bims-crepig 2021-05-09 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2021–05–09
twenty-six papers selected by
Connor Rogerson, University of Cambridge, MRC Cancer Unit

Differential chromatin binding of the lung lineage transcription factor NKX2-1 resolves opposing murine alveolar cell fates in vivo.
FACT interacts with Set3 HDAC and fine-tunes GAL1 transcription in response to environmental stimulation.
Decoding dynamic epigenetic landscapes in human oocytes using single-cell multi-omics sequencing.
The ZMYND8-regulated mevalonate pathway endows YAP-high intestinal cancer with metabolic vulnerability.
DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation.
Two bHLH transcription factors, bHLH48 and bHLH60, associate with phytochrome interacting factor 7 to regulate hypocotyl elongation in Arabidopsis.
The histone reader PHF7 cooperates with the SWI/SNF complex at cardiac super enhancers to promote direct reprogramming.
Hotspot identifies informative gene modules across modalities of single-cell genomics.
Single-nucleotide-level mapping of DNA regulatory elements that control fetal hemoglobin expression.
HOTAIR lncRNA promotes epithelial-mesenchymal transition by redistributing LSD1 at regulatory chromatin regions.
Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions.
Interplay of two transcription factors for recruitment of the chromatin remodeling complex modulates fungal nitrosative stress response.
HDAC3 controls male fertility through enzyme-independent transcriptional regulation at the meiotic exit of spermatogenesis.
Extended-representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells.
SMARCAD1-mediated active replication fork stability maintains genome integrity.
Co-occupancy identifies transcription factor co-operation for axon growth.
Learning a genome-wide score of human-mouse conservation at the functional genomics level.
The hierarchical packing of euchromatin domains can be described as multiplicative cascades.
A single-embryo, single-cell time-resolved model for mouse gastrulation.
Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells.
Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity.
PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing.
Comparison of the Hi-C, GAM and SPRITE methods using polymer models of chromatin.
Sox9 and Rbpj differentially regulate endothelial to mesenchymal transition and wound scarring in murine endovascular progenitors.
C/EBPβ is a MYB- and p300-cooperating pro-leukemogenic factor and promising drug target in acute myeloid leukemia.
CeLaVi: an interactive cell lineage visualization tool.

Nat Commun. 2021 05 04. 12(1): 2509

Differential chromatin binding of the lung lineage transcription factor NKX2-1 resolves opposing murine alveolar cell fates in vivo.

Danielle R Little, Anne M Lynch, Yun Yan, Haruhiko Akiyama, Shioko Kimura, Jichao Chen.

Differential transcription of identical DNA sequences leads to distinct tissue lineages and then multiple cell types within a lineage, an epigenetic process central to progenitor and stem cell biology. The associated genome-wide changes, especially in native tissues, remain insufficiently understood, and are hereby addressed in the mouse lung, where the same lineage transcription factor NKX2-1 promotes the diametrically opposed alveolar type 1 (AT1) and AT2 cell fates. Here, we report that the cell-type-specific function of NKX2-1 is attributed to its differential chromatin binding that is acquired or retained during development in coordination with partner transcriptional factors. Loss of YAP/TAZ redirects NKX2-1 from its AT1-specific to AT2-specific binding sites, leading to transcriptionally exaggerated AT2 cells when deleted in progenitors or AT1-to-AT2 conversion when deleted after fate commitment. Nkx2-1 mutant AT1 and AT2 cells gain distinct chromatin accessible sites, including those specific to the opposite fate while adopting a gastrointestinal fate, suggesting an epigenetic plasticity unexpected from transcriptional changes. Our genomic analysis of single or purified cells, coupled with precision genetics, provides an epigenetic basis for alveolar cell fate and potential, and introduces an experimental benchmark for deciphering the in vivo function of lineage transcription factors.

DOI: https://doi.org/10.1038/s41467-021-22817-6
Nucleic Acids Res. 2021 May 08. pii: gkab312. [Epub ahead of print]

FACT interacts with Set3 HDAC and fine-tunes GAL1 transcription in response to environmental stimulation.

He Leng, Shaofeng Liu, Yang Lei, Yuantao Tang, Shijia Gu, Jiazhi Hu, She Chen, Jianxun Feng, Qing Li.

The histone chaperone facilitates chromatin transactions (FACT) functions in various DNA transactions. How FACT performs these multiple functions remains largely unknown. Here, we found, for the first time, that the N-terminal domain of its Spt16 subunit interacts with the Set3 histone deacetylase complex (Set3C) and that FACT and Set3C function in the same pathway to regulate gene expression in some settings. We observed that Spt16-G132D mutant proteins show defects in binding to Set3C but not other reported FACT interactors. At the permissive temperature, induction of the GAL1 and GAL10 genes is reduced in both spt16-G132D and set3Δ cells, whereas transient upregulation of GAL10 noncoding RNA (ncRNA), which is transcribed from the 3' end of the GAL10 gene, is elevated. Mutations that inhibit GAL10 ncRNA transcription reverse the GAL1 and GAL10 induction defects in spt16-G132D and set3Δ mutant cells. Mechanistically, set3Δ and FACT (spt16-G132D) mutants show reduced histone acetylation and increased nucleosome occupancy at the GAL1 promoter under inducing conditions and inhibition of GAL10 ncRNA transcription also partially reverses these chromatin changes. These results indicate that FACT interacts with Set3C, which in turn prevents uncontrolled GAL10 ncRNA expression and fine-tunes the expression of GAL genes upon a change in carbon source.

