 20 1 9 8 207 93 2 21 0 5 5 132 39 0 49 0 4 6 160 LF LF LF Almost no overlap in Myb activated genes in different cell types

Unique Activities in Each Cell Type MCF-7 199 genes 166 6 55 LE 75 genes 1 26 13 299 Three Myb proteins, three cell types, little overlap amongst activated genes LF 339 genes

MSI1 Gene Activation 7 Legend 6 A-Myb B-Myb c-myb Fold Activation 5 4 3 2 1 0 MCF-7 Lung Epith Lung Fibro

TGFBI Gene Activation 4.5 Legend 4.0 3.5 A-Myb B-Myb c-myb Fold Activation 3.0 2.5 2.0 1.5 1.0 0.5 0.0 MCF-7 Lung Epith Lung Fibro

HSPA6 Gene Activation 90 Legend 80 70 A-Myb B-Myb c-myb Fold Activation 60 50 40 30 20 10 0 MCF-7 Lung Epith Lung Fibro

IL8 Gene Activation 60 Legend 50 A-Myb B-Myb c-myb Fold Activation 40 30 20 10 0 MCF-7 Lung Epith Lung Fibro

120 100 B-Myb Gene Activation Legend A-Myb B-Myb c-myb Fold Activation 80 60 40 20 0 MCF-7 Lung Epith Lung Fibro

Mechanisms that Could Alter the Specificity of Myb Proteins Tissue-Specific Combinatorial Interactions Different cell types have different cooperating factors Myb proteins cooperate with other transcription factors Unique domains in Myb protein interact with different factors A Myb B X C Myb X

Mechanisms that Could Alter the Specificity of Myb Proteins Tissue-Specific Combinatorial Interactions Different cell types have different cooperating factors Myb proteins cooperate with other transcription factors Unique domains in Myb protein interact with different factors Context-Specific Post-Translational Modifications Myb proteins are modified at numerous sites Different cell types have different modifying enzymes Modifications may alter Myb activity or promote specific interactions

Myb Proteins are Subject to Multiple Modifications c-myb A-Myb B-Myb X X X X X X X X X X Myb proteins are modified by X*-ylation Do Differences in Modifications Alter Interactions with Cellular Co-Factors? X* Insert your favorite modification here

Multiple Modifications of c-myb CKII Pim-1 CDK6 MAPK c-myb p300/cbp

Context-Specific Modifications A Gene 1 Myb B Gene 1 Gene 2 Gene 2 Myb

Mutations Unmask the Oncogenic Potential of c-myb c-myb v-myb N-Terminal Deletion Point Mutations Contribute to Transforming Activity C-Terminal Deletion Removes Negative Regulatory Domain AMV v-myb is a mutated, oncogenic version of c-myb

The v-myb Mutations Could Deregulate c-myb Repressed c-myb v-myb + C-Terminal Deletion Removes Negative Regulatory Domain

Structures of A-Myb, c-myb and v-myb A-Myb CHA AHC c-myb MutMyb v-myb AMV v-myb is a mutated, oncogenic version of c-myb

Test the Activities of Myb Proteins on Endogenous Human Genes Myb Expression Vector Recombinant Adenoviruses Use Affymetrix GeneChips to measure changes in endogenous gene expression Human Cells

Selected Gene Tree: ACV36 std Colored by: ACV Myb U133A Feb 03 Default Interpretation Gene List: ACV 36 genes up 2.5X >2500 in A,C,v or MutMyb (36) 214079_at Homo sapiens cdna FLJ203... 206463_s_at HEP27 209343_at PP3051 209800_at KRT16 202376_at SERPINA3 204542_at STHM 210592_s_at SSAT 204805_s_at H1FX 218010_x_at MGC2479, FLJ21046, dj697... 204698_at HEM45 33304_at HEM45 207076_s_at ASS1, CTLN1 202017_at MEH, EPHX 218035_s_at FLJ20273 205353_s_at PBP 201508_at IGFBP4 211456_x_at MT1H 206461_x_at MT1 208581_x_at MT1 212185_x_at MT2 201506_at CSD, CDB1, CSD1, CSD2, C. 204326_x_at MT1 215438_x_at GSPT1 202949_s_at DRAL, SLIM3 208763_s_at DIP, GILZ, TSC-22R 217764_s_at RAB31 212071_s_at Homo sapiens cdna FLJ137... 200878_at EPAS1 202468_s_at CLLP 220161_s_at EHM2 201952_at MEMD, CD166 217906_at LCP 205938_at KIAA1072 200800_s_at 200664_s_at 206976_s_at HSP105A, KIAA0201, NY-CO- Comparing A-Myb, c-myb, v-myb Ø A CHA AHC C Mut V The DBD mutations do not affect some genes The v-myb DBD mutations affect a subset of the c-myb regulated genes The activity of v-myb is the most unique Some genes are preferentially activated by MutMyb or v-myb

Unique Specificity of v-myb Con C Mut V A AHC CHA Minor differences in Myb proteins cause dramatic changes in gene expression DSIPI TGFBI Hep27 actin Northern Blot

The v-myb Protein Has a Unique Transcriptional Activity c-myb MutMyb v-myb The mutations in v-myb make it qualitatively different from c-myb

Ness Lab: Adenoviruses, Genomics: Hematopoiesis, Lentiviruses: My Backyard John Rushton Wanli Lei Fan Liu Lisa Davis John O Rourke Pim-1 Activity: Louise Winn (Queen s U., Kingston) Collaborators: Achim Leutz, Berlin Kathy Weston, London Anton Berns, Netherlands Tim Bender, Virginia Scott A. Ness Molecular Genetics & Microbiology University of New Mexico HSC Albuquerque, NM USA