Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers

Two dominant processes organizing chromosomes are loop extrusion and the compartmental segregation of active and inactive chromatin. The molecular players involved in loop extrusion during interphase, cohesin and CTCF, have been extensively studied and experimentally validated. However, neither the molecular determinants nor the functional roles of compartmentalization are well understood. Here, we distinguish three inactive chromatin states using contact frequency profiling, comprising two types of heterochromatin and a previously uncharacterized inactive state exhibiting a neutral interaction preference. We find that heterochromatin marked by long continuous stretches of H3K9me3, HP1α and HP1β correlates with a conserved signature of strong compartmentalization and is abundant in HCT116 colon cancer cells. We demonstrate that disruption of DNA methyltransferase activity dramatically remodels genome compartmentalization as a consequence of the loss of H3K9me3 and HP1 binding. Interestingly, H3K9me3-HP1α/β is replaced by the neutral inactive state and retains late replication timing. Furthermore, we show that H3K9me3-HP1α/β heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF, explaining a paucity of visible loop extrusion-associated patterns in Hi-C. Accordingly, CTCF loop extrusion barriers are reactivated upon loss of H3K9me3-HP1α/β, not as a result of canonical demethylation of the CTCF binding motif but due to an intrinsic resistance of H3K9me3-HP1α/β heterochromatin to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the inactive portion of the genome and establishes new connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion.

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Capturing the complexity of topologically associating domains through multi-feature optimization

10.1101/2021.01.04.425264 ◽

2021 ◽

Author(s):

Natalie Sauerwald ◽

Carl Kingsford

Keyword(s):

Binding Sites ◽

Three Dimensional ◽

Replication Timing ◽

Biological Properties ◽

Ctcf Binding ◽

Dimensional Structure ◽

Biological Targets ◽

Topologically Associating Domains ◽

Contact Frequency ◽

Algorithmic Framework

AbstractThe three-dimensional structure of human chromosomes is tied to gene regulation and replication timing, but there is still a lack of consensus on the computational and biological definitions for chromosomal substructures such as topologically associating domains (TADs). TADs are described and identified by various computational properties leading to different TAD sets with varying compatibility with biological properties such as boundary occupancy of structural proteins. We unify many of these computational and biological targets into one algorithmic framework that jointly maximizes several computational TAD definitions and optimizes TAD selection for a quantifiable biological property. Using this framework, we explore the variability of TAD sets optimized for six different desirable properties of TAD sets: high occupancy of CTCF, RAD21, and H3K36me3 at boundaries, reproducibility between replicates, high intra- vs inter-TAD difference in contact frequencies, and many CTCF binding sites at boundaries. The compatibility of these biological targets varies by cell type, and our results suggest that these properties are better reflected as subpopulations or families of TADs rather than a singular TAD set fitting all TAD definitions and properties. We explore the properties that produce similar TAD sets (reproducibility and inter- vs intra-TAD difference, for example) and those that lead to very different TADs (such as CTCF binding sites and inter- vs intra-TAD contact frequency difference).

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Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer

Cell Death and Disease ◽

10.1038/s41419-021-03793-7 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Donghong Zhang ◽

Jinfeng Ning ◽

Imoh Okon ◽

Xiaoxu Zheng ◽

Ganesh Satyanarayana ◽

...

Keyword(s):

Lung Cancer ◽

Epigenetic Modification ◽

Suppressor Gene ◽

Ctcf Binding ◽

Binding Motif ◽

Loss Of Function ◽

Dna Hypermethylation ◽

Lung Cancer Patients ◽

Lkb1 Tumor Suppressor ◽

And Migration

AbstractOncogenic KRAS mutations combined with the loss of the LKB1 tumor-suppressor gene (KL) are strongly associated with aggressive forms of lung cancer. N6-methyladenosine (m6A) in mRNA is a crucial epigenetic modification that controls cancer self-renewal and progression. However, the regulation and role of m6A modification in this cancer are unclear. We found that decreased m6A levels correlated with the disease progression and poor survival for KL patients. The correlation was mediated by a special increase in ALKBH5 (AlkB family member 5) levels, an m6A demethylase. ALKBH5 gain- or loss-of function could effectively reverse LKB1 regulated cell proliferation, colony formation, and migration of KRAS-mutated lung cancer cells. Mechanistically, LKB1 loss upregulated ALKBH5 expression by DNA hypermethylation of the CTCF-binding motif on the ALKBH5 promoter, which inhibited CTCF binding but enhanced histone modifications, including H3K4me3, H3K9ac, and H3K27ac. This effect could successfully be rescued by LKB1 expression. ALKBH5 demethylation of m6A stabilized oncogenic drivers, such as SOX2, SMAD7, and MYC, through a pathway dependent on YTHDF2, an m6A reader protein. The above findings were confirmed in clinical KRAS-mutated lung cancer patients. We conclude that loss of LKB1 promotes ALKBH5 transcription by a DNA methylation mechanism, reduces m6A modification, and increases the stability of m6A target oncogenes, thus contributing to aggressive phenotypes of KRAS-mutated lung cancer.

