bims-crepig 2022-01-16 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2022–01–16
28 papers selected by
Connor Rogerson, University of Cambridge, MRC Cancer Unit

Distinct transcription kinetics of pluripotent cell states.
MyoD is a 3D genome structure organizer for muscle cell identity.
Identification of a modular super-enhancer in murine retinal development.
Identification and functional characterization of transcriptional activators in human cells.
INO80 requires a polycomb subunit to regulate the establishment of poised chromatin in murine spermatocytes.
MUC1-C Dictates JUN and BAF-mediated Chromatin Remodeling at Enhancer Signatures in Cancer Stem Cells.
HIF-1 Interacts with TRIM28 and DNA-PK to release paused RNA polymerase II and activate target gene transcription in response to hypoxia.
CUT&Tag-BS for simultaneous profiling of histone modification and DNA methylation with high efficiency and low cost.
Tcf1 is essential for initiation of oncogenic Notch1-driven chromatin topology in T-ALL.
2-Hydroxyglutarate destabilizes chromatin regulatory landscape and lineage fidelity to promote cellular heterogeneity.
Identification of sequence-specific promoters driving polycistronic transcription initiation by RNA polymerase II in trypanosomes.
Chromatin topology defines estradiol-primed progesterone receptor and PAX2 binding in endometrial cancer cells.
Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.
Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.
KAS-seq: genome-wide sequencing of single-stranded DNA by N3-kethoxal-assisted labeling.
Single-cell chromatin accessibility landscape in kidney identifies additional cell-of-origin in heterogenous papillary renal cell carcinoma.
Modeling uniquely human gene regulatory function via targeted humanization of the mouse genome.
A GATA4-regulated secretory program suppresses tumors through recruitment of cytotoxic CD8 T cells.
The activity of human enhancers is modulated by the splicing of their associated lncRNAs.
The CLASSY family controls tissue-specific DNA methylation patterns in Arabidopsis.
The transcription factor ZIP-1 promotes resistance to intracellular infection in Caenorhabditis elegans.
LuxRep: a technical replicate-aware method for bisulfite sequencing data analysis.
YB-1 is a positive regulator of KLF5 transcription factor in basal-like breast cancer.
G-quadruplex DNA structures in human stem cells and differentiation.
Canonical WNT signaling-dependent gating of MYC requires a noncanonical CTCF function at a distal binding site.
A deep generative model for multi-view profiling of single-cell RNA-seq and ATAC-seq data.
Single-cell analysis of human primary prostate cancer reveals the heterogeneity of tumor-associated epithelial cell states.
C/EBPB-dependent adaptation to palmitic acid promotes tumor formation in hormone receptor negative breast cancer.

Mol Syst Biol. 2022 Jan;18(1): e10407

Distinct transcription kinetics of pluripotent cell states.

Rui Shao, Banushree Kumar, Katja Lidschreiber, Michael Lidschreiber, Patrick Cramer, Simon J Elsässer.

  Mouse embryonic stem cells (mESCs) can adopt naïve, ground, and paused pluripotent states that give rise to unique transcriptomes. Here, we use transient transcriptome sequencing (TT-seq) to define both coding and non-coding transcription units (TUs) in these three pluripotent states and combine TT-seq with RNA polymerase II occupancy profiling to unravel the kinetics of RNA metabolism genome-wide. Compared to the naïve state (serum), RNA synthesis and turnover rates are globally reduced in the ground state (2i) and the paused state (mTORi). The global reduction in RNA synthesis goes along with a genome-wide decrease of polymerase elongation velocity, which is related to epigenomic features and alterations in the Pol II termination window. Our data suggest that transcription activity is the main determinant of steady state mRNA levels in the naïve state and that genome-wide changes in transcription kinetics invoke ground and paused pluripotent states.

Keywords:  mouse pluripotent stem cells; transcription termination; transcription unit annotation; transcription velocity; transient transcriptome sequencing

DOI:  https://doi.org/10.15252/msb.202110407
Nat Commun. 2022 Jan 11. 13(1): 205

MyoD is a 3D genome structure organizer for muscle cell identity.

Ruiting Wang, Fengling Chen, Qian Chen, Xin Wan, Minglei Shi, Antony K Chen, Zhao Ma, Guohong Li, Min Wang, Yachen Ying, Qinyao Liu, Hu Li, Xu Zhang, Jinbiao Ma, Jiayun Zhong, Meihong Chen, Michael Q Zhang, Yong Zhang, Yang Chen, Dahai Zhu.

The genome exists as an organized, three-dimensional (3D) dynamic architecture, and each cell type has a unique 3D genome organization that determines its cell identity. An unresolved question is how cell type-specific 3D genome structures are established during development. Here, we analyzed 3D genome structures in muscle cells from mice lacking the muscle lineage transcription factor (TF), MyoD, versus wild-type mice. We show that MyoD functions as a "genome organizer" that specifies 3D genome architecture unique to muscle cell development, and that H3K27ac is insufficient for the establishment of MyoD-induced chromatin loops in muscle cells. Moreover, we present evidence that other cell lineage-specific TFs might also exert functional roles in orchestrating lineage-specific 3D genome organization during development.

DOI: https://doi.org/10.1038/s41467-021-27865-6
Nat Commun. 2022 Jan 11. 13(1): 253

Identification of a modular super-enhancer in murine retinal development.

