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Single-chromosome transcriptional
               profiling reveals chromosomal
                gene expression regulation

                            Marshall J Levesque, Arjun Raj
                                Systems Biology Lab
                               UPenn Bioengineering

                         Nature Methods (2013) doi:10.1038/nmeth.2372




Thursday, April 11, 13
Our genome is organized
                         into pairs of chromosomes




                           By 718 Bot at en.wikipedia [Public domain], from Wikimedia Commons. http://en.wikipedia.org/wiki/Karyotype




Thursday, April 11, 13
Chromosome rearrangements
                      are a hallmark of cancer




        Macville et al. Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping. Cancer Res (1999) vol. 59 (1) pp. 141-50




Thursday, April 11, 13
Does chromosome structure
                           affect gene expression?
                                           A HeLa Karyotype
                                                                                                                   t(13;19)
                                                                                                                   t(13;19)
                                                                                                                Chr 13
                                                                                                                Chr 13
                                                                                                                q-arm
                                                                                                                q-arm
                                                                                                                         t(6;19)
                                                                                                                         t(6;19)



                                                                                                             Chr 19
                                                                                                             Chr 19
                                                                                                    Chr 19
                                                                                                    Chr 19                    Chr 6
                                                                                                                              Chr 6
                                                                                                    p-arm
                                                                                                    p-arm                     q-arm
                                                                                                                              q-arm




                                                                                                    Chr 19
                                                                                                    Chr 19
                                                                                                    q-arm
                                                                                                    q-arm
                                                                                                                              Chr 6
                                                                                                                              Chr 6
              RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery                                 p-arm
                                                                                                                              p-arm



                                                                                                C
Thursday, April 11, 13
Does chromosome structure
                           affect gene expression?
                                           A HeLa Karyotype
                                                                                                                   t(13;19)
                                                                                                                   t(13;19)
                                                                                                                Chr 13
                                                                                                                Chr 13
                                                                                                                q-arm
                                                                                                                q-arm
                                                                                                                         t(6;19)
                                                                                                                         t(6;19)



                                                                                                             Chr 19
                                                                                                             Chr 19
                                                                                                    Chr 19
                                                                                                    Chr 19                    Chr 6
                                                                                                                              Chr 6
                                                                                                    p-arm
                                                                                                    p-arm                     q-arm
                                                                                                                              q-arm




                                                                                                    Chr 19
                                                                                                    Chr 19
                                                                                                    q-arm
                                                                                                    q-arm
                                                                                                                              Chr 6
                                                                                                                              Chr 6
              RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery                                 p-arm
                                                                                                                              p-arm



                                                                                                C
Thursday, April 11, 13
Does chromosome structure
                           affect gene expression?
                                           A HeLa Karyotype
                                                                                                                   t(13;19)
                                                                                                                   t(13;19)
                                                                                                                Chr 13
                                                                                                                Chr 13
                                                                                                                q-arm
                                                                                                                q-arm
                                                                                                                         t(6;19)
                                                                                                                         t(6;19)



                                                                                                             Chr 19
                                                                                                             Chr 19
                                                                                                    Chr 19
                                                                                                    Chr 19                    Chr 6
                                                                                                                              Chr 6
                                                                                                    p-arm
                                                                                                    p-arm                     q-arm
                                                                                                                              q-arm




                                                                                                    Chr 19
                                                                                                    Chr 19
                                                                                                    q-arm
                                                                                                    q-arm
                                                                                                                              Chr 6
                                                                                                                              Chr 6
              RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery                                 p-arm
                                                                                                                              p-arm



                                                                                                C
Thursday, April 11, 13
RNA counting cannot tell us
                         where the RNA comes from
                                         A HeLa Karyotype
                                                                                                                   t(13;19)
                                                                                                                   t(13;19)
                                                                                                                Chr 13
                                                                                                                Chr 13
                                                                                                                q-arm
                                                                                                                q-arm
                                                                                                                         t(6;19)
                                                                                                                         t(6;19)



                                                                                                             Chr 19
                                                                                                             Chr 19
                                                                                                    Chr 19
                                                                                                    Chr 19                    Chr 6
                                                                                                                              Chr 6
                                                                                                    p-arm
                                                                                                    p-arm                     q-arm
                                                                                                                              q-arm




                                                                                                    Chr 19
                                                                                                    Chr 19
                                                                                                    q-arm
                                                                                                    q-arm
                                                                                                                              Chr 6
                                                                                                                              Chr 6
              RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery                                 p-arm
                                                                                                                              p-arm



