Publications by authors named "Lihua Julie Zhu"

64 Publications

Deficient spermiogenesis in mice lacking .

Elife 2021 Feb 23;10. Epub 2021 Feb 23.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, United States.

The X-linked gene plays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemic knockout (KO) die around implantation, male KO mice appear healthy and are fertile. Here, we report an important role for in testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of the gene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditional cKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions of in male reproduction specifically in round spermatids during spermiogenesis.
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http://dx.doi.org/10.7554/eLife.63556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935487PMC
February 2021

Consequences of aneuploidy in human fibroblasts with trisomy 21.

Proc Natl Acad Sci U S A 2021 Feb;118(6)

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605;

An extra copy of chromosome 21 causes Down syndrome, the most common genetic disease in humans. The mechanisms contributing to aneuploidy-related pathologies in this syndrome, independent of the identity of the triplicated genes, are not well defined. To characterize aneuploidy-driven phenotypes in trisomy 21 cells, we performed global transcriptome, proteome, and phenotypic analyses of primary human fibroblasts from individuals with Patau (trisomy 13), Edwards (trisomy 18), or Down syndromes. On average, mRNA and protein levels were increased by 1.5-fold in all trisomies, with a subset of proteins enriched for subunits of macromolecular complexes showing signs of posttranscriptional regulation. These results support the lack of evidence for widespread dosage compensation or dysregulation of chromosomal domains in human autosomes. Furthermore, we show that several aneuploidy-associated phenotypes are present in trisomy 21 cells, including lower viability and increased dependency on serine-driven lipid synthesis. Our studies establish a critical role of aneuploidy, independent of triplicated gene identity, in driving cellular defects associated with trisomy 21.
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http://dx.doi.org/10.1073/pnas.2014723118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017964PMC
February 2021

Prostaglandin E2 stimulates cAMP signaling and resensitizes human leukemia cells to glucocorticoid-induced cell death.

Blood 2021 01;137(4):500-512

Department of Molecular, Cell, and Cancer Biology.

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.
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http://dx.doi.org/10.1182/blood.2020005712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845005PMC
January 2021

UPR scales mitochondrial network expansion with protein synthesis via mitochondrial import in Caenorhabditis elegans.

Nat Commun 2021 01 20;12(1):479. Epub 2021 Jan 20.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, 01605, USA.

As organisms develop, individual cells generate mitochondria to fulfill physiological requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth. The mitochondrial unfolded protein response (UPR) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, using the model organism Caenorhabditis elegans we demonstrate that ATFS-1 mediates an adaptable mitochondrial network expansion program that is active throughout normal development. Mitochondrial network expansion requires the relatively inefficient MTS in ATFS-1, which allows the transcription factor to be responsive to parameters that impact protein import capacity of the mitochondrial network. Increasing the strength of the ATFS-1 MTS impairs UPR activity by increasing accumulation within mitochondria. Manipulations of TORC1 activity increase or decrease ATFS-1 activity in a manner that correlates with protein synthesis. Lastly, expression of mitochondrial-targeted GFP is sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPR activation.
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http://dx.doi.org/10.1038/s41467-020-20784-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817664PMC
January 2021

dagLogo: An R/Bioconductor package for identifying and visualizing differential amino acid group usage in proteomics data.

PLoS One 2020 6;15(11):e0242030. Epub 2020 Nov 6.

Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

Sequence logos have been widely used as graphical representations of conserved nucleic acid and protein motifs. Due to the complexity of the amino acid (AA) alphabet, rich post-translational modification, and diverse subcellular localization of proteins, few versatile tools are available for effective identification and visualization of protein motifs. In addition, various reduced AA alphabets based on physicochemical, structural, or functional properties have been valuable in the study of protein alignment, folding, structure prediction, and evolution. However, there is lack of tools for applying reduced AA alphabets to the identification and visualization of statistically significant motifs. To fill this gap, we developed an R/Bioconductor package dagLogo, which has several advantages over existing tools. First, dagLogo allows various formats for input sets and provides comprehensive options to build optimal background models. It implements different reduced AA alphabets to group AAs of similar properties. Furthermore, dagLogo provides statistical and visual solutions for differential AA (or AA group) usage analysis of both large and small data sets. Case studies showed that dagLogo can better identify and visualize conserved protein sequence patterns from different types of inputs and can potentially reveal the biological patterns that could be missed by other logo generators.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0242030PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647101PMC
December 2020

The Lipid Handling Capacity of Subcutaneous Fat Is Programmed by mTORC2 during Development.