Victoria Honnell, Jackie L Norrie, Anand G Patel, Cody Ramirez, Jiakun Zhang, Yu-Hsuan Lai, Shibiao Wan, Michael A Dyer.

Super-enhancers are expansive regions of genomic DNA comprised of multiple putative enhancers that contribute to the dynamic gene expression patterns during development. This is particularly important in neurogenesis because many essential transcription factors have complex developmental stage- and cell-type specific expression patterns across the central nervous system. In the developing retina, Vsx2 is expressed in retinal progenitor cells and is maintained in differentiated bipolar neurons and Müller glia. A single super-enhancer controls this complex and dynamic pattern of expression. Here we show that deletion of one region disrupts retinal progenitor cell proliferation but does not affect cell fate specification. The deletion of another region has no effect on retinal progenitor cell proliferation but instead leads to a complete loss of bipolar neurons. This prototypical super-enhancer may serve as a model for dissecting the complex gene expression patterns for neurogenic transcription factors during development. Moreover, it provides a unique opportunity to alter expression of individual transcription factors in particular cell types at specific stages of development. This provides a deeper understanding of function that cannot be achieved with traditional knockout mouse approaches.

DOI: https://doi.org/10.1038/s41467-021-27924-y
Mol Cell. 2022 Jan 05. pii: S1097-2765(21)01070-4. [Epub ahead of print]

Identification and functional characterization of transcriptional activators in human cells.

Nader Alerasool, He Leng, Zhen-Yuan Lin, Anne-Claude Gingras, Mikko Taipale.

  Transcription is orchestrated by thousands of transcription factors (TFs) and chromatin-associated proteins, but how these are causally connected to transcriptional activation is poorly understood. Here, we conduct an unbiased proteome-scale screen to systematically uncover human proteins that activate transcription in a natural chromatin context. By combining interaction proteomics and chemical inhibitors, we delineate the preference of these transcriptional activators for specific co-activators, highlighting how even closely related TFs can function via distinct cofactors. We also identify potent transactivation domains among the hits and use AlphaFold2 to predict and experimentally validate interaction interfaces of two activation domains with BRD4. Finally, we show that many novel activators are partners in fusion events in tumors and functionally characterize a myofibroma-associated fusion between SRF and C3orf62, a potent p300-dependent activator. Our work provides a functional catalog of potent transactivators in the human proteome and a platform for discovering transcriptional regulators at genome scale.

Keywords:  AlphaFold2; activation domain; cancer; functional genomics; functional proteomics; fusion proteins; transcription; transcriptional activator

DOI:  https://doi.org/10.1016/j.molcel.2021.12.008
Development. 2022 Jan 01. pii: dev200089. [Epub ahead of print]149(1):

INO80 requires a polycomb subunit to regulate the establishment of poised chromatin in murine spermatocytes.

Prabuddha Chakraborty, Terry Magnuson.

  INO80 is the catalytic subunit of the INO80-chromatin remodeling complex that is involved in DNA replication, repair and transcription regulation. Ino80 deficiency in murine spermatocytes (Ino80cKO) results in pachytene arrest of spermatocytes due to incomplete synapsis and aberrant DNA double-strand break repair, which leads to apoptosis. RNA-seq on Ino80cKO spermatocytes revealed major changes in transcription, indicating that an aberrant transcription program arises upon INO80 depletion. In Ino80WT spermatocytes, genome-wide analysis showed that INO80-binding sites were mostly promoter proximal and necessary for the regulation of spermatogenic gene expression, primarily of premeiotic and meiotic genes. Furthermore, most of the genes poised for activity, as well as those genes that are active, shared INO80 binding. In Ino80cKO spermatocytes, most poised genes demonstrated de-repression due to reduced H3K27me3 enrichment and, in turn, showed increased expression levels. INO80 interacts with the core PRC2 complex member SUZ12 and promotes its recruitment. Furthermore, INO80 mediates H2A.Z incorporation at the poised promoters, which was reduced in Ino80cKO spermatocytes. Taken together, INO80 is emerging as a major regulator of the meiotic transcription program by mediating poised chromatin establishment through SUZ12 binding.

Keywords:  Chromatin remodeler; Germ cells; INO80; Poised chromatin; Polycomb subunits; Spermatogenesis

DOI:  https://doi.org/10.1242/dev.200089
Mol Cancer Res. 2022 Jan 12. pii: molcanres.MCR-21-0672-E.2021. [Epub ahead of print]

MUC1-C Dictates JUN and BAF-mediated Chromatin Remodeling at Enhancer Signatures in Cancer Stem Cells.

Atrayee Bhattacharya, Atsushi Fushimi, Nami Yamashita, Masayuki Hagiwara, Yoshihiro Morimoto, Hasan Rajabi, Mark D Long, Maha Abdulla, Rehan Ahmad, Kelly Street, Song Liu, Tao Liu, Donald Kufe.