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Functional Roles of the Conserved Threonine 250 in the Target Recognition Domain ofHhaI DNA Methyltransferase

Journal of Biological Chemistry ◽

10.1074/jbc.m005278200 ◽

2000 ◽

Vol 275 (49) ◽

pp. 38722-38730 ◽

Cited By ~ 36

Author(s):

Giedrius Vilkaitis ◽

Aiping Dong ◽

Elmar Weinhold ◽

Xiaodong Cheng ◽

Saulius Klimašauskas

Keyword(s):

Dna Methyltransferase ◽

Target Recognition ◽

Functional Roles

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Topologically associating domain boundaries are enriched in early firing origins and restrict replication fork progression

10.1101/2020.10.21.348946 ◽

2020 ◽

Author(s):

Emilia Puig Lombardi ◽

Madalena Tarsounas

Keyword(s):

Binding Sites ◽

Replication Fork ◽

Replication Timing ◽

Replication Initiation ◽

Ctcf Binding ◽

Origin Firing ◽

Replication Fork Progression ◽

Fork Stalling ◽

Genomic Regions ◽

The Impact

ABSTRACTTopologically associating domains (TADs) are units of the genome architecture defined by binding sites for the CTCF transcription factor and cohesin-mediated loop extrusion. Genomic regions containing DNA replication initiation sites have been mapped in the proximity of TAD boundaries. However, the factors that determine this positioning have not been identified. Moreover, the impact of TADs on the directionality of replication fork progression remains unknown. Here we use EdU-seq technology to map origin firing sites at 10 kb resolution and to monitor replication fork progression after restart from hydroxyurea arrest. We show that origins firing in early/mid S-phase within TAD boundaries map to two distinct peaks flanking the centre of the boundary, which is occupied by CTCF and cohesin. When transcription is inhibited chemically or deregulated by oncogene overexpression, replication origins become repositioned to the centre of the TAD. Furthermore, we demonstrate the strikingly asymmetric fork progression initiating from origins located within TAD boundaries. Divergent CTCF binding sites and neighbouring TADs with different replication timing (RT) cause fork stalling in regions external to the TAD. Thus, our work assigns for the first time a role to transcription within TAD boundaries in promoting replication origin firing and demonstrates how genomic regions adjacent to the TAD boundaries could restrict replication progression.

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Metastasis-related methyltransferase 1 (Merm1) represses the methyltransferase activity of Dnmt3a and facilitates RNA polymerase I transcriptional elongation

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjy023 ◽

2018 ◽

Vol 11 (1) ◽

pp. 78-90

Author(s):

Guoliang Lyu ◽

Le Zong ◽

Chao Zhang ◽

Xiaoke Huang ◽

Wenbing Xie ◽

...

Keyword(s):

Rna Polymerase ◽

Dna Methyltransferase ◽

Rna Polymerase I ◽

Rrna Genes ◽

Binding Motif ◽

Rrna Gene ◽

Transcriptional Elongation ◽

Methyltransferase Activity ◽

Polymerase I ◽

Methyltransferase 1

Abstract Stimulatory regulators for DNA methyltransferase activity, such as Dnmt3L and some Dnmt3b isoforms, affect DNA methylation patterns, thereby maintaining gene body methylation and maternal methylation imprinting, as well as the methylation landscape of pluripotent cells. Here we show that metastasis-related methyltransferase 1 (Merm1), a protein deleted in individuals with Williams–Beuren syndrome, acts as a repressive regulator of Dnmt3a. Merm1 interacts with Dnmt3a and represses its methyltransferase activity with the requirement of the binding motif for S-adenosyl-L-methionine. Functional analysis of gene regulation revealed that Merm1 is capable of maintaining hypomethylated rRNA gene bodies and co-localizes with RNA polymerase I in the nucleolus. Dnmt3a recruits Merm1, and in return, Merm1 ensures the binding of Dnmt3a to hypomethylated gene bodies. Such interplay between Dnmt3a and Merm1 facilitates transcriptional elongation by RNA polymerase I. Our findings reveal a repressive factor for Dnmt3a and uncover a molecular mechanism underlying transcriptional elongation of rRNA genes.