Cell Rep 2020 10;33(1):108223

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

Overweight and obesity are associated with type 2 diabetes, non-alcoholic fatty liver disease, cardiovascular disease and cancer, but all fat is not equal, as storing excess lipid in subcutaneous white adipose tissue (SWAT) is more metabolically favorable than in visceral fat. Here, we uncover a critical role for mTORC2 in setting SWAT lipid handling capacity. We find that subcutaneous white preadipocytes differentiating without the essential mTORC2 subunit Rictor upregulate mature adipocyte markers but develop a striking lipid storage defect resulting in smaller adipocytes, reduced tissue size, lipid re-distribution to visceral and brown fat, and sex-distinct effects on systemic metabolic fitness. Mechanistically, mTORC2 promotes transcriptional upregulation of select lipid metabolism genes controlled by PPARγ and ChREBP, including genes that control lipid uptake, synthesis, and degradation pathways as well as Akt2, which encodes a major mTORC2 substrate and insulin effector. Further exploring this pathway may uncover new strategies to improve insulin sensitivity.
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http://dx.doi.org/10.1016/j.celrep.2020.108223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607535PMC
October 2020

OneStopRNAseq: A Web Application for Comprehensive and Efficient Analyses of RNA-Seq Data.

Genes (Basel) 2020 Oct 2;11(10). Epub 2020 Oct 2.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.

Over the past decade, a large amount of RNA sequencing (RNA-seq) data were deposited in public repositories, and more are being produced at an unprecedented rate. However, there are few open source tools with point-and-click interfaces that are versatile and offer streamlined comprehensive analysis of RNA-seq datasets. To maximize the capitalization of these vast public resources and facilitate the analysis of RNA-seq data by biologists, we developed a web application called OneStopRNAseq for the one-stop analysis of RNA-seq data. OneStopRNAseq has user-friendly interfaces and offers workflows for common types of RNA-seq data analyses, such as comprehensive data-quality control, differential analysis of gene expression, exon usage, alternative splicing, transposable element expression, allele-specific gene expression quantification, and gene set enrichment analysis. Users only need to select the desired analyses and genome build, and provide a Gene Expression Omnibus (GEO) accession number or Dropbox links to sequence files, alignment files, gene-expression-count tables, or rank files with the corresponding metadata. Our pipeline facilitates the comprehensive and efficient analysis of private and public RNA-seq data.
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http://dx.doi.org/10.3390/genes11101165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650687PMC
October 2020

Selenium detoxification is required for cancer-cell survival.

Nat Metab 2020 07 6;2(7):603-611. Epub 2020 Jul 6.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA.

The micronutrient selenium is incorporated via the selenocysteine biosynthesis pathway into the rare amino acid selenocysteine, which is required in selenoproteins such as glutathione peroxidases and thioredoxin reductases. Here, we show that selenophosphate synthetase 2 (SEPHS2), an enzyme in the selenocysteine biosynthesis pathway, is essential for survival of cancer, but not normal, cells. SEPHS2 is required in cancer cells to detoxify selenide, an intermediate that is formed during selenocysteine biosynthesis. Breast and other cancer cells are selenophilic, owing to a secondary function of the cystine/glutamate antiporter SLC7A11 that promotes selenium uptake and selenocysteine biosynthesis, which, by allowing production of selenoproteins such as GPX4, protects cells against ferroptosis. However, this activity also becomes a liability for cancer cells because selenide is poisonous and must be processed by SEPHS2. Accordingly, we find that SEPHS2 protein levels are elevated in samples from people with breast cancer, and that loss of SEPHS2 impairs growth of orthotopic mammary-tumour xenografts in mice. Collectively, our results identify a vulnerability of cancer cells and define the role of selenium metabolism in cancer.
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http://dx.doi.org/10.1038/s42255-020-0224-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455022PMC
July 2020

KLF7 promotes pancreatic cancer growth and metastasis by up-regulating ISG expression and maintaining Golgi complex integrity.

Proc Natl Acad Sci U S A 2020 06 19;117(22):12341-12351. Epub 2020 May 19.