DOI: https://doi.org/10.1093/nar/gkab312
Cell Stem Cell. 2021 Apr 27. pii: S1934-5909(21)00169-7. [Epub ahead of print]

Decoding dynamic epigenetic landscapes in human oocytes using single-cell multi-omics sequencing.

Rui Yan, Chan Gu, Di You, Zhongying Huang, Jingjing Qian, Qiuyun Yang, Xin Cheng, Lin Zhang, Hongmei Wang, Ping Wang, Fan Guo.

Developing female human germ cells undergo genome-wide epigenetic reprogramming, but de novo DNA methylation dynamics and their interplay with chromatin states and transcriptional activation in developing oocytes is poorly understood. Here, we developed a single-cell multi-omics sequencing method, scChaRM-seq, that enables simultaneous profiling of the DNA methylome, transcriptome, and chromatin accessibility in single human oocytes and ovarian somatic cells. We observed a global increase in DNA methylation during human oocyte growth that correlates with chromatin accessibility, whereas increases of DNA methylation at specific features were associated with active transcription. Integrated analyses of multi-omics data from humans and mice revealed species-specific gene expression, and promoter accessibility contributes to gene body methylation programs. Alu elements retained low DNA methylation levels and high accessibility in early growing oocytes and were located near developmental genes in humans and mice. Together, these findings show how scChaRM-seq can provide insight into DNA methylation pattern establishment.

DOI: https://doi.org/10.1016/j.stem.2021.04.012
Mol Cell. 2021 Apr 22. pii: S1097-2765(21)00314-2. [Epub ahead of print]

The ZMYND8-regulated mevalonate pathway endows YAP-high intestinal cancer with metabolic vulnerability.

Qiang Pan, Shanshan Zhong, Hanling Wang, Xuege Wang, Ni Li, Yaqi Li, Guoying Zhang, Huairui Yuan, Yannan Lian, Qilong Chen, Ying Han, Jiacheng Guo, Qiuli Liu, Tong Qiu, Jun Jiang, Qintong Li, Minjia Tan, Huiyong Yin, Junjie Peng, Yichuan Xiao, Jun Qin.

  Cholesterol metabolism is tightly associated with colorectal cancer (CRC). Nevertheless, the clinical benefit of statins, the inhibitor of cholesterol biogenesis mevalonate (MVA) pathway, is inconclusive, possibly because of a lack of patient stratification criteria. Here, we describe that YAP-mediated zinc finger MYND-type containing 8 (ZMYND8) expression sensitizes intestinal tumors to the inhibition of the MVA pathway. We show that the oncogenic activity of YAP relies largely on ZMYND8 to enhance intracellular de novo cholesterol biogenesis. Disruption of the ZMYND8-dependent MVA pathway greatly restricts the self-renewal capacity of Lgr5+ intestinal stem cells (ISCs) and intestinal tumorigenesis. Mechanistically, ZMYND8 and SREBP2 drive the enhancer-promoter interaction to facilitate the recruitment of Mediator complex, thus upregulating MVA pathway genes. Together, our results establish that the epigenetic reader ZMYND8 endows YAP-high intestinal cancer with metabolic vulnerability.

Keywords:  Colorectal cancer; Enhancer-promoter contact; Intestinal stem cell (ISC); Mevalonate (MVA) pathway; YAP; ZMYND8

DOI:  https://doi.org/10.1016/j.molcel.2021.04.009
Nat Commun. 2021 05 03. 12(1): 2490

DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation.

Wendan Ren, Huitao Fan, Sara A Grimm, Jae Jin Kim, Linhui Li, Yiran Guo, Christopher James Petell, Xiao-Feng Tan, Zhi-Min Zhang, John P Coan, Jiekai Yin, Dae In Kim, Linfeng Gao, Ling Cai, Nelli Khudaverdyan, Burak Çetin, Dinshaw J Patel, Yinsheng Wang, Qiang Cui, Brian D Strahl, Or Gozani, Kyle M Miller, Seán E O'Leary, Paul A Wade, Gang Greg Wang, Jikui Song.

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically 'recognizes' H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1's activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.

DOI: https://doi.org/10.1038/s41467-021-22665-4
Cell Rep. 2021 May 04. pii: S2211-1247(21)00370-3. [Epub ahead of print]35(5): 109054

Two bHLH transcription factors, bHLH48 and bHLH60, associate with phytochrome interacting factor 7 to regulate hypocotyl elongation in Arabidopsis.

Chuanwei Yang, Sha Huang, Yue Zeng, Chang Liu, Qinyi Ma, Jose Pruneda-Paz, Steve A Kay, Lin Li.