The oncogenic MUC1-C protein promotes dedifferentiation of castrate-resistant prostate cancer (CRPC) and triple-negative breast cancer (TNBC) cells. Chromatin remodeling is critical for the cancer stem cell (CSC) state; however, there is no definitive evidence that MUC1-C regulates chromatin accessibility and thereby expression of stemness-associated genes. We demonstrate that MUC1-C drives global changes in chromatin architecture in the dedifferentiation of CRPC and TNBC cells. Our results show that MUC1-C induces differentially accessible regions (DARs) across their genomes, which are significantly associated with differentially expressed genes (DEGs). Motif and cistrome analysis further demonstrated MUC1-C-induced DARs align with genes regulated by the JUN/AP-1 family of transcription factors. MUC1-C activates the BAF chromatin remodeling complex, which is recruited by JUN in enhancer selection. In studies of the NOTCH1 gene, which is required for CRPC and TNBC cell self-renewal, we demonstrate that MUC1-C is necessary for (i) occupancy of JUN and ARID1A/BAF, (ii) increases in H3K27ac and H3K4me3 signals, and (iii) opening of chromatin accessibility on a proximal enhancer-like signature. Studies of the EGR1 and LY6E stemness-associated genes further demonstrate that MUC1-C-induced JUN/ARID1A complexes regulate chromatin accessibility on proximal and distal enhancer-like signatures. These findings uncover a role for MUC1-C in chromatin remodeling that is mediated at least in part by JUN/AP-1 and ARID1A/BAF in association with driving the CSC state. Implications: These findings show that MUC1-C, which is necessary for the CRPC and TNBC CSC state, activates a novel pathway involving JUN/AP-1 and ARID1A/BAF that regulates chromatin accessibility of stemness-associated gene enhancers.

DOI: https://doi.org/10.1158/1541-7786.MCR-21-0672
Nat Commun. 2022 Jan 14. 13(1): 316

HIF-1 Interacts with TRIM28 and DNA-PK to release paused RNA polymerase II and activate target gene transcription in response to hypoxia.

Yongkang Yang, Haiquan Lu, Chelsey Chen, Yajing Lyu, Robert N Cole, Gregg L Semenza.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a regulator of oxygen (O2) homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions but pauses after approximately 30-60 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1α and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain as well as the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies reveal a molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.

DOI: https://doi.org/10.1038/s41467-021-27944-8
Cell Rep Methods. 2021 Dec 20. pii: 100118. [Epub ahead of print]1(8):

CUT&Tag-BS for simultaneous profiling of histone modification and DNA methylation with high efficiency and low cost.

Ruifang Li, Sara A Grimm, Paul A Wade.

It remains a challenge to decipher the complex relationship between DNA methylation, histone modification, and the underlying DNA sequence with limited input material. Here, we developed an efficient, low-input, and low-cost method for the simultaneous profiling of genomic localization of histone modification and methylation status of the underlying DNA at single-base resolution from the same cells in a single experiment by integrating cleavage under targets and tagmentation (CUT&Tag) with tagmentation-based bisulfite sequencing (CUT&Tag-BS). We demonstrated the validity of our method using representative histone modifications of euchromatin and constitutive and facultative heterochromatin (H3K4me1, H3K9me3, and H3K27me3, respectively). Similar histone modification enrichment patterns were observed in CUT&Tag-BS compared with non-bisulfite-treated control, and H3K4me1-marked regions were found to mostly be CpG poor, lack methylation concordance, and exhibit prevalent DNA methylation heterogeneity among mouse embryonic stem cells (mESCs). We anticipate that CUT&Tag-BS will be widely applied to directly address the genomic relationship between DNA methylation and histone modification, especially in low-input scenarios with precious biological samples.

DOI: https://doi.org/10.1016/j.crmeth.2021.100118
Blood. 2022 Jan 12. pii: blood.2021012077. [Epub ahead of print]

Tcf1 is essential for initiation of oncogenic Notch1-driven chromatin topology in T-ALL.

Mateusz Antoszewski, Nadine Fournier, Gustavo A Ruiz Buendía, Joao Lourenco, Yuanlong Liu, Tara Sugrue, Christelle Dubey, Marianne Nkosi, Colin E J Pritchard, Ivo J Huijbers, Gabriela C Segat, Sandra Alonso-Moreno, Elisabeth Serracanta, Laura Belver, Adolfo A Ferrando, Giovanni Ciriello, Andrew P Weng, Ute Koch, Freddy Radtke.

NOTCH1 is a well-established lineage specifier for T cells and amongst the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). How oncogenic NOTCH1 signaling launches a leukemia-prone chromatin landscape during T-ALL initiation is unknown. Here we demonstrate an essential role for the high-mobility-group transcription factor Tcf1 in orchestrating chromatin accessibility and topology allowing aberrant Notch1 signaling to convey its oncogenic function. Although essential, Tcf1 is not sufficient to initiate leukemia. The formation of a leukemia-prone epigenetic landscape at the distal Notch1-regulated Myc enhancer, which is fundamental to this disease, is Tcf1-dependent and occurs within the earliest progenitor stage even before cells adopt a T lymphocyte or leukemic fate. Moreover, we discovered a unique evolutionarily conserved Tcf1-regulated enhancer element in the distal Myc-enhancer, which is important for the transition of pre-leukemic cells to full-blown disease.

DOI: https://doi.org/10.1182/blood.2021012077
Cell Rep. 2022 Jan 11. pii: S2211-1247(21)01724-1. [Epub ahead of print]38(2): 110220

2-Hydroxyglutarate destabilizes chromatin regulatory landscape and lineage fidelity to promote cellular heterogeneity.

Meena Kusi, Maryam Zand, Li-Ling Lin, Meizhen Chen, Anthony Lopez, Chun-Lin Lin, Chiou-Miin Wang, Nicholas D Lucio, Nameer B Kirma, Jianhua Ruan, Tim H-M Huang, Kohzoh Mitsuya.

