Cohesin composition and dosage independently affect early development in zebrafish.

Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed 'cohesinopathies' are characterized by germline variants of cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear whether mutations in individual cohesin subunits have independent developmental consequences. Here, we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single-cell RNA sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21, mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies.

Cohesin Mutations in Cancer: Emerging Therapeutic Targets.

The cohesin complex is crucial for mediating sister chromatid cohesion and for hierarchal three-dimensional organization of the genome. Mutations in cohesin genes are present in a range of cancers. Extensive research over the last few years has shown that cohesin mutations are key events that contribute to neoplastic transformation. Cohesin is involved in a range of cellular processes; therefore, the impact of cohesin mutations in cancer is complex and can be cell context dependent. Candidate targets with therapeutic potential in cohesin mutant cells are emerging from functional studies. Here, we review emerging targets and pharmacological agents that have therapeutic potential in cohesin mutant cells.

A non-coding genetic variant maximally associated with serum urate levels is functionally linked to HNF4A-dependent PDZK1 expression.

The precise molecular mechanisms by which urate-associated genetic variants affect urate levels are unknown. Here, we tested for functional linkage of the maximally associated genetic variant rs1967017 at the PDZK1 locus to elevated PDZK1 expression. We performed expression quantitative trait loci (eQTL) and likelihood analyses and gene expression assays. Zebrafish were used to evaluate tissue-specific gene expression. Luciferase assays in HEK293 and HepG2 cells measured the effect of rs1967017 on transcription amplitude. Probabilistic Annotation Integrator analysis revealed rs1967017 as most likely to be causal and rs1967017 was an eQTL for PDZK1 in the intestine. The region harboring rs1967017 was capable of directly driving green fluorescent protein expression in the kidney, liver and intestine of zebrafish embryos, consistent with a conserved ability to confer tissue-specific expression. Small interfering RNA depletion of HNF4A reduced endogenous PDZK1 expression in HepG2 cells. Luciferase assays showed that the T allele of rs1967017 gains enhancer activity relative to the urate-decreasing C allele, with T allele enhancer activity abrogated by HNF4A depletion. HNF4A physically binds the rs1967017 region, suggesting direct transcriptional regulation of PDZK1 by HNF4A. Computational prediction of increased motif strength, together with our functional assays, suggests that the urate-increasing T allele of rs1967017 strengthens a binding site for the transcription factor HNF4A. Our and other data predict that the urate-raising T allele of rs1967017 enhances HNF4A binding to the PDZK1 promoter, thereby increasing PDZK1 expression. As PDZK1 is a scaffold protein for many ion channel transporters, increased expression can be predicted to increase activity of urate transporters and alter excretion of urate.

GWAS on prolonged gestation (post-term birth): analysis of successive Finnish birth cohorts.

BACKGROUND: Gestation is a crucial timepoint in human development. Deviation from a term gestational age correlates with both acute and long-term adverse health effects for the child. Both being born preterm and post-term, that is, having short and long gestational ages, are heritable and influenced by the prenatal and perinatal environment. Despite the obvious heritable component, specific genetic influences underlying differences in gestational age are poorly understood. METHODS: We investigated the genetic architecture of gestational age in 9141 individuals, including 1167 born post-term, across two Northern Finland cohorts born in 1966 or 1986. RESULTS: Here we identify one globally significant intronic genetic variant within the ADAMTS13 gene that is associated with prolonged gestation (p=4.85×10-8). Additional variants that reached suggestive levels of significance were identified within introns at the ARGHAP42 and TKT genes, and in the upstream (5') intergenic regions of the B3GALT5 and SSBP2 genes. The variants near the ADAMTS13, B3GALT5, SSBP2 and TKT loci are linked to alterations in gene expression levels (cis-eQTLs). Luciferase assays confirmed the allele specific enhancer activity for the BGALT5 and TKT loci. CONCLUSIONS: Our findings provide the first evidence of a specific genetic influence associated with prolonged gestation. This study forms a foundation for a better understanding of the genetic and long-term health risks faced by induced and post-term individuals. The long-term risks for induced individuals who have a previously overlooked post-term potential may be a major issue for current health providers.

Intergenic GWAS SNPs are key components of the spatial and regulatory network for human growth.

