Kinetics and mechanisms of Ikaros-mediated transcriptional regulation

Abstract

The Ikaros family of zinc finger transcription factors is essential for B cell development, and frequently mutated in B cell malignancies. Our lab has previously identified Ikaros target genes in pre-B cells by combining Ikaros ChIP-seq binding data and gene expression profiling. To address the kinetics and mechanisms of Ikaros-mediated transcriptional regulation, I have used an inducible Ikaros system, which allows for the monitoring of cellular and molecular changes during Ikaros-mediated gene silencing at high temporal resolution. Within minutes of Ikaros induction, the Ikaros-regulated model loci Igll1 and Myc showed decreased promoter accessibility and RNA polymerase II (RNAPII) occupancy. These early events were followed by changes in nucleosome composition, including an increased histone H2B/H3 ratio, the deposition of the histone variant H2A.Z, and decreased active histone acetylation marks. Histone deacetylation was not required to initiate down-regulation of Igll1 and Myc transcription, since treatment with the histone deacetylase inhibitor Trichostatin A did not interfere with Ikaros-mediated gene silencing. I next elucidated the mechanistic relationship between the early events of decreased promoter accessibility and decreased RNAPII occupancy. Addition of Triptolide resulted in the removal of RNAPII from the Igll1 and Myc promoters, but did not affect nucleosome occupancy and its regulation mediated by Ikaros. This suggested that Ikaros regulates nucleosome positioning and occupancy directly, and not through effects on RNAPII. Consistent with this hypothesis, Ikaros-mediated gene silencing was delayed by RNAi-mediated knockdown of chromatin remodeler Mi-2β (Chd4), the ATPase subunit of the Mi-2/NuRD complex. Hence, Ikaros-initiated chromatin remodelling was identified as one of the earliest events during Ikaros-mediated gene silencing, and was required for rapid transcriptional down-regulation of Ikaros target genes.