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35233;

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Currently, there is no effective therapy for PDAC, and a detailed molecular and functional evaluation of PDACs is needed to identify and develop better therapeutic strategies. Here we show that the transcription factor Krüppel-like factor 7 (KLF7) is overexpressed in PDACs, and that inhibition of KLF7 blocks PDAC tumor growth and metastasis in cell culture and in mice. KLF7 expression in PDACs can be up-regulated due to activation of a MAP kinase pathway or inactivation of the tumor suppressor p53, two alterations that occur in a large majority of PDACs. ShRNA-mediated knockdown of KLF7 inhibits the expression of IFN-stimulated genes (ISGs), which are necessary for KLF7-mediated PDAC tumor growth and metastasis. KLF7 knockdown also results in the down-regulation of Discs Large MAGUK Scaffold Protein 3 (DLG3), resulting in Golgi complex fragmentation, and reduced protein glycosylation, leading to reduced secretion of cancer-promoting growth factors, such as chemokines. Genetic or pharmacologic activation of Golgi complex fragmentation blocks PDAC growth and metastasis similar to KLF7 inhibition. Our results demonstrate a therapeutically amenable, KLF7-driven pathway that promotes PDAC growth and metastasis by activating ISGs and maintaining Golgi complex integrity.
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http://dx.doi.org/10.1073/pnas.2005156117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275752PMC
June 2020

The coordinate actions of calcineurin and Hog1 mediate the stress response through multiple nodes of the cell cycle network.

PLoS Genet 2020 04 28;16(4):e1008600. Epub 2020 Apr 28.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

Upon exposure to environmental stressors, cells transiently arrest the cell cycle while they adapt and restore homeostasis. A challenge for all cells is to distinguish between stress signals and coordinate the appropriate adaptive response with cell cycle arrest. Here we investigate the role of the phosphatase calcineurin (CN) in the stress response and demonstrate that CN activates the Hog1/p38 pathway in both yeast and human cells. In yeast, the MAPK Hog1 is transiently activated in response to several well-studied osmostressors. We show that when a stressor simultaneously activates CN and Hog1, CN disrupts Hog1-stimulated negative feedback to prolong Hog1 activation and the period of cell cycle arrest. Regulation of Hog1 by CN also contributes to inactivation of multiple cell cycle-regulatory transcription factors (TFs) and the decreased expression of cell cycle-regulated genes. CN-dependent downregulation of G1/S genes is dependent upon Hog1 activation, whereas CN inactivates G2/M TFs through a combination of Hog1-dependent and -independent mechanisms. These findings demonstrate that CN and Hog1 act in a coordinated manner to inhibit multiple nodes of the cell cycle-regulatory network. Our results suggest that crosstalk between CN and stress-activated MAPKs helps cells tailor their adaptive responses to specific stressors.
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http://dx.doi.org/10.1371/journal.pgen.1008600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7209309PMC
April 2020

Distinct features of nucleolus-associated domains in mouse embryonic stem cells.

Chromosoma 2020 06 26;129(2):121-139. Epub 2020 Mar 26.

Department of Molecular, Cellular and Cancer Biology, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA, 01605, USA.

Heterochromatin in eukaryotic interphase cells frequently localizes to the nucleolar periphery (nucleolus-associated domains (NADs)) and the nuclear lamina (lamina-associated domains (LADs)). Gene expression in somatic cell NADs is generally low, but NADs have not been characterized in mammalian stem cells. Here, we generated the first genome-wide map of NADs in mouse embryonic stem cells (mESCs) via deep sequencing of chromatin associated with biochemically purified nucleoli. As we had observed in mouse embryonic fibroblasts (MEFs), the large type I subset of NADs overlaps with constitutive LADs and is enriched for features of constitutive heterochromatin, including late replication timing and low gene density and expression levels. Conversely, the type II NAD subset overlaps with loci that are not lamina-associated, but in mESCs, type II NADs are much less abundant than in MEFs. mESC NADs are also much less enriched in H3K27me3 modified regions than are NADs in MEFs. Additionally, comparision of MEF and mESC NADs revealed enrichment of developmentally regulated genes in cell-type-specific NADs. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin. These studies implicate association with the nucleolar periphery as a mechanism for developmentally regulated gene expression and will facilitate future studies of NADs during mESC differentiation.
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http://dx.doi.org/10.1007/s00412-020-00734-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265113PMC
June 2020

Genomic Characterization of Endothelial Enhancers Reveals a Multifunctional Role for NR2F2 in Regulation of Arteriovenous Gene Expression.