                                                                                                C
Thursday, April 11, 13
iceFISH assay detects active
                     genes on single chromosomes
                                                                       mRNA
                                                         Export and
                                                         Translation
                             Spliced mRNA


                 Exon                                    Exon
                                   Exon      Intron
                         Intron




                           Introns degraded before
                         leaving site of transcription




 Raj et al. Imaging individual mRNA molecules using
 multiple singly labeled probes. Nat Methods (2008)


Thursday, April 11, 13
iceFISH assay detects active
                     genes on single chromosomes
                                                                       intron
                                                         Export and
                                                         Translation
                             Spliced mRNA


                 Exon                                    Exon
                                   Exon      Intron
                         Intron




                           Introns degraded before
                         leaving site of transcription




Thursday, April 11, 13
Pseudocoloring provides 20+ unique
         tags to trace a chromosome
                          Intron
     A                                                B                  Chromosome 19



                                                                Intron
                                               A                                     B   Chromosome 19



   5 base colors
                                               5 base colors




                                                               2 colors 3 colors
                         2 colors 3 colors                     20 pseudocolors

                         20 pseudocolors                                      DAPI                 ATTO488           Cy3
                                                C                                    D                       E

     C                                  DAPI   D                                      ATTO488            E

                                                F                        Alexa 594   G           ATTO647N    H   ATTO700




     F
Thursday, April 11, 13
                                   Alexa 594   G  I                  Chr 19
                                                                                            J
                                                                                     ATTO647N            H             ATT
Pseudocoloring provides 20+ unique    F                                                                                          G                                                                                    H
                                                                                                Alexa 594                                                                                          ATTO647N



         tags to trace a chromosome
                          Intron
     A                                                                                        B                      Chromosome 19


                                           I                                                  Chr 19
                                                                                                            Intron                                                                                     J
                                                                                     A                                                            B                     Chromosome 19

                                                  Chr 19

   5 base colors
                                                                                     5 base colors




                                                                                                           2 colors 3 colors
                         2 colors 3 colors                                                                 20 pseudocolors
                                                                                                                                                                                                            4




                                                                                                                                                                                                   z (µm)
                         20 pseudocolors                                                                                            DAPI                                                          ATTO488                             Cy3
                                                                                      C                                                        D                                                            2   E

     C                                             DAPI                              D                                                                ATTO488                                           0
                                                                                                                                                                                                            E
                                                                                                                                                                                                       16
                                                                                                                                                                                                                    12
                                                                                                                                                                                                                              8
                                   0 Mb                                                               Chromosome 19                                                              59.13 Mb
                                                                                                                                                                                                                         y (µm)       4
                                                                                      F                              Alexa 594                 G                                                 ATTO647N       H                 ATTO700
                                   PTBP1

                                           EEF2




                                                                                                                                                              SLC1A5

                                                                                                                                                                       PPP2R1A
                                                                                                                                                                                 RPS9
                                                                                                                                                                                        ZNF444
                                                      DNMT1
                                                              ILF3




                                                                                                                     UBA2


                                                                                                                                    SUPT5H
                                                                                                                                       FBL
                                                                                                                                     EGLN2
                                                                                                                                              RPS19
                                                                                                                                             TOMM40
                                                                                                                                                      MARK4
                                                                     DHPS


                                                                            RPL18A

                                                                                      UBA52


                                                                                              ZNF91




                                                                                                                            EIF3K




     F
Thursday, April 11, 13
                                   Alexa 594                                         G   I                       Chr 19
                                                                                                                                                     J
                                                                                                                                              ATTO647N                                                      H                             ATT
iceFISH assigns gene activity to
     normal or derivative copies of chr19
                             A       HeLa Karyotype                              B
                                                t(13;19)                                                 t(6;19)
                                                                                                                                                        Chr 19
                                             Chr 13
                                             q-arm
                                                        t(6;19)


                                                                                    Fusion
                                          Chr 19                                 chromosomes

                                 Chr 19                                                           t(13;19)
                                                             Chr 6
                                 p-arm
                                                             q-arm
                                                                                                                                                                   Chr 19




                                 Chr 19
                                 q-arm
                                                             Chr 6
                                                                         0 Mb                                                           Chromosome 19                                                           59.13 Mb
                                                             p-arm
                                                                         PTBP1

                                                                                 EEF2




                                                                                                                                                                   SUPT5H
                                                                                                                                                                      FBL
                                                                                                                                                                    EGLN2
                                                                                                                                                                             RPS19
                                                                                                                                                                            TOMM40
                                                                                                                                                                                     MARK4
                                                                                                                                                                                             SLC1A5

