BSci 3230 Past Paper PDF

NOTE ! These files are provided for the sole purpose of assisting BSci 3230 students to study for exams in the class. Some of the material in these files may be copyrighted, and it is not OK for you to share these files with anyone who is not a student in this class or to use them for any purpose ot...

NOTE ! These files are provided for the sole purpose of assisting BSci 3230 students to study for exams in the class. Some of the material in these files may be copyrighted, and it is not OK for you to share these files with anyone who is not a student in this class or to use them for any purpose other than to study for BSci 3230. Thanks, Carl Johnson  BSci 3230 Gene expression outputs of the mammalian clockwork The TTFL leads to pervasive circadian transcription via CLOCK/BMAL1 activation Methods to understand circadian transcription: DNA microarray, RNA-seq, ChIP “Transcriptional Landscapes” and what’s with that transcriptional delay?  What % of the genome is regulated by the clock in different species? This was the story as of ~2004, but now most of these estimates are higher %  The mammalian TTFL: transcriptional outputs (CCGs) Extracellular signals Nucleus Cytoplasm Cell membrane ROR REV-ERBa RORE Bmal1 Ca2+ ROR cAMP CLOCK BMAL1 CREB/MAPK Rors REV-ERBa signaling pathway E-box CLOCK BMAL1 E-box Rev-erba bTrCP P U P PER Repression P P CLOCK BMAL1 CK1e/d LRE E-box Per1/2 PER PER 26S proteasome CLOCK BMAL1 P P AMPK E-box Cry1/Cry2 CRY CRY P P P U P PER CRY P P CRY CK1e/d PER CRY FBXL3 Nuclear translocation CK1e/d CLOCK BMAL1 Clock output/rhythmic E-box CCG biological processes P Phosphate U Ubiquitin Figure 6.1. Model of the mammalian cell-autonomous oscillator as described in the text. Abbreviations: CCG, clock-controlled gene; P, phosphate; U, ubiquitin. “CCG” = clock-controlled gene Lowrey and Takahashi, Advances in Genetics 2011  Transcriptional Control of mRNAs Genome sequencing and DNA microchip arrays ushered in an era of “Circadian Genomics” DNA microarray technology of poly-A RNA: Hybridize to DNA chip With probes for many genes Extract and Label Total Quantify RNA at Temporal Different Patterns of CTs Gene Expression  Transcriptional Control of mRNAs In mice two tissues were initially sampled – SCN and liver Out of ~7000 genes assayed, 337 were rhythmic in SCN, and 335 were rhythmic in liver Genes were “phase specific” peak time of these binned data Panda et al 2002  Transcriptional Control of mRNAs Rhythmic genes were also tissue specific Out of more that 300 cycling genes in each tissue only 28 (including core clock genes) were rhythmic in both of peak expression Panda et al 2002  Transcriptional Control of RNAs (including non-coding RNAs) Zhang et al. 2014 RNA-seq Technique: used RNA-seq to quantify the transcriptomes of 12 mouse organs over time. RNA-seq benchwork RNA-seq data analysis  Transcriptional Control of RNAs (including non-coding RNAs) Zhang et al. used RNA-seq to quantify the transcriptomes of 12 mouse organs over time. They found 43% of all protein- coding genes showed circadian rhythms in transcription somewhere in the body, largely in an organ-specific manner. RNA-seq analyses: number of protein-coding genes in each organ with circadian expression. Blue marks indicate the number of genes with at least one spliceform detected by RNA-seq. Orange marks indicate the number of genes with at least two spliceforms detected by RNA-seq. Blue numbers to the right of each bar list the percentage of protein-coding genes with rhythmic expression in each organ. Percentages of each transcript class that did or did not oscillate in at least one organ. Zhang et al. PNAS 2014  Transcriptional Control of RNAs (including non-coding RNAs) Relationships among organ, oscillation amplitude, and