  The epigenome delineates lineage-specific transcriptional programs and restricts cell plasticity to prevent non-physiological cell fate transitions. Although cell diversification fosters tumor evolution and therapy resistance, upstream mechanisms that regulate the stability and plasticity of the cancer epigenome remain elusive. Here we show that 2-hydroxyglutarate (2HG) not only suppresses DNA repair but also mediates the high-plasticity chromatin landscape. A combination of single-cell epigenomics and multi-omics approaches demonstrates that 2HG disarranges otherwise well-preserved stable nucleosome positioning and promotes cell-to-cell variability. 2HG induces loss of motif accessibility to the luminal-defining transcriptional factors FOXA1, FOXP1, and GATA3 and a shift from luminal to basal-like gene expression. Breast tumors with high 2HG exhibit enhanced heterogeneity with undifferentiated epigenomic signatures linked to adverse prognosis. Further, ascorbate-2-phosphate (A2P) eradicates heterogeneity and impairs growth of high 2HG-producing breast cancer cells. These findings suggest 2HG as a key determinant of cancer plasticity and provide a rational strategy to counteract tumor cell evolution.

Keywords:  2-hydroxyglutarate; DNA repair; breast cancer; cancer cell plasticity; chromatin CyTOF; epigenome fluctuation; lineage fidelity; luminal-to-basal transition; oncometabolite; single-cell epigenomics

DOI:  https://doi.org/10.1016/j.celrep.2021.110220
Cell Rep. 2022 Jan 11. pii: S2211-1247(21)01725-3. [Epub ahead of print]38(2): 110221

Identification of sequence-specific promoters driving polycistronic transcription initiation by RNA polymerase II in trypanosomes.

Carlos Cordon-Obras, Claudia Gomez-Liñan, Sara Torres-Rusillo, Isabel Vidal-Cobo, Diana Lopez-Farfan, Alicia Barroso-Del Jesus, Domingo Rojas-Barros, Mark Carrington, Miguel Navarro.

  Protein-coding genes in trypanosomes occur in polycistronic transcription units (PTUs). How RNA polymerase II (Pol II) initiates transcription of PTUs has not been resolved; the current model favors chromatin modifications inducing transcription rather than sequence-specific promoters. Here, we uncover core promoters by functional characterization of Pol II peaks identified by chromatin immunoprecipitation sequencing (ChIP-seq). Two distinct promoters are located between divergent PTUs, each driving unidirectional transcription. Detailed analysis identifies a 75-bp promoter that is necessary and sufficient to drive full reporter expression and contains functional motifs. Analysis of further promoters suggests transcription initiation is regulated and promoters are either focused or dispersed. In contrast to the previous model of unregulated and promoter-independent transcription initiation, we find that sequence-specific promoters determine the initiation of Pol II transcription of protein-coding genes PTUs. These findings in Trypanosoma brucei suggest that in addition of chromatin modifications, promoter motifs-based regulation of gene expression is deeply conserved among eukaryotes.

Keywords:  African trypanosomes; ChIP-seq; RNA polymerase II; core promoter; gene expression; nucleotide motifs; polycistronic transcription; transcription initiation site; transcriptional activity

DOI:  https://doi.org/10.1016/j.celrep.2021.110221
Elife. 2022 Jan 12. pii: e66034. [Epub ahead of print]11

Chromatin topology defines estradiol-primed progesterone receptor and PAX2 binding in endometrial cancer cells.

Alejandro La Greca, Nicolás Bellora, François Le Dily, Rodrigo Jara, Ana Silvina Nacht, Javier Quilez Oliete, José Luis Villanueva, Enrique Vidal, Gabriela Merino, Cristóbal Fresno, Inti Tarifa Reischle, Griselda Vallejo, Guillermo Pablo Vicent, Elmer Fernández, Miguel Beato, Patricia Saragüeta.

  Estrogen (E2) and Progesterone (Pg), via their specific receptors (ERalpha and PR), are major determinants in the development and progression of endometrial carcinomas, However, their precise mechanism of action and the role of other transcription factors involved are not entirely clear. Using Ishikawa endometrial cancer cells, we report that E2 treatment exposes a set of progestin-dependent PR binding sites which include both E2 and progestin target genes. ChIP-seq results from hormone-treated cells revealed a non-random distribution of PAX2 binding in the vicinity of these estrogen-promoted PR sites. Altered expression of hormone regulated genes in PAX2 knockdown cells suggests a role for PAX2 in fine-tuning ERalpha and PR interplay in transcriptional regulation. Analysis of long-range interactions by Hi-C coupled with ATAC-seq data showed that these regions, that we call 'progestin control regions' (PgCRs), exhibited an open chromatin state even before hormone exposure and were non-randomly associated with regulated genes. Nearly 20% of genes potentially influenced by PgCRs were found to be altered during progression of endometrial cancer. Our findings suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with PAX2 to accessible chromatin regions. What maintains these regions open remains to be studied.

Keywords:  cancer biology; cell biology; human

DOI:  https://doi.org/10.7554/eLife.66034
Proc Natl Acad Sci U S A. 2022 Jan 18. pii: e2105898119. [Epub ahead of print]119(3):

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.

