E Lella for logistical support, Simon Correa and Kebbah Konteh for microscopy expertise; Julie Furze for laboratory assistance; Philip Bejon for advice on modelling parasite growth rates; Melissa Kapulu for assistance in qPCR and all the study participants. We thank the Sanaria Manufacturing, Quality Systems, Legal, and Operations Teams including Tao Li, Adam Richman, Abraham G. Eappen, Minglin Li, Adam Ruben, Anita Manoj, Alexander Hoffman, Robert Thompson, and Richard E. Stafford.Author ContributionsConceived and designed the experiments: SHS AJS RJL SLH AVSH. Performed the experiments: SHS AJS RJL NJE DK ARW IDP NAA. Analyzed the data: SHS AJS ADD RJL NJE MV. Contributed reagents/ materials/analysis tools: ERJ BKLS PFB SLH. Wrote the paper: SHS ADD. Project Management Quality Assurance: AL RR SK ERJ BKLS PFB AG.Mosquito Bite CHMI at same centre. Red dots: qPCRmeasured parasite density for each individual subject in current trial and unimmunised control subjects from three previous
Mammalian zinc finger protein 423 (mouse Zfp423, human ZNF423) is a transcriptional regulator important to development and disease. Mutations of Zfp423 in mice produce severe midline defects in developing brain, most notably loss of the cerebellar vermis [1?], as well as abnormalities in olfactory neurons [4] and brown fat [5]. The severity of these defects is highly variable and influenced by both modifier genes and non-genetic factors [6]. Germline mutations in human ZNF423 result in a range of nephronophthisis-related ciliopathy (NPHP-RC) phenotypes, including characteristic defects in cerebellar vermis and kidney, with cellular deficits in DNA damage response [7]. ZNF423 may also play a role in human cancers. Epigenetic loss or reduction of ZNF423 expression in human neuroblastoma corresponds with lower response to retinoic acid therapy [8] and ectopic activation of Zfp423 in bone marrow cells induced B-cell leukemia in a mouse model [9]. Zfp423 is composed of 30 C2H2 zinc fingers, clustered into multi-finger domains reported to bind DNA or other transcription factors. Zfp423 (also known as ROAZ, OAZ, or Ebfaz) was first identified as a binding partner that inhibits Early B-cell factor (EBF, also known as Olf1) subfamily of basic helix-loop-helix transcription factors through its last three 23148522 zinc fingers [10,11].Subsequent studies from a variety of contexts have identified additional interactions with transcription factors, including BMPactivated SMAD proteins [12], retinoic acid receptor RARb [8], Notch intracellular domain [13], and DNA damage response related proteins, including poly (ADP-ribose) polymerase PARP1 [14] centrosomal protein CEP290 [15]. Several of these interactions are mutually inhibitory [12,13]. Zfp423 has been proposed to regulate several target genes dependent on specific binding partners with their own DNA binding domains. Whether direct DNA binding by Zfp423 is required at the majority of these sites is not known. In order to Title Loaded From File identify direct targets of Zfp423, we initiated an in silico strategy based on cross-species conservation of clustered consensus Title Loaded From File motifs [16] to predict candidate target sites. We used chromatin immunoprecipitation (ChIP), quantitative PCR (qPCR) and massively parallel sequencing to test occupancy of predicted sites in a standard cell culture model. Surprisingly, we found enrichment of consensus sites in or near genes encoding Zfp423, its paralog Zfp521, and two of four Ebf genes. Each of two sites identified.E Lella for logistical support, Simon Correa and Kebbah Konteh for microscopy expertise; Julie Furze for laboratory assistance; Philip Bejon for advice on modelling parasite growth rates; Melissa Kapulu for assistance in qPCR and all the study participants. We thank the Sanaria Manufacturing, Quality Systems, Legal, and Operations Teams including Tao Li, Adam Richman, Abraham G. Eappen, Minglin Li, Adam Ruben, Anita Manoj, Alexander Hoffman, Robert Thompson, and Richard E. Stafford.Author ContributionsConceived and designed the experiments: SHS AJS RJL SLH AVSH. Performed the experiments: SHS AJS RJL NJE DK ARW IDP NAA. Analyzed the data: SHS AJS ADD RJL NJE MV. Contributed reagents/ materials/analysis tools: ERJ BKLS PFB SLH. Wrote the paper: SHS ADD. Project Management Quality Assurance: AL RR SK ERJ BKLS PFB AG.Mosquito Bite CHMI at same centre. Red dots: qPCRmeasured parasite density for each individual subject in current trial and unimmunised control subjects from three previous
Mammalian zinc finger protein 423 (mouse Zfp423, human ZNF423) is a transcriptional regulator important to development and disease. Mutations of Zfp423 in mice produce severe midline defects in developing brain, most notably loss of the cerebellar vermis [1?], as well as abnormalities in olfactory neurons [4] and brown fat [5]. The severity of these defects is highly variable and influenced by both modifier genes and non-genetic factors [6]. Germline mutations in human ZNF423 result in a range of nephronophthisis-related ciliopathy (NPHP-RC) phenotypes, including characteristic defects in cerebellar vermis and kidney, with cellular deficits in DNA damage response [7]. ZNF423 may also play a role in human cancers. Epigenetic loss or reduction of ZNF423 expression in human neuroblastoma corresponds with lower response to retinoic acid therapy [8] and ectopic activation of Zfp423 in bone marrow cells induced B-cell leukemia in a mouse model [9]. Zfp423 is composed of 30 C2H2 zinc fingers, clustered into multi-finger domains reported to bind DNA or other transcription factors. Zfp423 (also known as ROAZ, OAZ, or Ebfaz) was first identified as a binding partner that inhibits Early B-cell factor (EBF, also known as Olf1) subfamily of basic helix-loop-helix transcription factors through its last three 23148522 zinc fingers [10,11].Subsequent studies from a variety of contexts have identified additional interactions with transcription factors, including BMPactivated SMAD proteins [12], retinoic acid receptor RARb [8], Notch intracellular domain [13], and DNA damage response related proteins, including poly (ADP-ribose) polymerase PARP1 [14] centrosomal protein CEP290 [15]. Several of these interactions are mutually inhibitory [12,13]. Zfp423 has been proposed to regulate several target genes dependent on specific binding partners with their own DNA binding domains. Whether direct DNA binding by Zfp423 is required at the majority of these sites is not known. In order to identify direct targets of Zfp423, we initiated an in silico strategy based on cross-species conservation of clustered consensus motifs [16] to predict candidate target sites. We used chromatin immunoprecipitation (ChIP), quantitative PCR (qPCR) and massively parallel sequencing to test occupancy of predicted sites in a standard cell culture model. Surprisingly, we found enrichment of consensus sites in or near genes encoding Zfp423, its paralog Zfp521, and two of four Ebf genes. Each of two sites identified.