oscillation phase of circadian genes across organs. Above = histograms of amplitudes within each organ (number of circadian genes within each amplitude bin is shown on the horizontal axis, grouped by organ). Right = histograms of phases within each organ, with summary radial diagrams (number of circadian genes within each phase bin is shown on the vertical axis, grouped by organ). Center = Venn diagrams of the identities of the genes that oscillated within a given pair of organs. Zhang et al. PNAS 2014 Transcriptional Control of Proteins (TFs?) bound to DNA Chromatin Immunoprecipitation (ChIP) to BMAL1 assay temporal changes in transcription factor (TF) binding and chromatin dynamics: Extract chromatin from tissues at different phases BMAL1 and chemically crosslink DNA to proteins that are touching DNA Fragment DNA via sonication Ab Immunoprecipitate using various antibodies BMAL1 (Abs) against the transcription factor of interest (e.g., BMAL1, CLOCK) De-crosslink and extract DNA Ab BMAL1 Identify the DNA sequences and quantify the amount of bound DNA by high-throughput sequencing Koike et al. Science 2012 (mouse study) Transcriptional Control of Proteins (TFs?) bound to DNA Chromatin Immunoprecipitation (ChIP) to BMAL1 assay temporal changes in transcription factor (TF) binding and chromatin dynamics. TFs often form complexes on gene BMAL1 promoters and enhancers, for example: Ab BMAL1 Ab BMAL1 Koike et al. Science 2012 (mouse study)  Transcriptional Control of Proteins (TFs?) bound to DNA RESEARCH ARTICLE ChIP-seq analysis of the binding of 7 core circadian transcriptional regulators in mouse liver to the promoter and first two introns of the Dbp gene (a strongly rhythmic gene in the liver). Each track represents the normalized ChIP-seq read coverage at a single time point. For each circadian factor, six time points every 4 hours over a 24-h cycle exons are shown beginning at CT0 and ending at CT20. promoter of Dbp gene introns Koike et al. Science 2012 (mouse study)  Transcriptional Control of Proteins (TFs?) bound to DNA RESEARCH ARTICLE Circadian Time exons Promoter of Dbp gene introns Koike et al. Science 2012 (mouse study) Transcriptional Control of Proteins (TFs?) bound to DNA Rhythmic recruitment of circadian transcription factors to clock-controlled genes: Role of CRY1 and CRY2 may not be redundant in function because differently phased Koike et al. Science 2012 (mouse study)  Transcriptional Control of Proteins (TFs?) bound to DNA Analysis of intron- and exon-specific cycling mRNAs shows that circadian regulation of gene expression can occur at both transcriptional and post- transcriptional* levels (only 22% of cycling mRNAs are driven by transcription). Intron-specific mRNAs are an indicator of newly synthesized transcripts (therefore of transcription rate), whereas exon-specific mRNAs are an indicator of steady-state abundance CT 15 is the main (*post-transcriptional circadian regulation could be peak of transcription mediated by circadian changes in RNA splicing, polyadenylation, mRNA stability, etc.) in the liver Koike et al. Science 2012 (mouse study) Transcriptional Control of Proteins (TFs?) bound to DNA Circadian transcriptional “landscape:” phase distributions of clock transcription factor binding to promoters, intron cycling RNA transcripts, and histone modifications. RESEARCH ARTICLE Poised Derep. Activation Transcription Repression 26. D. Feng, M. A. Lazar, Mol. Cell 47, 1 27. F. Hatanaka et al., Mol. Cell. Biol. 30 30 28. G. Rey et al., PLoS Biol. 9, e1000595 CLOCK:BMAL1 CLOCK:BMAL1 Nascent Repression Phase 29. D. Feng et al., Science 331, 1315 (2 CRY1 repressed Activation Phase Transcription 30. H. Cho et al., Nature 485, 123 (201 RNAPII-Ser5P p300 Recruitment 31. A. Bugge et al., Genes Dev. 26, 657 Poised State H3K9 acetylation 32. W. J. Kent et al., Genome Res. 12, 9 33. Y. Zhang et al., Genome Biol. 9, R13 PER1 34. M. Salmon-Divon, H. Dvinge, K. Tamm 15.9 Derepression BMC Bioinformatics 11, 415 (2010). 