Circ Res 2020 03 17;126(7):875-888. Epub 2020 Feb 17.

From the Department of Molecular, Cell and Cancer Biology (S.S., J.Y., A.Y., R.L., L.J.Z., N.D.L.), University of Massachusetts Medical School, Worcester.

Rationale: Significant progress has revealed transcriptional inputs that underlie regulation of artery and vein endothelial cell fates. However, little is known concerning genome-wide regulation of this process. Therefore, such studies are warranted to address this gap.

Objective: To identify and characterize artery- and vein-specific endothelial enhancers in the human genome, thereby gaining insights into mechanisms by which blood vessel identity is regulated.

Methods And Results: Using chromatin immunoprecipitation and deep sequencing for markers of active chromatin in human arterial and venous endothelial cells, we identified several thousand artery- and vein-specific regulatory elements. Computational analysis revealed that NR2F2 (nuclear receptor subfamily 2, group F, member 2) sites were overrepresented in vein-specific enhancers, suggesting a direct role in promoting vein identity. Subsequent integration of chromatin immunoprecipitation and deep sequencing data sets with RNA sequencing revealed that NR2F2 regulated 3 distinct aspects related to arteriovenous identity. First, consistent with previous genetic observations, NR2F2 directly activated enhancer elements flanking cell cycle genes to drive their expression. Second, NR2F2 was essential to directly activate vein-specific enhancers and their associated genes. Our genomic approach further revealed that NR2F2 acts with ERG (ETS-related gene) at many of these sites to drive vein-specific gene expression. Finally, NR2F2 directly repressed only a small number of artery enhancers in venous cells to prevent their activation, including a distal element upstream of the artery-specific transcription factor, (hes related family bHLH transcription factor with YRPW motif 2). In arterial endothelial cells, this enhancer was normally bound by ERG, which was also required for arterial expression. By contrast, in venous endothelial cells, NR2F2 was bound to this site, together with ERG, and prevented its activation.

Conclusions: By leveraging a genome-wide approach, we revealed mechanistic insights into how NR2F2 functions in multiple roles to maintain venous identity. Importantly, characterization of its role at a crucial artery enhancer upstream of established a novel mechanism by which artery-specific expression can be achieved.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.316075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212523PMC
March 2020

Adenine base editing in an adult mouse model of tyrosinaemia.

Nat Biomed Eng 2020 01 25;4(1):125-130. Epub 2019 Feb 25.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA.

In contrast to traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals.
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http://dx.doi.org/10.1038/s41551-019-0357-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986236PMC
January 2020

Prominin2 Drives Ferroptosis Resistance by Stimulating Iron Export.

Dev Cell 2019 12 14;51(5):575-586.e4. Epub 2019 Nov 14.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

Ferroptosis, regulated cell death characterized by the iron-dependent accumulation of lethal lipid reactive oxygen species, contributes to tissue homeostasis and numerous pathologies, and it may be exploited for therapy. Cells differ in their sensitivity to ferroptosis, however, and a key challenge is to understand mechanisms that contribute to resistance. Using RNA-seq to identify genes that contribute to ferroptosis resistance, we discovered that pro-ferroptotic stimuli, including inhibition of the lipid hydroperoxidase GPX4 and detachment from the extracellular matrix, induce expression of prominin2, a pentaspanin protein implicated in regulation of lipid dynamics. Prominin2 facilitates ferroptosis resistance in mammary epithelial and breast carcinoma cells. Mechanistically, prominin2 promotes the formation of ferritin-containing multivesicular bodies (MVBs) and exosomes that transport iron out of the cell, inhibiting ferroptosis. These findings reveal that ferroptosis resistance can be driven by a prominin2-MVB-exosome-ferritin pathway and have broad implications for iron homeostasis, intracellular trafficking, and cancer.
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http://dx.doi.org/10.1016/j.devcel.2019.10.007DOI Listing
December 2019

Alveolar progenitor cells in the mammary gland are dependent on the β4 integrin.