  The basic helix-loop-helix (bHLH) transcription factor PHYTOCHROME-INTERACTING FACTOR 7 (PIF7) is a central regulator that promotes stem growth by activating growth-related gene expression during shade-avoidance responses. Studying the co-factors of PIF7 can facilitate understanding of the mechanism of PIFs and light signal transduction. Here, we describe the identification of two bHLH transcription factors, bHLH48 and bHLH60 (bHLH48/bHLH60), as essential partners for PIF7-dependent modulation of hypocotyl elongation and function downstream of phytochrome B. These two bHLH factors display DNA binding activity and interact with PIF7. Genetic analysis indicated that bHLH48/bHLH60 and PIF7 are interdependent in promoting hypocotyl elongation. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis identified the substantially overlapping downstream targets of bHLH60 and PIF7. Biochemical analysis revealed that bHLH48/bHLH60 enhance the DNA binding ability of PIF7. These results provide evidence that bHLH48/bHLH60 act as positive partners of PIF7 for mutual benefit in the regulation of hypocotyl elongation.

Keywords:  DNA binding ability; PIF7; bHLH48/bHLH60; hypocotyl elongation; transcriptional activity

DOI:  https://doi.org/10.1016/j.celrep.2021.109054
Nat Cell Biol. 2021 May 03.

The histone reader PHF7 cooperates with the SWI/SNF complex at cardiac super enhancers to promote direct reprogramming.

Glynnis A Garry, Svetlana Bezprozvannaya, Kenian Chen, Huanyu Zhou, Hisayuki Hashimoto, Maria Gabriela Morales, Ning Liu, Rhonda Bassel-Duby, Eric N Olson.

Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through overexpression of the transcription factors Gata4, Mef2c and Tbx5; later, Hand2 and Akt1 were found to further enhance this process1-5. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogramme adult fibroblasts6,7. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor8. Mechanistically, PHF7 localizes to cardiac super enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, it increases chromatin accessibility and transcription factor binding at these sites. Furthermore, PHF7 recruits cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 achieves efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic readers, such as PHF7, in harnessing chromatin remodelling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.

DOI: https://doi.org/10.1038/s41556-021-00668-z
Cell Syst. 2021 Apr 29. pii: S2405-4712(21)00114-9. [Epub ahead of print]

Hotspot identifies informative gene modules across modalities of single-cell genomics.

David DeTomaso, Nir Yosef.

  Two fundamental aims that emerge when analyzing single-cell RNA-seq data are identifying which genes vary in an informative manner and determining how these genes organize into modules. Here, we propose a general approach to these problems, called "Hotspot," that operates directly on a given metric of cell-cell similarity, allowing for its integration with any method (linear or non-linear) for identifying the primary axes of transcriptional variation between cells. In addition, we show that when using multimodal data, Hotspot can be used to identify genes whose expression reflects alternative notions of similarity between cells, such as physical proximity in a tissue or clonal relatedness in a cell lineage tree. In this manner, we demonstrate that while Hotspot is capable of identifying genes that reflect nuanced transcriptional variability between T helper cells, it can also identify spatially dependent patterns of gene expression in the cerebellum as well as developmentally heritable expression programs during embryogenesis. Hotspot is implemented as an open-source Python package and is available for use at http://www.github.com/yoseflab/hotspot. A record of this paper's transparent peer review process is included in the supplemental information.

Keywords:  bioinformatics; genomics; multimodal single-cell; single-cell RNA-seq; software; spatial transcriptomics

DOI:  https://doi.org/10.1016/j.cels.2021.04.005
Nat Genet. 2021 May 06.

Single-nucleotide-level mapping of DNA regulatory elements that control fetal hemoglobin expression.

Li Cheng, Yichao Li, Qian Qi, Peng Xu, Ruopeng Feng, Lance Palmer, Jingjing Chen, Ruiqiong Wu, Tiffany Yee, Jingjing Zhang, Yu Yao, Akshay Sharma, Ross C Hardison, Mitchell J Weiss, Yong Cheng.

Pinpointing functional noncoding DNA sequences and defining their contributions to health-related traits is a major challenge for modern genetics. We developed a high-throughput framework to map noncoding DNA functions with single-nucleotide resolution in four loci that control erythroid fetal hemoglobin (HbF) expression, a genetically determined trait that modifies sickle cell disease (SCD) phenotypes. Specifically, we used the adenine base editor ABEmax to introduce 10,156 separate A•T to G•C conversions in 307 predicted regulatory elements and quantified the effects on erythroid HbF expression. We identified numerous regulatory elements, defined their epigenomic structures and linked them to low-frequency variants associated with HbF expression in an SCD cohort. Targeting a newly discovered γ-globin gene repressor element in SCD donor CD34+ hematopoietic progenitors raised HbF levels in the erythroid progeny, inhibiting hypoxia-induced sickling. Our findings reveal previously unappreciated genetic complexities of HbF regulation and provide potentially therapeutic insights into SCD.

DOI: https://doi.org/10.1038/s41588-021-00861-8
EMBO Rep. 2021 May 06. e50193

HOTAIR lncRNA promotes epithelial-mesenchymal transition by redistributing LSD1 at regulatory chromatin regions.

