Search

Genome sequencing (GS) is a powerful clinical tool used for the comprehensive diagnosis of germline disorders. GS library preparation typically involves mechanical DNA fragmentation, end repair, and bead-based library size selection followed by adapter ligation, which can require a large amount of input genomic DNA. Tagmentation using bead-linked transposomes can simplify the library preparation process and reduce the DNA input requirement. Here we describe the clinical validation of tagmentation-based PCR-free GS as a clinical test for rare germline disorders. Compared with the Genome-in-a-Bottle Consortium benchmark variant sets, GS had a recall >99.7% and a precision of 99.8% for single nucleotide variants and small insertion-deletions. GS also exhibited 100% sensitivity for clinically reported sequence variants and the copy number variants examined. Furthermore, GS detected mitochondrial sequence variants above 5% heteroplasmy and showed reliable detection of disease-relevant repeat expansions and SMN1 homozygous loss. Our results indicate that while lowering DNA input requirements and reducing library preparation time, GS enables uniform coverage across the genome as well as robust detection of various types of genetic alterations. With the advantage of comprehensive profiling of multiple types of genetic alterations, GS is positioned as an ideal first-tier diagnostic test for germline disorders., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Long noncoding RNAs have been implicated in many of the hallmarks of cancer. Herein, we found that the expression of lncRNA152 (lnc152; a.k.a. DRAIC), which we annotated previously, is highly upregulated in luminal breast cancer (LBC) and downregulated in triple-negative breast cancer (TNBC). Knockdown of lnc152 promotes cell migration and invasion in LBC cell lines. In contrast, ectopic expression of lnc152 inhibits growth, migration, invasion, and angiogenesis in TNBC cell lines. In mice, lnc152 inhibited the growth of TNBC cell xenografts, as well as metastasis of TNBC cells in an intracardiac injection model. Transcriptome analysis of the xenografts indicated that lnc152 downregulates genes controlling angiogenesis. Using pull down assays followed by LC/MS-MS, we identified RBM47, a known tumor suppressor in breast cancer, as a lnc152-interacting protein. The effects of lnc152 in TNBC cells are mediated, in part, by regulating the expression of RBM47. Collectively, our results demonstrate that lnc152 is an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC., Implications: This study identifies lncRNA152 as an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC by upregulating the expression of the tumor suppressor RBM47. As such, lncRNA152 may serve as a biomarker to track aggressiveness of breast cancer, as well as therapeutic target for treating TNBC., (©2022 American Association for Cancer Research.)

Recent studies have identified cancer-associated mutations in histone genes that lead to the expression of mutant versions of core histones called oncohistones. Many oncohistone mutations occur at Asp and Glu residues, two amino acids known to be ADP-ribosylated (ADPRylated) by PARP1. We screened 25 Glu or Asp oncohistone mutants for their effects on cell growth in breast and ovarian cancer cells. Ectopic expression of six mutants of three different core histones (H2B, H3, and H4) altered cell growth in at least two different cell lines. Two of these sites, H2B-D51 and H4-D68, were indeed sites of ADPRylation in wild-type (unmutated) histones, and mutation of these sites inhibited ADPRylation. Mutation of H2B-D51 dramatically altered chromatin accessibility at enhancers and promoters, as well as gene expression outcomes, whereas mutation of H4-D68 did not. Additional biochemical, cellular, proteomic, and genomic analyses demonstrated that ADPRylation of H2B-D51 inhibits p300-mediated acetylation of H2B at many Lys residues. In breast cancer cell xenografts in mice, H2B-D51A promoted tumor growth, but did not confer resistance to the cytotoxic effects of PARP inhibition. Collectively, these results demonstrate that functional Asp and Glu ADPRylation sites on histones are mutated in cancers, allowing cancer cells to escape the growth-regulating effects of post-translational modifications via distinct mechanisms., Significance: This study identifies cancer-driving mutations in histones as sites of PARP1-mediated ADP-ribosylation in breast and ovarian cancers, providing a molecular pathway by which cancers may subvert the growth-regulating effects of PARP1., (©2022 American Association for Cancer Research.)