35. S. Panda et al., Cell 109, 307 (2002 20 delay? RNAPII PER2 36. E. E. Zhang et al., Cell 139, 199 (20 8WG16 17.3 37. I. Schmutz, J. A. Ripperger, S. Baerisw U. Albrecht, Genes Dev. 24, 345 (20 % of peaks 14.5 BMAL1 NPAS2 38. K. A. Lamia et al., Nature 480, 552 8.1 CRY2 6.1 39. C. Kramer, J. J. Loros, J. C. Dunlap, S 15.4 Nature 421, 948 (2003). 40. A. Ameur et al., Nat. Struct. Mol. Bio CBP RNAPII H3K9ac 20.1 41. K. F. Storch et al., Nature 417, 78 (2 Ser5P p300 6.6 RNA 42. H. R. Ueda et al., Nature 418, 534 ( K36me3 5.0 CLOCK (intron) M. E. Hughes et al., PLoS Genet. 5, e 10 0.6 CRY1 7.3 15.1 H3K4me3 19.8 43. 0.4 17.8 44. J. Wuarin et al., J. Cell Sci. Suppl. 16 45. F. Gachon, F. F. Olela, O. Schaad, P. U. Schibler, Cell Metab. 4, 25 (2006 H3K27ac 46. D. Staiger, T. Köster, Cell. Mol. Life S H3K4me1 17.1 K79me2 47. S. Kojima, D. L. Shingle, C. B. Green, 5.8 22.4 (2011). CBP 48. R. J. Sims 3rd, R. Belotserkovskaya, D. R 18, 2437 (2004). 0 49. N. J. Fuda, M. B. Ardehali, J. T. Lis, Natu 50. J. C. Jones et al., J. Biol. Chem. 279, 0 4 8 12 16 20 24 51. R. D. Chapman et al., Science 318, 1 Circadian Time (hr) 52. A. Barski et al., Cell 129, 823 (2007 53. C. T. Ong, V. G. Corces, Nat. Rev. Gen Fig. 5. Circadian landscape of the cistrome and epigenome of theKoike liver. Phaseet al. Science distributions 2012 of circadian 54. (mouse study) M. P. Creyghton et al., Proc. Natl. Ac transcriptional regulators, intron cycling RNA transcripts, and histone modifications as shown in Figs. 21931 (2010).  Transcriptional Control of Proteins (TFs?) bound to DNA A complementary, but different perspective: repressed transcription allowed repressed CRY2 CRY1 alone displays a “blocking” type on-DNA repression around ZT0 PER2 in combination with CRY2 leads to a “displacement” type repression around ZT20 Ye et al. (Genes & Dev. 2014) mouse study  in nucleosome A simple model to explain signal and no the disconnect rhythmicity, between the Theindicating that was surprisingly amplitude of binding (Feng et highal. (greater 2011; Bugg The of uniformCLOCK:BMAL1 phase H2A.Z signal nucleosome is removal directly strongly at CLOCK: contributes rhythmic tothan at nucleosomeCLOCK: fourfold for many re- sites), suggesting In that contrast HNF6 to af- CLOCK CLOCK:BMAL1 is a pioneer transcription factor whose binding BMAL1BMAL1 sites and the moval (Fig. heterogeneous phases DNA-binding 1A; sites:ofhigh Supplemental transcrip- Fig.from tion is that other transcription factors bind to the open2). fects CLOCK:BMAL1 ZT02 Consistentto ZT14 with andtranscriptional output. Importantly this rhythmic binding does not occur REV-ERBa sites at control sites; e.g.,had in then decreasing during the night to reach a trough at then facilitates binding of other transcriptionaffected chromatin at these sites. To address this possibility, we ZT2231(Fig. first analyzed 3A). published previously Importantly, factorsin Bmal1 ChIP-seqincorporation mouse of Assuming (Fig. 4D). H2A.Z that nals at all time HNF6 sites without nearby CLOCK:BMAL1-binding sitespoints. is the activity of other transcription!/! liv severely compromised in Bmal1!/! mice, and levels do transcription and expre not exceed the trough levels observed in wild-type mice (Fig. 1C; Supplemental at ZT22 (Fig. 3A; Supplemental Fig. 8). Furthermore, Menet et al. 2012). The there is a striking correlation between H2A.Z signal and mic transcription facto CLOCK:BMAL1-mediated decrease First: CLK:BMAL1 binds signal in nucleosome in signal at TSSs 1st and suggest that CLOC (Fig. 3B). In Bmal1!