Cotranscriptional pre-mRNA splicing events detected by ChIP and ChRIP assays. (A) Schematic diagram of ChIP (adapted with permission from Listerman et al³⁶ ); antibodies against splicing factors (SF) pull down chromatin regions attached to splicing factors through the nascent RNA and pol II (pol). CBC indicates cap-binding complex. (B) 4.1R DNA sequences detected in ChIP assays by the use of anti-SF2/ASF Ab. (Top panel) the 4.1R gene sequences amplified by indicated primer sets comprises the regions spanning intron upstream of exon 2′ and exon 2 (In/Ex2), exon 2′ and exon 2 (Ex2′/Ex2), and within exon 2 (Ex2/Ex2). DNA regions upstream of 1A promoter were amplified with primer sets up1/up2 and up3/up4. (Bottom panel) PCR. (C) Schematic diagram of ChRIP (adapted with permission from Listerman et al³⁶ ); antibodies against AcH4 pull down the nascent RNA attached to the active chromatin through pol II. (D) Analyses of 4.1R pre-mRNA transcripts detected in anti-AcH4 (active chromatin) or anti-H3K4me1 (silenced chromatin) antibody precipitates. (Top panel) the region of primer annealing for RT-PCR is region. (Middle panel) 4.1R pre-mRNA spanning the region between the intron upstream of exon 2′ and exon 2 detected in input lysate, anti-AcH4, or IgG precipitates in the presence or absence of RT reaction. (Bottom panel) 4.1R pre-mRNA spanning the same region detected in input lysate, anti-H3K4me1, or IgG precipitates in the presence or absence of RT reaction. (E) 4.1R mRNA detected in anti-AcH4 or anti-H3K4me1 precipitates. (Top panel) the primer at exon 17 was used for RT and amplified by primer sets located at exons 13 and 17. (Middle panel) amplified products with or without exon 16 detected in anti-AcH4 precipitates in the presence or absence of RT reaction. Input lysate and IgG precipitates served as controls. (Bottom panel) amplified products with or without exon 16 detected in anti-H3K4me1 precipitates in the presence or absence of RT reaction. Input lysate and IgG precipitates served as controls.