title: Kaiso mediates human ICR1 methylation maintenance and H19 transcriptional fine regulation.
abstract: BACKGROUND
  Genomic imprinting evolved in a common ancestor to marsupials and eutherian mammals and ensured the transcription of developmentally important genes from defined parental alleles. The regulation of imprinted genes is often mediated by differentially methylated imprinting control regions (ICRs) that are bound by different proteins in an allele-specific manner, thus forming unique chromatin loops regulating enhancer-promoter interactions. Factors that maintain the allele-specific methylation therefore are essential for the proper transcriptional regulation of imprinted genes. Binding of CCCTC-binding factor (CTCF) to the IGF2/H19-ICR1 is thought to be the key regulator of maternal ICR1 function. Disturbances of the allele-specific CTCF binding are causative for imprinting disorders like the Silver-Russell syndrome (SRS) or the Beckwith-Wiedemann syndrome (BWS), the latter one being associated with a dramatically increased risk to develop nephroblastomas.
  
  
  METHODS
  Kaiso binding to the human ICR1 was detected and analyzed by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA). The role of Kaiso-ICR1 binding on DNA methylation was tested by lentiviral Kaiso knockdown and CRISPR/Cas9 mediated editing of a Kaiso binding site.
  
  
  RESULTS
  We find that another protein, Kaiso (ZBTB33), characterized as binding to methylated CpG repeats as well as to unmethylated consensus sequences, specifically binds to the human ICR1 and its unmethylated Kaiso binding site (KBS) within the ICR1. Depletion of Kaiso transcription as well as deletion of the ICR1-KBS by CRISPR/Cas9 genome editing results in reduced methylation of the paternal ICR1. Additionally, Kaiso affects transcription of the lncRNA H19 and specifies a role for ICR1 in the transcriptional regulation of this imprinted gene.
  
  
  CONCLUSIONS
  Kaiso binding to unmethylated KBS in the human ICR1 is necessary for ICR1 methylation maintenance and affects transcription rates of the lncRNA H19.
authors.affiliation: Centre for Paediatrics and Adolescent Medicine, University Medical Centre, Langenbeckstr. 1, 55101 Mainz, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Bohne
authors.affiliation: Centre for Paediatrics and Adolescent Medicine, University Medical Centre, Langenbeckstr. 1, 55101 Mainz, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Langer
authors.affiliation: Centre for Paediatrics and Adolescent Medicine, University Medical Centre, Langenbeckstr. 1, 55101 Mainz, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Martiné
authors.affiliation: Centre for Paediatrics and Adolescent Medicine, University Medical Centre, Langenbeckstr. 1, 55101 Mainz, Germany.
authors.forename: Claudia S
authors.initials: CS
authors.lastname: Eider
authors.affiliation: Department of Biochemistry and the Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, Quebec H3G 1Y6 Canada.
authors.forename: Regina
authors.initials: R
authors.lastname: Cencic
authors.affiliation: Institute of Human Genetics, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Begemann
authors.affiliation: Institute of Human Genetics, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany.
authors.forename: Miriam
authors.initials: M
authors.lastname: Elbracht
authors.affiliation: Institute of Human Genetics, University Medical Centre, Langenbeckstr. 1, 55101 Mainz, Germany.
authors.forename: Luzie
authors.initials: L
authors.lastname: Bülow
authors.affiliation: Institute of Human Genetics, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Eggermann
authors.affiliation: Institute of Human Genetics, University Medical Centre, Langenbeckstr. 1, 55101 Mainz, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Zechner
authors.affiliation: Department of Biochemistry and the Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, Quebec H3G 1Y6 Canada.
authors.forename: Jerry
authors.initials: J
authors.lastname: Pelletier
authors.affiliation: Centre for Paediatrics and Adolescent Medicine, University Medical Centre, Mathildenstr. 1, 79106 Freiburg, Germany.
authors.forename: Bernhard Ulrich
authors.initials: BU
authors.lastname: Zabel
authors.affiliation: Centre for Paediatrics and Adolescent Medicine, University Medical Centre, Langenbeckstr. 1, 55101 Mainz, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Enklaar
authors.affiliation: Centre for Paediatrics and Adolescent Medicine, University Medical Centre, Langenbeckstr. 1, 55101 Mainz, Germany.|Centre for Paediatrics and Adolescent Medicine, University Medical Centre, Obere Zahlbacher Str. 63, 55131 Mainz, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Prawitt
mesh_terms: D001665:Binding Sites; D002478:Cells, Cultured; D019175:DNA Methylation; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018392:Genomic Imprinting; D057809:HEK293 Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D062085:RNA, Long Noncoding / Q000737:chemistry / Q000235:genetics*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D014157:Transcription Factors; C492254:ZBTB33 protein, human