                                                                                                                                                                                                      PPP2R1A
                                                                                                                                                                                                                RPS9
                                                                                                                                                                                                                       ZNF444
                                                                                        DNMT1
                                                                                                ILF3
                                                                                                       DHPS


                                                                                                               RPL18A

                                                                                                                        UBA52


                                                                                                                                ZNF91




                                                                                                                                                    UBA2
                                                                                                                                                           EIF3K
                             C
                                                                                                                                                                                        Chr 19
                                                                                                                                                                                        Chr 13
                         1            Translocation         Centromere                                                                                                                  t(13;19)
                                                                                                                                                                                        t(6;19)
                                    t(13;19) t(13;19)                                                         ***
                  some




Thursday, April 11, 13 0.8        N=50                                              N=30                                                                                                                        ***
p-arm
                                                                                                                                                       q-arm
                                                                                                                                                                                                                                                                                                      Chr 19




      Translocations can lead to whole
    chromosome increases in expression                                                  Chr 19
                                                                                        q-arm
                                                                                                                                                       Chr 6
                                                                                                                                                       p-arm                         0 Mb                                                                                  Chromosome 19                                                           59.13 Mb




                                                                                                                                                                                     PTBP1

                                                                                                                                                                                                  EEF2




                                                                                                                                                                                                                                                                                                                RPS19
                                                                                                                                                                                                                                                                                                               TOMM40
                                                                                                                                                                                                                                                                                                                        MARK4
                                                                                                                                                                                                                      DNMT1
                                                                                                                                                                                                                                 ILF3




                                                                                                                                                                                                                                                                                       UBA2


                                                                                                                                                                                                                                                                                                      SUPT5H
                                                                                                                                                                                                                                                                                                         FBL
                                                                                                                                                                                                                                                                                                       EGLN2




                                                                                                                                                                                                                                                                                                                                SLC1A5

                                                                                                                                                                                                                                                                                                                                         PPP2R1A
                                                                                                                                                                                                                                                                                                                                                   RPS9
                                                                                                                                                                                                                                                                                                                                                          ZNF444
                                                                                                                                                                                                                                        DHPS


                                                                                                                                                                                                                                                RPL18A

                                                                                                                                                                                                                                                         UBA52


                                                                                                                                                                                                                                                                   ZNF91




                                                                                                                                                                                                                                                                                              EIF3K
                                                                                C
                                                                                                                                                                                                                                                                                                                           Chr 19
A       HeLa Karyotype                                                          B                                                                                                                                                                                                                                          Chr 13
                                                                         1                              Translocation                                Centromere                                                                                                                                                            t(13;19)
                   t(13;19)                                                                               t(6;19)
                                                                                                                                                                                                                                                                                                                           t(6;19)
                                                                                                  t(13;19) t(13;19)                                          Chr 19                                                                            ***
                                        iceFISH spots per chromosome




                Chr 13
                q-arm
                           t(6;19)
                                                                       0.8                 N=50                                                                                                        N=30                                                                                                                                        ***
                                                                                                                                                                   ***                                                                                                                           ***
                                                                                   Fusion
             Chr 19                                                    0.6      chromosomes

    Chr 19
                                Chr 6
                                                                                        **         t(13;19)
                                                                                                                                                     ***                                             ** ***                                                                                                    ***
    p-arm
                                q-arm                                  0.4
                                                                                                               *                                                        Chr 19                                                                                                      **

                                                                       0.2


    Chr 19                                                               0
                                                                                        MTZ1 DIAPH3 RPL18A                                   UBA52   ZNF91              UBA2      EIF3K           SUPT5H                        FBL            EGLN2             RPS19 TOMM40 MARK4 SLC1A5 PPP2R1A                              RPS9               ZNF444
    q-arm
                                Chr 6                                                    Chr. 13                                                                                             Chromosome 19 gene name
                                                                        0 Mb                                                                 Chromosome 19                                                           59.13 Mb
                                p-arm                                                  gene name
                                                                        PTBP1

                                                                                EEF2




                                                                                                                                                                        SUPT5H
                                                                                                                                                                           FBL
                                                                                                                                                                         EGLN2
                                                                                                                                                                                  RPS19
                                                                                                                                                                                 TOMM40
                                                                                                                                                                                          MARK4
                                                                                         DNMT1
                                                                                                 ILF3
                                                                                                        DHPS