Dev Biol 2020 01 3;457(1):13-19. Epub 2019 Oct 3.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA, 01605, USA. Electronic address:

Understanding how progenitor cell function is regulated in the mammary gland is an important developmental problem that has significant implications for breast cancer. Although it had been assumed that the expression the α6β4 integrin (β4) is restricted to the basal lineage, we report that alveolar progenitor cells in the mouse mammary gland also express this integrin based on analysis of single cell RNA-Seq data. Subsequent experiments using a mouse mammary epithelial cell line (NMuMG) confirmed this finding and revealed that β4 is essential for maintaining progenitor function as assessed by serial passage mammosphere assays. These data were substantiated by analyzing the alveolar progenitor population isolated from nulliparous mouse mammary glands. Based on the finding that the alveolar progenitor cells express Whey Acidic Protein (WAP), WAP-Cre mice were crossed with itgβ4 mice to generate conditional knock-out of β4 in alveolar progenitor cells. These itgβ4WAP-Cre mice exhibited significant defects in alveologenesis and milk production during pregnancy compared to itgβ4WAP-Cre mice, establishing a novel role for the β4 integrin in alveolar progenitor function and alveologenesis.
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http://dx.doi.org/10.1016/j.ydbio.2019.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687040PMC
January 2020

Conserved microRNA mediates heating tolerance in germ cells versus surrounding somatic cells.

RNA Biol 2019 10 15;16(10):1494-1503. Epub 2019 Jul 15.

National Engineering Laboratory for Animal Breeding; Key Laboratory of Animal Genetics, Breeding, and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University , Beijing , China.

Mammalian fertility is reduced during heat exposure in the summer, but is regained as temperatures decrease in the autumn again. However, the mechanism underlying the phenomenon remains unknown. We investigated heat stress tolerance of germ cells and their surrounding somatic cells, and discovered that microRNA ssc-ca-1 was upregulated after heat stress in cultured porcine granulosa cells (GCs), but not in serum-starved GCs. Ssc-ca-1 inhibited heat shock protein 70 (Hsp70) expression through its 3'- and 5'-UTRs. Although mRNA transcription was induced in GCs by exposure to heat in the summer, ssc-ca-1 inhibited Hsp70 expression. In ovarian cultures, heat stress-induced Hsp70 expression in primordial but not in growing follicles; ssc-ca-1 expression did not change in primordial follicles, but increased in growing follicles. Consistently, ssc-ca-1 was present in testicular cells and exhibited the same function as in ovarian cells. It modulated the different Hsp70 expression between spermatogonial stem cells and Sertoli cells after scrotal heat stress. This mechanism is of relevance to mammalian fertility in parts of the world dominated by heat stress associated with global climate change.
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http://dx.doi.org/10.1080/15476286.2019.1639311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779383PMC
October 2019

Gene expression signature of atypical breast hyperplasia and regulation by SFRP1.

Breast Cancer Res 2019 06 27;21(1):76. Epub 2019 Jun 27.

Pioneer Valley Life Sciences Institute, Springfield, MA, 01199, USA.

Background: Atypical breast hyperplasias (AH) have a 10-year risk of progression to invasive cancer estimated at 4-7%, with the overall risk of developing breast cancer increased by ~ 4-fold. AH lesions are estrogen receptor alpha positive (ERα+) and represent risk indicators and/or precursor lesions to low grade ERα+ tumors. Therefore, molecular profiles of AH lesions offer insights into the earliest changes in the breast epithelium, rendering it susceptible to oncogenic transformation.

Methods: In this study, women were selected who were diagnosed with ductal or lobular AH, but no breast cancer prior to or within the 2-year follow-up. Paired AH and histologically normal benign (HNB) tissues from patients were microdissected. RNA was isolated, amplified linearly, labeled, and hybridized to whole transcriptome microarrays to determine gene expression profiles. Genes that were differentially expressed between AH and HNB were identified using a paired analysis. Gene expression signatures distinguishing AH and HNB were defined using AGNES and PAM methods. Regulation of gene networks was investigated using breast epithelial cell lines, explant cultures of normal breast tissue and mouse tissues.

Results: A 99-gene signature discriminated the histologically normal and AH tissues in 81% of the cases. Network analysis identified coordinated alterations in signaling through ERα, epidermal growth factor receptors, and androgen receptor which were associated with the development of both lobular and ductal AH. Decreased expression of SFRP1 was also consistently lower in AH. Knockdown of SFRP1 in 76N-Tert cells resulted altered expression of 13 genes similarly to that observed in AH. An SFRP1-regulated network was also observed in tissues from mice lacking Sfrp1. Re-expression of SFRP1 in MCF7 cells provided further support for the SFRP1-regulated network. Treatment of breast explant cultures with rSFRP1 dampened estrogen-induced progesterone receptor levels.