Yiji Liao, Chen-Hao Chen, Tengfei Xiao, Bárbara de la Peña Avalos, Eloise V Dray, Changmeng Cai, Shuai Gao, Neel Shah, Zhao Zhang, Avery Feit, Pengya Xue, Zhijie Liu, Mei Yang, Ji Hoon Lee, Han Xu, Wei Li, Shenglin Mei, Roodolph S Pierre, Shaokun Shu, Teng Fei, Melissa Duarte, Jin Zhao, James E Bradner, Kornelia Polyak, Philip W Kantoff, Henry Long, Steven P Balk, X Shirley Liu, Myles Brown, Kexin Xu.

  Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

Keywords:  DNA damage repair; EZH2 inhibitors; cancer therapy; mechanism of drug action

DOI:  https://doi.org/10.1073/pnas.2105898119
Cell Rep. 2022 Jan 11. pii: S2211-1247(21)01749-6. [Epub ahead of print]38(2): 110240

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Yingying Han, Alvaro Villarreal-Ponce, Guadalupe Gutierrez, Quy Nguyen, Peng Sun, Ting Wu, Benjamin Sui, Geert Berx, Thomas Brabletz, Kai Kessenbrock, Yi Arial Zeng, Kazuhide Watanabe, Xing Dai.

  Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

Keywords:  Axin2; EMT; Ovol2; Wnt signaling; Zeb1; basal stem cell; epithelial-mesenchymal transition; mammary gland; quiescence

DOI:  https://doi.org/10.1016/j.celrep.2021.110240
Nat Protoc. 2022 Jan 10.

KAS-seq: genome-wide sequencing of single-stranded DNA by N3-kethoxal-assisted labeling.

Ruitu Lyu, Tong Wu, Allen C Zhu, Diana C West-Szymanski, Xiaocheng Weng, Mengjie Chen, Chuan He.

Transcription and its dynamics are crucial for gene expression regulation. However, very few methods can directly read out transcriptional activity with low-input material and high temporal resolution. This protocol describes KAS-seq, a robust and sensitive approach for capturing genome-wide single-stranded DNA (ssDNA) profiles using N3-kethoxal-assisted labeling. We developed N3-kethoxal, an azido derivative of kethoxal that reacts with deoxyguanosine bases of ssDNA in live cells within 5-10 min at 37 °C, allowing the capture of dynamic changes. Downstream biotinylation of labeled DNA occurs via copper-free click chemistry. Altogether, the KAS-seq procedure involves N3-kethoxal labeling, DNA isolation, biotinylation, fragmentation, affinity pull-down, library preparation, sequencing and bioinformatics analysis. The pre-library construction labeling and enrichment can be completed in as little as 3-4 h and is applicable to both animal tissue and as few as 1,000 cultured cells. Our recent study shows that ssDNA signals measured by KAS-seq simultaneously reveal the dynamics of transcriptionally engaged RNA polymerase (Pol) II, transcribing enhancers, RNA Pol I and Pol III activities and potentially non-canonical DNA structures with high analytical sensitivity. In addition to the experimental protocol, we also introduce here KAS-pipe, a user-friendly integrative data analysis pipeline for KAS-seq.

DOI: https://doi.org/10.1038/s41596-021-00647-6
Nat Commun. 2022 Jan 10. 13(1): 31

Single-cell chromatin accessibility landscape in kidney identifies additional cell-of-origin in heterogenous papillary renal cell carcinoma.

Qi Wang, Yang Zhang, Bolei Zhang, Yao Fu, Xiaozhi Zhao, Jing Zhang, Ke Zuo, Yuexian Xing, Song Jiang, Zhaohui Qin, Erguang Li, Hongqian Guo, Zhihong Liu, Jingping Yang.

Papillary renal cell carcinoma (pRCC) is the most heterogenous renal cell carcinoma. Patient survival varies and no effective therapies for advanced pRCC exist. Histological and molecular characterization studies have highlighted the heterogeneity of pRCC tumours. Recent studies identified the proximal tubule (PT) cell as a cell-of-origin for pRCC. However, it remains elusive whether other pRCC subtypes have different cell-of-origin. Here, by obtaining genome-wide chromatin accessibility profiles of normal human kidney cells using single-cell transposase-accessible chromatin-sequencing and comparing the profiles with pRCC samples, we discover that besides PT cells, pRCC can also originate from kidney collecting duct principal cells. We show pRCCs with different cell-of-origin exhibit different molecular characteristics and clinical behaviors. Further, metabolic reprogramming appears to mediate the progression of pRCC to the advanced state. Here, our results suggest that determining cell-of-origin and monitoring origin-dependent metabolism could potentially be useful for early diagnosis and treatment of pRCC.

DOI: https://doi.org/10.1038/s41467-021-27660-3
Nat Commun. 2022 Jan 13. 13(1): 304

Modeling uniquely human gene regulatory function via targeted humanization of the mouse genome.

Emily V Dutrow, Deena Emera, Kristina Yim, Severin Uebbing, Acadia A Kocher, Martina Krenzer, Timothy Nottoli, Daniel B Burkhardt, Smita Krishnaswamy, Angeliki Louvi, James P Noonan.

