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Hematopoietic Differentiation

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Hematopoietic Differentiation Hematopoietic Differentiation A number of lineages of mature cells are derived by a series of steps from a common stem cell precursor. Myeloid cells including neutrophils and monocytes are important for early defense against infection and because of the occurrence of myeloid leukemias. Myeloid differentiation and activation offers a favorable system for experimental investigation. Some Models of Gene Expression in Myeloid Cells 1. Compare resting neutrophils with neutrophils exposed for two hours to either. – A. Non-pathogenic E. coli. – B. Yersinia pestis D27 (plague bacillus). – C. Non-pathogenic Yersinia D28. 2. Induced differentiation of MPRO promyelocytic cells. NeutrophilsNeutrophils Major frontline defense cells against invading pathogens. – Constitute 70% of the total blood leukocytes. – Terminally differentiated professional phagocytic cells with an antibacterial arsenal. – Use both oxygen dependent and oxygen independent killing mechanisms. Play a central role in 2000X mag inflammation. Distribution of IL-8 transcripts on neutrophils (in situ hybridization) A combination of two Cy3 (red)- A labeled oligonucleotides complementary to IL-8 transcripts were used for in situ hybridization with neutrophils incubated in the absence (A) or presence (B) of ten E. coli K12 organisms per neutrophil. BB Methods of Analysis 1. mRNA analysis. – A. 3’ end restriction fragment gel display. – B. Affymetrix 60k or 42k oligonucleotide arrays. 2. Protein analysis. 2D gel electrophoresis using a variety of ph ranges for isoelectric focusing. MALDI-TOF identification of proteins. Representative Segments of Display Gels of cDNA Fragments: (Left) Neutrophils and Monocytes Exposed to E. coli for 2 H; (Right) Neutrophils Exposed to Various Bacteria for 2 H Ec: E. coli K12; Yp (KIM5): Y. pestis KIM5 (pCD1+); Yp (KIM6): Y. pestis KIM6 (pCD1-). [Gene Symbols] PPIF: peptidylprolyl isomerase F (cyclophilin F) RPL3: ribosomal protein L3 NBS1: Nijmegen breakage syndrome 1 (nibrin) ATP6C: ATPase, H+ transporting, lysosomal (vacuolar proton pump) 42kD SAT: spermidine/spermine N1-acetyltransferase XIP: hepatitis B virus x-interacting protein (9.6kD) RPS4X: ribosomal protein S4, X-linked RGS2: regulator of G-protein signalling 2, 24kD PAI2: Plasminogen activator inhibitor, type II (arginine-serpin) IL8RA: interleukin 8 receptor, alpha Correlation Between Band Intensities and PhosphorImager Quantification 106 105 0.5612x 104 y = 3722.6e R2 = 0.8647 PhosphorImager Signal 103 12345678 Band Intensity Comparison of the expression patterns of two clusters of genes from neutrophils stimulated with various bacteria 0.6 0.4 0.2 0.0 -0.2 Normalized-0.4 Band Intensity -0.6 0.6 0.4 0.2 0.0 -0.2 Normalized-0.4 Band Intensity -0.6 LHLH Control HLHL E.coli K12 Y. pestis (D27) Y. pestis (D28) Gene clusters separated by Principal Component Analysis (PCA) LLLH LLHL LLHH LHLL LHLH LHHH HLLL HLHL HLHH HHLL HHLH 1.0 0.5 0.0 cn1 -0.5 -1.0 -1.5 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 cn2 Genes expressed by neutrophils differently regulated by KIM5 (pCD1+) and KIM6 (pCD1-) strains of Y pestis KIM5-responsive KIM6-responsive