7_11 Genome Organisation and Function (3).pdf

Genome Organisation and Function The Genome: How does it all fit? • different distinct environments of density (different amounts of condensed DNA) • Nucleolus - cell biology (rRNA) Physical compaction of genomes: • each chromosomes has its own ‘space or neighbourhood’ that it resides in - but can int...

Genome Organisation and Function The Genome: How does it all fit? • different distinct environments of density (different amounts of condensed DNA) • Nucleolus - cell biology (rRNA) Physical compaction of genomes: • each chromosomes has its own ‘space or neighbourhood’ that it resides in - but can interact with others too • evolutionarily controlled process - packaging of DNA into a particular space • one chromosome can have different levels of compaction within it (eg one area is euchromatin and one is heterochromatin) Lessons from microscopy: properties of nuclear architecture • interphase chromosomes occupy discrete territories • different functions occur in discrete locations (eg chromosomes towards the outside of the nucleus are generally not genetically active, whereas towards the middle it’s the opposite) Genome organisation and function: • how are genes expressed at the right time and place during development ? • gene regulation: - transcription factors - epigenetic (methylation, histone modification, chromatin structure/loops) Long-range gene regulation: • enhancers can be brought into close proximity physically so that expression can be driven which wasn’t close to the gene before (located far away from their target gene, up to 1Mb) = accessible enhancer • insulated enhancer is when the movement prevents the gene interacting with the enhancer (suppressing transcription) • chromatin loops • how do they know which gene to regulate? another level of chromosome structure ‘restricts’ their reach • enhancers can be involved with multiple promoters both simultaneously and sequentially Biological importance of TADs/structure: Functions of TADs: • DNA replication • recombination • gene regulation Alterations of TADs: • developmental degects • congenital disease • cancer Cohesin, CTCF, regulators of chromosome structure: Cohesin: • ring-like protein that holds sister chromatids together until ready for replication (S/G2 phase) • also repurposed in G1 phase to hold the bottom of loops CTCF: • has multiple zinc fingers - recognises 11 nucleotides • only when the CTCF molecules are facing a particular way and sitting on the right place, is where the cohesin ring can hold (convergent motifs) Information that tells your genome which way to fold The methylation of a CTCF site dictates if the CTCF can bind and if cohesion can join UCL Cancer Institute Faculty of Medical Sciences Genome Organisation & Function Sam Weeks [email protected] MSc Cancer 2 Part 1 Loops within Loops Learning Outcomes Following this topic, students should be able to: • Appreciate the layers of chromatin organisation. • Link DNA looping and insulation to gene regulation. • Recall the key factors and their functions involved in DNA looping. 3 The Genome: How does it all fit? • The Genome: • • • 2.05m (male) • Majority of time, genome is decondensed 6469.66 Mb (diploid state) How does this all fit within a ~10um diameter nucleus? 4 5 3D Hierarchical Structuring Ø The genome is organised from 250 megabased-sized chromosome territories, down to 150bp-sized nucleosomes. Adapted from Doğan and Liu, 2018 Ø Each hierarchy relies on a range of structuring mechanisms. Cohesin CTCF TF Ø All levels of genome structures are associated with influences on transcription by affecting accessibility to gene promoters. Ø A compartments = Euchromatin, Ø B compartments = Heterochromatin. Ø Genome structuring is dynamic between cell types (and within individual cells). Cohesin CTCF TF Covalent DNA Modifications Chromatin Remodelling Enzymes 6 Topologically Associated Domains (TADs) Ø Chromatin loops bring distant regions of chromatin into close 3D space, creating domains of topological association of high interaction frequency. Ø TADs can be measured through Chromatin Conformation Capture (3C) techniques. Ø Chromatin contacts can be measured at different hierarchies. Szabo et al. 2019 Bonev et al. 2017 Lieberman-Aiden et al. 2009 An Epigenetic Regulator of Gene Expression Interaction frequency between regions of DNA can influence how likely a Cis-acting Regulatory Element + Trans-acting Regulatory Element can have on a gene promoter. Distal on Chromatin Enhancer Promoter CTCF CTCF Cohesin at CTCF-anchored stems (TAD Boarders) er E n h an c Proximal in 3D Space TSS TSS er E n h an c Genome Organisation TF Transcription Factors localising Cohesin (Within TADs) 7 Dynamic Topology & Gene Expression Changes over differentiation Majority of TADs are conserved between cell types, however interactions within TADs (intraTAD loops) are more variant. What are the TREs and CREs that regulate this? 8 9 CTCF: A TRE insulator protein CTCFmediated insulation Ø Act as a chromatin barrier between TADs, creating insulation to TAD contacts. Ø ‘CTCF Code’: x11 Zinc Finger protein gives multivalence to CTCF’s recognition sequence, which fine-tunes its insulator ‘strength’. Ø Zinc Fingers 4-7 cluster together to bind to conserved insulator (CRE) consensus sequence CCGCGNGGNGGCAG. Ø Methylation of CpG islands protects insulator CREs from CTCF binding. 