The evolution of uniquely human traits likely entailed changes in developmental gene regulation. Human Accelerated Regions (HARs), which include transcriptional enhancers harboring a significant excess of human-specific sequence changes, are leading candidates for driving gene regulatory modifications in human development. However, insight into whether HARs alter the level, distribution, and timing of endogenous gene expression remains limited. We examined the role of the HAR HACNS1 (HAR2) in human evolution by interrogating its molecular functions in a genetically humanized mouse model. We find that HACNS1 maintains its human-specific enhancer activity in the mouse embryo and modifies expression of Gbx2, which encodes a transcription factor, during limb development. Using single-cell RNA-sequencing, we demonstrate that Gbx2 is upregulated in the limb chondrogenic mesenchyme of HACNS1 homozygous embryos, supporting that HACNS1 alters gene expression in cell types involved in skeletal patterning. Our findings illustrate that humanized mouse models provide mechanistic insight into how HARs modified gene expression in human evolution.

DOI: https://doi.org/10.1038/s41467-021-27899-w
Nat Commun. 2022 Jan 11. 13(1): 256

A GATA4-regulated secretory program suppresses tumors through recruitment of cytotoxic CD8 T cells.

Rupesh S Patel, Rodrigo Romero, Emma V Watson, Anthony C Liang, Megan Burger, Peter M K Westcott, Kim L Mercer, Roderick T Bronson, Eric C Wooten, Arjun Bhutkar, Tyler Jacks, Stephen J Elledge.

The GATA4 transcription factor acts as a master regulator of development of multiple tissues. GATA4 also acts in a distinct capacity to control a stress-inducible pro-inflammatory secretory program that is associated with senescence, a potent tumor suppression mechanism, but also operates in non-senescent contexts such as tumorigenesis. This secretory pathway is composed of chemokines, cytokines, growth factors, and proteases. Since GATA4 is deleted or epigenetically silenced in cancer, here we examine the role of GATA4 in tumorigenesis in mouse models through both loss-of-function and overexpression experiments. We find that GATA4 promotes non-cell autonomous tumor suppression in multiple model systems. Mechanistically, we show that Gata4-dependent tumor suppression requires cytotoxic CD8 T cells and partially requires the secreted chemokine CCL2. Analysis of transcriptome data in human tumors reveals reduced lymphocyte infiltration in GATA4-deficient tumors, consistent with our murine data. Notably, activation of the GATA4-dependent secretory program combined with an anti-PD-1 antibody robustly abrogates tumor growth in vivo.

DOI: https://doi.org/10.1038/s41467-021-27731-5
PLoS Comput Biol. 2022 Jan 11. 18(1): e1009722

The activity of human enhancers is modulated by the splicing of their associated lncRNAs.

Jennifer Yihong Tan, Ana Claudia Marques.

Pervasive enhancer transcription is at the origin of more than half of all long noncoding RNAs in humans. Transcription of enhancer-associated long noncoding RNAs (elncRNA) contribute to their cognate enhancer activity and gene expression regulation in cis. Recently, splicing of elncRNAs was shown to be associated with elevated enhancer activity. However, whether splicing of elncRNA transcripts is a mere consequence of accessibility at highly active enhancers or if elncRNA splicing directly impacts enhancer function, remains unanswered. We analysed genetically driven changes in elncRNA splicing, in humans, to address this outstanding question. We showed that splicing related motifs within multi-exonic elncRNAs evolved under selective constraints during human evolution, suggesting the processing of these transcripts is unlikely to have resulted from transcription across spurious splice sites. Using a genome-wide and unbiased approach, we used nucleotide variants as independent genetic factors to directly assess the causal relationship that underpin elncRNA splicing and their cognate enhancer activity. We found that the splicing of most elncRNAs is associated with changes in chromatin signatures at cognate enhancers and target mRNA expression. We provide evidence that efficient and conserved processing of enhancer-associated elncRNAs contributes to enhancer activity.

DOI: https://doi.org/10.1371/journal.pcbi.1009722
Nat Commun. 2022 Jan 11. 13(1): 244

The CLASSY family controls tissue-specific DNA methylation patterns in Arabidopsis.

Ming Zhou, Ceyda Coruh, Guanghui Xu, Laura M Martins, Clara Bourbousse, Alice Lambolez, Julie A Law.

DNA methylation shapes the epigenetic landscape of the genome, plays critical roles in regulating gene expression, and ensures transposon silencing. As is evidenced by the numerous defects associated with aberrant DNA methylation landscapes, establishing proper tissue-specific methylation patterns is critical. Yet, how such differences arise remains a largely open question in both plants and animals. Here we demonstrate that CLASSY1-4 (CLSY1-4), four locus-specific regulators of DNA methylation, also control tissue-specific methylation patterns, with the most striking pattern observed in ovules where CLSY3 and CLSY4 control DNA methylation at loci with a highly conserved DNA motif. On a more global scale, we demonstrate that specific clsy mutants are sufficient to shift the epigenetic landscape between tissues. Together, these findings reveal substantial epigenetic diversity between tissues and assign these changes to specific CLSY proteins, elucidating how locus-specific targeting combined with tissue-specific expression enables the CLSYs to generate epigenetic diversity during plant development.

DOI: https://doi.org/10.1038/s41467-021-27690-x
Nat Commun. 2022 Jan 10. 13(1): 17

The transcription factor ZIP-1 promotes resistance to intracellular infection in Caenorhabditis elegans.

Vladimir Lažetić, Fengting Wu, Lianne B Cohen, Kirthi C Reddy, Ya-Ting Chang, Spencer S Gang, Gira Bhabha, Emily R Troemel.