NC NC NC (slightly Up-regulated Down-regulated E.coli K12 up-regulated) KIM5 Up- Down- NC (slightly NC (slightly NC regulated regulated up-regulated) up-regulated) KIM6 NC NC Up-regulated Up-regulated Down-regulated Transcription Modulators XIP BRF2 DSIPI CROC4 — — COPEB HDAC3 NFKB1 TRIP8 ZNF220 BTF3 TOM1 Genes expressed by neutrophils differently regulated by KIM5 (pCD1 +) and KIM6 (pCD1 -) strains of Y. pestis Genes Similarly Regulated by Both KIM5 (Pcd1 +) and KIM6 (Pcd1 -) Strains of Y. pestis Some Salient Observations in Neutrophil Activation Experiments 1. Activated neutrophils actively synthesize a variety of cytokines that may attract other neutrophils, monocytes, and perhaps other inflammatory cells. 2. A variety of anti-apoptotic genes are activated, presumably promoting the survival of activated neutrophils. 3. There is substantial overlap in early response genes seen in neutorphils and other cell types such as fibroblasts. Some Salient Observations in Neutrophil Activation Experiments 4. Responses to different non-pathogenic gram-negative bacteria are very similar but not necessarily identical. 5. The early response is entirely ablated by pathogenic Yersinia and a different more limited set of genes are activated. 2. MPRO differentiation experiment A: 0h B: 24h C: 48h D: 72h Morphology of MPRO cells during differentiation induced with ATRA Known Genes (50%) No Hits (21%) ESTs (20%) Mitochondrial Sequences (2%) rRNAs(1%) Distribution of genes identified by differential display assay Early Middle Late Time Category Up-regulation LHHH (n=10) LLHH (n=6) LLLH (n=13) Mad P2rx1 Itgb2 Pira1 Cybb Pfc Pira5 Il1a Csf1r Ii Ctsl S100a8 L- Il1r2 Lcn2 Itpr5 Cd53 Ifngr2 CCR Ctss Aldo1 Rac2 Fpr1 Cebpb H2-D Etohi6 Ctsd Ubb Ptmb4 Zyx Down-regulation HLLL (n=11) HHLL (n=1) HHHL (n=37) Tcrg-V4 Ly64 Ctsg Mpo Actx Irf2 EL2 Rpl19 Actb Spi2-1 Mcpt8 Myc Ly6e Atf1 Hist2 Psma2 Gnas Myb Tlr4 Npm1 Erh Zfp36 Il4ra Ltbr Shfdg1 Max Hsp60 Rps8 Csf2rb1 Slpi Tctex1 Tpi Btf3 Cntf Gys3 Slc10a1 Ctsb Sepp1 Rtn3 Ccnb2 S100a9 Cfl1 Hist5-2ax Rela Copa Gstm1 Gnb2-rs1 Grn RPL8 Transient LLHL (n=9) Sell Klf2 Pira6 Pirb Lst1 Ltf Sema4d Stat6 Mmp9 LHHL (n=17) Cebpa Lyzs Fcgr3 Arf5 Lamp1 Stat3 Csf2ra Osi Actg Sfpi1 Gpx3 Ptprc Prtn3 Irf1 Rps6ka1 Ltb4r Myln Genes differently regulated during the different stages of MPRO differentiation process Gene clusters in the first two principal component space Northern blot analysis of selected mRNAs AD Value By Arrays Intens ity By DD Gene Gene Symbol Accession 0-HR 24-HR 48-HR 72-HR 0-HR 24-HR 48-HR 72-HR Cebpa M62362 33 212 182 44 //// Cebpb X62600 390 1248 1380 1903 //// Cebpd X61800 157 262 168 430 //// Cebpe / / / / / //// Myb M12848 892 356 230 435 //// Slpi U73004 617 501 783 402 1233 Prg3 W45834 153 259 339 345 5112 Gnb2-rs X75313 4231 3623 3215 3403 4411 Ly6e U04268 3061 5391 2844 1282 3211 Lsp1 M90316 65 376 840 28 2356 Actb X03765 3095 3588 3976 2434 1232 Expression patterns of genes detected by Northern blot assay Transcription modulators presented during myeloid differentiation Maximal fold Gene Gene AD value by array change symbol accession 0h 24h 48h 72h 4 or more, Cebpb X62600 390 1248 1380 1903 less than 5 Stra14 Y07836 223 383 510 936 5 or more Cebpa M62362 33 212 182 44 Grg X73359 99 565 916 1005 Mad X83106 0 111 167 327 Myc L00039 314 112 62 173 Etohi6 W89667 169 386 313 1003 TBX1 AA542220 0 0 1 2 A Few Salient Observations on Gene Expression During MPRO DIfferentiation 1. A number of the activating transcription factors whose levels change have previously been partially studied in the context of myeloid differentiation. 