/! mice, atohigher DNA motfis H2A.Z daytime, displacing tion is special. is associated with a stronger decrease in nucleosome nucleosomes & causing Interestingly, CLOCK signal compared with intergenic regions or gene bodies lower nucleosome removal is more pron (Figs. 1F, 3B). Conversely, a signal higher at amplitude site of H2A.Z intergenic regions than signal occurs in wild-type mice within intergenic regions and gene bodies, which is associated with a stronger effect (Fig. 1D–F; Supplement observation are the nu liver chromatin, which genomic location of the B depleted of nucleosome whereas nucleosome de genic regions is much le The low TSS nucleosom CLOCK:BMAL1 bindin Binding of and ZT22 as well as in B histone variant Figure 4. CLOCK:BMAL1-mediated the literature; i.e., TSSs rhythmic nucleosome removal promotes the rhythmic binding of transcription factors to DNA. (A) H2A.Z factors Coassociation between transcription to depleted in mouse livers. Thirty-one publicly available mouse liver ChIP-seq data because sets were analyzed by pairs of int using the Genome Structure transcription start Correction statistic as previously described (Dunham et al. 2012). Black rectangles denote core clock genes and nuclear receptors (see the textsite for(TSS) theChIP-seq more details). (B) Percentage of overlap between 31 publicly available mouse liver presence of the t data sets. Black etmore rectangles denote transcription factors that exhibit an overlap superior to 40% with core clock genes (see the text for al.details). 2012; nucleosome signal at a CLOCK:BMAL1 DNA-binding site located near HNF6 DNA-binding sites. Nucleosome signal is displayed for wild-type Iyer 2012; T (C) Rhythmic. (A) mice at time of high (average ZT6 and ZT10; green) or low (average ZT18 Menet et al. and ZT22; red)Genes Dev. DNA CLOCK:BMAL1 2014 (Drosophila nucleosome binding. study) a removal The HNF6 ChIP-seq signal from Faure et al. (2012) is shown in gray. Genomic locations of CLOCK:BMAL1 (blue) and HNF6 (black) consensus sequences are also iver displayed. (D) HNF6 ChIP-seq signal in mouse livers at ZT08 (white) and ZT20 (black) at several specific DNA-binding intergenic regions) sites (n = 4 mice per time is m  in nucleosome A simple model to explain signal and no the disconnect rhythmicity, between the Theindicating that was surprisingly amplitude of binding (Feng et highal. (greater 2011; Bugg The of uniformCLOCK:BMAL1 phase H2A.Z signal nucleosome is removal directly strongly at CLOCK: contributes rhythmic tothan at nucleosomeCLOCK: fourfold for many re- sites), suggesting In that contrast HNF6 to af- CLOCK CLOCK:BMAL1 is a pioneer transcription factor whose binding BMAL1BMAL1 sites and the moval (Fig. heterogeneous phases DNA-binding 1A; sites:ofhigh Supplemental transcrip- Fig.from tion is that other transcription factors bind to the open2). fects CLOCK:BMAL1 ZT02 Consistentto ZT14 with andtranscriptional output. Importantly this rhythmic binding does not occur REV-ERBa sites at control sites; e.g.,had in then decreasing during the night to reach a trough at then facilitates binding of other transcriptionaffected chromatin at these sites. To address this possibility, we ZT2231(Fig. first analyzed 3A). published previously Importantly, factorsin Bmal1 ChIP-seqincorporation mouse of Assuming (Fig. 4D). H2A.Z that nals at all time HNF6 sites without nearby CLOCK:BMAL1-binding sitespoints. is the activity of other transcription!/! liv severely compromised in Bmal1!/! mice, and levels do transcription and expre not exceed the trough levels observed in wild-type mice (Fig. 1C; Supplemental at ZT22 (Fig. 3A; Supplemental Fig. 8). Furthermore, Menet et al. 2012). The there is a striking correlation between H2A.Z signal and mic transcription facto CLOCK:BMAL1-mediated decrease First: CLK:BMAL1 in nucleosome signal and suggest that CLOC (Fig. 3B). In Bmal1!