During gestation, the female reproductive tract must maintain pregnancy while concurrently preparing for parturition. Here, we explore the transitions in gene expression and protein turnover (fractional synthesis rates [FSR]) by which the cervix implements a transition from rigid to compliant. Shifts in gene transcription to achieve immune tolerance and alter epithelial cell programs begin in early pregnancy. Subsequently, in mid-to-late pregnancy transcriptional programs emerge that promote structural reorganization of the extracellular matrix (ECM). Stable isotope labeling revealed a striking slowdown of overall FSRs across the proteome on gestation day 6 that reverses in mid-to-late pregnancy. An exception was soluble fibrillar collagens and proteins of collagen assembly, which exhibit high turnover in nonpregnant cervix compared with other tissues and FSRs that continue throughout pregnancy. This finding provides a mechanism to explain how cross-linked collagen is replaced by newly synthesized, less cross-linked collagens, which allows increased tissue compliance during parturition. The rapid transition requires a reservoir of newly synthesized, less cross-linked collagens, which is assured by the high FSR of soluble collagens in the cervix. These findings suggest a previously unrecognized form of "metabolic flexibility" for ECM in the cervix that underlies rapid transformation in compliance to allow parturition., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Miscarriage is a common complication of pregnancy for which there are few clinical interventions. Deficiency in endometrial stromal cell decidualization is considered a major contributing factor to pregnancy loss; however, our understanding of the underlying mechanisms of decidual deficiency are incomplete. ADP ribosylation by PARP-1 and PARP-2 has been linked to physiological processes essential to successful pregnancy outcomes. Here, we report that the catalytic inhibition or genetic ablation of PARP-1 and PARP-2 in the uterus lead to pregnancy loss in mice. Notably, the absence of PARP-1 and PARP-2 resulted in increased p53 signaling and an increased population of senescent decidual cells. Molecular and histological analysis revealed that embryo attachment and the removal of the luminal epithelium are not altered in uterine Parp1 , Parp2 knockout mice, but subsequent decidualization failure results in pregnancy loss. These findings provide evidence for a previously unknown function of PARP-1 and PARP-2 in mediating decidualization for successful pregnancy establishment., Competing Interests: Competing interest statement: W.L.K. is a founder, consultant, and Scientific Advisory Board member for Ribon Therapeutics, Inc. and ARase Therapeutics, Inc. He is also coholder of US Patent 9,599,606 covering the ADP-ribose detection reagent used herein, which has been licensed to and is sold by EMD Millipore.

Although ADP-ribosylation of histones by PARP-1 has been linked to genotoxic stress responses, its role in physiological processes and gene expression has remained elusive. We found that NAD + -dependent ADP-ribosylation of histone H2B-Glu35 by small nucleolar RNA (snoRNA)-activated PARP-1 inhibits AMP kinase-mediated phosphorylation of adjacent H2B-Ser36, which is required for the proadipogenic gene expression program. The activity of PARP-1 on H2B requires NMNAT-1, a nuclear NAD + synthase, which directs PARP-1 catalytic activity to Glu and Asp residues. ADP-ribosylation of Glu35 and the subsequent reduction of H2B-Ser36 phosphorylation inhibits the differentiation of adipocyte precursors in cultured cells. Parp1 knockout in preadipocytes in a mouse lineage-tracing genetic model increases adipogenesis, leading to obesity. Collectively, our results demonstrate a functional interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation that controls adipogenesis., Competing Interests: Declaration of Interests W.L.K. is a founder and consultant for Ribon Therapeutics. He is also a co-holder of U.S. Patent 9,599,606 covering the ADP-ribose detection reagents used herein, which have been licensed to and are sold by EMD Millipore. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)