Defense against intracellular infection has been extensively studied in vertebrate hosts, but less is known about invertebrate hosts; specifically, the transcription factors that induce defense against intracellular intestinal infection in the model nematode Caenorhabditis elegans remain understudied. Two different types of intracellular pathogens that naturally infect the C. elegans intestine are the Orsay virus, which is an RNA virus, and microsporidia, which comprise a phylum of fungal pathogens. Despite their molecular differences, these pathogens induce a common host transcriptional response called the intracellular pathogen response (IPR). Here we show that zip-1 is an IPR regulator that functions downstream of all known IPR-activating and regulatory pathways. zip-1 encodes a putative bZIP transcription factor, and we show that zip-1 controls induction of a subset of genes upon IPR activation. ZIP-1 protein is expressed in the nuclei of intestinal cells, and is at least partially required in the intestine to upregulate IPR gene expression. Importantly, zip-1 promotes resistance to infection by the Orsay virus and by microsporidia in intestinal cells. Altogether, our results indicate that zip-1 represents a central hub for triggers of the IPR, and that this transcription factor has a protective function against intracellular pathogen infection in C. elegans.

DOI: https://doi.org/10.1038/s41467-021-27621-w
BMC Bioinformatics. 2022 Jan 14. 23(1): 41

LuxRep: a technical replicate-aware method for bisulfite sequencing data analysis.

Maia H Malonzo, Viivi Halla-Aho, Mikko Konki, Riikka J Lund, Harri Lähdesmäki.

   BACKGROUND: DNA methylation is commonly measured using bisulfite sequencing (BS-seq). The quality of a BS-seq library is measured by its bisulfite conversion efficiency. Libraries with low conversion rates are typically excluded from analysis resulting in reduced coverage and increased costs.
RESULTS: We have developed a probabilistic method and software, LuxRep, that implements a general linear model and simultaneously accounts for technical replicates (libraries from the same biological sample) from different bisulfite-converted DNA libraries. Using simulations and actual DNA methylation data, we show that including technical replicates with low bisulfite conversion rates generates more accurate estimates of methylation levels and differentially methylated sites. Moreover, using variational inference speeds up computation time necessary for whole genome analysis.
CONCLUSIONS: In this work we show that taking into account technical replicates (i.e. libraries) of BS-seq data of varying bisulfite conversion rates, with their corresponding experimental parameters, improves methylation level estimation and differential methylation detection.

Keywords:  Bisulfite sequencing; Methylation; Probabilistic

DOI:  https://doi.org/10.1186/s12859-021-04546-1
Cell Death Differ. 2022 Jan 13.

YB-1 is a positive regulator of KLF5 transcription factor in basal-like breast cancer.

Dewei Jiang, Ting Qiu, Junjiang Peng, Siyuan Li, Tala, Wenlong Ren, Chuanyu Yang, Yi Wen, Chuan-Huizi Chen, Jian Sun, Yingying Wu, Rong Liu, Jun Zhou, Kongming Wu, Wen Liu, Xiaoyun Mao, Zhongmei Zhou, Ceshi Chen.

Y-box binding protein 1 (YB-1) is a well-known oncogene highly expressed in various cancers, including basal-like breast cancer (BLBC). Beyond its role as a transcription factor, YB-1 is newly defined as an epigenetic regulator involving RNA 5-methylcytosine. However, its specific targets and pro-cancer functions are poorly defined. Here, based on clinical database, we demonstrate a positive correlation between Kruppel-like factor 5 (KLF5) and YB-1 expression in breast cancer patients, but a negative correlation with that of Dachshund homolog 1 (DACH1). Mechanistically, YB-1 enhances KLF5 expression not only through transcriptional activation that can be inhibited by DACH1, but also by stabilizing KLF5 mRNA in a RNA 5-methylcytosine modification-dependent manner. Additionally, ribosomal S6 kinase 2 (RSK2) mediated YB-1 phosphorylation at Ser102 promotes YB-1/KLF5 transcriptional complex formation, which co-regulates the expression of BLBC specific genes, Keratin 16 (KRT16) and lymphocyte antigen 6 family member D (Ly6D), to promote cancer cell proliferation. The RSK inhibitor, LJH685, suppressed BLBC cell tumourigenesis in vivo by disturbing YB-1-KLF5 axis. Our data suggest that YB-1 positively regulates KLF5 at multiple levels to promote BLBC progression. The novel RSK2-YB-1-KLF5-KRT16/Ly6D axis provides candidate diagnostic markers and therapeutic targets for BLBC.

DOI: https://doi.org/10.1038/s41418-021-00920-x
Nat Commun. 2022 Jan 10. 13(1): 142

G-quadruplex DNA structures in human stem cells and differentiation.

Katherine G Zyner, Angela Simeone, Sean M Flynn, Colm Doyle, Giovanni Marsico, Santosh Adhikari, Guillem Portella, David Tannahill, Shankar Balasubramanian.

The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation.

DOI: https://doi.org/10.1038/s41467-021-27719-1
Nat Commun. 2022 Jan 11. 13(1): 204

Canonical WNT signaling-dependent gating of MYC requires a noncanonical CTCF function at a distal binding site.

Ilyas Chachoua, Ilias Tzelepis, Hao Dai, Jia Pei Lim, Anna Lewandowska-Ronnegren, Felipe Beccaria Casagrande, Shuangyang Wu, Johanna Vestlund, Carolina Diettrich Mallet de Lima, Deeksha Bhartiya, Barbara A Scholz, Mirco Martino, Rashid Mehmood, Anita Göndör.