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Active enhancers strengthen TAD insulation by bRNA mediated CTCF enrichment at the TAD boundaries

10.1101/2021.07.13.452118 ◽

2021 ◽

Author(s):

Zubairul Islam ◽

Bharath Saravanan ◽

Kaivalya Walavalkar ◽

Umer Farooq ◽

Anurag Kumar Singh ◽

...

Keyword(s):

Gene Transcription ◽

Transcriptional Activity ◽

Ctcf Binding ◽

Functional Roles ◽

Topologically Associating Domains ◽

Genome Wide ◽

Direct Association ◽

Per Se

The genome is partitioned into Topologically Associating Domains (TADs). About half of the boundaries of these TADs exhibit transcriptional activity and are correlated with better TAD insulation. However, the role of these transcripts per se in TAD insulation, enhancer:promoter interactions and transcription remain unknown. Here we investigate the functional roles of these bRNAs (boundary-RNA) in boundary insulation and consequent effects on enhancer-promoter interactions and TAD transcription genome-wide and on disease relevant INK4a/ARF TAD. Using series of CTCF sites deletion and bRNA knockdown approaches at this TAD boundary, we show a direct association of CTCF with bidirectional bRNAs where the loss of bRNA triggers the concomitant loss of: CTCF clustering at TAD boundary, its insulation, enhancer:promoter interactions and gene transcription within the targeted TAD. In search of what regulates bRNA expression itself, we used another series of enhancer deletions and CRISPRi on promoters within INK4a/ARF TAD and observed that indeed, enhancers interact with boundaries and positively regulate the bRNA transcription at TAD boundaries. In return, the bRNAs recruit/stabilise the CTCF even on weaker motifs within these boundaries and supports CTCF binding in clusters, therefore enhancing TAD insulation which favors the intra-TAD enhancer:promoter interactions and robust gene transcription. Functionally, eRNAs within the boundaries are repurposed as more stable bRNAs and their knockdown exactly mimics the boundary loss. Furthermore, transcribing boundaries exhibit high TAD transcription in TCGA tumor datasets. Together, these results show that active enhancers directly mediate better insulation of TADs by activating the transcription at TAD boundaries. These transcripts trigger CTCF clustering at the boundary resulting in better insulation which favours robust intra-TAD enhancer:promoter interactions to activate the gene transcription.

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LKB1 Loss Upregulates ALKBH5 and Contributes to Aggressive Phenotypes of KRAS Mutated Lung Cancer

10.21203/rs.3.rs-105982/v1 ◽

2020 ◽

Author(s):

Donghong Zhang ◽

Jinfeng Ning ◽

Imoh Okon ◽

Xiaoxu Zheng ◽

Ganesh Satyanarayana ◽

...

Keyword(s):

Lung Cancer ◽

Disease Progression ◽

Epigenetic Modification ◽

Suppressor Gene ◽

Ctcf Binding ◽

Luciferase Reporter ◽

Binding Motif ◽

Kras Mutations ◽

Bioinformatics Analyses ◽

Lung Cancer Patients

Abstract Background: Oncogenic KRAS mutations combined with loss of the LKB1 tumor-suppressor gene (KL) are strongly associated with aggressive forms of lung cancer. N6-methyladenosine (m6A) in mRNA is a crucial epigenetic modification that controls cancer self-renewal and progression. However, the function, regulation and mechanism of m6A in this aggressive phenotypes remain largely unclear.Methods: The clinic-pathological role of m6A was evaluated in a cohort of lung cancer tissues and further validated by public databases and integrating bioinformatics analyses. We examined the upstream and downstream regulation of ALKBH5 (AlkB family member 5, an m6A demethylase) using quantitative real-time PCR, western blot, bisulfite genome sequencing, luciferase reporter assay, methylated DNA immunoprecipitation, m6A-RNA Immunoprecipitation (m6A-RIP) and m6A-seq data analysis. Results: We found that LKB1 loss decreased m6A levels and correlated with disease progression and poor survival for KRAS mutant lung cancer patients. The effect on m6A levels and the disease progression and survival was mediated by increasing levels of ALKBH5. LKB1 inactivation increased ALKBH5 transcription in multiple KRAS mutated tumor types, including colon, pancreas, and lung. Conversely, LKB1 overexpression decreased DNA methylation of the CTCF-binding motif on the ALKBH5 promoter, which enhanced CTCF binding and inhibited histone modifications, including H3K4me3, H3K9ac, and H3K27ac. ALKBH5 demethylation of m6A stabilized oncogenic drivers, such as SOX2, SMAD7, and MYC, through a pathway dependent on YTHDF2, an m6A reader protein. Conclusions: Loss of LKB1 in KRAS mutated cancers promoted ALKBH5 transcription, decreased m6A levels, and increased the stability of m6A target oncogenes, thus contributing to aggressive phenotypes of KRAS mutated lung cancer.

