B1 SINE-binding ZFP266 impedes reprogramming through suppression of chromatin opening mediated by pioneering factors

Induced pluripotent stem cell reprogramming is inherently inefficient and understanding the molecular mechanisms underlying this inefficiency holds the key to successfully control cellular identity. Here, we report 16 novel reprogramming roadblock genes identified by CRISPR/Cas9-mediated genome-wide knockout (KO) screening. Of these, depletion of the predicted KRAB zinc finger protein (KRAB-ZFP) Zfp266 strongly and consistently enhanced iPSC generation in several iPSC reprogramming settings, emerging as the most robust roadblock. Further analyses revealed that ZFP266 binds Short Interspersed Nuclear Elements (SINEs) adjacent to binding sites of pioneering factors, OCT4 (POU5F1), SOX2 and KLF4, and impedes chromatin opening. Replacing the KRAB co-suppressor with a co-activator domain converted ZFP266 from a reprogramming inhibitor to a potent reprogramming facilitator. This work proposes SINE-KRAB-ZFP interaction to be a critical regulator of chromatin accessibility at enhancers for efficient cellular identity changes and also serves as a resource to further illuminate molecular mechanisms hindering reprogramming.

[1]  B. Ozkan,et al.  Dissecting OCT4 defines the role of nucleosome binding in pluripotency , 2021, Nature Cell Biology.

[2]  D. Trono,et al.  KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine lineage , 2020, eLife.

[3]  E. Zueva,et al.  Specific subfamilies of transposable elements contribute to different domains of T lymphocyte enhancers , 2020, Proceedings of the National Academy of Sciences.

[4]  N. Luscombe,et al.  Nucleosome positioning stability is a modulator of germline mutation rate variation across the human genome , 2020, Nature Communications.

[5]  Xiaohua Shen,et al.  Genomic Repeats Categorize Genes with Distinct Functions for Orchestrated Regulation , 2020, Cell reports.

[6]  K. Woltjen,et al.  N-Terminal Amino Acids Determine KLF4 Protein Stability in 2A Peptide-Linked Polycistronic Reprogramming Constructs , 2020, Stem cell reports.

[7]  Ahmed E. Radwan,et al.  Direct Induction of the Three Pre-implantation Blastocyst Cell Types from Fibroblasts , 2019, Cell stem cell.

[8]  R. Jaenisch,et al.  Hominoid-Specific Transposable Elements and KZFPs Facilitate Human Embryonic Genome Activation and Control Transcription in Naive Human ESCs , 2019, Cell stem cell.

[9]  G. Bourque,et al.  Ten things you should know about transposable elements , 2018, Genome Biology.

[10]  Gordon K Smyth,et al.  The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads , 2018, bioRxiv.

[11]  Xiaohua Shen,et al.  A LINE1-Nucleolin Partnership Regulates Early Development and ESC Identity , 2018, Cell.

[12]  S. Linnarsson,et al.  Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming , 2017, Cell stem cell.

[13]  Yixuan Wang,et al.  The Role of KRAB-ZFPs in Transposable Element Repression and Mammalian Evolution. , 2017, Trends in genetics : TIG.

[14]  R. Nitsch,et al.  CRISPR-UMI: single-cell lineage tracing of pooled CRISPR–Cas9 screens , 2017, Nature Methods.

[15]  Nicholas A. Sinnott-Armstrong,et al.  An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues , 2017, Nature Methods.

[16]  D. Trono,et al.  KRAB zinc finger proteins , 2017, Development.

[17]  Boon Siang Nicholas Tan,et al.  Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo , 2017, bioRxiv.

[18]  D. Trono,et al.  KRAB zinc-finger proteins contribute to the evolution of gene regulatory networks , 2017, Nature.

[19]  W. Skarnes,et al.  Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells , 2017, Development.

[20]  J. Ernst,et al.  Cooperative Binding of Transcription Factors Orchestrates Reprogramming , 2017, Cell.

[21]  Julio Saez-Rodriguez,et al.  A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia , 2016, Cell reports.

[22]  Fidel Ramírez,et al.  deepTools2: a next generation web server for deep-sequencing data analysis , 2016, Nucleic Acids Res..

[23]  Helen M. Rowe,et al.  Transposable Elements and Their KRAB-ZFP Controllers Regulate Gene Expression in Adult Tissues. , 2016, Developmental cell.