Conclusions: The alterations in gene expression were observed in both ductal and lobular AH suggesting shared underlying mechanisms predisposing to AH. Loss of SFRP1 expression is a significant regulator of AH transcriptional profiles driving previously unidentified changes affecting responses to estrogen and possibly other pathways. The gene signature and pathways provide insights into alterations contributing to AH breast lesions.
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http://dx.doi.org/10.1186/s13058-019-1157-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598287PMC
June 2019

The VEGF receptor neuropilin 2 promotes homologous recombination by stimulating YAP/TAZ-mediated Rad51 expression.

Proc Natl Acad Sci U S A 2019 07 24;116(28):14174-14180. Epub 2019 Jun 24.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605;

Vascular endothelial growth factor (VEGF) signaling in tumor cells mediated by neuropilins (NRPs) contributes to the aggressive nature of several cancers, including triple-negative breast cancer (TNBC), independently of its role in angiogenesis. Understanding the mechanisms by which VEGF-NRP signaling contributes to the phenotype of such cancers is a significant and timely problem. We report that VEGF-NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double-strand break repair. Mechanistically, we provide evidence that VEGF-NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ-TEAD transcriptional target. We also discovered that VEGF-NRP2-YAP/TAZ signaling contributes to the resistance of TNBC cells to cisplatin and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF-NRP2 or YAP/TAZ. These findings reveal roles for VEGF-NRP2 and YAP/TAZ in DNA repair, and they indicate a unified mechanism involving VEGF-NRP2, YAP/TAZ, and Rad51 that contributes to resistance to platinum chemotherapy.
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http://dx.doi.org/10.1073/pnas.1821194116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628806PMC
July 2019

Two contrasting classes of nucleolus-associated domains in mouse fibroblast heterochromatin.

Genome Res 2019 08 14;29(8):1235-1249. Epub 2019 Jun 14.

Department of Molecular, Cellular and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining nucleolus-associated domains (NADs) and lamina-associated domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called and demonstrated that it identifies NADs more accurately than other peak-calling tools, owing to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3, and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Type II NADs are enriched in distinct gene classes, including factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, and present in MEFs but not in undifferentiated embryonic stem (ES) cells.
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http://dx.doi.org/10.1101/gr.247072.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6673712PMC
August 2019

foxc1 is required for embryonic head vascular smooth muscle differentiation in zebrafish.

Dev Biol 2019 09 11;453(1):34-47. Epub 2019 Jun 11.

Alberta Children's Hospital Research Institute, University of Calgary, Canada; Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada, T2N 4N1. Electronic address:

Vascular smooth muscle of the head derives from neural crest, but developmental mechanisms and early transcriptional drivers of the vSMC lineage are not well characterized. We find that in early development, the transcription factor foxc1b is expressed in mesenchymal cells that associate with the vascular endothelium. Using timelapse imaging, we observe that foxc1b expressing mesenchymal cells differentiate into acta2 expressing vascular mural cells. We show that in zebrafish, while foxc1b is co-expressed in acta2 positive smooth muscle cells that associate with large diameter vessels, it is not co-expressed in capillaries where pdgfrβ positive pericytes are located. In addition to being an early marker of the lineage, foxc1 is essential for vSMC differentiation; we find that foxc1 loss of function mutants have defective vSMC differentiation and that early genetic ablation of foxc1b or acta2 expressing populations blocks vSMC differentiation. Furthermore, foxc1 is expressed upstream of acta2 and is required for acta2 expression in vSMCs. Using RNA-Seq we determine an enriched intersectional gene expression profile using dual expression of foxc1b and acta2 to identify novel vSMC markers. Taken together, our data suggests that foxc1 is a marker of vSMCs and plays a critical functional role in promoting their differentiation.
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http://dx.doi.org/10.1016/j.ydbio.2019.06.005DOI Listing
September 2019

trackViewer: a Bioconductor package for interactive and integrative visualization of multi-omics data.

Nat Methods 2019 06;16(6):453-454

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA.

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http://dx.doi.org/10.1038/s41592-019-0430-yDOI Listing
June 2019

Prosurvival kinase PIM2 is a therapeutic target for eradication of chronic myeloid leukemia stem cells.