                                                                                                                   RPL18A

                                                                                                                             UBA52


                                                                                                                                     ZNF91




                                                                                                                                                         UBA2




                                                                                                                                                                                                  SLC1A5

                                                                                                                                                                                                           PPP2R1A
                                                                                                                                                                                                                     RPS9
                                                                                                                                                                                                                              ZNF444
                                                                                                                                                                EIF3K




C                                                                        1
                                                                                                                            N=30
                                                                                                                                                  t(6;19)
                                                                                                                                                                                             Chr 19
                                                                       0.8                                                                                                                   Chr 13
         Translocation         Centromere                                                                                                                                                    t(13;19)
                                                                                                                                                                                             t(6;19)
       t(13;19) t(13;19)
                                                                       0.6                                     ***
     N=50
Thursday, April 11, 13
                                                                                   N=30                                                                                                                              ***
q-arm




                                                                                                                                          iceFISH spots per chromo
                                                                                                                                                                                                                                                                    Chr 19
                                                                                                                                                                                                                                                                                                                ***
                                Translocations do not necessarily***lead
                                                    **
                                               Chr 19
                                                           ***  **
                                                                                                                                                                     0.6




                                 to changes in transcriptional activity
                                               q-arm                                                                                                                 0.4
                                                       *                                    Chr 6
                                               Chr 19                                       p-arm             0 Mb                                                                                                          Chromosome 19                                                                                                                      59.13 Mb
                                               q-arm
                                                                                            Chr 6                                                                    0.2




                                                                                                              PTBP1

                                                                                                                       EEF2



                                                                                                                                DNMT1 DNMT1
                                                                                                                                                           ILF3
                                                                                                                                                                     DHPS


                                                                                                                                                                              RPL18A RPL18A

                                                                                                                                                                                                  UBA52


                                                                                                                                                                                                                 ZNF91




                                                                                                                                                                                                                                                                    SUPT5H SUPT5H
                                                                                                                                                                                                                                                                              FBL
                                                                                                                                                                                                                                                                     EGLN2 EGLN2
                                                                                                                                                                                                                                                                                         RPS19 RPS19
                                                                                                                                                                                                                                                                                        TOMM40 TOMM40
                                                                                                                                                                                                                                                                                                        MARK4 MARK4
                                                                                                                                                                                                                                                     UBA2




                                                                                                                                                                                                                                                                                                                      SLC1A5 SLC1A5

                                                                                                                                                                                                                                                                                                                                                     PPP2R1A
                                                                                                                                                                                                                                                                                                                                                               RPS9
                                                                                                                                                                                                                                                                                                                                                                      ZNF444 ZNF444
                                                                                                                                                                                                                                                            EIF3K
                                                                                                              0 Mb                                                                                                          Chromosome 19                                                                                                                      59.13 Mb

                                           C                                                p-arm




                                                                                                              PTBP1

                                                                                                                       EEF2




                                                                                                                                                                                                                                                                       FBL
                                                                                                                                                           ILF3
                                                                                                                                                                     DHPS




                                                                                                                                                                                                  UBA52


                                                                                                                                                                                                                 ZNF91




                                                                                                                                                                                                                                                     UBA2




                                                                                                                                                                                                                                                                                                                                              PPP2R1A
                                                                                                                                                                                                                                                                                                                                                               RPS9
                                                                                                                                                                                                                                                            EIF3K
                                                                                                                                                                        0
                                           C                                                                                                                                                              MTZ1 DIAPH3 RPL18A
                                                                                                                                                                                                    Chr. 13
                                                                                                                                                                                                                                                             UBA52                 ZNF91
                                                                                                                                                                                                                                                                                                               Chr 19 EIF3K SUPT5H FBL EGLN2 RPS19 TOMM
                                                                                                                                                                                                                                                                                                                UBA2
                                                                                                                                                                                                                                                                                                                           Chromosome 19 gene name
                                                                                                                                                                                                                                                                                                               Chr 13
                                                                                                                                                                                                  gene name
                                      1             Translocation
                                                                      A              Centromere
                                                                                   HeLa Karyotype                                                                     B                                                                                                                                        Chr 19
                                                                                                                                                                                                                                                                                                               t(13;19)
                                                                                                                                                                                                                                                                                                               Chr 13
                                                                                                                                                                                                                                                                                                               t(6;19)
                                                  t(13;19) t(13;19)                                                                                                          ***
iceFISH spots spots per chromosome




                                                                                             t(13;19)                                                                   1                                   t(6;19)
                                                    Translocation                         Centromere                                                                                                                                                                                                           t(13;19)
                                       1
                                     0.8        N=50                                                                      N=30                                                                                                            N=30                              Chr 19
                                                                                                                                                                                                                                                                                                               t(6;19)
                                                                                                                                                                                                                                                                                                                                                               ***
                                                  t(13;19) t(13;19)                         Chr 13
                                                                                                        t(6;19)                                                              ***                                                                               ***
                                                                                                                                                                                                                                                                           t(6;19)