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Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells

10.1101/175448 ◽

2017 ◽

Author(s):

Ioanna Pavlaki ◽

France Docquier ◽

Igor Chernukhin ◽

Georgia Kita ◽

Svetlana Gretton ◽

...

Keyword(s):

Gene Expression ◽

Molecular Mass ◽

Molecular Mechanisms ◽

Apparent Molecular Mass ◽

Ctcf Binding ◽

Binding Motif ◽

Cellular Functions ◽

Altered Expression ◽

Cycle Arrest ◽

Breast Cells

AbstractCTCF is an evolutionarily conserved and ubiquitously expressed architectural protein regulating a plethora of cellular functions via different molecular mechanisms. CTCF can undergo a number of post-translational modifications which change its properties and functions. One such modifications linked to cancer is poly(ADP-ribosyl)ation (PARylation). The highly PARylated CTCF form has an apparent molecular mass of 180 kDa (referred to as CTCF180), which can be distinguished from hypo- and non-PARylated CTCF with the apparent molecular mass of 130 kDa (referred to as CTCF130). The existing data accumulated so far have been mainly related to CTCF130. However, the properties of CTCF180 are not well understood despite its abundance in a number of primary tissues. In this study we performed ChIP-seq and RNA-seq analyses in human breast cells 226LDM, which display predominantly CTCF130 when proliferating, but CTCF180 upon cell cycle arrest. We observed that in the arrested cells the majority of sites lost CTCF, whereas fewer sites gained CTCF or remain bound (i.e. common sites). The classical CTCF binding motif was found in the lost and common, but not in the gained sites. The changes in CTCF occupancies in the lost and common sites were associated with increased chromatin densities and altered expression from the neighboring genes. Based on these results we propose a model integrating the CTCF130/180 transition with CTCF-DNA binding and gene expression changes. This study also issues an important cautionary note concerning the design and interpretation of any experiments using cells and tissues where CTCF180 may be present.

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Rif1 functions in a tissue-specific manner to control replication timing through its PP1-binding motif

10.1101/870451 ◽

2019 ◽

Author(s):

Robin L. Armstrong ◽

Souradip Das ◽

Christina A. Hill ◽

Robert J. Duronio ◽

Jared T. Nordman

Keyword(s):

Cell Cycle ◽

Genome Stability ◽

Cell Lineage ◽

Replication Timing ◽

Cell Types ◽

Replication Initiation ◽

Control Factor ◽

Binding Motif ◽

Tissue Specific ◽

Specific Manner

AbstractReplication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early and late replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster. We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs.

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Chromatin loop anchors are associated with genome instability in cancer and recombination hotspots in the germline

10.1101/166934 ◽

2017 ◽

Author(s):

Vera B Kaiser ◽

Colin A Semple

Keyword(s):

Genome Instability ◽

Large Fraction ◽

Replication Timing ◽

Basic Unit ◽

Mutational Bias ◽

Binding Motif ◽

Chromatin Loop ◽

Single Nucleotide Variants ◽

Chromatin Loops ◽

Evolutionary Innovation

ABSTRACTChromatin loops form a basic unit of interphase nuclear organisation, providing contacts between regulatory regions and target promoters, and forming higher level patterns defining self interacting domains. Recent studies have shown that mutations predicted to alter chromatin loops and domains are frequently observed in tumours and can result in the upregulation of oncogenes, but the combinations of selection and mutational bias underlying these observations remains unknown. Here, we explore the unusual mutational landscape associated with chromatin loop anchor points (LAPs), which are located at the base of chromatin loops and form a kinetic trap for cohesin. We show that LAPs are strongly depleted for single nucleotide variants (SNVs) in tumours, which is consistent with their relatively early replication timing. However, despite low SNV rates, LAPs emerge as sites of evolutionary innovation showing enrichment for structural variants (SVs). They harbour an excess of SV breakpoints in cancers, are prone to double strand breaks in somatic cells, and are bound by DNA repair complex proteins. Recurrently disrupted LAPs are often associated with genes annotated with functions in cell cycle transitions. An unexpectedly large fraction of LAPs (16%) also overlap known meiotic recombination hotspot (HSs), and are enriched for the core PRDM9 binding motif, suggesting that LAPs have been foci for diversity generated during recent human evolution. We suggest that the unusual chromatin structure at LAPs underlies the elevated SV rates observed, marking LAPs as sites of regulatory importance but also genomic fragility.

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