10 Cohesin as a regulator for dynamic topology Smc1a Sister Chromatid Cohesion Smc3 Double Stand Break Repair Sister)Chroa'd) Cohesion) Rad21 c.) Stag Ø Highly conserved multi-protein complex. Ø Topologically embraces DNA- has no sequence specificity. Ø Relies on a range of accessory proteins to load, stabilise, and unload it from chromatin. Ø Able to interact with DNA in trans and cis, giving it versatility in functions. a.) Chroma'n)loop)) Forma'on! Cohesin)Cycle) Genome Organisation b.) Double)Strand)DNA)) Break)Repair)) DNA Replication DNA)Replica'on! Replication Complex Replica'on)) Complex) Figure)1:)Roles&of&Cohesin&during&the&Cell&Cycle.!a.! During!the!G1!phase,!cohesin!associates!with! single! stranded! chroma8n! to! form! chroma8n! loop! structures! that! are! integral! for! genome! organisa8on! and! cell:specific! gene! expression.! b.) In! S:phase,! cohesin! associates! at! replica8on! Summary • Hierarchical genome organisation is essential for compacting the genome and regulating gene accessibility to transcription machinery. • DNA looping influences contact frequencies between distal regions of chromatin. • Looping is versatile between cell fates. • Cohesin and CTCF anchor DNA loops. 11 UCL Cancer Institute Faculty of Medical Sciences Genome Organisation & Function Sam Weeks [email protected] MSc Cancer 2 Part 2 Functional Experiments Learning Outcomes Following this topic, students should be able to: • Describe techniques used to validate cohesin and CTCF roles. • Understand the limitations of whole-population experiments when looking at genome organisation. 3 Cohesin&CTCF in Genome Organisation Smc1a Smc3 Distal on Chromatin Stag Enhancer Promoter CTCF CTCF TSS TSS Cohesin at CTCF-anchored stems (TAD Boarders) Proximal in 3D Space er E n h an c Genome Organisation er E n h an c Rad21 TF Transcription Factors localising Cohesin (Within TADs) 4 CTCF and Cohesin Co-localise at TAD boarders 5 Ø ChIP-Seq used in conjunction with Chromatin Conformation Capture techniques highlight cohesinCTCF co-localise at TAD boundaries. Ø CTCF CTD associates with the Stag protein of cohesin. Ø Looping forms between convergent CTCF sites, thus regulating structure specificity. CTCF CTCF CCGCGNGGNGGCAG GACGGNGGNGCGCC De Wit et al, 2015 Pezic & Varsally, unpublished 6 Static and Transient loops Ø CTCF-independent contacts within TADs (intra-TAD DNA loops) are more variable between cell types. Ø Cohesin able to associate with Mediator complex and cell-specific Transcription Factors to influence specific gene expression. Kagey et al. 2010 Bonev et al. 2017 Lieberman-Aiden et al. 2009 How is Cohesin loaded? 7 Ø Population-based Chromatin Conformation Capture techniques create an average of most ‘stable’ domain structure. Ø However, cohesin association/dissociation with chromatin is far more dynamic than this would suggest. Ø Chromatin residence time of 20mins. Ø Nipbl does not localise at CTCF sites, suggesting cohesin must travel on chromatin to reach CTCF. Ø Chromatin loop extrusion model suggests cohesin is loaded onto chromatin and translocate along it until they reach a boundary element eg. CTCF. If TADs are so dynamic, do they actually do anything? Experimental Evidence for Genome Structuring Loss of insulation between TADs Loss of insulation between TADs Cohesin and CTCF knock-down cause loss of intra-TAD contact frequency on the TAD level, but A/B compartments remain unaffected. Model for continuous loading and dissociation of cohesin supports theory of ‘stable’ TAD structures. 8 Experimental Evidence for Fate Determination WT 9 Scrambled Stag1 siRNA SP siRNA Stag1 Stag2 Nanog Tubulin P value = <0.05 D.Pezic and W.Varsally, unpublished Stag1 promotes pro-pluripotency in ESCs Stag2 is sensitive to Stag1 expression Is this a direct result of regulating gene expression, or does Cohesin/Stag determine cell fate in another way? Summary • Cohesin is suggested to be loaded onto chromatin via loop extrusion. • Various forms of experimental techniques/modeling support the link between genome organisation and gene expression and regulation of this is in part performed by CTCF and Cohesin. • BUT we don’t know everything… 10 UCL Cancer Institute Faculty of Medical Sciences Genome Organisation & Function Sam Weeks [email protected] MSc Cancer 2 Part 3 Cohesinopathies & Cancer Learning Outcomes Following this topic, students should be able to: • Define cohesinopathies. • Recall the prevalence of cohesin mutations in pan-cancers. • Explain the complexity of assigning a molecular pathology of cohesin mutations in pan-cancers. • Describe ways in which cohesin mutations can act as a therapeutic target. 