Abnormal WNT signaling increases MYC expression in colon cancer cells in part via oncogenic super-enhancer-(OSE)-mediated gating of the active MYC to the nuclear pore in a poorly understood process. We show here that the principal tenet of the WNT-regulated MYC gating, facilitating nuclear export of the MYC mRNA, is regulated by a CTCF binding site (CTCFBS) within the OSE to confer growth advantage in HCT-116 cells. To achieve this, the CTCFBS directs the WNT-dependent trafficking of the OSE to the nuclear pore from intra-nucleoplasmic positions in a stepwise manner. Once the OSE reaches a peripheral position, which is triggered by a CTCFBS-mediated CCAT1 eRNA activation, its final stretch (≤0.7 μm) to the nuclear pore requires the recruitment of AHCTF1, a key nucleoporin, to the CTCFBS. Thus, a WNT/ß-catenin-AHCTF1-CTCF-eRNA circuit enables the OSE to promote pathological cell growth by coordinating the trafficking of the active MYC gene within the 3D nuclear architecture.

DOI: https://doi.org/10.1038/s41467-021-27868-3
Genome Biol. 2022 Jan 12. 23(1): 20

A deep generative model for multi-view profiling of single-cell RNA-seq and ATAC-seq data.

Gaoyang Li, Shaliu Fu, Shuguang Wang, Chenyu Zhu, Bin Duan, Chen Tang, Xiaohan Chen, Guohui Chuai, Ping Wang, Qi Liu.

Here, we present a multi-modal deep generative model, the single-cell Multi-View Profiler (scMVP), which is designed for handling sequencing data that simultaneously measure gene expression and chromatin accessibility in the same cell, including SNARE-seq, sci-CAR, Paired-seq, SHARE-seq, and Multiome from 10X Genomics. scMVP generates common latent representations for dimensionality reduction, cell clustering, and developmental trajectory inference and generates separate imputations for differential analysis and cis-regulatory element identification. scMVP can help mitigate data sparsity issues with imputation and accurately identify cell groups for different joint profiling techniques with common latent embedding, and we demonstrate its advantages on several realistic datasets.

DOI: https://doi.org/10.1186/s13059-021-02595-6
Nat Commun. 2022 Jan 10. 13(1): 141

Single-cell analysis of human primary prostate cancer reveals the heterogeneity of tumor-associated epithelial cell states.

Hanbing Song, Hannah N W Weinstein, Paul Allegakoen, Marc H Wadsworth, Jamie Xie, Heiko Yang, Ethan A Castro, Kevin L Lu, Bradley A Stohr, Felix Y Feng, Peter R Carroll, Bruce Wang, Matthew R Cooperberg, Alex K Shalek, Franklin W Huang.

Prostate cancer is the second most common malignancy in men worldwide and consists of a mixture of tumor and non-tumor cell types. To characterize the prostate cancer tumor microenvironment, we perform single-cell RNA-sequencing on prostate biopsies, prostatectomy specimens, and patient-derived organoids from localized prostate cancer patients. We uncover heterogeneous cellular states in prostate epithelial cells marked by high androgen signaling states that are enriched in prostate cancer and identify a population of tumor-associated club cells that may be associated with prostate carcinogenesis. ERG-negative tumor cells, compared to ERG-positive cells, demonstrate shared heterogeneity with surrounding luminal epithelial cells and appear to give rise to common tumor microenvironment responses. Finally, we show that prostate epithelial organoids harbor tumor-associated epithelial cell states and are enriched with distinct cell types and states from their parent tissues. Our results provide diagnostically relevant insights and advance our understanding of the cellular states associated with prostate carcinogenesis.

DOI: https://doi.org/10.1038/s41467-021-27322-4
Nat Commun. 2022 Jan 10. 13(1): 69

C/EBPB-dependent adaptation to palmitic acid promotes tumor formation in hormone receptor negative breast cancer.

Xiao-Zheng Liu, Anastasiia Rulina, Man Hung Choi, Line Pedersen, Johanna Lepland, Sina T Takle, Noelly Madeleine, Stacey D'mello Peters, Cara Ellen Wogsland, Sturla Magnus Grøndal, James B Lorens, Hani Goodarzi, Per E Lønning, Stian Knappskog, Anders Molven, Nils Halberg.

Epidemiological studies have established a positive association between obesity and the incidence of postmenopausal breast cancer. Moreover, it is known that obesity promotes stem cell-like properties of breast cancer cells. However, the cancer cell-autonomous mechanisms underlying this correlation are not well defined. Here we demonstrate that obesity-associated tumor formation is driven by cellular adaptation rather than expansion of pre-existing clones within the cancer cell population. While there is no correlation with specific mutations, cellular adaptation to obesity is governed by palmitic acid (PA) and leads to enhanced tumor formation capacity of breast cancer cells. This process is governed epigenetically through increased chromatin occupancy of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPB). Obesity-induced epigenetic activation of C/EBPB regulates cancer stem-like properties by modulating the expression of key downstream regulators including CLDN1 and LCN2. Collectively, our findings demonstrate that obesity drives cellular adaptation to PA drives tumor initiation in the obese setting through activation of a C/EBPB dependent transcriptional network.

DOI: https://doi.org/10.1038/s41467-021-27734-2