/! mice, abinds higherto DNA H2A.Zmotfis in signal at TSSs 1st daytime, displacing tion is special. is associated with a stronger decrease in nucleosome nucleosomes & causing Interestingly, CLOCK signal compared with intergenic regions or gene bodies lower nucleosome removal is more pron (Figs. 1F, 3B). Conversely, a signal higher amplitude of H2A.Z2nd at site intergenic regions than signal occurs in wild-type mice within intergenic regions and gene bodies, which is associated with a stronger effect (Fig. 1D–F; Supplement observation are the nu liver chromatin, which genomic location of the B Second: CLK:BMAL1 binding depleted of nucleosome associated with histone whereas nucleosome de variant H2A.Z*(orange nucleosomes labeled with genic regions is much le stars) occupancy at TSS The low TSS nucleosom decreases nucleosome CLOCK:BMAL1 bindin Binding of density and facilitates and ZT22 as well as in B histone variant subsequent binding of later the literature; i.e., TSSs Figure 4. CLOCK:BMAL1-mediated rhythmic nucleosome removal promotes the rhythmic binding of transcription factors to DNA. (A) H2A.Z factors Coassociation between transcription to in mouseTFs (e.g., livers. “X”)publicly available mouse liver ChIP-seq data Thirty-one depleted because sets were analyzed by pairs of int transcription start using the Genome Structure Correction statistic as previously described (Dunham et nuclear receptors (see the textsite for(TSS) *H2A.Z establishes a the al. 2012). Black rectangles chromatin more details). (B) Percentage of overlap between 31 publicly available mouse liver denote core ChIP-seq environment clock presence genes and of the t data sets. Black that rectangles denote transcription factors that exhibit an overlap superior to 40% with core enhances clock RNA genes (see the textpolymerase etmore for II(C) al.details). 2012; recruitment Iyer 2012; T Rhythmic nucleosome signal at a CLOCK:BMAL1 DNA-binding site located near HNF6 DNA-binding sites. Nucleosome signal is displayed for wild-type. (A) mice at time of high (average ZT6 and ZT10; green) or low (average ZT18 Menet et al. and ZT22; red)Genes Dev. DNA CLOCK:BMAL1 2014 (Drosophila nucleosome binding. study) a removal The HNF6 ChIP-seq signal from Faure et al. (2012) is shown in gray. Genomic locations of CLOCK:BMAL1 (blue) and HNF6 (black) consensus sequences are also iver displayed. (D) HNF6 ChIP-seq signal in mouse livers at ZT08 (white) and ZT20 (black) at several specific DNA-binding intergenic regions) sites (n = 4 mice per time is m  in nucleosome A simple model to explain signal and no the disconnect rhythmicity, between the Theindicating that was surprisingly amplitude of binding (Feng et highal. (greater 2011; Bugg The of uniformCLOCK:BMAL1 phase H2A.Z signal nucleosome is removal directly strongly at CLOCK: contributes rhythmic tothan at nucleosomeCLOCK: fourfold for many re- sites), suggesting In that contrast HNF6 to af- CLOCK BMAL1BMAL1 CLOCK:BMAL1 is a pioneer transcription factor whose binding sites and the moval (Fig. heterogeneous phases DNA-binding 1A; sites:ofhigh Supplemental transcrip- Fig.from tion is that other transcription factors bind to the open2). fects CLOCK:BMAL1 ZT02 Consistentto ZT14 with andtranscriptional output. Importantly this rhythmic binding does not occur REV-ERBa sites at control sites; e.g.,had in then decreasing during the night to reach a trough at first analyzedthen facilitates binding of other transcriptionaffected chromatin at these sites. To address this possibility, we ZT2231(Fig. 3A). published previously Importantly, ChIP-seqincorporation mouse factorsin Bmal1 of Assuming (Fig. 4D). H2A.Z that nals at all time HNF6 sites without nearby CLOCK:BMAL1-binding sitespoints. is the activity of other transcription!