Julien Jarroux, Dominika Foretek, Claire Bertrand, Marc Gabriel, Ugo Szachnowski, Zohra Saci, Shuling Guo, Arturo Londoño-Vallejo, Marina Pinskaya, Antonin Morillon.

  Epithelial-to-mesenchymal transition (EMT) describes the loss of epithelial traits and gain of mesenchymal traits by normal cells during development and by neoplastic cells during cancer metastasis. The long noncoding RNA HOTAIR triggers EMT, in part by serving as a scaffold for PRC2 and thus promoting repressive histone H3K27 methylation. In addition to PRC2, HOTAIR interacts with the LSD1 lysine demethylase, an epigenetic regulator of cell fate during development and differentiation, but little is known about the role of LSD1 in HOTAIR function during EMT. Here, we show that HOTAIR requires its LSD1-interacting domain, but not its PRC2-interacting domain, to promote the migration of epithelial cells. This activity is suppressed by LSD1 overexpression. LSD1-HOTAIR interactions induce partial reprogramming of the epithelial transcriptome altering LSD1 distribution at promoter and enhancer regions. Thus, we uncover an unexpected role of HOTAIR in EMT as an LSD1 decommissioning factor, counteracting its activity in the control of epithelial identity.

Keywords:  EMT; HOTAIR; LSD1; enhancer; promoter

DOI:  https://doi.org/10.15252/embr.202050193
Nat Methods. 2021 May 06.

Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions.

Haiqing Xiong, Yingjie Luo, Qianhao Wang, Xianhong Yu, Aibin He.

Deciphering mechanisms in cell-fate decisions requires single-cell holistic reconstructions of multidimensional epigenomic states in transcriptional regulation. Here we develop CoTECH, a combinatorial barcoding method allowing high-throughput single-cell joint detection of chromatin occupancy and transcriptome. We used CoTECH to examine bivalent histone marks (H3K4me3 and H3K27me3) with transcription from naive to primed mouse embryonic stem cells. We also derived concurrent bivalent marks in pseudosingle cells using transcriptome as an anchor for resolving pseudotemporal bivalency trajectories and disentangling a context-specific interplay between H3K4me3/H3K27me3 and transcription level. Next, we revealed the regulatory basis of endothelial-to-hematopoietic transition in two waves of hematopoietic cells and distinctive enhancer-gene-linking schemes guiding hemogenic endothelial cell emergence, indicating a unique epigenetic control of transcriptional regulation for hematopoietic stem cell priming. CoTECH provides an efficient framework for single-cell coassay of chromatin occupancy and transcription, thus enabling higher-dimensional epigenomic reconstructions.

DOI: https://doi.org/10.1038/s41592-021-01129-z
Nat Commun. 2021 May 06. 12(1): 2576

Interplay of two transcription factors for recruitment of the chromatin remodeling complex modulates fungal nitrosative stress response.

Yunqing Jian, Zunyong Liu, Haixia Wang, Yun Chen, Yanni Yin, Youfu Zhao, Zhonghua Ma.

Nitric oxide (NO) is a diffusible signaling molecule that modulates animal and plant immune responses. In addition, reactive nitrogen species derived from NO can display antimicrobial activities by reacting with microbial cellular components, leading to nitrosative stress (NS) in pathogens. Here, we identify FgAreB as a regulator of the NS response in Fusarium graminearum, a fungal pathogen of cereal crops. FgAreB serves as a pioneer transcription factor for recruitment of the chromatin-remodeling complex SWI/SNF at the promoters of genes involved in the NS response, thus promoting their transcription. FgAreB plays important roles in fungal infection and growth. Furthermore, we show that a transcription repressor (FgIxr1) competes with the SWI/SNF complex for FgAreB binding, and negatively regulates the NS response. NS, in turn, promotes the degradation of FgIxr1, thus enhancing the recruitment of the SWI/SNF complex by FgAreB.

DOI: https://doi.org/10.1038/s41467-021-22831-8
Nucleic Acids Res. 2021 May 03. pii: gkab313. [Epub ahead of print]

HDAC3 controls male fertility through enzyme-independent transcriptional regulation at the meiotic exit of spermatogenesis.

Huiqi Yin, Zhenlong Kang, Yingwen Zhang, Yingyun Gong, Mengrou Liu, Yanfeng Xue, Wenxiu He, Yanfeng Wang, Shuya Zhang, Qiushi Xu, Kaiqiang Fu, Bangjin Zheng, Jie Xie, Jinwen Zhang, Yuanyuan Wang, Mingyan Lin, Yihan Zhang, Hua Feng, Changpeng Xin, Yichun Guan, Chaoyang Huang, Xuejiang Guo, P Jeremy Wang, Joseph A Baur, Ke Zheng, Zheng Sun, Lan Ye.