                                                                                                                                 iceFISH spots per chromosome
                                                                                                     ***
      iceFISH per chromosome




                                                                                            q-arm

                                     0.8        N=50                                                                      N=30                                       0.8                                                                                                                                                                                       ***
                                     0.6
                                                                                                     ***                                                                 Fusion                                                                                ***
                                     0.6
                                               **                                         ***
                                                                                        Chr 19
                                                                                                                         ** ***
                                                                                                                              0.6
                                                                                                                                                                      chromosomes                                                                                                   ***
                                                                               Chr 19
                                                                                                                                                                                                                                                  **
                                                                                                                         ** ***                                                                                                                                                     ***
                                                                                                              Chr 6                                                                                  t(13;19)
                                     0.4
                                               **          *                   p-arm
                                                                                          ***                 q-arm
                                                                                                                                                                                                                                                                                        Chr 19


                                     0.4
                                                           *
                                                                                                                                                                     0.4                                                                          **
                                     0.2

                                     0.2                                                                                                                             0.2
                                       0                                       Chr 19
                                               MTZ1 DIAPH3 RPL18A               UBA52     ZNF91      UBA2   EIF3K      SUPT5H                          FBL                   EGLN2                        RPS19 TOMM40 MARK4 SLC1A5 PPP2R1A                                                                           RPS9                                     ZNF444
                                             Chr. 13
                                                                               q-arm
                                                                                                                      Chromosome 19 gene name
                                                                                                                                                                                                                                                                                                                         to Chr 6 centromere
                                                                                                               Chr 6
                                      0                                                                                       0 Mb 0                                                                                                              Chromosome 19                                                                                                                 59.13 Mb
                                           gene name
                                             MTZ1 DIAPH3              RPL18A    UBA52     ZNF91      UBA2
                                                                                                               p-arm
                                                                                                            EIF3K SUPT5H                               FBL                   EGLN2 PTBP1
                                                                                                                                                                                    RPS19 TOMM40DNMT1
                                                                                                                                                                                          EEF2         ILF3   DHPS
                                                                                                                                                                                                 MARK4 SLC1A5 PPP2R1A                                                                                                 RPS9                                     ZNF444
                                             Chr. 13                                                                                  Chromosome 19 gene name
                                                                                                                      Chromosome 19 gene name
                                                                                                                                                        PTBP1

                                                                                                                                                                      EEF2




                                                                                                                                                                                                                                                                                         SUPT5H
                                                                                                                                                                                                                                                                                            FBL
                                                                                                                                                                                                                                                                                          EGLN2
                                                                                                                                                                                                                                                                                                                RPS19
                                                                                                                                                                                                                                                                                                               TOMM40
                                                                                                                                                                                                                                                                                                                                      MARK4
                                                                                                                                                                                          DNMT1
                                                                                                                                                                                                   ILF3
                                                                                                                                                                                                          DHPS


                                                                                                                                                                                                                         RPL18A

                                                                                                                                                                                                                                  UBA52


                                                                                                                                                                                                                                          ZNF91




                                                                                                                                                                                                                                                                         UBA2




                                                                                                                                                                                                                                                                                                                                                     SLC1A5

                                                                                                                                                                                                                                                                                                                                                                  PPP2R1A
                                                                                                                                                                                                                                                                                                                                                                                 RPS9
                                                                                                                                                                                                                                                                                                                                                                                        ZNF444
                                                                                                                                                                                                                                                                                EIF3K
                                      1
                                           gene name                  C
                                                                  N=30
                                                                                        t(6;19)
                                                                                                                                                                                                                                                                                                                                         Chr 19
                                       1
                                     0.8                         N=30                                                                                                                                                                                                                                                                    Chr 13
                                                                 1                    t(6;19)
                                                                                    Translocation           Centromere                                                                                                                                                                                                                   t(13;19)
                                                                                                                                                                                                                                                                                                                                         t(6;19)
                                     0.8                                          t(13;19) t(13;19)                                                                                                              ***
                                                        osome




   Thursday, April 11, 13
                                     0.6                        0.8             N=50                                                                                        N=30                                                                                                                                                                                            ***
Are chromosomes built to
                 regulate their genes?