[24]  S. Linnarsson,et al.  Reprogramming Roadblocks Are System Dependent , 2015, Stem cell reports.

[25]  G. Pan,et al.  The oncogene c-Jun impedes somatic cell reprogramming , 2015, Nature Cell Biology.

[26]  William Stafford Noble,et al.  The MEME Suite , 2015, Nucleic Acids Res..

[27]  M. Pellegrini,et al.  Pioneer Transcription Factors Target Partial DNA Motifs on Nucleosomes to Initiate Reprogramming , 2015, Cell.

[28]  S. Yamanaka,et al.  KLF4 N-Terminal Variance Modulates Induced Reprogramming to Pluripotency , 2015, Stem cell reports.

[29]  Matthew E. Ritchie,et al.  limma powers differential expression analyses for RNA-sequencing and microarray studies , 2015, Nucleic acids research.

[30]  A. Brunet,et al.  Inhibition of pluripotency networks by the Rb tumor suppressor restricts reprogramming and tumorigenesis. , 2015, Cell stem cell.

[31]  Ron Weiss,et al.  Highly-efficient Cas9-mediated transcriptional programming , 2014, Nature Methods.

[32]  Jun S. Liu,et al.  MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens , 2014, Genome Biology.

[33]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[34]  S. Yamanaka,et al.  Dynamic regulation of human endogenous retroviruses mediates factor-induced reprogramming and differentiation potential , 2014, Proceedings of the National Academy of Sciences.

[35]  T. Rana,et al.  Genome-wide functional analysis reveals factors needed at the transition steps of induced reprogramming. , 2014, Cell reports.

[36]  Yilong Li,et al.  Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library , 2013, Nature Biotechnology.

[37]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[38]  Greg Donahue,et al.  Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome , 2012, Cell.

[39]  Muneef Ayyash,et al.  The H3K27 demethylase Utx regulates somatic and germ cell epigenetic reprogramming , 2012, Nature.

[40]  Eric S. Lander,et al.  Chromatin modifying enzymes as modulators of reprogramming , 2012, Nature.

[41]  Marcel Martin.  Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[42]  A. Bradley,et al.  A hyperactive piggyBac transposase for mammalian applications , 2011, Proceedings of the National Academy of Sciences.

[43]  Helga Thorvaldsdóttir,et al.  Integrative Genomics Viewer , 2011, Nature Biotechnology.

[44]  C. Glass,et al.  Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. , 2010, Molecular cell.

[45]  Aaron R. Quinlan,et al.  BIOINFORMATICS APPLICATIONS NOTE , 2022 .

[46]  Matthew D. Young,et al.  Gene ontology analysis for RNA-seq: accounting for selection bias , 2010, Genome Biology.

[47]  Yun-Fai Chris Lau,et al.  Epigenetic Gene Silencing by the SRY Protein Is Mediated by a KRAB-O Protein That Recruits the KAP1 Co-repressor Machinery* , 2009, The Journal of Biological Chemistry.

[48]  C. Pereira,et al.  Senescence impairs successful reprogramming to pluripotent stem cells. , 2009, Genes & development.

[49]  Manuel Serrano,et al.  A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity , 2009, Nature.

[50]  M. Blasco,et al.  The Ink4/Arf locus is a barrier for iPS cell reprogramming , 2009, Nature.

[51]  J. Utikal,et al.  Immortalization eliminates a roadblock during cellular reprogramming into iPS cells , 2009, Nature.

[52]  T. Ichisaka,et al.  Suppression of induced pluripotent stem cell generation by the p53–p21 pathway , 2009, Nature.

[53]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[54]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[55]  Wei Wang,et al.  piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells , 2009, Nature.

[56]  K. Woltjen,et al.  Virus free induction of pluripotency and subsequent excision of reprogramming factors , 2009, Nature.

[57]  Xiaolei Yin,et al.  Two supporting factors greatly improve the efficiency of human iPSC generation. , 2008, Cell stem cell.

[58]  Clifford A. Meyer,et al.  Model-based Analysis of ChIP-Seq (MACS) , 2008, Genome Biology.

[59]  Bas van Steensel,et al.  Detection of in vivo protein–DNA interactions using DamID in mammalian cells , 2007, Nature Protocols.

[60]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[61]  H. Thiesen,et al.  Krüppel-associated boxes are potent transcriptional repression domains. , 1994, Proceedings of the National Academy of Sciences of the United States of America.