Proc Natl Acad Sci U S A 2019 05 8;116(21):10482-10487. Epub 2019 May 8.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605;

A major obstacle to curing chronic myeloid leukemia (CML) is the intrinsic resistance of CML stem cells (CMLSCs) to the drug imatinib mesylate (IM). Prosurvival genes that are preferentially expressed in CMLSCs compared with normal hematopoietic stem cells (HSCs) represent potential therapeutic targets for selectively eradicating CMLSCs. However, the discovery of such preferentially expressed genes has been hampered by the inability to completely separate CMLSCs from HSCs, which display a very similar set of surface markers. To overcome this challenge, and to minimize confounding effects of individual differences in gene expression profiles, we performed single-cell RNA-seq on CMLSCs and HSCs that were isolated from the same patient and distinguished based on the presence or absence of BCR-ABL. Among genes preferentially expressed in CMLSCs is , which encodes a prosurvival serine-threonine kinase that phosphorylates and inhibits the proapoptotic protein BAD. We show that IM resistance of CMLSCs is due, at least in part, to maintenance of BAD phosphorylation by PIM2. We find that in CMLSCs, expression is promoted by both a BCR-ABL-dependent (IM-sensitive) STAT5-mediated pathway and a BCR-ABL-independent (IM-resistant) STAT4-mediated pathway. Combined treatment with IM and a PIM inhibitor synergistically increases apoptosis of CMLSCs, suppresses colony formation, and significantly prolongs survival in a mouse CML model, with a negligible effect on HSCs. Our results reveal a therapeutically targetable mechanism of IM resistance in CMLSCs. The experimental approach that we describe can be generally applied to other malignancies that harbor oncogenic fusion proteins or other characteristic genetic markers.
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http://dx.doi.org/10.1073/pnas.1903550116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535024PMC
May 2019

Enhanced Cas12a editing in mammalian cells and zebrafish.

Nucleic Acids Res 2019 05;47(8):4169-4180

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.
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http://dx.doi.org/10.1093/nar/gkz184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486634PMC
May 2019

Mitochondrial UPR repression during infection requires the bZIP protein ZIP-3.

Proc Natl Acad Sci U S A 2019 03 8;116(13):6146-6151. Epub 2019 Mar 8.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605;

Mitochondria generate most cellular energy and are targeted by multiple pathogens during infection. In turn, metazoans employ surveillance mechanisms such as the mitochondrial unfolded protein response (UPR) to detect and respond to mitochondrial dysfunction as an indicator of infection. The UPR is an adaptive transcriptional program regulated by the transcription factor ATFS-1, which induces genes that promote mitochondrial recovery and innate immunity. The bacterial pathogen produces toxins that disrupt oxidative phosphorylation (OXPHOS), resulting in UPR activation. Here, we demonstrate that exploits an intrinsic negative regulatory mechanism mediated by the bZIP protein ZIP-3 to repress UPR activation. Strikingly, worms lacking were impervious to -mediated UPR repression and resistant to infection. Pathogen-secreted phenazines perturbed mitochondrial function and were the primary cause of UPR activation, consistent with these molecules being electron shuttles and virulence determinants. Surprisingly, unable to produce phenazines and thus elicit UPR activation were hypertoxic in -deletion worms. These data emphasize the significance of virulence-mediated UPR repression and the potency of the UPR as an antibacterial response.
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http://dx.doi.org/10.1073/pnas.1817259116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442607PMC
March 2019

Publisher Correction: Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing.

Nat Commun 2018 12 10;9(1):5294. Epub 2018 Dec 10.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA.

The original version of this Article contained errors in the author affiliations. Mehmet Fatih Bolukbasi was incorrectly associated with Bluebird Bio., Cambridge, MA, USA and Ankit Gupta was incorrectly associated with Exonics Therapeutics, Watertown, MA, USA. This has now been corrected in the HTML version of the Article. The PDF version of the Article was correct at the time of publication.
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http://dx.doi.org/10.1038/s41467-018-07776-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6288159PMC
December 2018

NmeCas9 is an intrinsically high-fidelity genome-editing platform.

Genome Biol 2018 12 5;19(1):214. Epub 2018 Dec 5.

RNA Therapeutics Institute, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA, 01605, USA.

Background: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.

Results: Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5'-NGATT-3'), for NmeCas9 genome editing in human cells.