                               EEF2 intron   EEF2 mRNA   Chr. 19




Thursday, April 11, 13
Single chromosome transcription
             profiles unravel gene interactions
                    Correlated Expression                                          Intra-chromosomal (cis) interactions

                                                                                      Nucleus

                                                                                                              Gene A
                                                                                                Chr. 19

                                                                                                                Gene B



              Anti-Correlated Expression




                                                                                   PPP2R1A
                                                                                   TOMM40
                Intra-chromosomal (cis) interactions                               Inter-chromosomal (trans) interactions




                                                                                   SUPT5H
                                                                                   RPL18A




                                                                                   SLC1A5



                                                                                   ZNF444
                                                                                   MARK4
                                                                                   DNMT1




                                                                                   EGLN2
                                                                                   PTBP1




                                                                                   UBA52




                                                                                   RPS19
                                                                                   ZNF91

                                                                                   EIF3K
                                                                                   DHPS




                                                                                   UBA2




                                                                                   RPS9
                                                                                   EEF2

                                                                                   ILF3




                                                                                   FBL
                         Nucleus                                                      Nucleus                                    PTBP1
                 Uncorrelated Expression
                               Gene A                                                                         Gene A             EEF2
                                   Chr. 19                                                      Chr. 19                          DNMT1
                                                              reskin fibroblasts



                                                                                                                                 ILF3
                                                 Gene B                                                          Gene B          DHPS
                                                                                                                                 RPL18A
                                                                                                                                 UBA52
                                                                                                                                 ZNF91
                                                                                                                                 UBA2
                                                                                                                                 EIF3K
                                                                                                                                 SUPT5H
                                                    1A




                                                                                                                        1A
                                                   40




                                                                                                                       40
Thursday, April 11, 13
                                             H




                                                                                                          H
                               A




                                                    5




                                                                                                A




                                                                                                                        5
                                                          4




                                                                                                                             4
                                                   4




                                                                                                                       4
                     1




                                                                                      1
Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation
Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation
Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation
Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation
Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation
Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation
Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation
Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation
Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation

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Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation

  • 1. Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation Marshall J Levesque, Arjun Raj Systems Biology Lab UPenn Bioengineering Nature Methods (2013) doi:10.1038/nmeth.2372 Thursday, April 11, 13
  • 2. Our genome is organized into pairs of chromosomes By 718 Bot at en.wikipedia [Public domain], from Wikimedia Commons. http://en.wikipedia.org/wiki/Karyotype Thursday, April 11, 13
  • 3. Chromosome rearrangements are a hallmark of cancer Macville et al. Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping. Cancer Res (1999) vol. 59 (1) pp. 141-50 Thursday, April 11, 13
  • 4. Does chromosome structure affect gene expression? A HeLa Karyotype t(13;19) t(13;19) Chr 13 Chr 13 q-arm q-arm t(6;19) t(6;19) Chr 19 Chr 19 Chr 19 Chr 19 Chr 6 Chr 6 p-arm p-arm q-arm q-arm Chr 19 Chr 19 q-arm q-arm Chr 6 Chr 6 RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery p-arm p-arm C Thursday, April 11, 13
  • 5. Does chromosome structure affect gene expression? A HeLa Karyotype t(13;19) t(13;19) Chr 13 Chr 13 q-arm q-arm t(6;19) t(6;19) Chr 19 Chr 19 Chr 19 Chr 19 Chr 6 Chr 6 p-arm p-arm q-arm q-arm Chr 19 Chr 19 q-arm q-arm Chr 6 Chr 6 RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery p-arm p-arm C Thursday, April 11, 13
  • 6. Does chromosome structure affect gene expression? A HeLa Karyotype t(13;19) t(13;19) Chr 13 Chr 13 q-arm q-arm t(6;19) t(6;19) Chr 19 Chr 19 Chr 19 Chr 19 Chr 6 Chr 6 p-arm p-arm q-arm q-arm Chr 19 Chr 19 q-arm q-arm Chr 6 Chr 6 RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery p-arm p-arm C Thursday, April 11, 13
  • 7. RNA counting cannot tell us where the RNA comes from A HeLa Karyotype t(13;19) t(13;19) Chr 13 Chr 13 q-arm q-arm t(6;19) t(6;19) Chr 19 Chr 19 Chr 19 Chr 19 Chr 6 Chr 6 p-arm p-arm q-arm q-arm Chr 19 Chr 19 q-arm q-arm Chr 6 Chr 6 RNA FISH (fluorescence in situ hybridization) from Raj Lab website image gallery p-arm p-arm C Thursday, April 11, 13
  • 8. iceFISH assay detects active genes on single chromosomes mRNA Export and Translation Spliced mRNA Exon Exon Exon Intron Intron Introns degraded before leaving site of transcription Raj et al. Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods (2008) Thursday, April 11, 13
  • 9. iceFISH assay detects active genes on single chromosomes intron Export and Translation Spliced mRNA Exon Exon Exon Intron Intron Introns degraded before leaving site of transcription Thursday, April 11, 13
  • 10. Pseudocoloring provides 20+ unique tags to trace a chromosome Intron A B Chromosome 19 Intron A B Chromosome 19 5 base colors 5 base colors 2 colors 3 colors 2 colors 3 colors 20 pseudocolors 20 pseudocolors DAPI ATTO488 Cy3 C D E C DAPI D ATTO488 E F Alexa 594 G ATTO647N H ATTO700 F Thursday, April 11, 13 Alexa 594 G I Chr 19 J ATTO647N H ATT
  • 11. Pseudocoloring provides 20+ unique F G H Alexa 594 ATTO647N tags to trace a chromosome Intron A B Chromosome 19 I Chr 19 Intron J A B Chromosome 19 Chr 19 5 base colors 5 base colors 2 colors 3 colors 2 colors 3 colors 20 pseudocolors 4 z (µm) 20 pseudocolors DAPI ATTO488 Cy3 C D 2 E C DAPI D ATTO488 0 E 16 12 8 0 Mb Chromosome 19 59.13 Mb y (µm) 4 F Alexa 594 G ATTO647N H ATTO700 PTBP1 EEF2 SLC1A5 PPP2R1A RPS9 ZNF444 DNMT1 ILF3 UBA2 SUPT5H FBL EGLN2 RPS19 TOMM40 MARK4 DHPS RPL18A UBA52 ZNF91 EIF3K F Thursday, April 11, 13 Alexa 594 G I Chr 19 J ATTO647N H ATT
  • 12. iceFISH assigns gene activity to normal or derivative copies of chr19 A HeLa Karyotype B t(13;19) t(6;19) Chr 19 Chr 13 q-arm t(6;19) Fusion Chr 19 chromosomes Chr 19 t(13;19) Chr 6 p-arm q-arm Chr 19 Chr 19 q-arm Chr 6 0 Mb Chromosome 19 59.13 Mb p-arm PTBP1 EEF2 SUPT5H FBL EGLN2 RPS19 TOMM40 MARK4 SLC1A5 PPP2R1A RPS9 ZNF444 DNMT1 ILF3 DHPS RPL18A UBA52 ZNF91 UBA2 EIF3K C Chr 19 Chr 13 1 Translocation Centromere t(13;19) t(6;19) t(13;19) t(13;19) *** some Thursday, April 11, 13 0.8 N=50 N=30 ***