Meta-analysis of genome-wide association studies has resulted in the identification of hundreds of genetic variants associated with growth and stature. Determining how these genetic variants influence growth is important, but most are non-coding, and there is little understanding of how these variants contribute to adult height. To determine the mechanisms by which human variation contributes to growth, we combined spatial genomic connectivity (high-throughput conformation capture) with functional (gene expression, expression Quantitative Trait Loci) data to determine how non-genic loci associated with infant length, pubertal and adult height and contribute to gene regulatory networks. This approach identified intergenic single-nucleotide polymorphisms (SNPs) ∼85 kb upstream of FBXW11 that spatially connect with distant loci. These regulatory connections are reinforced by evidence of SNP-enhancer effects and altered expression in genes influencing the action of human growth hormone. Functional assays provided evidence for enhancer activity of the intergenic region near FBXW11 that harbors SNP rs12153391, which is associated with an expression Quantitative Trait Loci. Our results suggest that variants in this locus have genome-wide effects as key modifiers of growth (both overgrowth and short stature) acting through a regulatory network. We believe that the genes and pathways connected with this regulatory network are potential targets that could be investigated for diagnostic, prenatal and carrier testing for growth disorders. Finally, the regulatory networks we generated illustrate the power of using existing datasets to interrogate the contribution of intergenic SNPs to common syndromes/diseases.

HDAC8 Inhibition Blocks SMC3 Deacetylation and Delays Cell Cycle Progression without Affecting Cohesin-dependent Transcription in MCF7 Cancer Cells.

Cohesin, a multi-subunit protein complex involved in chromosome organization, is frequently mutated or aberrantly expressed in cancer. Multiple functions of cohesin, including cell division and gene expression, highlight its potential as a novel therapeutic target. The SMC3 subunit of cohesin is acetylated (ac) during S phase to establish cohesion between replicated chromosomes. Following anaphase, ac-SMC3 is deacetylated by HDAC8. Reversal of SMC3 acetylation is imperative for recycling cohesin so that it can be reloaded in interphase for both non-mitotic and mitotic functions. We blocked deacetylation of ac-SMC3 using an HDAC8-specific inhibitor PCI-34051 in MCF7 breast cancer cells, and examined the effects on transcription of cohesin-dependent genes that respond to estrogen. HDAC8 inhibition led to accumulation of ac-SMC3 as expected, but surprisingly, had no influence on the transcription of estrogen-responsive genes that are altered by siRNA targeting of RAD21 or SMC3. Knockdown of RAD21 altered estrogen receptor α (ER) recruitment at SOX4 and IL20, and affected transcription of these genes, while HDAC8 inhibition did not. Rather, inhibition of HDAC8 delayed cell cycle progression, suppressed proliferation and induced apoptosis in a concentration-dependent manner. We conclude that HDAC8 inhibition does not change the estrogen-specific transcriptional role of cohesin in MCF7 cells, but instead, compromises cell cycle progression and cell survival. Our results argue that candidate inhibitors of cohesin function may differ in their effects depending on the cellular genotype and should be thoroughly tested for predicted effects on cohesin's mechanistic roles.

Cohesin modulates transcription of estrogen-responsive genes.

The cohesin complex has essential roles in cell division, DNA damage repair and gene transcription. The transcriptional function of cohesin is thought to derive from its ability to connect distant regulatory elements with gene promoters. Genome-wide binding of cohesin in breast cancer cells frequently coincides with estrogen receptor alpha (ER), leading to the hypothesis that cohesin facilitates estrogen-dependent gene transcription. We found that cohesin modulates the expression of only a subset of genes in the ER transcription program, either activating or repressing transcription depending on the gene target. Estrogen-responsive genes most significantly influenced by cohesin were enriched in pathways associated with breast cancer progression such as PI3K and ErbB1. In MCF7 breast cancer cells, cohesin depletion enhanced transcription of TFF1 and TFF2, and was associated with increased ER binding and increased interaction between TFF1 and its distal enhancer situated within TMPRSS3. In contrast, cohesin depletion reduced c-MYC mRNA and was accompanied by reduced interaction between a distal enhancer of c-MYC and its promoters. Our data indicates that cohesin is not a universal facilitator of ER-induced transcription and can even restrict enhancer-promoter communication. We propose that cohesin modulates transcription of estrogen-dependent genes to achieve appropriate directionality and amplitude of expression.

Cohesin and CTCF differentially regulate spatiotemporal runx1 expression during zebrafish development.

Runx1 is a transcription factor essential for definitive hematopoiesis. In all vertebrates, the Runx1 gene is transcribed from two promoters: a proximal promoter (P2), and a distal promoter (P1). We previously found that runx1 expression in a specific hematopoietic cell population in zebrafish embryos depends on cohesin. Here we show that zebrafish runx1 is directly bound by cohesin and CCCTC binding factor (CTCF) at the P1 and P2 promoters, and within the intron between P1 and P2. Cohesin initiates expression of runx1 in the posterior lateral mesoderm and influences promoter use, while CTCF represses its expression in the newly emerging cells of the tail bud. The intronic binding sites for cohesin and CTCF coincide with histone modifications that confer enhancer-like properties, and two of the cohesin/CTCF sites behaved as insulators in an in vivo assay. The identified cohesin and CTCF binding sites are likely to be cis-regulatory elements (CREs) for runx1 since they also recruit RNA polymerase II (RNAPII). CTCF depletion excluded RNAPII from two intronic CREs but not the promoters of runx1. We propose that cohesin and CTCF have distinct functions in the regulation of runx1 during zebrafish embryogenesis, and that these regulatory functions are likely to involve runx1 intronic CREs. Cohesin (but not CTCF) depletion enhanced RUNX1 expression in a human leukemia cell line, suggesting conservation of RUNX1 regulation through evolution.