The transition from meiotic spermatocytes to postmeiotic haploid germ cells constitutes an essential step in spermatogenesis. The epigenomic regulatory mechanisms underlying this transition remain unclear. Here, we find a prominent transcriptomic switch from the late spermatocytes to the early round spermatids during the meiotic-to-postmeiotic transition, which is associated with robust histone acetylation changes across the genome. Among histone deacetylases (HDACs) and acetyltransferases, we find that HDAC3 is selectively expressed in the late meiotic and early haploid stages. Three independent mouse lines with the testis-specific knockout of HDAC3 show infertility and defects in meiotic exit with an arrest at the late stage of meiosis or early stage of round spermatids. Stage-specific RNA-seq and histone acetylation ChIP-seq analyses reveal that HDAC3 represses meiotic/spermatogonial genes and activates postmeiotic haploid gene programs during meiotic exit, with associated histone acetylation alterations. Unexpectedly, abolishing HDAC3 catalytic activity by missense mutations in the nuclear receptor corepressor (NCOR or SMRT) does not cause infertility, despite causing histone hyperacetylation as HDAC3 knockout, demonstrating that HDAC3 enzyme activity is not required for spermatogenesis. Motif analysis of the HDAC3 cistrome in the testes identified SOX30, which has a similar spatiotemporal expression pattern as HDAC3 during spermatogenesis. Depletion of SOX30 in the testes abolishes the genomic recruitment of the HDAC3 to the binding sites. Collectively, these results establish the SOX30/HDAC3 signaling as a key regulator of the transcriptional program in a deacetylase-independent manner during the meiotic-to-postmeiotic transition in spermatogenesis.

DOI: https://doi.org/10.1093/nar/gkab313
Nat Biotechnol. 2021 May 06.

Extended-representation bisulfite sequencing of gene regulatory elements in multiplexed samples and single cells.

Sarah J Shareef, Samantha M Bevill, Ayush T Raman, Martin J Aryee, Peter van Galen, Volker Hovestadt, Bradley E Bernstein.

The biological roles of DNA methylation have been elucidated by profiling methods based on whole-genome or reduced-representation bisulfite sequencing, but these approaches do not efficiently survey the vast numbers of non-coding regulatory elements in mammalian genomes. Here we present an extended-representation bisulfite sequencing (XRBS) method for targeted profiling of DNA methylation. Our design strikes a balance between expanding coverage of regulatory elements and reproducibly enriching informative CpG dinucleotides in promoters, enhancers and CTCF binding sites. Barcoded DNA fragments are pooled before bisulfite conversion, allowing multiplex processing and technical consistency in low-input samples. Application of XRBS to single leukemia cells enabled us to evaluate genetic copy number variations and methylation variability across individual cells. Our analysis highlights heterochromatic H3K9me3 regions as having the highest cell-to-cell variability in their methylation, likely reflecting inherent epigenetic instability of these late-replicating regions, compounded by differences in cell cycle stages among sampled cells.

DOI: https://doi.org/10.1038/s41587-021-00910-x
Sci Adv. 2021 May;pii: eabe7804. [Epub ahead of print]7(19):

SMARCAD1-mediated active replication fork stability maintains genome integrity.

Calvin Shun Yu Lo, Marvin van Toorn, Vincent Gaggioli, Mariana Paes Dias, Yifan Zhu, Eleni Maria Manolika, Wei Zhao, Marit van der Does, Chirantani Mukherjee, João G S C Souto Gonçalves, Martin E van Royen, Pim J French, Jeroen Demmers, Ihor Smal, Hannes Lans, David Wheeler, Jos Jonkers, Arnab Ray Chaudhuri, Jurgen A Marteijn, Nitika Taneja.

The stalled fork protection pathway mediated by breast cancer 1/2 (BRCA1/2) proteins is critical for replication fork stability. However, it is unclear whether additional mechanisms are required to maintain replication fork stability. We describe a hitherto unknown mechanism, by which the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily-A containing DEAD/H box-1 (SMARCAD1) stabilizes active replication forks, that is essential to maintaining resistance towards replication poisons. We find that SMARCAD1 prevents accumulation of 53BP1-associated nucleosomes to preclude toxic enrichment of 53BP1 at the forks. In the absence of SMARCAD1, 53BP1 mediates untimely dissociation of PCNA via the PCNA-unloader ATAD5, causing frequent fork stalling, inefficient fork restart, and accumulation of single-stranded DNA. Although loss of 53BP1 in SMARCAD1 mutants rescues these defects and restores genome stability, this rescued stabilization also requires BRCA1-mediated fork protection. Notably, fork protection-challenged BRCA1-deficient naïve- or chemoresistant tumors require SMARCAD1-mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation.

DOI: https://doi.org/10.1126/sciadv.abe7804
Nat Commun. 2021 05 05. 12(1): 2555

Co-occupancy identifies transcription factor co-operation for axon growth.

Ishwariya Venkatesh, Vatsal Mehra, Zimei Wang, Matthew T Simpson, Erik Eastwood, Advaita Chakraborty, Zac Beine, Derek Gross, Michael Cabahug, Greta Olson, Murray G Blackmore.