  • 13. p-arm q-arm Chr 19 Translocations can lead to whole chromosome increases in expression Chr 19 q-arm Chr 6 p-arm 0 Mb Chromosome 19 59.13 Mb PTBP1 EEF2 RPS19 TOMM40 MARK4 DNMT1 ILF3 UBA2 SUPT5H FBL EGLN2 SLC1A5 PPP2R1A RPS9 ZNF444 DHPS RPL18A UBA52 ZNF91 EIF3K C Chr 19 A HeLa Karyotype B Chr 13 1 Translocation Centromere t(13;19) t(13;19) t(6;19) t(6;19) t(13;19) t(13;19) Chr 19 *** iceFISH spots per chromosome Chr 13 q-arm t(6;19) 0.8 N=50 N=30 *** *** *** Fusion Chr 19 0.6 chromosomes Chr 19 Chr 6 ** t(13;19) *** ** *** *** p-arm q-arm 0.4 * Chr 19 ** 0.2 Chr 19 0 MTZ1 DIAPH3 RPL18A UBA52 ZNF91 UBA2 EIF3K SUPT5H FBL EGLN2 RPS19 TOMM40 MARK4 SLC1A5 PPP2R1A RPS9 ZNF444 q-arm Chr 6 Chr. 13 Chromosome 19 gene name 0 Mb Chromosome 19 59.13 Mb p-arm gene name PTBP1 EEF2 SUPT5H FBL EGLN2 RPS19 TOMM40 MARK4 DNMT1 ILF3 DHPS RPL18A UBA52 ZNF91 UBA2 SLC1A5 PPP2R1A RPS9 ZNF444 EIF3K C 1 N=30 t(6;19) Chr 19 0.8 Chr 13 Translocation Centromere t(13;19) t(6;19) t(13;19) t(13;19) 0.6 *** N=50 Thursday, April 11, 13 N=30 ***
  • 14. q-arm iceFISH spots per chromo Chr 19 *** Translocations do not necessarily***lead ** Chr 19 *** ** 0.6 to changes in transcriptional activity q-arm 0.4 * Chr 6 Chr 19 p-arm 0 Mb Chromosome 19 59.13 Mb q-arm Chr 6 0.2 PTBP1 EEF2 DNMT1 DNMT1 ILF3 DHPS RPL18A RPL18A UBA52 ZNF91 SUPT5H SUPT5H FBL EGLN2 EGLN2 RPS19 RPS19 TOMM40 TOMM40 MARK4 MARK4 UBA2 SLC1A5 SLC1A5 PPP2R1A RPS9 ZNF444 ZNF444 EIF3K 0 Mb Chromosome 19 59.13 Mb C p-arm PTBP1 EEF2 FBL ILF3 DHPS UBA52 ZNF91 UBA2 PPP2R1A RPS9 EIF3K 0 C MTZ1 DIAPH3 RPL18A Chr. 13 UBA52 ZNF91 Chr 19 EIF3K SUPT5H FBL EGLN2 RPS19 TOMM UBA2 Chromosome 19 gene name Chr 13 gene name 1 Translocation A Centromere HeLa Karyotype B Chr 19 t(13;19) Chr 13 t(6;19) t(13;19) t(13;19) *** iceFISH spots spots per chromosome t(13;19) 1 t(6;19) Translocation Centromere t(13;19) 1 0.8 N=50 N=30 N=30 Chr 19 t(6;19) *** t(13;19) t(13;19) Chr 13 t(6;19) *** *** t(6;19) iceFISH spots per chromosome *** iceFISH per chromosome q-arm 0.8 N=50 N=30 0.8 *** 0.6 *** Fusion *** 0.6 ** *** Chr 19 ** *** 0.6 chromosomes *** Chr 19 ** ** *** *** Chr 6 t(13;19) 0.4 ** * p-arm *** q-arm Chr 19 0.4 * 0.4 ** 0.2 0.2 0.2 0 Chr 19 MTZ1 DIAPH3 RPL18A UBA52 ZNF91 UBA2 EIF3K SUPT5H FBL EGLN2 RPS19 TOMM40 MARK4 SLC1A5 PPP2R1A RPS9 ZNF444 Chr. 13 q-arm Chromosome 19 gene name to Chr 6 centromere Chr 6 0 0 Mb 0 Chromosome 19 59.13 Mb gene name MTZ1 DIAPH3 RPL18A UBA52 ZNF91 UBA2 p-arm EIF3K SUPT5H FBL EGLN2 PTBP1 RPS19 TOMM40DNMT1 EEF2 ILF3 DHPS MARK4 SLC1A5 PPP2R1A RPS9 ZNF444 Chr. 13 Chromosome 19 gene name Chromosome 19 gene name PTBP1 EEF2 SUPT5H FBL EGLN2 RPS19 TOMM40 MARK4 DNMT1 ILF3 DHPS RPL18A UBA52 ZNF91 UBA2 SLC1A5 PPP2R1A RPS9 ZNF444 EIF3K 1 gene name C N=30 t(6;19) Chr 19 1 0.8 N=30 Chr 13 1 t(6;19) Translocation Centromere t(13;19) t(6;19) 0.8 t(13;19) t(13;19) *** osome Thursday, April 11, 13 0.6 0.8 N=50 N=30 ***
  • 15. Are chromosomes built to regulate their genes? EEF2 intron EEF2 mRNA Chr. 19 Thursday, April 11, 13
  • 16. Single chromosome transcription profiles unravel gene interactions Correlated Expression Intra-chromosomal (cis) interactions Nucleus Gene A Chr. 19 Gene B Anti-Correlated Expression PPP2R1A TOMM40 Intra-chromosomal (cis) interactions Inter-chromosomal (trans) interactions SUPT5H RPL18A SLC1A5 ZNF444 MARK4 DNMT1 EGLN2 PTBP1 UBA52 RPS19 ZNF91 EIF3K DHPS UBA2 RPS9 EEF2 ILF3 FBL Nucleus Nucleus PTBP1 Uncorrelated Expression Gene A Gene A EEF2 Chr. 19 Chr. 19 DNMT1 reskin fibroblasts ILF3 Gene B Gene B DHPS RPL18A UBA52 ZNF91 UBA2 EIF3K SUPT5H 1A 1A 40 40 Thursday, April 11, 13 H H A 5 A 5 4 4 4 4 1 1