2. In contrast, several transcription regulators that may be involved in decreasing expression of specific genes have not been previously described in the context of myeloid cells. 2DE of MPRO cells during differentiation Analysis of MPRO Gels with Melanie II 2-D Software A: 0 hour B: 24 hours AB C: 48 hours D: 72 hours CD 2DE of uninduced and induced MPRO cells 2DE of uninduced and induced MPRO cells 2DE of MPRO cells in basic pH range 2. Cycloheximide inhibition experiment of MPRO cells 2-DE of MPRO cells of cycloheximide inhibition experiment (pH 4-7) Up regulated Down regulated Magnified image of highlighted area in 2DE of MPRO cells (pH 4-7) 2DE of MPRO cells of cycloheximide inhibition experiment (pH 6-11) Magnified image of the highlighted area in 2DE of MPRO of cycloheximide inhibition experiment (pH 6-11) Time-course Profile and Functional Classification of Genes Expressed by E coli K12–stimulated Neutrophils 3. Heparin-agarose experiment A B A: HL 60 NE; B: Heparin-agarose purified proteins from HL 60 NE AB A: HL 60 NE; B: Heparin-agarose purified proteins from HL 60 NE. Red arrows: Enriched products.; Blue arrows: Depleted products. Comparison of Expression Patterns in Monocytes and Neutrophils 1. Many mRNAs are present in both monocytes and neutrophils or induced in activated cells, although some are cell type specific. 2. Strikingly there are many differences in the relative abundance of shared mRNAs between these tow related cell types. “PANOMICS” Arrays of probes covering the unique sequence regions of human DNA are “in the works.” Professor Snyder has already prepared arrays of most of the yeast proteins and the same type of array is becoming feasible for human cells. With these arrays a variety of analyses could be done in a global fashion. Some Applications of Genomic Arrays 1. Detect transcripts by mRNA hybridization. 2. Detect sequence polymorphisms. 3. Detect chromatin associated proteins. 4. Detect sites of initiation of DNA replication. 5. Detect hetrochromatin and imprinted regions. TDG Reaction: Removing Thymine from G.T, forming an Apurinic Site E + DNA (G.T) Kd E-DNA (G.T) kcat T E-DNA (G.Ap) koff E + DNA (G.Ap) Modified from JBC, 1999, 274 (1): 67-74 hTDG Mediated Cleavage of Mismatch DNA Containing G/T Pairing 60 bp Duplex Pa Ma Pb Mb 315 bp CG/GC CG/GT AG/TC AG/TT Perfect Pairing Marker (bp) hTDG(ul) 0 1 5 0 1 5 0 1 5 0 1 5 0 1 5 315 60 37 30 P: Perfect match, M: Mismatch 16 a: Template set (a) b: Template set (b) Chromatin Immunoprecipitation 4.5 4 4.5 GATA-1 antibody was 3.5 4 3 used ot precipitate 3.5 2.5 3 cross-linked 2 2.5 1.5 chromatin. DNA was 2 1 amplified from the 1.5 0.5 1 0 preciitate with 0.5 HS4 HS3 HS2 HS1 random primers and 0 HS4-globin HS3 locusHS2LCR HS1 annealed to an arraay -globin locusLCR covering the globin locus control region A B A: HL 60 NE; B: Heparin-agarose purified proteins from HL 60 NE 2DE of nuclear extracts purified by heparin-agarose column.
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