/! liv severely compromised in Bmal1!/! mice, and levels do transcription and expre not exceed the trough levels observed in wild-type mice (Fig. 1C; Supplemental at ZT22 (Fig. 3A; Supplemental Fig. 8). Furthermore, Menet et al. 2012). The there is a striking correlation between H2A.Z signal and mic transcription facto CLOCK:BMAL1-mediated decrease First: CLK:BMAL1 in nucleosome signal and suggest that CLOC (Fig. 3B). In Bmal1!/! mice, abinds higherto DNA H2A.Zmotfis in signal at TSSs 1st daytime, displacing tion is special. is associated with a stronger decrease in nucleosome nucleosomes & causing Interestingly, CLOCK signal compared with intergenic regions or gene bodies lower nucleosome removal is more pron (Figs. 1F, 3B). Conversely, a signal higher amplitude of H2A.Z2nd at site intergenic regions than signal occurs in wild-type mice within intergenic regions and gene bodies, which is associated with a stronger effect (Fig. 1D–F; Supplement observation are the nu 3rdliver chromatin, which genomic location of the B Second: CLK:BMAL1 depleted of nucleosome binding associated with whereas nucleosome de histone variant H2A.Z occupancy at TSS genic regions is much le decreases nucleosome The low TSS nucleosom density and facilitates CLOCK:BMAL1 bindin Binding of subsequent binding of and ZT22 as well as in B histone variant later TFs the literature; i.e., TSSs Figure 4. CLOCK:BMAL1-mediated rhythmic nucleosome removal promotes the rhythmic binding of transcription factors to DNA. (A) H2A.Z factors Coassociation between transcription to Third: in mouse livers. Thirty-one CLK:BMAL1 publicly available mousebinding opens liver ChIP-seq depleted data upwere sets because theanalyzed chromatinby pairs of int using the Genome Structure transcription start Correction statistic as previously described (Dunham et al. 2012). Black rectangles denote core clock genes and nuclear receptors (see the textsite for(TSS) for other TFs (X,Y,Z) – directly and more details). (B) Percentage of overlap between 31 publicly available mouse liver indirectly theChIP-seq presence regulates of the t data sets. Black rectangles denote transcription factors that exhibit an overlap superiorexpression to 40% with core(tissue specific, clock genes different (see the text for al.phases) etmore 2012; details). Iyer 2012; T (C) Rhythmic nucleosome signal at a CLOCK:BMAL1 DNA-binding site located near HNF6 DNA-binding sites. Nucleosome signal is displayed for wild-type. (A) mice at time of high (average ZT6 and ZT10; green) or low (average ZT18 Menet et al. and ZT22; red)Genes Dev. DNA CLOCK:BMAL1 2014 (Drosophila nucleosome binding. study) a removal The HNF6 ChIP-seq signal from Faure et al. (2012) is shown in gray. Genomic locations of CLOCK:BMAL1 (blue) and HNF6 (black) consensus sequences are also iver displayed. (D) HNF6 ChIP-seq signal in mouse livers at ZT08 (white) and ZT20 (black) at several specific DNA-binding intergenic regions) sites (n = 4 mice per time is m Gene Ontology: KEGG Metabolic Pathways Slide courtesy of Dr. Joseph Takahashi Gene Ontology: BMAL1-regulated KEGG Metabolic Pathways Slide courtesy of Dr. Joseph Takahashi  Food for Thought If gene regulation by the CLOCK/BMAL1 heterodimeric transcriptional factor is so pervasive, what are the implications? Will a Bmal1-knockout animal be sick? What about a Cry1/2- double knockout animal? And if sick, does this mean that the clock disruption is the reason why the animal is sick? How would you test if clock control of a particular transcriptional cascade is important? What are alternative explanations for the “delay” between the phase of maximal CLOCK/BMAL1 binding to the regulatory elements of a gene and the initiation of its transcription ?