3 Coheisn in Disease 4 Ø Cohesinopathies: Collection of cohesin-related diseases that primarily manifest as developmental disorders. Ø Eg. Cornelia de Lange Syndrome: sporadic mutation of Nipbl causes wide-ranging (but low level) gene expression changes. Although not generally classed as cohesin-specific diseases, cohesin abberrations are prevalent in a wide range of disease, such as Downs Syndrome and infertility. 5 Cohesin mutations in Pan-Cancers Ø Cohesin mutations are widespread in cancers, but are almost always mutually exclusive of each other. Coheisn-Stag1 Mitotic Cells Ø X-linked STAG2 is one of only 12 genes frequently mutated in 4 or more human cancer types. Coheisn-Stag2 Meiotic Cells Coheisn-Stag3 Ø Mutations of the core cohesin ring are almost always heterozygote. Stag mutations in Tumour Heterogeneity Ø Lack of clonal heterogeneity for Stag mutations suggests it is an early hit/trunk mutation in pan-cancers. Ø Stag2 mutations in Down Syndrome can be seen as a ‘pre-leukemic’ state. 6 What is the Molecular Pathology of Cohesin in Cancer? Cohesin performs so many integral parts of genome stability, which role is promoting pathogenesis? Sister Chromatid Cohesion Double Stand Break Repair Mutations of the cohesin complex may cause loss of expression, loss of function or have a dominant negative effect. Sister)Chroa'd) Cohesion) c.) a.) Chroma'n)loop)) Forma'on! Cohesin)Cycle) Genome Organisation b.) Double)Strand)DNA)) Break)Repair)) DNA Replication Replication Complex DNA)Replica'on! Replica'on)) Complex) Figure)1:)Roles&of&Cohesin&during&the&Cell&Cycle.!a.! During!the!G1!phase,!cohesin!associates!with! single! stranded! chroma8n! to! form! chroma8n! loop! structures! that! are! integral! for! genome! 7 Kim et al. 2016 8 Cohesin mutation and Aneuploidy Hypothesis: Cohesin mutation leads to premature sister chromatin separation, causing aneuploidy and chromosome instability. A promising start… But, looking wider at mutant cohesin in pan-cancers, few display abnormal chromosome number. Kim et al. 2016 Solomon et al. 2011 How are cells able to survive at all without any STAG2? 9 STAG1 partially compensates for STAG2 loss Ø Stag1 and Stag2 are paralogs of eachother, sharing >75% sequence homology. 70% Homology N-term AT-Hook ‘KRKRGRP’ SA SA SCD SCD Stag1 Stag2 C-term Ser/Thr NLS Kim et al. 2016 Cohesin Mutation and Gene Expression 10 Hypothesis: STAG mutation leads to deregulation of 3D chromatin contacts, causing aberrant gene expression. Minimal change is observed between TAD boarders But, lack of STAG2 at specific developmental genes cannot be compensated by STAG1 in HSPCs, causing significant gene expression change. Cuadrado et al. 2019 Little/inconsistent change in global gene expression Viny et al. 2019 Casa et al. 2019 Are the generally modest expression changes a direct result of STAG2 loss? 11 Cohesin Mutation and Cell Fate Hypothesis: Cohesin mutation deregulates cell identity through an as yet undefined mechanism and gene expression changes are an indirect consequence of this. STAG2 KO in HSPCs promotes stemness STAG levels appear important for ES cell fate changes; this may be due to STAG1/2 interaction with cell fate regulators Epiblast mESC 24h 48h Stag1 Stag2 Smc3 Actin Viny et al. 2019 Stag1 Weeks et al. Unpublished Oncogenic Addiction to STAG1 as a Therapeutic Target for STAG2 Cancers DNA Replication 12 Synthetic Lethality: Aberration of Stag1 function in cells already Stag2 deficient promotes cell death Bladder Cancer Cell lines Coheisn-STAG1 Coheisn-STAG2 Faithful Sister Chromatid Cohesion Oncogenic Addiction: DNA Replication Coheisn-STAG1 Mitotic Catastrophe Van der Lelij et al. 2017 Summary 13 • Cohesinopathies are diseases comprised of cohesin-related aberrations, but cohesin mutations are also prevalent in a range of disease. • The multi-functionality of cohesin makes assigning a pathology to these diseases complex. There are potentially many mechanisms of action that are tissue/gene expression profile-specific. • Non-redundancy of STAG proteins leave STAG2 pan-cancers vulnerable to synthetic lethality. Further Reading Cohesion and cohesin-dependent chromatin organization https://doi.org/10.1016/j.ceb.2018.11.006 More to cohesin than meets the eye: complex diversity for fine-tuning of function https://doi.org/10.1016/j.gde.2017.01.004 Emerging themes in cohesin cancer biology https://doi.org/10.1016/j.ceb.2018.11.006 14