title: Isl1 and Pou4f2 form a complex to regulate target genes in developing retinal ganglion cells.
abstract: Precise regulation of gene expression during biological processes, including development, is often achieved by combinatorial action of multiple transcription factors. The mechanisms by which these factors collaborate are largely not known. We have shown previously that Isl1, a Lim-Homeodomain transcription factor, and Pou4f2, a class IV POU domain transcription factor, co-regulate a set of genes required for retinal ganglion cell (RGC) differentiation. Here we further explore how these two factors interact to precisely regulate gene expression during RGC development. By GST pulldown assays, co-immunoprecipitation, and electrophoretic mobility shift assays, we show that Isl1 and Pou4f2 form a complex in vitro and in vivo, and identify the domains within these two proteins that are responsible for this interaction. By luciferase assay, in situ hybridization, and RNA-seq, we further demonstrate that the two factors contribute quantitatively to gene expression in the developing RGCs. Although each factor alone can activate gene expression, both factors are required to achieve optimal expression levels. Finally, we discover that Isl1 and Pou4f2 can interact with other POU and Lim-Homeodomain factors respectively, indicating the interactions between these two classes of transcription factors are prevalent in development and other biological processes.
authors.affiliation: Department of Ophthalmology/Ross Eye Institute, University of Buffalo, Buffalo, New York, United States of America; Developmental Genomics Group, New York State Center of Excellence in Bioinformatics and Life Sciences, University of Buffalo, Buffalo, New York, United States of America; SUNY Eye Institute, University of Buffalo, Buffalo, New York, United States of America.
authors.forename: Renzhong
authors.initials: R
authors.lastname: Li
authors.affiliation: Department of Ophthalmology/Ross Eye Institute, University of Buffalo, Buffalo, New York, United States of America; Developmental Genomics Group, New York State Center of Excellence in Bioinformatics and Life Sciences, University of Buffalo, Buffalo, New York, United States of America; SUNY Eye Institute, University of Buffalo, Buffalo, New York, United States of America.
authors.forename: Fuguo
authors.initials: F
authors.lastname: Wu
authors.affiliation: Department of Ophthalmology/Ross Eye Institute, University of Buffalo, Buffalo, New York, United States of America; Developmental Genomics Group, New York State Center of Excellence in Bioinformatics and Life Sciences, University of Buffalo, Buffalo, New York, United States of America; SUNY Eye Institute, University of Buffalo, Buffalo, New York, United States of America.
authors.forename: Raili
authors.initials: R
authors.lastname: Ruonala
authors.affiliation: Department of Ophthalmology/Ross Eye Institute, University of Buffalo, Buffalo, New York, United States of America; Department of Biochemistry, University of Buffalo, Buffalo, New York, United States of America; Developmental Genomics Group, New York State Center of Excellence in Bioinformatics and Life Sciences, University of Buffalo, Buffalo, New York, United States of America; SUNY Eye Institute, University of Buffalo, Buffalo, New York, United States of America.
authors.forename: Darshan
authors.initials: D
authors.lastname: Sapkota
authors.affiliation: Department of Ophthalmology/Ross Eye Institute, University of Buffalo, Buffalo, New York, United States of America; Department of Biostatistics, University of Buffalo, Buffalo, New York, United States of America; Department of Medicine, University of Buffalo, Buffalo, New York, United States of America; Center of Computational Research, New York State Center of Excellence in Bioinformatics and Life Sciences, University of Buffalo, Buffalo, New York, United States of America; SUNY Eye Institute, University of Buffalo, Buffalo, New York, United States of America.
authors.forename: Zihua
authors.initials: Z
authors.lastname: Hu
authors.affiliation: Department of Ophthalmology/Ross Eye Institute, University of Buffalo, Buffalo, New York, United States of America; Department of Biochemistry, University of Buffalo, Buffalo, New York, United States of America; Developmental Genomics Group, New York State Center of Excellence in Bioinformatics and Life Sciences, University of Buffalo, Buffalo, New York, United States of America; SUNY Eye Institute, University of Buffalo, Buffalo, New York, United States of America; CCSG Cancer Genetics Program, Roswell Park Cancer Institute, Buffalo, New York, United States of America.
authors.forename: Xiuqian
authors.initials: X
authors.lastname: Mu
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D024202:Electrophoretic Mobility Shift Assay; D004622:Embryo, Mammalian; D018507:Gene Expression Regulation, Developmental; D057809:HEK293 Cells; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D060850:LIM-Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011485:Protein Binding; D020218:Response Elements; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction; D050820:Transcription Factor Brn-3B / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D053823:Hedgehog Proteins; D018398:Homeodomain Proteins; D060850:LIM-Homeodomain Proteins; C085559:Pou4f2 protein, mouse; C465570:Shh protein, mouse; D050820:Transcription Factor Brn-3B; D014157:Transcription Factors; C063843:insulin gene enhancer binding protein Isl-1