Targeted DNA Methylation Editing Using an All-in-One System Establishes Paradoxical Activation of EBF3.

Cutaneous melanoma is rapidly on the rise globally, surpassing the growth rate of other cancers, with metastasis being the primary cause of death in melanoma patients. Consequently, understanding the mechanisms behind this metastatic process and exploring innovative treatments is of paramount importance. Recent research has shown promise in unravelling the role of epigenetic factors in melanoma progression to metastasis. While DNA hypermethylation at gene promoters typically suppresses gene expression, we have contributed to establishing the newly understood mechanism of paradoxical activation of genes via DNA methylation, where high methylation coincides with increased gene activity. This mechanism challenges the conventional paradigm that promoter methylation solely silences genes, suggesting that, for specific genes, it might actually activate them. Traditionally, altering DNA methylation in vitro has involved using global demethylating agents, which is insufficient for studying the mechanism and testing the direct consequence of gene methylation changes. To investigate promoter hypermethylation and its association with gene activation, we employed a novel approach utilising a CRISPR-SunTag All-in-one system. Here, we focused on editing the DNA methylation of a specific gene promoter segment (EBF3) in melanoma cells using the All-in-one system. Using bisulfite sequencing and qPCR with RNA-Seq, we successfully demonstrated highly effective methylation and demethylation of the EBF3 promoter, with subsequent gene expression changes, to establish and validate the paradoxical role of DNA methylation. Further, our study provides novel insights into the function of the EBF3 gene, which remains largely unknown. Overall, this study challenges the conventional view of methylation as solely a gene-silencing mechanism and demonstrates a potential function of EBF3 in IFN pathway signalling, potentially uncovering new insights into epigenetic drivers of malignancy and metastasis.

Transcriptional Regulation of RUNX1: An Informatics Analysis.

The RUNX1/AML1 gene encodes a developmental transcription factor that is an important regulator of haematopoiesis in vertebrates. Genetic disruptions to the RUNX1 gene are frequently associated with acute myeloid leukaemia. Gene regulatory elements (REs), such as enhancers located in non-coding DNA, are likely to be important for Runx1 transcription. Non-coding elements that modulate Runx1 expression have been investigated over several decades, but how and when these REs function remains poorly understood. Here we used bioinformatic methods and functional data to characterise the regulatory landscape of vertebrate Runx1. We identified REs that are conserved between human and mouse, many of which produce enhancer RNAs in diverse tissues. Genome-wide association studies detected single nucleotide polymorphisms in REs, some of which correlate with gene expression quantitative trait loci in tissues in which the RE is active. Our analyses also suggest that REs can be variant in haematological malignancies. In summary, our analysis identifies features of the RUNX1 regulatory landscape that are likely to be important for the regulation of this gene in normal and malignant haematopoiesis.

Cohesin mutations are synthetic lethal with stimulation of WNT signaling.

Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21, and STAG2 and screened for synthetic lethality with 3009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top 'hits' was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of ß-catenin in cohesin-mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunits stag2b and rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin-mutant cancers.

Corrigendum: Functional Urate-Associated Genetic Variants Influence Expression of lincRNAs LINC01229 and MAFTRR.

[This corrects the article DOI: 10.3389/fgene.2018.00733.].

Functional Urate-Associated Genetic Variants Influence Expression of lincRNAs LINC01229 and MAFTRR.

Genetic variation in the genomic regulatory landscape likely plays a crucial role in the pathology of disease. Non-coding variants associated with disease can influence the expression of long intergenic non-coding RNAs (lincRNAs), which in turn function in the control of protein-coding gene expression. Here, we investigate the function of two independent serum urate-associated signals (SUA1 and SUA2) in close proximity to lincRNAs and an enhancer that reside ∼60 kb and ∼300 kb upstream of MAF, respectively. Variants within SUA1 are expression quantitative trait loci (eQTL) for LINC01229 and MAFTRR, both co-expressed with MAF. We have also identified that variants within SUA1 are trans-eQTL for genes that are active in kidney- and serum urate-relevant pathways. Serum urate-associated variants rs4077450 and rs4077451 within SUA2 lie within an enhancer that recruits the transcription factor HNF4α and forms long range interactions with LINC01229 and MAFTRR. The urate-raising alleles of rs4077450 and rs4077451 increase enhancer activity and associate with increased expression of LINC01229. We show that the SUA2 enhancer region drives expression in the zebrafish pronephros, recapitulating endogenous MAF expression. Depletion of MAFTRR and LINC01229 in HEK293 cells in turn lead to increased MAF expression. Collectively, our results are consistent with serum urate variants mediating long-range transcriptional regulation of the lincRNAs LINC01229 and MAFTRR and urate relevant genes (e.g., SLC5A8 and EHHADH) in trans.