Transcription factors (TFs) act as powerful levers to regulate neural physiology and can be targeted to improve cellular responses to injury or disease. Because TFs often depend on cooperative activity, a major challenge is to identify and deploy optimal sets. Here we developed a bioinformatics pipeline, centered on TF co-occupancy of regulatory DNA, and used it to predict factors that potentiate the effects of pro-regenerative Klf6 in vitro. High content screens of neurite outgrowth identified cooperative activity by 12 candidates, and systematic testing in a mouse model of corticospinal tract (CST) damage substantiated three novel instances of pairwise cooperation. Combined Klf6 and Nr5a2 drove the strongest growth, and transcriptional profiling of CST neurons identified Klf6/Nr5a2-responsive gene networks involved in macromolecule biosynthesis and DNA repair. These data identify TF combinations that promote enhanced CST growth, clarify the transcriptional correlates, and provide a bioinformatics approach to detect TF cooperation.

DOI: https://doi.org/10.1038/s41467-021-22828-3
Nat Commun. 2021 05 03. 12(1): 2495

Learning a genome-wide score of human-mouse conservation at the functional genomics level.

Soo Bin Kwon, Jason Ernst.

Identifying genomic regions with functional genomic properties that are conserved between human and mouse is an important challenge in the context of mouse model studies. To address this, we develop a method to learn a score of evidence of conservation at the functional genomics level by integrating information from a compendium of epigenomic, transcription factor binding, and transcriptomic data from human and mouse. The method, Learning Evidence of Conservation from Integrated Functional genomic annotations (LECIF), trains neural networks to generate this score for the human and mouse genomes. The resulting LECIF score highlights human and mouse regions with shared functional genomic properties and captures correspondence of biologically similar human and mouse annotations. Analysis with independent datasets shows the score also highlights loci associated with similar phenotypes in both species. LECIF will be a resource for mouse model studies by identifying loci whose functional genomic properties are likely conserved.

DOI: https://doi.org/10.1038/s41467-021-22653-8
PLoS Comput Biol. 2021 May 05. 17(5): e1008974

The hierarchical packing of euchromatin domains can be described as multiplicative cascades.

Amra Noa, Hui-Shun Kuan, Vera Aschmann, Vasily Zaburdaev, Lennart Hilbert.

The genome is packed into the cell nucleus in the form of chromatin. Biochemical approaches have revealed that chromatin is packed within domains, which group into larger domains, and so forth. Such hierarchical packing is equally visible in super-resolution microscopy images of large-scale chromatin organization. While previous work has suggested that chromatin is partitioned into distinct domains via microphase separation, it is unclear how these domains organize into this hierarchical packing. A particular challenge is to find an image analysis approach that fully incorporates such hierarchical packing, so that hypothetical governing mechanisms of euchromatin packing can be compared against the results of such an analysis. Here, we obtain 3D STED super-resolution images from pluripotent zebrafish embryos labeled with improved DNA fluorescence stains, and demonstrate how the hierarchical packing of euchromatin in these images can be described as multiplicative cascades. Multiplicative cascades are an established theoretical concept to describe the placement of ever-smaller structures within bigger structures. Importantly, these cascades can generate artificial image data by applying a single rule again and again, and can be fully specified using only four parameters. Here, we show how the typical patterns of euchromatin organization are reflected in the values of these four parameters. Specifically, we can pinpoint the values required to mimic a microphase-separated state of euchromatin. We suggest that the concept of multiplicative cascades can also be applied to images of other types of chromatin. Here, cascade parameters could serve as test quantities to assess whether microphase separation or other theoretical models accurately reproduce the hierarchical packing of chromatin.

DOI: https://doi.org/10.1371/journal.pcbi.1008974
Cell. 2021 Apr 27. pii: S0092-8674(21)00439-6. [Epub ahead of print]

A single-embryo, single-cell time-resolved model for mouse gastrulation.

Markus Mittnenzweig, Yoav Mayshar, Saifeng Cheng, Raz Ben-Yair, Ron Hadas, Yoach Rais, Elad Chomsky, Netta Reines, Anna Uzonyi, Lior Lumerman, Aviezer Lifshitz, Zohar Mukamel, Ayelet-Hashahar Orenbuch, Amos Tanay, Yonatan Stelzer.

  Mouse embryonic development is a canonical model system for studying mammalian cell fate acquisition. Recently, single-cell atlases comprehensively charted embryonic transcriptional landscapes, yet inference of the coordinated dynamics of cells over such atlases remains challenging. Here, we introduce a temporal model for mouse gastrulation, consisting of data from 153 individually sampled embryos spanning 36 h of molecular diversification. Using algorithms and precise timing, we infer differentiation flows and lineage specification dynamics over the embryonic transcriptional manifold. Rapid transcriptional bifurcations characterize the commitment of early specialized node and blood cells. However, for most lineages, we observe combinatorial multi-furcation dynamics rather than hierarchical transcriptional transitions. In the mesoderm, dozens of transcription factors combinatorially regulate multifurcations, as we exemplify using time-matched chimeric embryos of Foxc1/Foxc2 mutants. Our study rejects the notion of differentiation being governed by a series of binary choices, providing an alternative quantitative model for cell fate acquisition.

Keywords:  cell fate decisions; chimera assay; developmental biology; mouse gastrulation; network flow model; scRNA-seq; tetraploid complementation assay; trajectory inference

DOI:  https://doi.org/10.1016/j.cell.2021.04.004
Nat Commun. 2021 05 04. 12(1): 2505

Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells.