Conclusions: Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.
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http://dx.doi.org/10.1186/s13059-018-1591-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282386PMC
December 2018

Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing.

Nat Commun 2018 11 19;9(1):4856. Epub 2018 Nov 19.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA.

The development of robust, versatile and accurate toolsets is critical to facilitate therapeutic genome editing applications. Here we establish RNA-programmable Cas9-Cas9 chimeras, in single- and dual-nuclease formats, as versatile genome engineering systems. In both of these formats, Cas9-Cas9 fusions display an expanded targeting repertoire and achieve highly specific genome editing. Dual-nuclease Cas9-Cas9 chimeras have distinct advantages over monomeric Cas9s including higher target site activity and the generation of predictable precise deletion products between their target sites. At a therapeutically relevant site within the BCL11A erythroid enhancer, Cas9-Cas9 nucleases produced precise deletions that comprised up to 97% of all sequence alterations. Thus Cas9-Cas9 chimeras represent an important tool that could be particularly valuable for therapeutic genome editing applications where a precise cleavage position and defined sequence end products are desirable.
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http://dx.doi.org/10.1038/s41467-018-07310-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242970PMC
November 2018

Opposing Roles of FANCJ and HLTF Protect Forks and Restrain Replication during Stress.

Cell Rep 2018 09;24(12):3251-3261

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

The DNA helicase FANCJ is mutated in hereditary breast and ovarian cancer and Fanconi anemia (FA). Nevertheless, how loss of FANCJ translates to disease pathogenesis remains unclear. We addressed this question by analyzing proteins associated with replication forks in cells with or without FANCJ. We demonstrate that FANCJ-knockout (FANCJ-KO) cells have alterations in the replisome that are consistent with enhanced replication stress, including an aberrant accumulation of the fork remodeling factor helicase-like transcription factor (HLTF). Correspondingly, HLTF contributes to fork degradation in FANCJ-KO cells. Unexpectedly, the unrestrained DNA synthesis that characterizes HLTF-deficient cells is FANCJ dependent and correlates with S1 nuclease sensitivity and fork degradation. These results suggest that FANCJ and HLTF promote replication fork integrity, in part by counteracting each other to keep fork remodeling and elongation in check. Indicating one protein compensates for loss of the other, loss of both HLTF and FANCJ causes a more severe replication stress response.
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http://dx.doi.org/10.1016/j.celrep.2018.08.065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218949PMC
September 2018

Pharmacological reactivation of inactive X-linked in cerebral cortical neurons of living mice.

Proc Natl Acad Sci U S A 2018 07 16;115(31):7991-7996. Epub 2018 Jul 16.

Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908;

Rett syndrome (RTT) is a genetic disorder resulting from a loss-of-function mutation in one copy of the X-linked gene methyl-CpG-binding protein 2 (). Typical RTT patients are females and, due to random X chromosome inactivation (XCI), ∼50% of cells express mutant MECP2 and the other ∼50% express wild-type MECP2. Cells expressing mutant MECP2 retain a wild-type copy of MECP2 on the inactive X chromosome (Xi), the reactivation of which represents a potential therapeutic approach for RTT. Previous studies have demonstrated reactivation of Xi-linked in cultured cells by biological or pharmacological inhibition of factors that promote XCI (called "XCI factors" or "XCIFs"). Whether XCIF inhibitors in living animals can reactivate Xi-linked in cerebral cortical neurons, the cell type most therapeutically relevant to RTT, remains to be determined. Here, we show that pharmacological inhibitors targeting XCIFs in the PI3K/AKT and bone morphogenetic protein signaling pathways reactivate Xi-linked in cultured mouse fibroblasts and human induced pluripotent stem cell-derived postmitotic RTT neurons. Notably, reactivation of Xi-linked corrects characteristic defects of human RTT neurons including reduced soma size and branch points. Most importantly, we show that intracerebroventricular injection of the XCIF inhibitors reactivates Xi-linked in cerebral cortical neurons of adult living mice. In support of these pharmacological results, we also demonstrate genetic reactivation of Xi-linked in cerebral cortical neurons of living mice bearing a homozygous XCIF deletion. Collectively, our results further establish the feasibility of pharmacological reactivation of Xi-linked as a therapeutic approach for RTT.
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http://dx.doi.org/10.1073/pnas.1803792115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6077728PMC
July 2018