Regulation of the interferon-gamma (IFN-γ) pathway by p63 and Δ133p53 isoform in different breast cancer subtypes.

The TP53 family consists of three sets of transcription factor genes, TP53, TP63 and TP73, each of which expresses multiple RNA variants and protein isoforms. Of these, TP53 is mutated in 25-30% of breast cancers. How TP53 mutations affect the interaction of TP53 family members and their isoforms in breast cancer is unknown. To investigate this, 3 independent breast cancer cohorts were stratified into 4 groups based on oestrogen receptor (ER) and TP53 mutation status. Using bioinformatic methodologies, principal signalling pathways associated with the expression of TP53 family members were identified. Results show an enrichment of IFN-γ signalling associated with TP63 RNA in wild type TP53 (wtTP53), ER negative (ER-) tumours and with Δ133TP53 RNA in mutant TP53 (mTP53) ER positive (ER+) tumours. Moreover, tumours with low IFN-γ signalling were associated with significantly poorer patient outcome. The predicted changes in expression of a subset of RNAs involved in IFN-γ signalling were confirmed in vitro. Our data show that different members of the TP53 family can drive transcription of genes involved in IFN-γ signalling in different breast cancer subgroups.

KDM4B-mediated reduction of H3K9me3 and H3K36me3 levels improves somatic cell reprogramming into pluripotency.

Correct reprogramming of epigenetic marks is essential for somatic cells to regain pluripotency. Repressive histone (H) lysine (K) methylation marks are known to be stable and difficult to reprogram. In this study, we generated transgenic mice and mouse embryonic fibroblasts (MEFs) for the inducible expression of KDM4B, a demethylase that removes H3 K9 and H3K36 trimethylation (me3) marks (H3K9/36me3). Upon inducing Kdm4b, H3K9/36me3 levels significantly decreased compared to non-induced controls. Concurrently, H3K9me1 levels significantly increased, while H3K9me2 and H3K27me3 remained unchanged. The global transcriptional impact of Kdm4b-mediated reduction in H3K9/36me3 levels was examined by comparative microarray analysis and mRNA-sequencing of three independent transgenic MEF lines. We identified several commonly up-regulated targets, including the heterochromatin-associated zinc finger protein 37 and full-length endogenous retrovirus repeat elements. Following optimized zona-free somatic nuclear transfer, reduced H3K9/36me3 levels were restored within hours. Nevertheless, hypo-methylated Kdm4b MEF donors reprogrammed six-fold better into cloned blastocysts than non-induced donors. They also reprogrammed nine-fold better into induced pluripotent stem cells that gave rise to teratomas and chimeras. In summary, we firmly established H3K9/36me3 as a major roadblock to somatic cell reprogramming and identified transcriptional targets of derestricted chromatin that could contribute towards improving this process in mouse.

Insights from space: potential role of diet in the spatial organization of chromosomes.

We can now sequence and identify genome wide epigenetic patterns and perform a variety of "genomic experiments" within relatively short periods of time-ranging from days to weeks. Yet, despite these technological advances, we have a poor understanding of the inter-relationships between epigenetics, genome structure-function, and nutrition. Perhaps this limitation lies, in part, in our propensity to study epigenetics in terms of the linear arrangement of elements and genes. Here we propose that a more complete understanding of how nutrition impacts on epigenetics and cellular development resides within the inter-relationships between DNA and histone modification patterns and genome function, in the context of spatial organization of chromatin and the epigenome.

Transient JMJD2B-mediated reduction of H3K9me3 levels improves reprogramming of embryonic stem cells into cloned embryos.

Correct reprogramming of epigenetic marks in the donor nuclei is crucial for successful cloning by nuclear transfer. Specific epigenetic modifications, such as repressive histone lysine methylation marks, are known to be very stable and difficult to reprogram. The discovery of histone lysine demethylases has opened up opportunities to study the effects of removing repressive histone lysine methylation marks in donor cells prior to nuclear transfer. In this study, we generated mouse embryonic stem (ES) cells for the inducible expression of JMJD2B (also known as KDM4B), a demethylase that primarily removes the histone-3 lysine-9 trimethylation (H3K9me3) mark. Induction of jmjd2b in the ES cells decreased total levels of H3K9me3 by 63%. When these cells were used for nuclear transfer, H3K9me3 levels were normalized within minutes following fusion with an enucleated oocyte. This transient reduction of H3K9me3 levels improved in vitro development into cloned embryos by 30%.