Elena Enzo, Alessia Secone Seconetti, Mattia Forcato, Elena Tenedini, Maria Pia Polito, Irene Sala, Sonia Carulli, Roberta Contin, Clelia Peano, Enrico Tagliafico, Silvio Bicciato, Sergio Bondanza, Michele De Luca.

Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.

DOI: https://doi.org/10.1038/s41467-021-22779-9
Nature. 2021 May 05.

Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity.

Gabriel K Griffin, Jingyi Wu, Arvin Iracheta-Vellve, James C Patti, Jeffrey Hsu, Thomas Davis, Deborah Dele-Oni, Peter P Du, Aya G Halawi, Jeffrey J Ishizuka, Sarah Y Kim, Susan Klaeger, Nelson H Knudsen, Brian C Miller, Tung H Nguyen, Kira E Olander, Malvina Papanastasiou, Suzanna Rachimi, Emily J Robitschek, Emily M Schneider, Mitchell D Yeary, Margaret D Zimmer, Jacob D Jaffe, Steven A Carr, John G Doench, W Nicholas Haining, Kathleen B Yates, Robert T Manguso, Bradley E Bernstein.

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape1,2. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape3-5. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution6. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.

DOI: https://doi.org/10.1038/s41586-021-03520-4
Nucleic Acids Res. 2021 May 03. pii: gkab319. [Epub ahead of print]

PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing.

Gue-Ho Hwang, You Kyeong Jeong, Omer Habib, Sung-Ah Hong, Kayeong Lim, Jin-Soo Kim, Sangsu Bae.

Prime editing technology is capable of generating targeted insertions, deletions, and base conversions. However, the process of designing prime editing guide RNAs (pegRNAs), which contain a primer binding site and a reverse-transcription template at the 3' end, is more complex than that for the single guide RNAs used with CRISPR nucleases or base editors. Furthermore, the assessment of high-throughput sequencing data after prime editors (PEs) have been employed should consider the unique feature of PEs; thus, pre-existing assessment tools cannot directly be adopted for PEs. Here, we present two user-friendly web-based tools for PEs, named PE-Designer and PE-Analyzer. PE-Designer, a dedicated tool for pegRNA selection, provides all possible target sequences, pegRNA extension sequences, and nicking guide RNA sequences together with useful information, and displays the results in an interactive image. PE-Analyzer, a dedicated tool for PE outcome analysis, accepts high-throughput sequencing data, summarizes mutation-related information in a table, and provides interactive graphs. PE-Analyzer was mainly written using JavaScript so that it can analyze several data sets without requiring that huge sequencing data (>100MB) be uploaded to the server, reducing analysis time and increasing personal security. PE-Designer and PE-Analyzer are freely available at http://www.rgenome.net/pe-designer/ and http://www.rgenome.net/pe-analyzer/ without a login process.

DOI: https://doi.org/10.1093/nar/gkab319
Nat Methods. 2021 May;18(5): 482-490

Comparison of the Hi-C, GAM and SPRITE methods using polymer models of chromatin.

Luca Fiorillo, Francesco Musella, Mattia Conte, Rieke Kempfer, Andrea M Chiariello, Simona Bianco, Alexander Kukalev, Ibai Irastorza-Azcarate, Andrea Esposito, Alex Abraham, Antonella Prisco, Ana Pombo, Mario Nicodemi.

Hi-C, split-pool recognition of interactions by tag extension (SPRITE) and genome architecture mapping (GAM) are powerful technologies utilized to probe chromatin interactions genome wide, but how faithfully they capture three-dimensional (3D) contacts and how they perform relative to each other is unclear, as no benchmark exists. Here, we compare these methods in silico in a simplified, yet controlled, framework against known 3D structures of polymer models of murine and human loci, which can recapitulate Hi-C, GAM and SPRITE experiments and multiplexed fluorescence in situ hybridization (FISH) single-molecule conformations. We find that in silico Hi-C, GAM and SPRITE bulk data are faithful to the reference 3D structures whereas single-cell data reflect strong variability among single molecules. The minimal number of cells required in replicate experiments to return statistically similar contacts is different across the technologies, being lowest in SPRITE and highest in GAM under the same conditions. Noise-to-signal levels follow an inverse power law with detection efficiency and grow with genomic distance differently among the three methods, being lowest in GAM for genomic separations >1 Mb.

DOI: https://doi.org/10.1038/s41592-021-01135-1
Nat Commun. 2021 May 07. 12(1): 2564

Sox9 and Rbpj differentially regulate endothelial to mesenchymal transition and wound scarring in murine endovascular progenitors.

Jilai Zhao, Jatin Patel, Simranpreet Kaur, Seen-Ling Sim, Ho Yi Wong, Cassandra Styke, Isabella Hogan, Sam Kahler, Hamish Hamilton, Racheal Wadlow, James Dight, Ghazaleh Hashemi, Laura Sormani, Edwige Roy, Mervin C Yoder, Mathias Francois, Kiarash Khosrotehrani.

Endothelial to mesenchymal transition (EndMT) is a leading cause of fibrosis and disease, however its mechanism has yet to be elucidated. The endothelium possesses a profound regenerative capacity to adapt and reorganize that is attributed to a population of vessel-resident endovascular progenitors (EVP) governing an endothelial hierarchy. Here, using fate analysis, we show that two transcription factors SOX9 and RBPJ specifically affect the murine EVP numbers and regulate lineage specification. Conditional knock-out of Sox9 from the vasculature (Sox9fl/fl/Cdh5-CreER RosaYFP) depletes EVP while enhancing Rbpj expression and canonical Notch signalling. Additionally, skin wound analysis from Sox9 conditional knock-out mice demonstrates a significant reduction in pathological EndMT resulting in reduced scar area. The converse is observed with Rbpj conditionally knocked-out from the murine vasculature (Rbpjfl/fl/Cdh5-CreER RosaYFP) or inhibition of Notch signaling in human endothelial colony forming cells, resulting in enhanced Sox9 and EndMT related gene (Snail, Slug, Twist1, Twist2, TGF-β) expression. Similarly, increased endothelial hedgehog signaling (Ptch1fl/fl/Cdh5-CreER RosaYFP), that upregulates the expression of Sox9 in cells undergoing pathological EndMT, also results in excess fibrosis. Endothelial cells transitioning to a mesenchymal fate express increased Sox9, reduced Rbpj and enhanced EndMT. Importantly, using topical administration of siRNA against Sox9 on skin wounds can substantially reduce scar area by blocking pathological EndMT. Overall, here we report distinct fates of EVPs according to the relative expression of Rbpj or Notch signalling and Sox9, highlighting their potential plasticity and opening exciting avenues for more effective therapies in fibrotic diseases.

DOI: https://doi.org/10.1038/s41467-021-22717-9
Oncogene. 2021 May 06.

C/EBPβ is a MYB- and p300-cooperating pro-leukemogenic factor and promising drug target in acute myeloid leukemia.

Maria V Yusenko, Amke Trentmann, Debora A Casolari, Luca Abdel Ghani, Mairin Lenz, Melanie Horn, Wolfgang Dörner, Stefan Klempnauer, Henning D Mootz, Maria Francisca Arteaga, Jan-Henrik Mikesch, Richard J D' Andrea, Thomas J Gonda, Carsten Müller-Tidow, Thomas J Schmidt, Karl-Heinz Klempnauer.

Transcription factor MYB has recently emerged as a promising drug target for the treatment of acute myeloid leukemia (AML). Here, we have characterized a group of natural sesquiterpene lactones (STLs), previously shown to suppress MYB activity, for their potential to decrease AML cell proliferation. Unlike what was initially thought, these compounds inhibit MYB indirectly via its cooperation partner C/EBPβ. C/EBPβ-inhibitory STLs affect the expression of a large number of MYB-regulated genes, suggesting that the cooperation of MYB and C/EBPβ broadly shapes the transcriptional program of AML cells. We show that expression of GFI1, a direct MYB target gene, is controlled cooperatively by MYB, C/EBPβ, and co-activator p300, and is down-regulated by C/EBPβ-inhibitory STLs, exemplifying that they target the activity of composite MYB-C/EBPβ-p300 transcriptional modules. Ectopic expression of GFI1, a zinc-finger protein that is required for the maintenance of hematopoietic stem and progenitor cells, partially abrogated STL-induced myelomonocytic differentiation, implicating GFI1 as a relevant target of C/EBPβ-inhibitory STLs. Overall, our data identify C/EBPβ as a pro-leukemogenic factor in AML and suggest that targeting of C/EBPβ may have therapeutic potential against AML.

DOI: https://doi.org/10.1038/s41388-021-01800-x
Nucleic Acids Res. 2021 May 06. pii: gkab325. [Epub ahead of print]

CeLaVi: an interactive cell lineage visualization tool.

Irepan Salvador-Martínez, Marco Grillo, Michalis Averof, Maximilian J Telford.

Recent innovations in genetics and imaging are providing the means to reconstruct cell lineages, either by tracking cell divisions using live microscopy, or by deducing the history of cells using molecular recorders. A cell lineage on its own, however, is simply a description of cell divisions as branching events. A major goal of current research is to integrate this description of cell relationships with information about the spatial distribution and identities of the cells those divisions produce. Visualizing, interpreting and exploring these complex data in an intuitive manner requires the development of new tools. Here we present CeLaVi, a web-based visualization tool that allows users to navigate and interact with a representation of cell lineages, whilst simultaneously visualizing the spatial distribution, identities and properties of cells. CeLaVi's principal functions include the ability to explore and manipulate the cell lineage tree; to visualise the spatial distribution of cell clones at different depths of the tree; to colour cells in the 3D viewer based on lineage relationships; to visualise various cell qualities on the 3D viewer (e.g. gene expression, cell type) and to annotate selected cells/clones. All these capabilities are demonstrated with four different example data sets. CeLaVi is available at http://www.celavi.pro.

DOI: https://doi.org/10.1093/nar/gkab325